The disordered region 1 of Mdm2 weakens p53-binding in both its unmodified and hyperphospho-forms

The disordered region 1 of Mdm2 weakens p53-binding in both its unmodified and hyperphospho-forms | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article The disordered region 1 of Mdm2 weakens p53-binding in both its unmodified and hyperphospho-forms François-Xavier Theillet, Yingyue Luo, Chafiaa Bouguechtouli, and 3 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-9014206/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted You are reading this latest preprint version Abstract Mdm2, the main negative regulator of the "tumor-suppressor" p53, is an attractive target for anti-cancer treatments, but the molecular mechanisms of its action are still elusive. Indeed, producing purified samples for its structural analysis is difficult: Mdm2 is a multidomain protein, alternating folded and disordered regions, the latter being moreover natively hyperphosphorylated. Here, we report the structural study of Mdm2 constructs including its folded p53-binding domain (p53-BD) and Zinc-Finger, and their spacer, a 190-residue long disordered region (IDR1) hyperphosphorylated by CK1. Using NMR spectroscopy, we revealed that IDR1 establish ultraweak intramolecular contacts with p53-BD in the μs timescale, which provokes the partial release of the N-terminal tail from the p53-binding groove of Mdm2. Then, using ITC, we established that these Mdm2 intramolecular interactions decrease the affinity for p53 by up to 25-fold compared to the isolated p53-binding domain. Surprisingly, the identified 15 CK1-established phosphosites between Mdm2-S192 and -S286 do not improve the affinity for p53 despite their binding to the positively charged DNA-binding domain. Structural studies on multidomain proteins are scarce, and even more rare on natively phosphorylated species. This report shows how disordered regions can interfere with folded domains, and markedly affect binding between cognate cellular partners. Biological sciences/Structural biology/NMR spectroscopy/Solution-state NMR Biological sciences/Biophysics/Intrinsically disordered proteins Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Introduction The E3-ubiquitin ligase Mdm2 is an oncoprotein overexpressed in many different types of cancers 1,2 . Its most famous function is to be the main negative regulator of the transcription factor p53, the so-called "tumor suppressor" found mutated in about half of all cancers 3,4 . Given their importance, these proteins have been extensively studied, generating a wealth of information on their partners, activities and regulation. The sum of this knowledge is often summarized by the words "complexity and context" by cell biologists 4,5 . From the structural biology standpoint, the complexity has been simplified. These proteins have been dissected, studied domain by domain, even though p53 has been characterized eventually in its full-length version using stabilizing mutations in its DNA-binding domain 6,7 . Unfortunately, protocols have been missing to produce milligram-scale samples for structural biology of Mdm2 constructs including more than one domain, to the best of our knowledge. The N-terminal p53-binding domain (p53BD) of Mdm2 (aa26-109, Mdm2-p53BD) has focused the attention of the community: it plays an anchoring role in p53-tethering, an interaction whose inhibition has raised many hopes for treatments 8 . Much less structural biology reports exist on the intrinsically disordered regions (IDRs) intercalated between the folded p53BD, the central Zinc-finger (aa299-328, Mdm2-ZF) binding immature ribosome particles 9 , and the C-terminal E3-ubiquitin effector Mdm2-RING domain 7 . We sought to provide structural information on Mdm2-IDR1 (aa110-298) and its possible contacts with its surrounding folded domains, i.e. the Mdm2-p53BD and the Mdm2-ZF. This follows our earlier report on the IDR2 of Mdm2 (aa329-438) and the mapping of its phosphorylation sites by DNA-PK/ATM/ATR 10 . Mdm2-IDR1 is fairly conserved among vertebrates for an IDR, especially on its acidic domain (AD, aa220-296, ~45% identity between human and zebrafish) containing 28 Asp/Glu (Supplementary Fig. 1) 11 . Mdm2-AD's structural behavior has been scarcely characterized experimentally 12–14 , confirming by NMR the prediction of a disordered peptide behavior. We can also mention a few crystal structures of IDR1's short peptides : aa145-150 and aa225-229 both extended in complex with the deubiquitinase USP7 15,16 , and aa164-167(pS166) and aa183-188(pS186) both extended in complex with 14-3-3s 17,18 . IDR1 has nevertheless important roles in Mdm2 activities: i) it contains both a nuclear localization sequence NLS(aa179-186) and a nuclear export signal NES(aa190-202); ii) the AD plays an important auxiliary role in p53 binding 7,19 ; iii) it has been reported to interact directly with multiple noteworthy partners, among which the Polycomb Repressor Complex 2 20 , PARP-1 21 , the tumor suppressor p14ARF 13 , the ubiquitin-ligase complex SCF 22 , tau 23 , or b-arrestin 1 24 . Moreover, the corresponding IDR1 of the homolog MdmX has been shown to establish intramolecular contacts with its own p53BD 25–27 and even with the Mdm2-p53BD 28 , hence competing in binding with p53 N-terminal (p53-NT, aa17-29). These contacts engage the so-called WW and WF motifs present in MdmX-IDR1 but absent in Mdm2-IDR1; hydrophobic motifs appear at similar localizations in Mdm2-IDR1 though (Supplementary Fig. 2). At the opposite, Mdm2-IDR1 has been shown to be a secondary binding site of p53, whose affinity has been reported to be tuned by phosphorylation 22,29–35 -the Mdm2-p53BD remaining the primary anchor for tethering p53. A structural understanding of all these conflicting interactions is still missing, to the best of our knowledge. Studying Mdm2 constructs containing IDR1 has certainly been impeded by the fact that its native form is hyperphosphorylated in absence of stress, which makes the production of pure samples more difficult. According to the literature, Mdm2-IDR1's phosphorylation improves its binding to p53, while DNA-damage was reported to provoke a hypophosphorylation of Mdm2-IDR1 favoring p53 stabilization 29–32,34,35 . The corresponding phosphorylation schemes have not been intensively mapped, probably because the amino acid composition of the Mdm2-AD hampers a proper coverage by mass-spectrometry proteomics (see https://www.uniprot.org/uniprotkb/Q00987/feature-viewer). NMR spectroscopy is an appropriate complementary technique is such cases, not only because of its ability to provide residue-specific information on IDPs 36–38 , but also because of its capacities in poly-phosphorylation mapping and monitoring 10,39–43 . Here, we report our structural investigations on Mdm2-IDR1, either isolated or in more relevant contexts, i.e. in constructs containing also its neighbor folded domains Mdm2-p53BD and Mdm2-ZF. We carried out parallel studies on the structural behavior of IDR1 in the various constructs and on the resulting affinities for p53, using NMR and ITC, respectively. We also characterized the hyperphosphorylation executed by CK1, the main kinase active on Mdm2 in absence of stress, and its consequences on Mdm2 structure and interaction with p53. Because these peptides carry abundant electrostatic charges, we performed all experiments in phosphate-buffered saline (PBS) to favor the adoption of close-to-native conformational and binding behaviors. Results Mdm2-IDR1 is disordered before and after phosphorylation by CK1 d on 15 sites First, we verified experimentally the predicted intrinsic disorder of Mdm2-IDR1 in constructs of increasing sizes, from aa111-180, aa111-230, aa111-293, aa111-333, aa1-333, i.e. incorporating progressively segments of human Mdm2 including the folded Mdm2-p53BD(aa26-109) and the Mdm2-ZF(aa296-333) (Fig. 1a). We started using 1 H- 15 N NMR at 283 K, a standard approach to obtain residue-specific information. The low temperature limits water-amide 1 H-exchange, which is too fast at 310 K in IDRs to obtain exploitable 1 H- 15 N spectra 10 . Interestingly the folded Mdm2-p53BD was invisible and the Mdm2-ZF barely detectable in Mdm2(aa1-333). Mdm2-IDR1 residues were all in the random coil window of chemical shifts (Fig. 1b), which was later confirmed by the backbone assignment giving access to the Ca-Cb chemical shifts and the associated secondary structure propensities (Fig. 1c, Supplementary Fig. 3) 44 . We used cysteine-to-alanine mutants for the longest constructs, ensuring their production and stability during the hours-long NMR acquisitions: even in presence of DTT or TCEP, the 4 cysteines of IDR1 were rapidly forming multiple disulfide bonds, and the wild-type Mdm2 constructs were expressed mainly as aggregates. We verified that the four cysteine mutations C127A-C200A-C206-C207A in Mdm2-IDR1 did not alter its disordered behavior (Supplementary Fig. 3). We achieved the NMR backbone assignment of Mdm2-IDR1 with and without cysteine mutations (BMRB codes: 52016 for Mdm2(aa111-293), 52028 for Mdm2(aa111-230_4C4A)). We generated a few point mutations and recorded the resulting spectra to confirm the assignment (Supplementary Fig. 4-5). Then, we carried out the phosphorylation of Mdm2-IDR1 by CK1d. The native polyphosphorylation of IDR1 has been reported to be executed by CK1, CK2 and GSK-3b 22,29–32,34,35 : these kinases are attracted by negatively charged residues, which are abundant in Mdm2-IDR1. We decided to use the isoform CK1d, because i) the recombinant production of an active form of this kinase is feasible in sufficient quantities for NMR structural studies 41,45 , and ii) the literature was suggesting it to be dominant in establishing Mdm2-IDR1 polyphosphorylation 22,29–32,34,35 . Using 1 H- 15 N NMR correlation spectroscopy, we detected 15 phosphosites upon incubation of 15 N-Mdm2-IDR1 with an "NMR-invisible" 14 N-CK1d (Figure 1a-1b). We mapped them by assigning the cross-peaks of phospho-Mdm2-IDR1 ( 13 C/ 15 N labeled version, BMRB code: 52289) after its purification and separation from CK1d, whose loose binding provokes otherwise poor-quality spectra. The identified phosphosites were S192, S215, T218, S220, S229, S232, S240, S242, S246, S253, S256, S260, S262, T279, and S286. This shows that CK1d is attracted by the negatively charged region of Mdm2-IDR1, more than by its classical consensus motifs pS/pT/D/E-x-x-S/T (Figure 1a) 46,47 . We noticed some supplementary very weak crosspeaks from minor phosphosites, which we could not assign due to low signal levels except S166. Then, we monitored the reaction in real-time, recording time-series of 1 H- 15 N NMR spectra. To extract site-specific information, we also had to assign cross-peaks appearing transiently over time: these are due to intermediate species containing close, non-stoichiometric phosphosites, which can generate multiple combinations of chemical environments 10,39–43 . We met some cumbersome cases with 3 close phosphosites, which forced us to obtain the assignments from synthesized peptides containing the various combinations of one or two phosphosites (Supplementary Fig. 6a). Altogether, we observed that all sites were phosphorylated in an independent, distributive manner. We also recorded phosphorylation kinetics of Ser/Thr to Ala mutants, which provided supplementary confirmations of the assignments (Supplementary Fig. 7). These mutants exhibited often slower phosphorylation rates in the vicinity of the mutated residue (Supplementary Fig. 8a). Nevertheless, the final phosphorylation states remained unaffected, indicating that phosphorylation at a given site is not a prerequisite for phosphorylation at adjacent sites, and supporting a distributive mechanism for CK1δ on Mdm2-IDR1. We grouped the phosphosites arbitrarily into five clusters with similar kinetics, from the fastest to the slowest ones: S192-S215–S242–S246–S253, S220–S229–S232–S240–S256, T218–S260, S262, and S192-T279–S286 (Fig. 1d, Supplementary Fig. 8b). The backbone assignment of phospho-Mdm2-IDR1 (BMRB 52289) provided also the 13 Ca- 13 Cb chemical shifts, which, in turn, gave access to the secondary structure content (Fig. 1c). Interestingly, the hyperphosphorylated Mdm2-IDR1 shows more extended average backbone conformations than the non-phosphorylated form, which is probably due to electrostatic repulsion. Hence, the Mdm2-AD remains highly flexible and disordered upon phosphorylation, but behaves less like a random coil than its non-phospho counterpart. The rest of Mdm2-IDR1 did not show any sign of conformational changes upon phosphorylation and remained highly disordered. Overall, the isolated Mdm2-IDR1 behaves as a classical IDR before and after phosphorylation by CK1d. This phosphorylation is distributive, with no conditional priming event, and occurs preferentially on the segments containing the highest densities of Asp/Glu of Mdm2-IDR1. NMR evidence for intramolecular interactions between Mdm2-IDR1 and Mdm2-p53BD Next, we incorporated the N-terminal Mdm2-p53BD in our analysis, by producing a longer construct Mdm2(aa1-333). We noticed immediately that some peak intensities were weaker in 1 H- 15 N NMR spectra (Fig. 2a-2b). Two segments were concerned: the most perturbed one around the region aa189-208, and a second one, a bit less affected, around aa242-255. These are the most hydrophobic segments of Mdm2-IDR1. The residue-specific relaxation parameters 15 N-R 1 , and 1 H- 15 N heteronuclear nOes did almost not change between Mdm2-IDR1 and Mdm2(aa1-333), revealing comparable conformational dynamics of Mdm2-IDR1 in both constructs at the nanosecond timescale (Supplementary Fig. 9a). The 15 N-R 2 relaxation rates showed a diffuse increase all along IDR1 in Mdm2(aa1-333), with very high rates between aa195 and aa205 (actually even non-measurable), and around aa250 (Fig. 2c). This differential variations between 1 H- 15 N nOes and 15 N-R 2 are only consistent with transient intramolecular interactions between the segment aa190-210 and Mdm2-p53BD at the ms-ms timescale, together with weaker transient interactions between the rest of Mdm2-IDR1 and Mdm2-p53BD. To confirm this hypothesis, we mixed 15 N-Mdm2(aa1-333) and an "NMR-invisible" 14 N-p53(aa14-29) in stoichiometric proportions. The latter peptide is the core binding element of p53 to Mdm2-p53BD. We observed the recovery of peak intensities from Mdm2-IDR1 residues (Fig. 2b-2d), which was consistently accompanied by a decrease of 15 N-R 2 rates almost back to the values obtained from Mdm2-IDR1 isolated (Fig. 2c). We conclude that the transient intramolecular interactions occur mainly between the hydrophobic segments of Mdm2-IDR1 and the hydrophobic groove of Mdm2-p53BD, the latter of which has still a much better affinity for its cognate partner p53(aa14-29). Out of curiosity, we evaluated the affinity between Mdm2-p53BD and Mdm2-IDR1 in an artificial situation, where they would be present as separate species. We titrated 15 N-Mdm2-IDR1 at 100 mM with 14 N-Mdm2(aa1-112), which revealed the same loose binding patches, i.e. aa195-205 and aa245-255 (Supplementary Fig. 10). According to the peak intensity losses and the extreme weak chemical shift perturbations (<0.06 ppm) evolving linearly up to [ 14 N-Mdm2(aa1-112)]=200 mM, the transient interactions occur in the ms-ms scale with affinities above 100 mM. Phosphorylation does not disrupt intramolecular interactions between Mdm2-IDR1 and Mdm2-p53BD Then, we carried out the same investigations on phospho-Mdm2(aa1-333). First, we monitored the phosphorylation kinetics of Mdm2(aa1-333) by CK1d, which turned out to be very similar to those obtained from Mdm2-IDR1 in isolation (Supplementary Fig. 8c). The 1 H- 15 N NMR spectra of phospho-Mdm2(aa1-333) and the relaxation parameters 15 N-R 1 , 15 N-R 2 and 1 H- 15 N heteronuclear nOes showed high similarities with the non-modified form (Fig. 2e-2f, Supplementary Fig. 9b). They revealed transient interactions at the ms-ms timescale between the Mdm2-p53BD and the segment Mdm2(aa190-210), with some weaker transient touches between Mdm2-p53BD and the rest of Mdm2-IDR1. Hence, CK1d-phosphorylated Mdm2-IDR1 establishes intramolecular contacts between its most hydrophobic segments and the p53-binding groove of Mdm2-p53BD. These do not change the phosphorylation kinetics by CK1d, nor does the resulting hyperphosphorylation affect these intramolecular interactions. Mdm2-IDR1 reverts and accelerates the "open & closed" equilibrium of the N-ter lid with the Mdm2-p53BD The characterization reported above was limited to the disordered Mdm2-IDR1. This is due to the fact that Mdm2-p53BD is invisible in 1 H- 15 N NMR spectra of Mdm2(aa1-333). Mdm2-p53BD has been regularly studied by 1 H- 15 N NMR in isolation by many labs including ours 48,49 , but its tumbling time is apparently very much slowed down in a more native context including IDR1, which provokes a very fast R 2 relaxation and a vanishing signal. This is consistent with the multiple intramolecular interactions that we detected, see above. We thought to produce a perdeuterated protein construct Mdm2(aa1-333) with only 13 CH 3 methyl labeling: 1 H- 13 C methyl NMR in a perdeuterated context can provide exploitable signals even for slow-tumbling species up to the MDa size 50 . This approach permitted recently to Nishida and colleagues to investigate the open-closed equilibrium of Mdm2-p53BD governed by the flexible N-terminal loop Mdm2(aa1-24), so called the "lid" 51 . We adopted their strategy and produced deuterated Mdm2(aa1-333) incorporating Ile d 1 -Met e ( 13 CH 3 ) labeling, and recorded 1 H- 13 C SOFAST-HMQC methyl TROSY spectra at 310 K. We paid a particular attention to the signals from Ile19 in the lid: Ile19 is a good reporter of the equilibrium between the open flexible state of the lid, and the closed state where it binds to the p53-binding groove of Mdm2-p53BD. In presence of p53(aa14-29), the lid is pushed away from the Mdm2-p53BD binding groove and is found 100% in the open state, yielding a 1 H- 13 C correlation spectrum matching the one published recently from Mdm2(aa1-109) by Nishida et al. 51 (Fig. 3a). In contrast, we observed strong differences between Mdm2(aa1-109) and Mdm2(aa1-333) in their apo forms. While Nishida and colleagues reported a 20:80 open:closed equilibrium in slow exchange (~1 Hz or less) for their construct Mdm2(aa1-109), the peaks of Ile19 and Met50 revealed a 70:30 ratio and a fast exchange (~10 6 Hz or more) in Mdm2(aa1-333) at 310 K (Fig. 3a-3b). Decreasing the temperature favored the displacement of these two peaks towards the chemical shifts of the closed state, but also their progressive disappearance (Fig. 3c). This corresponds to the exchange slowing down to an intermediate timescale (~10 3 -10 6 Hz), starting from 306 K. This is consistent with the interactions observed on the Mdm2-IDR1 side using 1 H- 15 N NMR at 283 K (see above). Interestingly, the peaks from Ile74 or Met102 shifted with the temperature in apo-Mdm2(aa1-333) (Fig. 3c), whereas they were not in apo-Mdm2(aa1-109) 51 or in Mdm2(aa1-333):p53(aa14-29) (Fig. 3d). This means that Mdm2-IDR1 establishes supplementary transient contacts with Mdm2-p53BD, which are mainly taking place in the p53-binding groove - but not exclusively, see Ile99 in Mdm2(aa1-333):p53(aa14-29). All these observations on Mdm2-p53BD from 1 H- 13 C spectra are fully consistent with our earlier observations in 1 H- 15 N spectra showing lower peak intensities of IDR1's residues in Mdm2(aa1-333) than in Mdm2(aa111-333), and a reemergence of these peaks upon adding p53(aa14-29) (Fig. 2). Also consistent with our earlier characterization using 1 H- 15 N spectra, the 1 H- 13 C SOFAST-HMQC methyl-TROSY spectra of phospho-Mdm2(aa1-333), phosphorylated by CK1d, were extremely similar to those obtained from the non-modified Mdm2(aa1-333) (Supplementary Fig. 11). This indicates that CK1δ-mediated phosphorylation does not significantly affect the open:closed equilibrium of the lid with p53BD. Hence, Mdm2-IDR1 affects considerably the open:closed equilibrium between the lid and the p53BD. It competes with the lid for binding the p53-binding groove, and provokes a much more dynamic environment than the one reported previously from truncated forms, which were comprising only the lid and the p53BD 48,51 . The incorporation of Mdm2-IDR1 pushed the open:closed ratio of the lid from 4 to 0.25 (an equivalent of almost 2 kcal.mol -1 of free energy of folding), and accelerated the related conformational exchange by at least 6 orders of magnitude at 310 K. p53(aa14-29) binding releases IDR1 but makes Mdm2(aa1-333) globally more compact The temperature-dependent chemical shifts observed on Ile d 1 -Met e ( 13 CH 3 ) of Mdm2(aa1-333) suggested temperature-dependent degrees of transient interactions between Mdm2-IDR1 and Mdm2-p53BD. We determined the hydrodynamic radius (R h ) as a function of temperature by measuring diffusion rates using the double echo PGSTE-WATERGATE sequence 52 with 1,4-dioxane as an internal reference. Mdm2(aa1-333) R h decreased with temperature, indicating that Mdm2(aa1-333) adopts a more compact conformation as temperature increases (Fig. 4), which is rather common for IDRs 53,54 . More surprising, we measured lower R h values upon the addition of p53(aa14-29), although this peptide forced the lid and Mdm2-IDR1 to leave their transient occupation of the p53-binding groove. Hence, the ensemble of "lid/IDR1-open" conformations is actually more compact than its "lid/IDR1-closed" counterpart. The Mdm2-lid is much shorter than Mdm2-IDR1, and these changes of hydrodynamic radii are probably mostly due to the conformational behavior of Mdm2-IDR1. Once released from the p53-binding groove, the most hydrophobic segments of Mdm2-IDR1 may form a more hydrophobic nucleus for the disordered molecular cloud, promoting its relative desolvation and a global compaction. This rationale appears to hold true even for a construct deleted from aa189-208, the most interacting segment of Mdm2-IDR1: the addition of p53(aa14-29) still provokes the adoption of a conformational ensemble with a smaller R h (Supplementary Fig. 12). Hence, upon binding the motif p53(aa14-29), the Mdm2-p53BD releases its transient interactions with Mdm2-IDR1 and the Mdm2-lid, which, counterintuitively, makes Mdm2-IDR1 more compact, and thus possibly less accessible to other interactions with the rest of p53. Mdm2-IDR1 weakens the affinity for p53 Finally, we asked whether the Mdm2-IDR1 had an effect on binding p53. We have shown that Mdm2-IDR1 interacts transiently with Mdm2-p53BD in a competing fashion with p53(aa14-29), which should have deleterious effects on the affinity between Mdm2 and p53. Concurrently, Mdm2-IDR1 is very negatively charged and may improve binding to p53, which contains a positively charged DNA-binding domain (p53-DBD). Which way does the balance tip? We chose isothermal calorimetry (ITC) to measure affinities: it allows for titrations in solution, avoiding the immobilization of one protein on a surface and the potential bias that can easily emerge in these semi-liquid conditions for the highly flexible IDRs. The measured values are summarized in Figure 5 and Table 1. Starting with truncated constructs, we measured a K D of 37 ± 8 nM between Mdm2-p53BD(aa18-112) and p53-NT(aa1-88). Incorporating the lid, i.e. using Mdm2(aa1-112), decreased the affinity by a factor 3 for p53-NT(aa1-88) at 105 ± 8 nM. This is consistent with the competing interaction between the lid and p53 for binding the p53BD-binding groove. Next, we incorporated Mmd2-IDR1 and Mdm2-ZF in Mdm2(aa1-333), which led the affinity for p53-NT(aa1-88) to 480 ± 5 nM. Even more deleterious, phosphoMdm2(aa1-333) affinity for p53-NT(aa1-88) rose to 1.0 ± 0.1 mM. This is most probably due to an electrostatic repulsion with p53-NT(aa1-88), which carries a total 16 negative charges (18 Asp/Glu vs 2 Arg/Lys). Interestingly, the removal of the segment Mdm2(aa190-210) from Mdm2(aa1-333) led to a weak but measurable strengthening of the affinity for p53-NT(aa1-88) at 320 ± 30 nM, in agreement with the NMR-revealed competition between Mdm2(aa190-210) and p53(aa14-29) for binding Mdm2-p53BD. Then, we carried out the same series of titrations with full-length p53 (p53FL). It is worth mentioning that it assembles as a tetramer at the working concentrations 55 . We measured a K D of 81 ± 6 nM between Mdm2-p53BD(aa18-112) and p53FL, weaker than with p53-NT(aa1-88). This is consistent with intramolecular interactions between p53-NT(aa1-88) and p53-DBD (reported earlier and confirmed by us 6,56,57 , see Supplementary Fig. 13), which can compete with Mdm2-p53BD(aa18-112). The loss of affinity due to the incorporation of the lid in Mdm2-p53BD(aa1-112), observed previously with p53-NT(aa1-88), was confirmed with p53FL: we measured a K D of 510 ± 40 nM. Then, the addition of Mmd2-IDR1 and Mdm2-ZF, resulting in Mdm2(aa1-333), affected weakly the affinity for p53FL, which was measured to be 310 ± 90 nM. Even the CK1d-mediated hyperphosphorylation did not have any strong effects on binding p53FL, the affinity being evaluated to 390 ± 20 nM. NMR spectra show that the negatively charged Mdm2-AD interacts with the positively charged p53-DBD in a salt-dependent manner (Supplementary Fig. 14). Even though we observed faint chemical shifts perturbations (Dd < 0.03 ppm) and disappearance of crosspeaks in 1 H- 15 N NMR spectra of 15 N-Mdm2-IDR1 in presence of 14 N-p53FL, we could not measure any affinity using ITC (data not shown), placing it probably in the millimolar range. Hence, this electrostatic interaction appears to be rather diffuse with no well-defined contacts, and seems to only counterbalance the deleterious contributions of Mdm2-IDR1 due to intramolecular contacts and diffuse shielding of the Mdm2-p53BD. Finally, we confirmed again the negative contribution of the interaction between Mdm2(aa190-210) and Mdm2-p53BD: the affinity between Mdm2(aa1-333_D189-208) and p53FL was measured to be 150 ± 10 nM. Altogether, Mdm2-IDR1 interferes strongly with the capacity of Mdm2-p53BD to bind p53, from 5- to 25-fold, depending on the peptide construct, i.e. p53FL or the short p53(aa1-88). Competing intramolecular contacts combine with electrostatic attractions and repulsions to tune positively or negatively the affinity between Mdm2 and p53. Importantly, the beneficial electrostatic interaction between the Mdm2-AD and p53-DBD is only moderate, and it does not overcome the unfavorable intramolecular contacts and the diffuse screening established by the IDR1. Discussion Although its N-terminal p53BD has been heavily scrutinized structurally, Mdm2's global structure is not yet well apprehended. Specifically, Mdm2-IDR1 is poorly described, even though its reported functions and partners are numerous 7,13,15–24 . These observations identified the Mdm2-AD as a nodal point for regulatory signals, through which the p53 pathway can integrate information from the cellular state. However, studying IDRs' structural behavior in their native contexts, inserted between folded domains, is still a challenge: only NMR spectroscopy can bring residue-specific information, but the current isotope-labeled recombinant expression in bacterial systems comes often with low yields for these multidomain protein constructs. It is even worse if we add a supplementary but necessary layer of biological context: IDRs carry abundant PTMs in their native states, eventually in a conditional fashion when related to cell signaling 58–60 . Producing pure hyperphosphorylated samples is not straightforward, and the present report is thus bringing novel information on such highly modified IDRs in a multidomain context. Earlier reports on p53 had shown that weak, transient contacts between its N-terminal region and its DNA-BD were capable of inhibiting DNA binding 6,56 . Here, we have shown that these p53 intramolecular contacts were also inhibiting the binding to Mdm2. More novel, we have shown that multiple intramolecular contacts existed also in Mdm2 (Fig. 2-3, Supplementary Fig. 9-11), and that they also affect negatively the interaction with p53 (Fig. 5). Interestingly, while Mdm2-AD and its ~20 negative charges bind loosely to the positively charged p53-DBD even at clost-to-physiological salt concentrations in PBS (Supplementary Fig. 14), it does not compensate the detrimental effects of the multiple intramolecular interactions that Mdm2-IDR1 generates with Mdm2-p53BD in a competing fashion with p53. These intramolecular contacts in Mdm2 are in correspondence with those reported on its homolog MdmX 25–28 : the most involved fragments in Mdm2 are aa189-208 and aa242-255, which are exactly aligned with the regions of MdmX binding to its own MdmX-p53BD (Supplementary Fig. 2). In MdmX, two equivalent peptide segments, containing multiple Trp residues, have been shown to have autoinhibitory capacities 25,61,62 . If measured with two separate peptides, MdmX-IDR1 has an affinity for MdmX-p53BD of about 1 mM, which provokes a 100-fold affinity loss of affinity for p53 in constructs containing MdmX-p53BD+IDR1 vs MdmX-p53BD 62 . MdmX-IDR1 has also a good affinity for Mdm2-p53BD, about 8 mM, which can also interfere with p53 binding to Mdm2 28 . Here, we evaluated that Mdm2-IDR1 would have a dissociation constant above 100 mM for Mdm2-p53BD as separate species, but their native concatenation generates transient contacts, which compete with p53 and tune the affinity between Mdm2 and p53 by a factor 2-3 depending on the Mdm2 construct. The functional consequences of these loose intramolecular contacts can be substantial. In the present case, the addition of those identified in p53 and Mdm2 lead to the a 10-fold decrease in affinity (comparison between Mdm2(aa18-112)+p53(aa1-88) vs Mdm2(aa1-333)+p53FL). This is most probably an under-evaluation of their pure effects, because electrostatic interactions between the Mdm2-AD and p53-DBD bring more favorable components to the global interaction. It appears anyway to be difficult to deconvolute all the by-products of loose binding events appearing upon merging folded and disordered domains. For example, upon binding to p53(aa14-29), the release of Mdm2-IDR1 from its transient binding to Mdm2-p53BD provoked a faint, but measurable compaction of Mdm2(aa1-333). This counterintuitive result shows that it is probably often difficult to predict IDRs' accessibility in their native context from isolated models. This calls for improved modeling tools adapted to multidomain constructs, if we want to progress in our understanding of native IDRs conformational ensembles and binding accessibility 58,63,64 . It calls also for developing further thermodynamic models to fully account for the complexity of IDRs and the appearing carousel of their multiple loose contacts in multidomain proteins 65 . Regarding the phosphorylation sites, one study had reported a list of CK1-phosphorylated residues in Mdm2-AD using mass-spectrometry without quantification: the list was impressive, containing S215, S220, S229, S232, S240, S242, S246, S256, S260, S269, S286, S288 et S290 22 . We confirmed most of these sites, except S269, S288 and S290. At the opposite, we found out that T218 and S253 were readily phosphorylated by CK1d, and that S192, S262 and T279 were eventually secondary targets. NMR spectroscopy cannot provide information on endogenous phosphosites, but it delivers a non-ambiguous residue-specific and quantitative information on phosphorylation reactions for purified material 36–38 , because of its capacities in poly-phosphorylation mapping and monitoring 10,39–43 . Here, it allowed us to show that all sites were phosphorylated in parallel but at different rates. These findings contradict the widely discussed conditional phosphorylation mechanisms associated to CK1δ, and instead support a fully distributive and independent mechanism. The data suggest that local electrostatics, driven by glutamate- and aspartate-rich sequences, may play a more significant role than canonical CK1δ motifs. These motifs were classically defined using peptide arrays 66 , which cannot account for the properties emerging from longer, native IDRs. Here again, studying IDRs in their native multidomain context brings information that is hardly predictable using the actual prediction tools. More experimental data from more native protein constructs will be necessary to improve these predictions. Quite surprisingly, the 15 phosphorylation events on Mdm2-AD did neither affect the intramolecular contacts with Mdm2-p53BD, nor did they strongly modulate the affinity for the positively charged p53-DBD. This is in stark contrast with all the possible effects and functions of IDRs' poly-phosphorylation, which have been reported on other proteins in the literature 67 . However, this result might not be definitive: other intramolecular contacts may appear upon integrating Mdm2-IDR2 and Mdm2-RING domains, a work that we are currently tempting to achieve. Altogether, it becomes clear that studying the isolated domains Mdm2-p53BD and p53-NT does not account for all the transient contacts, intra- or inter-molecular, emerging from incorporating the other neighboring domains. These modulate Mdm2:p53 affinity in the range of one or two orders of magnitude, with multiple layers of competitions counteracting each other. Interestingly, these unpredictable interactions and effects are comparable to the affinity differences measured between the isolated Mdm2-p53BD and p53-NT across vertebrates 68 . It is tempting to imagine that some intramolecular contacts might have appeared or disappeared through evolution, as a result of a need to tune the intrinsic affinities of the core binding event between Mdm2-p53BD and p53-NT. This report shows that Mdm2-IDR1 interferes with Mdm2 binding to p53, with negative contributions combining with weaker positive effects, if any deconvolution is really conceivable. The affinity of Mdm2(aa1-333) for p53FL converges towards ~400 nM, in the non-modified and phosphorylated forms, instead of the ~40 nM measured on the isolated Mdm2-p53BD and p53-NT. Only a handful of structural studies have been yet published on multidomain proteins incorporating folded and disordered domains 6,25,57,69–76 , which all report interdomain regulating contacts. These are not yet apprehended by machine-learning assisted predictions nor well-captured by the current molecular dynamics force-fields. Hence, obtaining more experimental data on IDR's transient intramolecular contacts is one of the important axes in structural biology to explore in the next years, and eventually to confirm in the native, crowded cellular environment 38,77–79 . Methods Peptide constructs Codon-optimized genes encoding truncated variants of human Mdm2, including Mdm2 (aa1-333), a deletion-mutant lacking residues 189-208 (D189-208), and additional truncated forms (aa1-112, aa18-112, aa111-180, aa111-230, aa111-333, aa111-237, aa230-333) were synthesized and cloned by Genscript (Supplementary Fig. 16). These constructs contained alanine substitutions at position Cys2, Cys127, Cys200, Cys206, and Cys207. The genes were cloned into the pET-26(b+) expression vector using NdeI and BamHI restriction sites. Additional constructs incorporating alanine substitutions at S215, T218, S220, S246, S253, S260, and S262 were mutated by Genscript, using the initial constructs as templates. Because p53-WT is poorly stable and aggregates progressively as a purified species, we used a stabilized version incorporating mutations in the p53-DBD introduced by different teams in the past. 80,81 The resulting construct included 10 mutations C135V-C141V-C182S-V203A-R209P-C229S-Y234F-N235K-Y236F-T253V, yielding a very stable p53-DBD (Tm~55 °C, in comparison with Tm~42°C for the WT). The crystallographic structure of this mutant p53-DBD(aa90-293) shows its proper folding and structure. All constructs were designed to include a hexahistidine (His6) tag, followed by a Tobacco Etch Virus (TEV) protease cleavage site (ENLYFQG) at the N-terminus of the peptide of interest. Mdm2 fragments expression and purification Recombinant Mdm2 constructs, including unlabeled and u - 15 N/ 13 C-labeled variants, were expressed in Escherichia coli BL21(DE3) cells transformed with the previously described plasmids. Cultures were grown in M9 minimal medium supplemented with either 13 C-glucose or 12 C-glucose (2 g/L) and 15 NH 4 Cl (0.5 g/L) as sole carbon and nitrogen sources, or in LB medium. All media were supplemented with 30 μg/mL kanamycin for plasmid selection. Protein expression was induced at an optical density at 600 nm (OD 600 ) of 0.8 by adding 0.25-0.5 mM isopropyl β-D-1-thiogalactopyranoside (IPTG), followed by incubation overnight at 20 °C. Cells were harvested 24 hours post-induction by centrifugation, and the resulting cell pellets were stored at -80 °C. Cell lysis was performed by sonication in a lysis buffer consisting of 20 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1 mM phenylmethylsulfonyl fluoride (PMSF), 10 mM dithiothreitol (DTT), 1 mM lysozyme, a 1× concentration of EDTA-free protease inhibitor cocktail (cOmplete, Roche), and 0.5 μL benzonase (Sigma-Aldrich). Lysates were centrifuged at 15,000 × g for 15 minutes at 4 °C to separate soluble and insoluble fractions. Mdm2 peptides were recovered from the soluble fraction. The soluble lysates were applied to a 5 mL His-Trap FF column (GE Healthcare) pre-equilibrated with lysis buffer. Bound proteins were eluted using an imidazole gradient. Eluted fractions were pooled, concentrated, and further purified by ion-exchange chromatography on a 6 mL Resource Q column (Cytiva) using a NaCl gradient. The resulting fractions were concentrated and incubated with TEV protease for 90 minutes at room temperature in the presence of 2 mM DTT and a 1x EDTA-free protease inhibitor cocktail. The final purification was achieved by size-exclusion chromatography using a Superdex 16/600 200 pg column (GE Healthcare) equilibrated with phosphate-buffer (PBS; 20 mM phosphate buffer, 150 mM NaCl) at pH 7.0 or 7.4. Fractions containing Mdm2 peptides were identified and stored at -80 °C for downstream applications. Ile δ 1-[ 13 CH 3 ]- and Met ε -[ 13 CH 3 ]- 15 N- 2 H-labeled Mdm2 NMR analysis For selective 13 CH₃ labeling of isoleucine (δ1) and methionine (ε) methyl groups in a 2 H/ 12 C background, E. coli cells were progressively adapted to D 2 O by growing in LB medium with increasing concentrations of D 2 O (0 %, 30 %, 70 %, 99 %). The final expression was carried out in 99% D 2 O-based M9 medium supplemented with 12 C, 2 D-glucose (2 g/L; Cortecnet), 15 NH 4 Cl (0.5 g/L), 2-ketobutyric acid-4- 13 C,3,3-d 2 (70 mg/L), and 13 C-methionine (100 mg/L). The overexpression and purification of the protein were executed using the protocol described above. Resonance assignments for Mdm2 were transferred from published data by Watanabe et al. (2024). The initial NMR sample contained 90 μM Ileδ1-[ 13 CH₃]- and Metε-[ 13 CH₃]-labeled Mdm2 in PBS (pH 7.4) with 1× EDTA-free protease inhibitor cocktail (cOmplete, Roche), 3% D 2 O, and 100 μM DSS. Phosphorylation was then initiated in PBS supplemented with 50 mM HEPES, 4 mM DTT, 10 mM ATP, 20 mM MgCl 2 , 1x EDTA-free protease inhibitor cocktail (Roche), by the addition of 1 μM unlabeled CK1δ kinase. An additional sample was prepared under the same conditions and supplemented with 90 μM unlabeled p53(aa14–29) peptide. NMR experiments were conducted on these three samples at multiple temperatures (283 K, 288 K, 293 K, 298 K, 303 K, 306 K, 308 K, 310 K, and 312 K) using the same 700 MHz Bruker Avance Neo spectrometer equipped with a cryogenically cooled TCI probe. 1 H- 13 C SOFAST Methyl-TROSY spectra were acquired with 2048 ( 1 H) × 192 ( 13 C) complex points, using sweep widths of 14.9 ppm ( 1 H) and 14.0 ppm ( 13 C). Data processing was performed using TopSpin 4.1.3 (Bruker). Synthesized peptides Synthesized phosphorylated peptides of Mdm2 were all purchased from Proteogenix (Illkirch, France) in al lyophilized form with no TFA, and resuspended in PBS at final millimolar concentrations; pH was adjusted to 7.0. These peptides are Mdm2(aa214-226) Ac-SESTG-T/pT-PpSNPDLDA-NH 2 and Mdm2(aa211-223) Ac-SSSEpSTG-T/pT-P-S/pS-NPD-NH 2 . The peptide p53(aa14-29) Ac-LSQETFSDLWKLLPEN-NH 2 was purchased and resuspended exactly in the same way. NMR assignments - Synthesized peptides: the assignments were achieved using 1 H- 1 H-TOCSY and 1 H- 1 H-ROESY experiments to obtain the 1 H N amide assignments, which was later correlated to the 1 H- 15 N crosspeaks from two-dimensional 1 H- 15 N HSQC spectra recorded at natural abundance. All experiments were recorded at pH 7.0 and 283 K in 3 mm diameter tubes, using a 600 MHz spectrometer equipped with a cryoprobe. The peptides being dissolved at ~ 3 mM concentrations in PBS in presence of protease inhibitors and DSS at 100 mM. 1 H- 1 H-TOCSY (mlevgpph19) and 1 H- 1 H-ROESY spectra (roesygpph19.2) were recorded with 2048 ( 1 H direct dimension) x 448 ( 1 H indirect) complex points and sweep widths of 16 ppm ( 1 H direct dimension) and 10 ppm ( 1 H indirect dimension), 8 scans and interscan delays of 1.5 s, and processed with a cosine apodization in the direct dimension. 1 H- 15 N HSQC (hsqcetfpf3gpsi2) spectra were recorded with 2048 ( 1 H) x 192 ( 15 N) complex points and sweep widths of 16 ppm ( 1 H) and 32 ppm ( 15 N), 128 scans and interscan delays of 1.25 s, and processed with a cosine apodization in both the direct and indirect dimensions, respectively. - u - 15 N/ 13 C-labeled Mdm2 and phosphorylated Mdm2: NMR assignments of backbone amide resonances of uniformly-labeled peptides ( 13 C/ 15 N) was achieved via HNCO, HN(CA)CO, HNCACB, (H)N(CA)NH and HNCA 3D experiments. [ u - 13 C/ 15 N]-Mdm2(aa111-293) was assigned at 600 mM in a buffer containing HEPES at 10 mM, sodium chloride at 50 mM, TCEP at 4 mM, 3% D 2 O, protease inhibitors (cOmplete EDTA-free, Roche) and DSS at 0.1 mM, at pH 6.2 and 283 K using a Bruker Avance III 700 MHz spectrometer equipped with a cryoprobe. CK1d-phosphorylated [ u - 13 C/ 15 N]-Mdm2(aa111-333_C127A-C200A-C206A-C207A) was assigned at 180 mM in a buffer containing sodium phosphate at 20 mM, sodium chloride at 150 mM, 3% D 2 O, protease inhibitors (cOmplete EDTA-free, Roche), DSS at 0.1 mM at pH 7.4 and 283K, using a Bruker Avance III 950 MHz spectrometer equipped with a cryoprobe. Assignments of unmodified and phosphorylated human Mdm2(aa111-293), Mdm2(aa111-230_C127A-C200A-C206A-C207A) and CK1d-phosphorylated Mdm2(aa111-333_C127A-C200A-C206A-C207A), as well as details of NMR experiments used to derive them have been deposited in the Biological Magnetic Resonance Data Bank (BMRB, accession numbers 52016, 52028 and 52289, respectively). As reported in the literature 10 , we observed two stable conformations of the Zn-finger corresponding to all-trans- and all-cis-prolines. The relative peak intensities of the two species were the same in the isolated Zn-finger and in the longer constructs aa111-333 or aa1-333 (trans:cis~2:1). Secondary structure propensities were obtained with the neighbor-corrected structural propensity calculator ncSCP 44 (https://www.jku.at/en/institute-of-biochemistry/protein-nmrorg/ , https://st-protein02.chem.au.dk/ncSPC/) using DSS referenced Ca and Cb chemical shifts as input, and the random coil values of phospho-residues reported by Kragelund and colleagues 82 . Assignments of synthesized peptides have also been deposited in the BMRB: Mdm2(aa214-226_pT218-pS220) (BMRB 52251), Mdm2(aa211-223_pS215-pT218) (BMRB 52255), Mdm2(aa214-226_pS220) (BMRB 52256), Mdm2(aa211-223_pS215) (BMRB 52257). Titration 15 N-Mdm2(aa111-333) vs 14 N-Mdm2(aa1-112) NMR samples were prepared by spiking 150 mL of 15 N-labeled Mdm2(aa111-333) at 100 mM with non-labeled Mdm2(aa1-112) at 475 mM, both in PBS at pH7.0 supplemented with 1x ETDA-Free protease inhibitors (cOmplete, Roche), 3% D₂O, and 100 μM DSS. The pH was adjusted to 7.00 very carefully for every sample. At the final point of the titration, the concentrations were 54 and 219 mM for 15 N-labeled Mdm2(aa111-333) and for non-labeled Mdm2(aa1-112), respectively. All NMR experiments were conducted on a 600 MHz Bruker Avance III-HD spectrometer equipped with a cryogenically cooled triple-resonance TCI probe. 1 H- 15 N HSQC (hsqcetfpf3gpsi2) experiments were recorded at 283 K with 2048 ( 1 H) x 256 ( 15 N) complex points and sweep widths of 16 ppm ( 1 H) and 26 ppm ( 15 N), 4 to 32 scans and interscan delays of 0.8 s, and processed in Topspin 4.5 with a cosine apodization in the direct dimension and no apodization in the indirect dimension, respectively. The chemical shift analysis was carried out using CcpNmr 3.1 83 . The peak intensities were normalized according to the dilution factors and plotted using Kaleidagraph5. The chemical shift perturbations were calculated from the combination of 1 H and 15 N chemical shift changes Dd=√((d 1H 2 +0.2*d 15N 2 )/2), and plotted using Kaleidagraph5. In Vitro CK1δ phosphorylation assays by NMR NMR samples were prepared by combining 450 μL of u - 15 N/ 13 C-labeled Mdm2 at a final concentration of 100 μM in PBS supplemented with 50 mM HEPES, 4 mM DTT, 10 mM ATP, 20 mM MgCl 2 , 1x ETDA-Free protease inhibitors (cOmplete, Roche), 3% D₂O, and 100 μM DSS. The pH was adjusted to 7.0. Phosphorylation reaction was initiated by the addition of 2 μM unlabeled CK1δ kinase. Reaction progress was monitored using NMR spectroscopy. All NMR experiments were conducted on a 700 MHz Bruker Avance Neo spectrometer equipped with a cryogenically cooled triple-resonance TCI probe. 1 H- 15 N HSQC experiments were recorded before and after phosphorylation at 283 K, 290 K, and 298 K to normalize the final phosphorylation levels and analyze temperature-dependent chemical shift variations. A time-resolved series of 1 H- 15 N SOFAST-HMQC spectra was recorded at 298 K until phosphorylation reached completion. After phosphorylation, the sample was purified using ion-exchange chromatography on a 6 mL Resource Q column (Cytiva) with a NaCl gradient, followed by size-exclusion chromatography on a Superdex 16/600 200 pg column (GE Healthcare) equilibrated with PBS (pH 7.4). Two-dimensional 1 H- 15 N HSQC experiments were recorded with 1536 ( 1 H) × 192 ( 15 N) complex points, sweep widths of 16.23 ppm ( 1 H) and 28 ppm ( 15 N), 8 scans (4 dummy scans), and an interscan delay of 1 s. Two-dimensional 1 H- 15 N SOFAST-HMQC spectra were recorded with 1536 ( 1 H) × 192 ( 15 N) complex points, sweep widths of 16.23 ppm ( 1 H) and 28 ppm ( 15 N), 16 scans (32 dummy scans), and a 0.2 s interscan delays, resulting in a total acquisition time of 15 minutes. Data were processed using TopSpin 4.1.3 (Bruker), and peak intensities were analyzed using CcpNmr Analysis 2.4. Peak positions were tracked to monitor chemical shift perturbations, and phosphorylation kinetics were normalized using relative intensity increase changes. Using Mdm2(aa111-237 WT/T218A/S220A), Mdm2(aa111-333 S215A), and unlabeled phosphorylated peptides of Mdm2(aa214-226): Ac-SESTG-T/pT-PpSNPDLDA-NH₂, and Mdm2(aa211-223): Ac-SSSEpSTG-T/pT-P-S/pS-NPD-NH₂, phosphorylation peaks corresponding of pS215, pT218, pS220, pS229, and pS232 were identified using 2D 1 H- 15 N SOFAST-HMQC and HSQC spectra. Similarly, phosphorylation peaks for pS253 and pS256 were identified using Mdm2(aa230-333 S253A); for pS240, pS242, and pS246 using Mdm2(aa111-333 S246A); and for pS260 and pS262 using Mdm2(aa111-333 S260A/S262A). In the case of Mdm2(aa111-333 WT), phosphorylation of residues pT218, pS240, pS242, pS256, pS260, and pT279 was monitored, along with adjacent residues including pG217 (for pS215), pD225 (for pS220), pV228 (for pS229), pG233, pD234 and pS232 (for pS232), pE248 and pS246 (for pS246), pV251, pD252 and pS253 (for pS253), pG265 (for pS262), and pQ277 and pT279 (for pT279). The intensities of these neighboring residues increased proportionally with the phosphorylation of the corresponding sites. To enhance the signal-to-noise ratio (S/N), the intensities of peaks corresponding to each phosphosite, along with those of adjacent residues were aggregated and used to trace phosphorylation kinetics. The percentage of phosphorylation (%phospho) was calculated using the equation %phospho final = 1 - I final /I 0 from unphospho-peaks, where intensities were extracted from 2D 1 H- 15 N HSQC spectra of Mdm2(aa111-333) and pMdm2(aa111-333) at 283 K. Normalization was achieved by combining %phospho values with peak intensities from SOFAST-HMQC spectra. Phosphorylation build-up curves were fitted using the equation %phospho(t)= 1 - (I/ (I final /%phospho final )*exp(-k*t)). Relaxation Experiments NMR relaxation experiments were recorded using the same 700 MHz Bruker Avance Neo spectrometer equipped with a cryoprobe. Data acquisition, processing, and spectral analysis were carried out with TopSpin 4.1.3 (Bruker) and CcpNmr Analysis 2.4. Two-dimensional 1 H- 15 N correlation spectra of u - 15 N-labeled Mdm2 were acquired using a standard HSQC pulse sequence at 283 K with protein concentrations ranging from 200 to 300 μM in PBS (pH 7.4). Duplicate experiments were acquired at each time point to estimate experimental error. - T₁ and T₂ Relaxation Measurements: 15 N amide-backbone relaxation rates, including longitudinal (T₁, 1/R₁) and transverse (T₂, 1/R₂), were determined using standard Bruker pulse sequences. T₁ values were measured at relaxation delay times of 40, 100, 200, 300, 400, 500, 600, 800, and 1200 ms and intensities were fitted to the exponential delay function: I(t)=I 0 *(1-e -t/T1 ). T₂ values were determined using a Carr-Purcell-Meiboom-Gill (CPMG) sequence with delays of 15.7, 31.5, 47, 62.5, 78.5, 110, 141, 188, and 376 ms. Apparent R 2 rates were derived via multi-exponential fitting to account for hydrogen exchange (HX) effects, following methodology described in Alik et al. (2020) 10 . - Hydrogen Exchange (HX) Rate Measurements: Hydrogen exchange rates (k HX ) were determined using the CLEANEX-PM experiment (Hwang et al., 1998) 84 with 1 H- 15 N HSQC detection. The fhsqccxf3gpph pulse sequence from Bruker was used with mixing times of 0.005 s, 0.01 s, and 0.02 s at pH 7.4. fhsqcf3gpph 1 H- 15 N HSQC spectra served as reference (V 0 ) in the analysis, and k HX were calculated using the equation described by Hwang et al. (1998) 84 . Data were acquired with 2048 complex points in the t 2 dimension and 256 complex points in t 1 , using a 2-seconds recycle delay. - Heteronuclear NOE Measurements: Heteronuclear NOEs (hetNOE) values were obtained by comparing peak intensities from spectra acquired with and without a 4-seconds proton saturation during a 5-seconds recycle delay. Residue-specific signal intensity ratios (I/I 0 ), corresponding to hetNOE values, were calculated for Mdm2. NMR Diffusion Experiments Double echo PGSTE-WATERGATE experiments (Zheng et al. 2009) 52 was conducted on 200 μM unlabeled Mdm2 dissolved in PBS (pH 7.4) supplemented with 1x EDTA-free protease inhibitor cocktail (Roche), 3% D 2 O, 100 μM DSS, and 0.03% 1,3-dioxane. Measurements were performed at multiple temperatures (283 K, 288 K, 293 K, 298 K, 303 K, 306 K, 308 K, 310 K, and 312 K) using a 600 MHz Bruker Avance Neo spectrometer equipped with a cryogenically cooled TXI probe. A series of 24 one-dimensional 1 H-NMR spectra were acquired with linearly incremented gradient strengths. Signal intensities were measured by integrating peaks from the 1D spectra. The diffusion coefficients of Mdm2 were determined by fitting the exponential decays of signal intensities in the methyl region (1-0.5 ppm) to the Stejskal-Tanner equation. The diffusion coefficient of 1,3-dioxane was determined independently by analyzing the intensity decay of its specific resonance. Peak integration and analysis were performed using TopSpin 4.1.3 (Bruker). For each pulsed-field gradient (PFG) NMR experiment, 16 scans were acquired, with the gradient strength varying from 10% to 100%. The gyromagnetic ratio (γ) was set to 2.68 × 10⁸ rad.s⁻¹.T⁻¹, with a diffusion time (∆) of 200 ms and gradient pulse duration (δ) of 5 or 7 ms. Isothermal Titration Calorimetry (ITC) ITC experiments were performed using both a VP-ITC and a PEAQ-ITC instruments (MicroCal). In a typical VP-ITC experiment, the sample cell was loaded with Mdm2 at 5 or 10 μM (aa1-112, aa18-112, aa1-333, or aa111-333), while the titrating syringe contained p53(aa1-88) at 50 or 100 μM. For PEAQ-ITC experiments, the cell was loaded with 15 or 20 μM phospho-MDM2(aa1-333) or MDM2(aa1-333del189-208), and the syringe contained p53(aa1-88) of p53FL at 150 or 200 μM. Raw data are shown in Supplementary Figure 15. All titrations were conducted in PBS buffer at pH7.4 and 20 °C. Data were analyzed using MicroCal Analysis software, with the binding isotherms fitted to a single-site binding model. Declarations Acknowledgements This work was supported by the CNRS and the CEA-Saclay (CEA/PSAC/DPRS/BE/TG/2021-410), by the French Infrastructure for Integrated Structural Biology (https://frisbi.eu/, grant number ANR-10-INSB-05-01, Acronym FRISBI) and by the French National Research Agency (ANR; research grants ANR-14-ACHN-0015 and ANR-20-CE92-0013). This work has been supported by the Fondation ARC pour la recherche sur le cancer (YL, ARCDOC42023120007483). Financial support from the IR INFRANALYTICS FR2054 for conducting the research is gratefully acknowledged, and we value the commitment and expertise of F. Giraud, E. Lescop and N. Morellet. We also thank the Platform PIM from the I2BC. Author contributions Y.L., J.P., C.B., A.A. and F.-X.T. produced protein samples, Y.L., J.P., C.B., M.A.-N. and F.-X.T. conducted the ITC experiments, Y.L. and F.-X.T. conducted and analyzed the NMR experiments, Y.L. and F.-X.T. wrote the manuscript, F.-X.T. secured funding, designed and supervised the project. Competing interests The authors declare no competing interests. Additional information Supplementary information The online version contains supplementary material. Correspondence and requests for materials should be addressed to Francois-Xavier Theillet. References Ge, Z. et al. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-9014206","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":600079953,"identity":"44501d18-e29a-46a0-aaa8-8bbc03e1a388","order_by":0,"name":"François-Xavier Theillet","email":"data:image/png;base64,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","orcid":"https://orcid.org/0000-0002-3264-210X","institution":"Université Paris Cité, CNRS, CiTCoM","correspondingAuthor":true,"prefix":"","firstName":"François-Xavier","middleName":"","lastName":"Theillet","suffix":""},{"id":600079954,"identity":"2809f941-6ccd-4264-934c-34db01ae35c8","order_by":1,"name":"Yingyue Luo","email":"","orcid":"https://orcid.org/0009-0006-7344-0885","institution":"CNRS-I2BC","correspondingAuthor":false,"prefix":"","firstName":"Yingyue","middleName":"","lastName":"Luo","suffix":""},{"id":600079955,"identity":"83777a8d-b024-4548-b5ea-77ef6cc07ca6","order_by":2,"name":"Chafiaa Bouguechtouli","email":"","orcid":"","institution":"CNRS-I2BC","correspondingAuthor":false,"prefix":"","firstName":"Chafiaa","middleName":"","lastName":"Bouguechtouli","suffix":""},{"id":600079956,"identity":"cbe606e6-0628-4ee9-b1d0-98ebe54b01e1","order_by":3,"name":"Jehanne Procaccia","email":"","orcid":"","institution":"Université Paris Cité - CNRS","correspondingAuthor":false,"prefix":"","firstName":"Jehanne","middleName":"","lastName":"Procaccia","suffix":""},{"id":600079957,"identity":"6333a205-7841-4648-8001-c04e15de2241","order_by":4,"name":"Ania Alik","email":"","orcid":"","institution":"CNRS-I2BC","correspondingAuthor":false,"prefix":"","firstName":"Ania","middleName":"","lastName":"Alik","suffix":""},{"id":600079958,"identity":"a79ab51e-a187-45bb-b5e6-fd7bb39a7a66","order_by":5,"name":"Magali Aumont-Nicaise","email":"","orcid":"","institution":"Institute for Integrative Biology of the Cell","correspondingAuthor":false,"prefix":"","firstName":"Magali","middleName":"","lastName":"Aumont-Nicaise","suffix":""}],"badges":[],"createdAt":"2026-03-02 23:20:30","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-9014206/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-9014206/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":104207986,"identity":"f1a8d1b3-bc52-415d-b1c4-cf9650ddda16","added_by":"auto","created_at":"2026-03-09 07:12:27","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":895593,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eExperimental evidence that IDR1 is disordered and hyperphosphorylated by CK1d a. \u003c/strong\u003ePrimary structure of Mdm2 showing folded regions (p53BD, ZF, RING, dark blue) and disordered regions (light blue), Mdm2-IDR1(aa109-296) containing the NLS-NES (grey) and the acidic domain (AD, purple); the NMR-assigned phosphosites are localized by circles using the color-code used in d., while the arrows indicate the positions of the classical CK1 consensus motifs; the fragment aa333-491 in pale blue is not studied in this report; \u003cstrong\u003eb. \u003c/strong\u003eOverlay of 2D \u003csup\u003e1\u003c/sup\u003eH-\u003csup\u003e15\u003c/sup\u003eN HSQC spectra of Mdm2(aa111-333) non-modified (blue) and phosphorylated by CK1d (magenta); peaks from the Zinc-Finger (ZF) are highlighted with dotted lines; peak assignments are given for residues showing large chemical shift perturbations, used for kinetics determination; \u003cstrong\u003ec.\u003c/strong\u003e Secondary structure propensities of Mdm2(aa111-293) (blue) and phosphoMdm2(aa111-333) as calculated from \u003csup\u003e13\u003c/sup\u003eCa/\u003csup\u003e13\u003c/sup\u003eCb chemical shifts using ncSCP \u003csup\u003e44\u003c/sup\u003e and the random coil values of phospho-residues reported by Kragelund and colleagues;\u003csup\u003e82\u003c/sup\u003e \u003cstrong\u003ed. \u003c/strong\u003eRepresentative kinetics for 5 classes of phosphosites and their corresponding colors; individual kinetics are shown in Supplementary Fig. 8.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-9014206/v1/157064d5337b0da829661989.png"},{"id":104207988,"identity":"774fff90-0a84-47e4-93fa-770564e1537f","added_by":"auto","created_at":"2026-03-09 07:12:28","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":1026040,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ea. \u003c/strong\u003eOverlay of 2D \u003csup\u003e1\u003c/sup\u003eH-\u003csup\u003e15\u003c/sup\u003eN HSQC spectra of Mdm2(aa111-333) and Mdm2(aa1-333) (blue and black, respectively); assignments are given for peaks showing decreased intensities in Mdm2(aa1-333); \u003cstrong\u003eb. \u003c/strong\u003eResidue-specific peak intensity ratios for Mdm2(aa1-333) or [Mdm2(aa111-333)+p53(aa14-29)] in comparison with Mdm2(aa111-333) (black and orange, respectively); \u003cstrong\u003ec.\u003c/strong\u003e Residue-specific \u003csup\u003e15\u003c/sup\u003eN R\u003csub\u003e2\u003c/sub\u003e relaxation, providing information on the ms-ms timescale environment exchange for Mdm2(aa111-333), Mdm2(aa1-333) and [Mdm2(aa111-333)+p53(aa14-29)] (blue, black, orange, respectively); the \u003csup\u003e15\u003c/sup\u003eN R\u003csub\u003e1\u003c/sub\u003e relaxation and \u003csup\u003e1\u003c/sup\u003eH-\u003csup\u003e15\u003c/sup\u003eN heteronuclear NOEs values in Supplementary Fig. 9 show that the high Mdm2-IDR1 flexibility is conserved at the ns-timescale; \u003cstrong\u003ed. \u003c/strong\u003eOverlay of 2D \u003csup\u003e1\u003c/sup\u003eH-\u003csup\u003e15\u003c/sup\u003eN HSQC spectra of Mdm2(aa111-333) and [Mdm2(aa111-333)+p53(aa14-29)] (blue and orange, respectively); \u003cstrong\u003ee. \u003c/strong\u003eResidue-specific peak intensity ratios for phospho-Mdm2(aa1-333) or [phospho-Mdm2(aa111-333)+p53(aa14-29)], after phosphorylation by CK1d; \u003cstrong\u003ef. \u003c/strong\u003eResidue-specific \u003csup\u003e15\u003c/sup\u003eN R\u003csub\u003e2\u003c/sub\u003e relaxation, providing information on the ms-ms timescale environment exchange for Mdm2(aa111-333), Mdm2(aa1-333) and phosphoMdm2(aa111-333) and phosphoMdm2(aa1-333) (blue, black, magenta, purple, respectively); the \u003csup\u003e15\u003c/sup\u003eN R\u003csub\u003e1\u003c/sub\u003e relaxation and \u003csup\u003e1\u003c/sup\u003eH-\u003csup\u003e15\u003c/sup\u003eN heteronuclear NOEs values in Supplementary Fig. 9 show that the high IDR flexibility is conserved at the ns-timescale; the positions of the phosphosites are indicated by circles using the color code from Fig. 1d.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-9014206/v1/382a2588af24dd35155387e1.png"},{"id":104207994,"identity":"550137a0-f857-489f-aea5-d52ec02e72a7","added_by":"auto","created_at":"2026-03-09 07:12:30","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":712952,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ea. \u003c/strong\u003eOverlay of \u003csup\u003e1\u003c/sup\u003eH-\u003csup\u003e13\u003c/sup\u003eC SOFAST-HMQC methyl-TROSY spectra (700 MHz, 310 K) of [\u003cem\u003eu\u003c/em\u003e-\u003csup\u003e2\u003c/sup\u003eD, Iled1-Mete-\u003csup\u003e13\u003c/sup\u003eCH\u003csub\u003e3\u003c/sub\u003e]-labeled Mdm2(aa1-333) and Mdm2(aa111-333)+p53(aa14-29) (black and orange, respectively); the position of Ile19d1(\u003csup\u003e13\u003c/sup\u003eCH\u003csub\u003e3\u003c/sub\u003e) in Mdm2(aa1-109) is shown as a grey circle, according to Nishida and colleagues \u003csup\u003e51\u003c/sup\u003e; \u003cstrong\u003eb. \u003c/strong\u003eSchematic sketch of Mdm2(aa1-109) showing \"open\" and \"closed\" positions of the Mdm2-lid (aa1-24) according to PDB structure 1Z1M, and the orders of magnitudes of exchange rates in Mdm2(aa1-109) and Mdm2(aa1-333); \u003cstrong\u003ec. \u003c/strong\u003eOverlay of \u003csup\u003e1\u003c/sup\u003eH-\u003csup\u003e13\u003c/sup\u003eC SOFAST-HMQC methyl-TROSY spectra of Mdm2(aa1-333) at varying temperatures; \u003cstrong\u003ed. \u003c/strong\u003eOverlay of \u003csup\u003e1\u003c/sup\u003eH-\u003csup\u003e13\u003c/sup\u003eC SOFAST-HMQC methyl-TROSY spectra of Mdm2(aa111-333)+p53(aa14-29) at varying temperatures.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-9014206/v1/44c31008668c3f64f06372ed.png"},{"id":104208018,"identity":"a8830b37-aec1-42a6-a79c-c8b5ad53221b","added_by":"auto","created_at":"2026-03-09 07:12:35","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":464451,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ea. \u003c/strong\u003eHydrodynamic radii (R\u003csub\u003eh\u003c/sub\u003e) of Mdm2(aa1-333) (blue) and of the complex Mdm2(aa1-333)+p53(aa14-29) (orange) as a function of temperature, as extracted from the NMR measurements of diffusion rates; \u003cstrong\u003eb.\u003c/strong\u003e Schemes showing the average spheres occupied by the molecular clouds formed by Mdm2(aa1-333) and of the complex Mdm2(aa1-333)+p53(aa14-29), according to the measured R\u003csub\u003eh\u003c/sub\u003e; the cartoon representation in the middle of the spheres shows the folded Mdm2-p53BD(aa24-109), which fills about 15% of these volumes.\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-9014206/v1/433a39db560e1f44e91b474c.png"},{"id":104207993,"identity":"17173d60-dc3e-495d-8637-ffc5ee2cdcd2","added_by":"auto","created_at":"2026-03-09 07:12:30","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":202524,"visible":true,"origin":"","legend":"\u003cp\u003eAffinities between Mdm2 and p53 constructs, as measured by ITC in PBS at pH7.4 and 293 K; the colors of the rectangles follow the color code at the top left; the pink circles correspond to CK1d-mediated phosphosites.\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-9014206/v1/6c5e4f00e71e3f0abf5c63f1.png"},{"id":104207981,"identity":"40183f6d-6844-4261-af58-748a57b4ba9a","added_by":"auto","created_at":"2026-03-09 07:12:25","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":157597,"visible":true,"origin":"","legend":"\u003cp\u003eScheme of the primary structures of p53 and Mdm2 showing the intermolecular interactions and their competing intramolecular contacts; folded domains are in dark blue and orange, IDRs are in light blue and yellow; phosphosites are localized with circles, using the same color code as in Fig. 1.\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-9014206/v1/64ad05cd2bdf1b0e888d9a5e.png"},{"id":104207984,"identity":"a920b119-a6d2-4bd3-8da8-97f4b7b6b3fa","added_by":"auto","created_at":"2026-03-09 07:12:26","extension":"pdf","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":13740559,"visible":true,"origin":"","legend":"Supplementary-figures","description":"","filename":"Suppinfo260225.pdf","url":"https://assets-eu.researchsquare.com/files/rs-9014206/v1/fc75527ca68f9f4d8d2070f6.pdf"}],"financialInterests":"There is \u003cb\u003eNO\u003c/b\u003e Competing Interest.","formattedTitle":"The disordered region 1 of Mdm2 weakens p53-binding in both its unmodified and hyperphospho-forms","fulltext":[{"header":"Introduction","content":"\u003cp\u003eThe E3-ubiquitin ligase Mdm2 is an oncoprotein overexpressed in many different types of cancers\u0026nbsp;\u003csup\u003e1,2\u003c/sup\u003e. Its most famous function is to be the main negative regulator of the transcription factor p53, the so-called \u0026quot;tumor suppressor\u0026quot; found mutated in about half of all cancers\u0026nbsp;\u003csup\u003e3,4\u003c/sup\u003e. Given their importance, these proteins have been extensively studied, generating a wealth of information on their partners, activities and regulation. The sum of this knowledge is often summarized by the words \u0026quot;complexity and context\u0026quot; by cell biologists\u0026nbsp;\u003csup\u003e4,5\u003c/sup\u003e.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eFrom the structural biology standpoint, the complexity has been simplified. These proteins have been dissected, studied domain by domain, even though p53 has been characterized eventually in its full-length version using stabilizing mutations in its DNA-binding domain\u0026nbsp;\u003csup\u003e6,7\u003c/sup\u003e. Unfortunately, protocols have been missing to produce milligram-scale samples for structural biology of Mdm2 constructs including more than one domain, to the best of our knowledge. The N-terminal p53-binding domain (p53BD) of Mdm2 (aa26-109, Mdm2-p53BD) has focused the attention of the community: it plays an anchoring role in p53-tethering, an interaction whose inhibition has raised many hopes for treatments\u0026nbsp;\u003csup\u003e8\u003c/sup\u003e. Much less structural biology reports exist on the intrinsically disordered regions (IDRs) intercalated between the folded p53BD, the central Zinc-finger (aa299-328, Mdm2-ZF) binding immature ribosome particles\u0026nbsp;\u003csup\u003e9\u003c/sup\u003e, and the C-terminal E3-ubiquitin effector Mdm2-RING domain\u0026nbsp;\u003csup\u003e7\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003eWe sought to provide structural information on Mdm2-IDR1 (aa110-298) and its possible contacts with its surrounding folded domains, i.e. the Mdm2-p53BD and the Mdm2-ZF. This follows our earlier report on the IDR2 of Mdm2 (aa329-438) and the mapping of its phosphorylation sites by DNA-PK/ATM/ATR \u003csup\u003e10\u003c/sup\u003e. Mdm2-IDR1 is fairly conserved among vertebrates for an IDR, especially on its acidic domain (AD, aa220-296, ~45% identity between human and zebrafish) containing 28 Asp/Glu (Supplementary Fig. 1)\u0026nbsp;\u003csup\u003e11\u003c/sup\u003e. Mdm2-AD\u0026apos;s structural behavior has been scarcely characterized experimentally\u0026nbsp;\u003csup\u003e12\u0026ndash;14\u003c/sup\u003e, confirming by NMR the prediction of a disordered peptide behavior. We can also mention a few crystal structures of IDR1\u0026apos;s short peptides : aa145-150 and aa225-229 both extended in complex with the deubiquitinase USP7\u0026nbsp;\u003csup\u003e15,16\u003c/sup\u003e, and aa164-167(pS166) and aa183-188(pS186) both extended in complex with 14-3-3s\u0026nbsp;\u003csup\u003e17,18\u003c/sup\u003e.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eIDR1 has nevertheless important roles in Mdm2 activities: i) it contains both a nuclear localization sequence NLS(aa179-186) and a nuclear export signal NES(aa190-202); ii) the AD plays an important auxiliary role in p53 binding\u0026nbsp;\u003csup\u003e7,19\u003c/sup\u003e; iii) it has been reported to interact directly with multiple noteworthy partners, among which the Polycomb Repressor Complex 2\u0026nbsp;\u003csup\u003e20\u003c/sup\u003e, PARP-1\u0026nbsp;\u003csup\u003e21\u003c/sup\u003e, the tumor suppressor p14ARF\u0026nbsp;\u003csup\u003e13\u003c/sup\u003e, the ubiquitin-ligase complex SCF\u0026nbsp;\u003csup\u003e22\u003c/sup\u003e, tau\u0026nbsp;\u003csup\u003e23\u003c/sup\u003e, or\u0026nbsp;b-arrestin 1\u0026nbsp;\u003csup\u003e24\u003c/sup\u003e. \u0026nbsp;Moreover, the corresponding IDR1 of the homolog MdmX has been shown to establish intramolecular contacts with its own p53BD\u0026nbsp;\u003csup\u003e25\u0026ndash;27\u003c/sup\u003e and even with the Mdm2-p53BD\u0026nbsp;\u003csup\u003e28\u003c/sup\u003e, hence competing in binding with p53 N-terminal (p53-NT, aa17-29). These contacts engage the so-called WW and WF motifs present in MdmX-IDR1 but absent in Mdm2-IDR1; hydrophobic motifs appear at similar localizations in Mdm2-IDR1 though (Supplementary Fig. 2). At the opposite, Mdm2-IDR1 has been shown to be a secondary binding site of p53, whose affinity has been reported to be tuned by phosphorylation\u0026nbsp;\u003csup\u003e22,29\u0026ndash;35\u003c/sup\u003e -the Mdm2-p53BD remaining the primary anchor for tethering p53. A structural understanding of all these conflicting interactions is still missing, to the best of our knowledge.\u003c/p\u003e\n\u003cp\u003eStudying Mdm2 constructs containing IDR1 has certainly been impeded by the fact that its native form is hyperphosphorylated in absence of stress, which makes the production of pure samples more difficult. According to the literature, Mdm2-IDR1\u0026apos;s phosphorylation improves its binding to p53, while DNA-damage was reported to provoke a hypophosphorylation of Mdm2-IDR1 favoring p53 stabilization\u0026nbsp;\u003csup\u003e29\u0026ndash;32,34,35\u003c/sup\u003e. The corresponding phosphorylation schemes have not been intensively mapped, probably because the amino acid composition of the Mdm2-AD hampers a proper coverage by mass-spectrometry proteomics (see https://www.uniprot.org/uniprotkb/Q00987/feature-viewer). NMR spectroscopy is an appropriate complementary technique is such cases, not only because of its ability to provide residue-specific information on IDPs\u0026nbsp;\u003csup\u003e36\u0026ndash;38\u003c/sup\u003e, but also because of its capacities in poly-phosphorylation mapping and monitoring\u0026nbsp;\u003csup\u003e10,39\u0026ndash;43\u003c/sup\u003e.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eHere, we report our structural investigations on Mdm2-IDR1, either isolated or in more relevant contexts, i.e. in constructs containing also its neighbor folded domains Mdm2-p53BD and Mdm2-ZF. We carried out parallel studies on the structural behavior of IDR1 in the various constructs and on the resulting affinities for p53, using NMR and ITC, respectively. We also characterized the hyperphosphorylation executed by CK1, the main kinase active on Mdm2 in absence of stress, and its consequences on Mdm2 structure and interaction with p53. Because these peptides carry abundant electrostatic charges, we performed all experiments in phosphate-buffered saline (PBS) to favor the adoption of close-to-native conformational and binding behaviors.\u003c/p\u003e\n"},{"header":"Results","content":"\u003cp\u003e\u003cstrong\u003eMdm2-IDR1 is disordered before and after phosphorylation by CK1\u003c/strong\u003e\u003cstrong\u003ed\u003c/strong\u003e\u003cstrong\u003e on 15 sites\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFirst, we verified experimentally the predicted intrinsic disorder of Mdm2-IDR1 in constructs of increasing sizes, from aa111-180, aa111-230, aa111-293, aa111-333, aa1-333, i.e. incorporating progressively segments of human Mdm2 including the folded Mdm2-p53BD(aa26-109) and the Mdm2-ZF(aa296-333) (Fig. 1a). We started using \u003csup\u003e1\u003c/sup\u003eH-\u003csup\u003e15\u003c/sup\u003eN NMR at 283 K, a standard approach to obtain residue-specific information. The low temperature limits water-amide \u003csup\u003e1\u003c/sup\u003eH-exchange, which is too fast at 310 K in IDRs to obtain exploitable \u003csup\u003e1\u003c/sup\u003eH-\u003csup\u003e15\u003c/sup\u003eN spectra \u003csup\u003e10\u003c/sup\u003e. Interestingly the folded Mdm2-p53BD was invisible and the Mdm2-ZF barely detectable in Mdm2(aa1-333). Mdm2-IDR1 residues were all in the random coil window of chemical shifts (Fig. 1b), which was later confirmed by the backbone assignment giving access to the Ca-Cb chemical shifts and the associated secondary structure propensities (Fig. 1c, Supplementary Fig. 3) \u003csup\u003e44\u003c/sup\u003e. We used cysteine-to-alanine mutants for the longest constructs, ensuring their production and stability during the hours-long NMR acquisitions: even in presence of DTT or TCEP, the 4 cysteines of IDR1 were rapidly forming multiple disulfide bonds, and the wild-type Mdm2 constructs were expressed mainly as aggregates. We verified that the four cysteine mutations C127A-C200A-C206-C207A in Mdm2-IDR1 did not alter its disordered behavior (Supplementary Fig. 3). We achieved the NMR backbone assignment of Mdm2-IDR1 with and without cysteine mutations (BMRB codes: 52016 for Mdm2(aa111-293), 52028 for Mdm2(aa111-230_4C4A)). We generated a few point mutations and recorded the resulting spectra to confirm the assignment (Supplementary Fig. 4-5).\u003c/p\u003e\n\u003cp\u003eThen, we carried out the phosphorylation of Mdm2-IDR1 by CK1d. The native polyphosphorylation of IDR1 has been reported to be executed by CK1, CK2 and GSK-3b \u003csup\u003e22,29\u0026ndash;32,34,35\u003c/sup\u003e: these kinases are attracted by negatively charged residues, which are abundant in Mdm2-IDR1. We decided to use the isoform CK1d, because i) the recombinant production of an active form of this kinase is feasible in sufficient quantities for NMR structural studies \u003csup\u003e41,45\u003c/sup\u003e, and ii) the literature was suggesting it to be dominant in establishing Mdm2-IDR1 polyphosphorylation \u003csup\u003e22,29\u0026ndash;32,34,35\u003c/sup\u003e. Using \u003csup\u003e1\u003c/sup\u003eH-\u003csup\u003e15\u003c/sup\u003eN NMR correlation spectroscopy, we detected 15 phosphosites upon incubation of \u003csup\u003e15\u003c/sup\u003eN-Mdm2-IDR1 with an \u0026quot;NMR-invisible\u0026quot; \u003csup\u003e14\u003c/sup\u003eN-CK1d (Figure 1a-1b). We mapped them by assigning the cross-peaks of phospho-Mdm2-IDR1 (\u003csup\u003e13\u003c/sup\u003eC/\u003csup\u003e15\u003c/sup\u003eN labeled version, BMRB code: 52289) after its purification and separation from CK1d, whose loose binding provokes otherwise poor-quality spectra. The identified phosphosites were S192, S215, T218, S220, S229, S232, S240, S242, S246, S253, S256, S260, S262, T279, and S286. This shows that CK1d is attracted by the negatively charged region of Mdm2-IDR1, more than by its classical consensus motifs pS/pT/D/E-x-x-S/T (Figure 1a) \u003csup\u003e46,47\u003c/sup\u003e. We noticed some supplementary very weak crosspeaks from minor phosphosites, which we could not assign due to low signal levels except S166.\u003c/p\u003e\n\u003cp\u003eThen, we monitored the reaction in real-time, recording time-series of \u003csup\u003e1\u003c/sup\u003eH-\u003csup\u003e15\u003c/sup\u003eN NMR spectra. To extract site-specific information, we also had to assign cross-peaks appearing transiently over time: these are due to intermediate species containing close, non-stoichiometric phosphosites, which can generate multiple combinations of chemical environments \u003csup\u003e10,39\u0026ndash;43\u003c/sup\u003e. We met some cumbersome cases with 3 close phosphosites, which forced us to obtain the assignments from synthesized peptides containing the various combinations of one or two phosphosites (Supplementary Fig. 6a). Altogether, we observed that all sites were phosphorylated in an independent, distributive manner. We also recorded phosphorylation kinetics of Ser/Thr to Ala mutants, which provided supplementary confirmations of the assignments (Supplementary Fig. 7). These mutants exhibited often slower phosphorylation rates in the vicinity of the mutated residue (Supplementary Fig. 8a). Nevertheless, the final phosphorylation states remained unaffected, indicating that phosphorylation at a given site is not a prerequisite for phosphorylation at adjacent sites, and supporting a distributive mechanism for CK1\u0026delta; on Mdm2-IDR1. We grouped the phosphosites arbitrarily into five clusters with similar kinetics, from the fastest to the slowest ones: S192-S215\u0026ndash;S242\u0026ndash;S246\u0026ndash;S253, S220\u0026ndash;S229\u0026ndash;S232\u0026ndash;S240\u0026ndash;S256, T218\u0026ndash;S260, S262, and S192-T279\u0026ndash;S286 (Fig. 1d, Supplementary Fig. 8b).\u003c/p\u003e\n\u003cp\u003eThe backbone assignment of phospho-Mdm2-IDR1 (BMRB 52289) provided also the \u003csup\u003e13\u003c/sup\u003eCa-\u003csup\u003e13\u003c/sup\u003eCb chemical shifts, which, in turn, gave access to the secondary structure content (Fig. 1c). Interestingly, the hyperphosphorylated Mdm2-IDR1 shows more extended average backbone conformations than the non-phosphorylated form, which is probably due to electrostatic repulsion. Hence, the Mdm2-AD remains highly flexible and disordered upon phosphorylation, but behaves less like a random coil than its non-phospho counterpart. The rest of Mdm2-IDR1 did not show any sign of conformational changes upon phosphorylation and remained highly disordered.\u003c/p\u003e\n\u003cp\u003eOverall, the isolated Mdm2-IDR1 behaves as a classical IDR before and after phosphorylation by CK1d. This phosphorylation is distributive, with no conditional priming event, and occurs preferentially on the segments containing the highest densities of Asp/Glu of Mdm2-IDR1.\u003c/p\u003e\n\n\u003cp\u003e\u003cstrong\u003eNMR evidence for intramolecular interactions between Mdm2-IDR1 and Mdm2-p53BD\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNext, we incorporated the N-terminal Mdm2-p53BD in our analysis, by producing a longer construct Mdm2(aa1-333). We noticed immediately that some peak intensities were weaker in \u003csup\u003e1\u003c/sup\u003eH-\u003csup\u003e15\u003c/sup\u003eN NMR spectra (Fig. 2a-2b). Two segments were concerned: the most perturbed one around the region aa189-208, and a second one, a bit less affected, around aa242-255. These are the most hydrophobic segments of Mdm2-IDR1. The residue-specific relaxation parameters \u003csup\u003e15\u003c/sup\u003eN-R\u003csub\u003e1\u003c/sub\u003e, and \u003csup\u003e1\u003c/sup\u003eH-\u003csup\u003e15\u003c/sup\u003eN heteronuclear nOes did almost not change between Mdm2-IDR1 and Mdm2(aa1-333), revealing comparable conformational dynamics of Mdm2-IDR1 in both constructs at the nanosecond timescale (Supplementary Fig. 9a). The \u003csup\u003e15\u003c/sup\u003eN-R\u003csub\u003e2\u003c/sub\u003e\u003csup\u003e \u003c/sup\u003erelaxation rates showed a diffuse increase all along IDR1 in Mdm2(aa1-333), with very high rates between aa195 and aa205 (actually even non-measurable), and around aa250 (Fig. 2c). This differential variations between \u003csup\u003e1\u003c/sup\u003eH-\u003csup\u003e15\u003c/sup\u003eN nOes and \u003csup\u003e15\u003c/sup\u003eN-R\u003csub\u003e2\u003c/sub\u003e are only consistent with transient intramolecular interactions between the segment aa190-210 and Mdm2-p53BD at the ms-ms timescale, together with weaker transient interactions between the rest of Mdm2-IDR1 and Mdm2-p53BD.\u003c/p\u003e\n\u003cp\u003eTo confirm this hypothesis, we mixed \u003csup\u003e15\u003c/sup\u003eN-Mdm2(aa1-333) and an \u0026quot;NMR-invisible\u0026quot; \u003csup\u003e14\u003c/sup\u003eN-p53(aa14-29) in stoichiometric proportions. The latter peptide is the core binding element of p53 to Mdm2-p53BD. We observed the recovery of peak intensities from Mdm2-IDR1 residues (Fig. 2b-2d), which was consistently accompanied by a decrease of \u003csup\u003e15\u003c/sup\u003eN-R\u003csub\u003e2\u003c/sub\u003e rates almost back to the values obtained from Mdm2-IDR1 isolated (Fig. 2c). We conclude that the transient intramolecular interactions occur mainly between the hydrophobic segments of Mdm2-IDR1 and the hydrophobic groove of Mdm2-p53BD, the latter of which has still a much better affinity for its cognate partner p53(aa14-29).\u003c/p\u003e\n\u003cp\u003eOut of curiosity, we evaluated the affinity between Mdm2-p53BD and Mdm2-IDR1 in an artificial situation, where they would be present as separate species. We titrated \u003csup\u003e15\u003c/sup\u003eN-Mdm2-IDR1 at 100 mM with \u003csup\u003e14\u003c/sup\u003eN-Mdm2(aa1-112), which revealed the same loose binding patches, i.e. aa195-205 and aa245-255 (Supplementary Fig. 10). According to the peak intensity losses and the extreme weak chemical shift perturbations (\u0026lt;0.06 ppm) evolving linearly up to [\u003csup\u003e14\u003c/sup\u003eN-Mdm2(aa1-112)]=200 mM, the transient interactions occur in the ms-ms scale with affinities above 100 mM.\u003c/p\u003e\n\n\u003cp\u003e\u003cstrong\u003ePhosphorylation does not disrupt intramolecular interactions between Mdm2-IDR1 and Mdm2-p53BD\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThen, we carried out the same investigations on phospho-Mdm2(aa1-333). First, we monitored the phosphorylation kinetics of Mdm2(aa1-333) by CK1d, which turned out to be very similar to those obtained from Mdm2-IDR1 in isolation (Supplementary Fig. 8c). The \u003csup\u003e1\u003c/sup\u003eH-\u003csup\u003e15\u003c/sup\u003eN NMR spectra of phospho-Mdm2(aa1-333) and the relaxation parameters \u003csup\u003e15\u003c/sup\u003eN-R\u003csub\u003e1\u003c/sub\u003e,\u003csup\u003e 15\u003c/sup\u003eN-R\u003csub\u003e2\u003c/sub\u003e and \u003csup\u003e1\u003c/sup\u003eH-\u003csup\u003e15\u003c/sup\u003eN heteronuclear nOes showed high similarities with the non-modified form (Fig. 2e-2f, Supplementary Fig. 9b). They revealed transient interactions at the ms-ms timescale between the Mdm2-p53BD and the segment Mdm2(aa190-210), with some weaker transient touches between Mdm2-p53BD and the rest of Mdm2-IDR1.\u003c/p\u003e\n\u003cp\u003eHence, CK1d-phosphorylated Mdm2-IDR1 establishes intramolecular contacts between its most hydrophobic segments and the p53-binding groove of Mdm2-p53BD. These do not change the phosphorylation kinetics by CK1d, nor does the resulting hyperphosphorylation affect these intramolecular interactions.\u003c/p\u003e\n\n\u003cp\u003e\u003cstrong\u003eMdm2-IDR1 reverts and accelerates the \u0026quot;open \u0026amp; closed\u0026quot; equilibrium of the N-ter lid with the Mdm2-p53BD\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe characterization reported above was limited to the disordered Mdm2-IDR1. This is due to the fact that Mdm2-p53BD is invisible in \u003csup\u003e1\u003c/sup\u003eH-\u003csup\u003e15\u003c/sup\u003eN NMR spectra of Mdm2(aa1-333). Mdm2-p53BD has been regularly studied by \u003csup\u003e1\u003c/sup\u003eH-\u003csup\u003e15\u003c/sup\u003eN NMR in isolation by many labs including ours \u003csup\u003e48,49\u003c/sup\u003e, but its tumbling time is apparently very much slowed down in a more native context including IDR1, which provokes a very fast R\u003csub\u003e2\u003c/sub\u003e relaxation and a vanishing signal. This is consistent with the multiple intramolecular interactions that we detected, see above. \u003c/p\u003e\n\u003cp\u003eWe thought to produce a perdeuterated protein construct Mdm2(aa1-333) with only \u003csup\u003e13\u003c/sup\u003eCH\u003csub\u003e3\u003c/sub\u003e methyl labeling: \u003csup\u003e1\u003c/sup\u003eH-\u003csup\u003e13\u003c/sup\u003eC methyl NMR in a perdeuterated context can provide exploitable signals even for slow-tumbling species up to the MDa size \u003csup\u003e50\u003c/sup\u003e. This approach permitted recently to Nishida and colleagues to investigate the open-closed equilibrium of Mdm2-p53BD governed by the flexible N-terminal loop Mdm2(aa1-24), so called the \u0026quot;lid\u0026quot; \u003csup\u003e51\u003c/sup\u003e. We adopted their strategy and produced deuterated Mdm2(aa1-333) incorporating Ile\u003csup\u003ed\u003c/sup\u003e\u003csup\u003e1\u003c/sup\u003e-Met\u003csup\u003ee\u003c/sup\u003e(\u003csup\u003e13\u003c/sup\u003eCH\u003csub\u003e3\u003c/sub\u003e) labeling, and recorded \u003csup\u003e1\u003c/sup\u003eH-\u003csup\u003e13\u003c/sup\u003eC SOFAST-HMQC methyl TROSY spectra at 310 K. We paid a particular attention to the signals from Ile19 in the lid: Ile19 is a good reporter of the equilibrium between the open flexible state of the lid, and the closed state where it binds to the p53-binding groove of Mdm2-p53BD.\u003c/p\u003e\n\u003cp\u003eIn presence of p53(aa14-29), the lid is pushed away from the Mdm2-p53BD binding groove and is found 100% in the open state, yielding a \u003csup\u003e1\u003c/sup\u003eH-\u003csup\u003e13\u003c/sup\u003eC correlation spectrum matching the one published recently from Mdm2(aa1-109) by Nishida et al. \u003csup\u003e51\u003c/sup\u003e (Fig. 3a). In contrast, we observed strong differences between Mdm2(aa1-109) and Mdm2(aa1-333) in their apo forms. While Nishida and colleagues reported a 20:80 open:closed equilibrium in slow exchange (~1 Hz or less) for their construct Mdm2(aa1-109), the peaks of Ile19 and Met50 revealed a 70:30 ratio and a fast exchange (~10\u003csup\u003e6\u003c/sup\u003e Hz or more) in Mdm2(aa1-333) at 310 K (Fig. 3a-3b). Decreasing the temperature favored the displacement of these two peaks towards the chemical shifts of the closed state, but also their progressive disappearance (Fig. 3c). This corresponds to the exchange slowing down to an intermediate timescale (~10\u003csup\u003e3\u003c/sup\u003e-10\u003csup\u003e6\u003c/sup\u003e Hz), starting from 306 K. This is consistent with the interactions observed on the Mdm2-IDR1 side using \u003csup\u003e1\u003c/sup\u003eH-\u003csup\u003e15\u003c/sup\u003eN NMR at 283 K (see above). \u003c/p\u003e\n\u003cp\u003eInterestingly, the peaks from Ile74 or Met102 shifted with the temperature in apo-Mdm2(aa1-333) (Fig. 3c), whereas they were not in apo-Mdm2(aa1-109) \u003csup\u003e51\u003c/sup\u003e or in Mdm2(aa1-333):p53(aa14-29) (Fig. 3d). This means that Mdm2-IDR1 establishes supplementary transient contacts with Mdm2-p53BD, which are mainly taking place in the p53-binding groove - but not exclusively, see Ile99 in Mdm2(aa1-333):p53(aa14-29). \u003c/p\u003e\n\u003cp\u003eAll these observations on Mdm2-p53BD from \u003csup\u003e1\u003c/sup\u003eH-\u003csup\u003e13\u003c/sup\u003eC spectra are fully consistent with our earlier observations in \u003csup\u003e1\u003c/sup\u003eH-\u003csup\u003e15\u003c/sup\u003eN spectra showing lower peak intensities of IDR1\u0026apos;s residues in Mdm2(aa1-333) than in Mdm2(aa111-333), and a reemergence of these peaks upon adding p53(aa14-29) (Fig. 2). Also consistent with our earlier characterization using \u003csup\u003e1\u003c/sup\u003eH-\u003csup\u003e15\u003c/sup\u003eN spectra, the \u003csup\u003e1\u003c/sup\u003eH-\u003csup\u003e13\u003c/sup\u003eC SOFAST-HMQC methyl-TROSY spectra of phospho-Mdm2(aa1-333), phosphorylated by CK1d, were extremely similar to those obtained from the non-modified Mdm2(aa1-333) (Supplementary Fig. 11). This indicates that CK1\u0026delta;-mediated phosphorylation does not significantly affect the open:closed equilibrium of the lid with p53BD.\u003c/p\u003e\n\u003cp\u003eHence, Mdm2-IDR1 affects considerably the open:closed equilibrium between the lid and the p53BD. It competes with the lid for binding the p53-binding groove, and provokes a much more dynamic environment than the one reported previously from truncated forms, which were comprising only the lid and the p53BD \u003csup\u003e48,51\u003c/sup\u003e. The incorporation of Mdm2-IDR1 pushed the open:closed ratio of the lid from 4 to 0.25 (an equivalent of almost 2 kcal.mol\u003csup\u003e-1\u003c/sup\u003e of free energy of folding), and accelerated the related conformational exchange by at least 6 orders of magnitude at 310 K.\u003c/p\u003e\n\n\u003cp\u003e\u003cstrong\u003ep53(aa14-29) binding releases IDR1 but makes Mdm2(aa1-333) globally more compact\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe temperature-dependent chemical shifts observed on Ile\u003csup\u003ed\u003c/sup\u003e\u003csup\u003e1\u003c/sup\u003e-Met\u003csup\u003ee\u003c/sup\u003e(\u003csup\u003e13\u003c/sup\u003eCH\u003csub\u003e3\u003c/sub\u003e) of Mdm2(aa1-333) suggested temperature-dependent degrees of transient interactions between Mdm2-IDR1 and Mdm2-p53BD. We determined the hydrodynamic radius (R\u003csub\u003eh\u003c/sub\u003e) as a function of temperature by measuring diffusion rates using the double echo PGSTE-WATERGATE sequence\u003csup\u003e52\u003c/sup\u003e with 1,4-dioxane as an internal reference. Mdm2(aa1-333) R\u003csub\u003eh\u003c/sub\u003e decreased with temperature, indicating that Mdm2(aa1-333) adopts a more compact conformation as temperature increases (Fig. 4), which is rather common for IDRs \u003csup\u003e53,54\u003c/sup\u003e. More surprising, we measured lower R\u003csub\u003eh\u003c/sub\u003e values upon the addition of p53(aa14-29), although this peptide forced the lid and Mdm2-IDR1 to leave their transient occupation of the p53-binding groove. Hence, the ensemble of \u0026quot;lid/IDR1-open\u0026quot; conformations is actually more compact than its \u0026quot;lid/IDR1-closed\u0026quot; counterpart. The Mdm2-lid is much shorter than Mdm2-IDR1, and these changes of hydrodynamic radii are probably mostly due to the conformational behavior of Mdm2-IDR1. Once released from the p53-binding groove, the most hydrophobic segments of Mdm2-IDR1 may form a more hydrophobic nucleus for the disordered molecular cloud, promoting its relative desolvation and a global compaction. This rationale appears to hold true even for a construct deleted from aa189-208, the most interacting segment of Mdm2-IDR1: the addition of p53(aa14-29) still provokes the adoption of a conformational ensemble with a smaller R\u003csub\u003eh\u003c/sub\u003e (Supplementary Fig. 12).\u003c/p\u003e\n\u003cp\u003eHence, upon binding the motif p53(aa14-29), the Mdm2-p53BD releases its transient interactions with Mdm2-IDR1 and the Mdm2-lid, which, counterintuitively, makes Mdm2-IDR1 more compact, and thus possibly less accessible to other interactions with the rest of p53.\u003c/p\u003e\n\n\u003cp\u003e\u003cstrong\u003eMdm2-IDR1 weakens the affinity for p53 \u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFinally, we asked whether the Mdm2-IDR1 had an effect on binding p53. We have shown that Mdm2-IDR1 interacts transiently with Mdm2-p53BD in a competing fashion with p53(aa14-29), which should have deleterious effects on the affinity between Mdm2 and p53. Concurrently, Mdm2-IDR1 is very negatively charged and may improve binding to p53, which contains a positively charged DNA-binding domain (p53-DBD). Which way does the balance tip? We chose isothermal calorimetry (ITC) to measure affinities: it allows for titrations in solution, avoiding the immobilization of one protein on a surface and the potential bias that can easily emerge in these semi-liquid conditions for the highly flexible IDRs. The measured values are summarized in Figure 5 and Table 1.\u003c/p\u003e\n\u003cp\u003eStarting with truncated constructs, we measured a K\u003csub\u003eD\u003c/sub\u003e of 37 \u0026plusmn; 8 nM between Mdm2-p53BD(aa18-112) and p53-NT(aa1-88). Incorporating the lid, i.e. using Mdm2(aa1-112), decreased the affinity by a factor 3 for p53-NT(aa1-88) at 105 \u0026plusmn; 8 nM. This is consistent with the competing interaction between the lid and p53 for binding the p53BD-binding groove. Next, we incorporated Mmd2-IDR1 and Mdm2-ZF in Mdm2(aa1-333), which led the affinity for p53-NT(aa1-88) to 480 \u0026plusmn; 5 nM. Even more deleterious, phosphoMdm2(aa1-333) affinity for p53-NT(aa1-88) rose to 1.0 \u0026plusmn; 0.1 mM. This is most probably due to an electrostatic repulsion with p53-NT(aa1-88), which carries a total 16 negative charges (18 Asp/Glu vs 2 Arg/Lys). Interestingly, the removal of the segment Mdm2(aa190-210) from Mdm2(aa1-333) led to a weak but measurable strengthening of the affinity for p53-NT(aa1-88) at 320 \u0026plusmn; 30 nM, in agreement with the NMR-revealed competition between Mdm2(aa190-210) and p53(aa14-29) for binding Mdm2-p53BD.\u003c/p\u003e\n\u003cp\u003eThen, we carried out the same series of titrations with full-length p53 (p53FL). It is worth mentioning that it assembles as a tetramer at the working concentrations \u003csup\u003e55\u003c/sup\u003e. We measured a K\u003csub\u003eD\u003c/sub\u003e of 81 \u0026plusmn; 6 nM between Mdm2-p53BD(aa18-112) and p53FL, weaker than with p53-NT(aa1-88). This is consistent with intramolecular interactions between p53-NT(aa1-88) and p53-DBD (reported earlier and confirmed by us \u003csup\u003e6,56,57\u003c/sup\u003e, see Supplementary Fig. 13), which can compete with Mdm2-p53BD(aa18-112). The loss of affinity due to the incorporation of the lid in Mdm2-p53BD(aa1-112), observed previously with p53-NT(aa1-88), was confirmed with p53FL: we measured a K\u003csub\u003eD\u003c/sub\u003e of 510 \u0026plusmn; 40 nM. Then, the addition of Mmd2-IDR1 and Mdm2-ZF, resulting in Mdm2(aa1-333), affected weakly the affinity for p53FL, which was measured to be 310 \u0026plusmn; 90 nM. Even the CK1d-mediated hyperphosphorylation did not have any strong effects on binding p53FL, the affinity being evaluated to 390 \u0026plusmn; 20 nM. NMR spectra show that the negatively charged Mdm2-AD interacts with the positively charged p53-DBD in a salt-dependent manner (Supplementary Fig. 14). Even though we observed faint chemical shifts perturbations (Dd \u0026lt; 0.03 ppm) and disappearance of crosspeaks in \u003csup\u003e1\u003c/sup\u003eH-\u003csup\u003e15\u003c/sup\u003eN NMR spectra of \u003csup\u003e15\u003c/sup\u003eN-Mdm2-IDR1 in presence of \u003csup\u003e14\u003c/sup\u003eN-p53FL, we could not measure any affinity using ITC (data not shown), placing it probably in the millimolar range. Hence, this electrostatic interaction appears to be rather diffuse with no well-defined contacts, and seems to only counterbalance the deleterious contributions of Mdm2-IDR1 due to intramolecular contacts and diffuse shielding of the Mdm2-p53BD.\u003c/p\u003e\n\u003cp\u003eFinally, we confirmed again the negative contribution of the interaction between Mdm2(aa190-210) and Mdm2-p53BD: the affinity between Mdm2(aa1-333_D189-208) and p53FL was measured to be 150 \u0026plusmn; 10 nM.\u003c/p\u003e\n\u003cp\u003eAltogether, Mdm2-IDR1 interferes strongly with the capacity of Mdm2-p53BD to bind p53, from 5- to 25-fold, depending on the peptide construct, i.e. p53FL or the short p53(aa1-88). Competing intramolecular contacts combine with electrostatic attractions and repulsions to tune positively or negatively the affinity between Mdm2 and p53. Importantly, the beneficial electrostatic interaction between the Mdm2-AD and p53-DBD is only moderate, and it does not overcome the unfavorable intramolecular contacts and the diffuse screening established by the IDR1.\u003c/p\u003e\n"},{"header":"Discussion","content":"\u003cp\u003eAlthough its N-terminal p53BD has been heavily scrutinized structurally, Mdm2\u0026apos;s global structure is not yet well apprehended. Specifically, Mdm2-IDR1 is poorly described, even though its reported functions and partners are numerous\u0026nbsp;\u003csup\u003e7,13,15\u0026ndash;24\u003c/sup\u003e. These observations identified the Mdm2-AD as a nodal point for regulatory signals, through which the p53 pathway can integrate information from the\u0026nbsp;cellular state. However, studying IDRs\u0026apos; structural behavior in their native contexts, inserted between folded domains, is still a challenge: only NMR spectroscopy can bring residue-specific information, but the current isotope-labeled recombinant expression in bacterial systems comes often with low yields for these multidomain protein constructs. It is even worse if we add a supplementary but necessary layer of biological context: IDRs carry abundant PTMs in their native states, eventually in a conditional fashion when related to cell signaling\u0026nbsp;\u003csup\u003e58\u0026ndash;60\u003c/sup\u003e. Producing pure hyperphosphorylated samples is not straightforward, and the present report is thus bringing novel information on such highly modified IDRs in a multidomain context.\u003c/p\u003e\n\u003cp\u003eEarlier reports on p53 had shown that weak, transient contacts between its N-terminal region and its DNA-BD were capable of inhibiting DNA binding\u0026nbsp;\u003csup\u003e6,56\u003c/sup\u003e. Here, we have shown that these p53 intramolecular contacts were also inhibiting the binding to Mdm2. More novel, we have shown that multiple intramolecular contacts existed also in Mdm2 (Fig. 2-3, Supplementary Fig. 9-11), and that they also affect negatively the interaction with p53 (Fig. 5). Interestingly, while Mdm2-AD and its ~20 negative charges bind loosely to the positively charged p53-DBD even at clost-to-physiological salt concentrations in PBS (Supplementary Fig. 14), it does not compensate the detrimental effects of the multiple intramolecular interactions that Mdm2-IDR1 generates with Mdm2-p53BD in a competing fashion with p53.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThese intramolecular contacts in Mdm2 are in correspondence with those reported on its homolog MdmX\u0026nbsp;\u003csup\u003e25\u0026ndash;28\u003c/sup\u003e: the most involved fragments in Mdm2 are aa189-208 and aa242-255, which are exactly aligned with the regions of MdmX binding to its own MdmX-p53BD (Supplementary Fig. 2). In MdmX, two equivalent peptide segments, containing multiple Trp residues, have been shown to have autoinhibitory capacities\u0026nbsp;\u003csup\u003e25,61,62\u003c/sup\u003e. If measured with two separate peptides, MdmX-IDR1 has an affinity for MdmX-p53BD of about 1\u0026nbsp;mM, which provokes a 100-fold affinity loss of affinity for p53 in constructs containing MdmX-p53BD+IDR1 vs MdmX-p53BD\u0026nbsp;\u003csup\u003e62\u003c/sup\u003e. MdmX-IDR1 has also a good affinity for Mdm2-p53BD, about 8\u0026nbsp;mM, which can also interfere with p53 binding to Mdm2\u0026nbsp;\u003csup\u003e28\u003c/sup\u003e. Here, we evaluated that Mdm2-IDR1 would have a dissociation constant above 100\u0026nbsp;mM for Mdm2-p53BD as separate species, but their native concatenation generates transient contacts, which compete with p53 and tune the affinity between Mdm2 and p53 by a factor 2-3 depending on the Mdm2 construct.\u003c/p\u003e\n\u003cp\u003eThe functional consequences of these loose intramolecular contacts can be substantial. In the present case, the addition of those identified in p53 and Mdm2 lead to the a 10-fold decrease in affinity (comparison between Mdm2(aa18-112)+p53(aa1-88) vs Mdm2(aa1-333)+p53FL). This is most probably an under-evaluation of their pure effects, because electrostatic interactions between the Mdm2-AD and p53-DBD bring more favorable components to the global interaction. It appears anyway to be difficult to deconvolute all the by-products of loose binding events appearing upon merging folded and disordered domains. For example, upon binding to p53(aa14-29), the release of Mdm2-IDR1 from its transient binding to Mdm2-p53BD provoked a faint, but measurable compaction of Mdm2(aa1-333). This counterintuitive result shows that it is probably often difficult to predict IDRs\u0026apos; accessibility in their native context from isolated models. This calls for improved modeling tools adapted to multidomain constructs, if we want to progress in our understanding of native IDRs conformational ensembles and binding accessibility\u0026nbsp;\u003csup\u003e58,63,64\u003c/sup\u003e. It calls also for developing further thermodynamic models to fully account for the complexity of IDRs and the appearing carousel of their multiple loose contacts in multidomain proteins\u0026nbsp;\u003csup\u003e65\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp; Regarding the phosphorylation sites, one study had reported a list of CK1-phosphorylated residues in Mdm2-AD using mass-spectrometry without quantification: the list was impressive, containing\u0026nbsp;S215, S220, S229, S232, S240, S242, S246, S256, S260, S269, S286, S288 et S290\u0026nbsp;\u003csup\u003e22\u003c/sup\u003e. We confirmed most of these sites, except S269, S288 and S290. At the opposite, we found out that T218 and S253 were readily phosphorylated by CK1d, and that S192, S262 and T279 were eventually secondary targets. NMR spectroscopy cannot provide information on endogenous phosphosites, but it delivers a non-ambiguous residue-specific and quantitative information on phosphorylation reactions for purified material\u0026nbsp;\u003csup\u003e36\u0026ndash;38\u003c/sup\u003e, because of its capacities in poly-phosphorylation mapping and monitoring\u0026nbsp;\u003csup\u003e10,39\u0026ndash;43\u003c/sup\u003e. Here, it allowed us to show that\u0026nbsp;all sites were phosphorylated in parallel but at different rates. These findings contradict the widely discussed conditional phosphorylation mechanisms associated to CK1\u0026delta;, and instead support a fully distributive and independent mechanism. The data suggest that local electrostatics, driven by glutamate- and aspartate-rich sequences, may play a more significant role than canonical CK1\u0026delta; motifs. These motifs were classically defined using peptide arrays\u0026nbsp;\u003csup\u003e66\u003c/sup\u003e, which cannot account for the properties emerging from longer, native IDRs. Here again, studying IDRs in their native multidomain context brings information that is hardly predictable using the actual prediction tools. More experimental data from more native protein constructs will be necessary to improve these predictions.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eQuite surprisingly, the 15 phosphorylation events on Mdm2-AD did neither affect the intramolecular contacts with Mdm2-p53BD, nor did they strongly modulate the affinity for the positively charged p53-DBD. This is in stark contrast with all the possible effects and functions of IDRs\u0026apos; poly-phosphorylation, which have been reported on other proteins in the literature\u0026nbsp;\u003csup\u003e67\u003c/sup\u003e. However, this result might not be definitive: other intramolecular contacts may appear upon integrating Mdm2-IDR2 and Mdm2-RING domains, a work that we are currently tempting to achieve.\u003c/p\u003e\n\u003cp\u003eAltogether, it becomes clear that studying the isolated domains Mdm2-p53BD and p53-NT does not account for all the transient contacts, intra- or inter-molecular, emerging from incorporating the other neighboring domains. These modulate Mdm2:p53 affinity in the range of one or two orders of magnitude, with multiple layers of competitions counteracting each other. Interestingly, these unpredictable interactions and effects are comparable to the affinity differences measured between the isolated Mdm2-p53BD and p53-NT across vertebrates\u0026nbsp;\u003csup\u003e68\u003c/sup\u003e. It is tempting to imagine that some intramolecular contacts might have appeared or disappeared through evolution, as a result of a need to tune the intrinsic affinities of the core binding event between Mdm2-p53BD and p53-NT.\u003c/p\u003e\n\u003cp\u003eThis report shows that Mdm2-IDR1 interferes with Mdm2 binding to p53, with negative contributions combining with weaker positive effects, if any deconvolution is really conceivable. The affinity of Mdm2(aa1-333) for p53FL converges towards ~400 nM, in the non-modified and phosphorylated forms, instead of the ~40 nM measured on the isolated Mdm2-p53BD and p53-NT. Only a handful of structural studies have been yet published on multidomain proteins incorporating folded and disordered domains\u0026nbsp;\u003csup\u003e6,25,57,69\u0026ndash;76\u003c/sup\u003e, which all report interdomain regulating contacts. These are not yet apprehended by machine-learning assisted predictions nor well-captured by the current molecular dynamics force-fields. Hence, obtaining more experimental data on IDR\u0026apos;s transient intramolecular contacts is one of the important axes in structural biology to explore in the next years, and eventually to confirm in the native, crowded cellular environment\u0026nbsp;\u003csup\u003e38,77\u0026ndash;79\u003c/sup\u003e.\u003c/p\u003e"},{"header":"Methods","content":"\u003cp\u003e\u003cstrong\u003ePeptide constructs\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCodon-optimized genes encoding truncated variants of human Mdm2, including Mdm2 (aa1-333), a deletion-mutant lacking residues 189-208 (D189-208), and additional truncated forms (aa1-112, aa18-112, aa111-180, aa111-230, aa111-333, aa111-237, aa230-333) were synthesized and cloned by Genscript (Supplementary Fig. 16). These constructs contained alanine substitutions at position Cys2, Cys127, Cys200, Cys206, and Cys207. The genes were cloned into the pET-26(b+) expression vector using NdeI and BamHI restriction sites. Additional constructs incorporating alanine substitutions at S215, T218, S220, S246, S253, S260, and S262 were mutated by Genscript, using the initial constructs as templates.\u003c/p\u003e\n\u003cp\u003eBecause p53-WT is poorly stable and aggregates progressively as a purified species, we used a stabilized version incorporating mutations in the p53-DBD introduced by different teams in the past.\u003csup\u003e80,81\u003c/sup\u003e The resulting construct included 10 mutations C135V-C141V-C182S-V203A-R209P-C229S-Y234F-N235K-Y236F-T253V, yielding a very stable p53-DBD (Tm~55 \u0026deg;C, in comparison with Tm~42\u0026deg;C for the WT). The crystallographic structure of this mutant p53-DBD(aa90-293) shows its proper folding and structure.\u003c/p\u003e\n\u003cp\u003eAll constructs were designed to include a hexahistidine (His6) tag, followed by a Tobacco Etch Virus (TEV) protease cleavage site (ENLYFQG) at the N-terminus of the peptide of interest.\u003c/p\u003e\n\n\u003cp\u003e\u003cstrong\u003eMdm2 fragments expression and purification\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eRecombinant Mdm2 constructs, including unlabeled and \u003cem\u003eu\u003c/em\u003e-\u003csup\u003e15\u003c/sup\u003eN/\u003csup\u003e13\u003c/sup\u003eC-labeled variants, were expressed in \u003cem\u003eEscherichia coli\u003c/em\u003e BL21(DE3) cells transformed with the previously described plasmids. Cultures were grown in M9 minimal medium supplemented with either \u003csup\u003e13\u003c/sup\u003eC-glucose or \u003csup\u003e12\u003c/sup\u003eC-glucose (2 g/L) and \u003csup\u003e15\u003c/sup\u003eNH\u003csub\u003e4\u003c/sub\u003eCl (0.5 g/L) as sole carbon and nitrogen sources, or in LB medium. All media were supplemented with 30 \u0026mu;g/mL kanamycin for plasmid selection. Protein expression was induced at an optical density at 600 nm (OD\u003csub\u003e600\u003c/sub\u003e) of 0.8 by adding 0.25-0.5 mM isopropyl \u0026beta;-D-1-thiogalactopyranoside (IPTG), followed by incubation overnight at 20 \u0026deg;C. Cells were harvested 24 hours post-induction by centrifugation, and the resulting cell pellets were stored at -80 \u0026deg;C.\u003c/p\u003e\n\u003cp\u003eCell lysis was performed by sonication in a lysis buffer consisting of 20 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1 mM phenylmethylsulfonyl fluoride (PMSF), 10 mM dithiothreitol (DTT), 1 mM lysozyme, a 1\u0026times; concentration of EDTA-free protease inhibitor cocktail (cOmplete, Roche), and 0.5 \u0026mu;L benzonase (Sigma-Aldrich). Lysates were centrifuged at 15,000 \u0026times; g for 15 minutes at 4 \u0026deg;C to separate soluble and insoluble fractions. Mdm2 peptides were recovered from the soluble fraction.\u003c/p\u003e\n\u003cp\u003eThe soluble lysates were applied to a 5 mL His-Trap FF column (GE Healthcare) pre-equilibrated with lysis buffer. Bound proteins were eluted using an imidazole gradient. Eluted fractions were pooled, concentrated, and further purified by ion-exchange chromatography on a 6 mL Resource Q column (Cytiva) using a NaCl gradient. The resulting fractions were concentrated and incubated with TEV protease for 90 minutes at room temperature in the presence of 2 mM DTT and a 1x EDTA-free protease inhibitor cocktail.\u003c/p\u003e\n\u003cp\u003eThe final purification was achieved by size-exclusion chromatography using a Superdex 16/600 200 pg column (GE Healthcare) equilibrated with phosphate-buffer (PBS; 20 mM phosphate buffer, 150 mM NaCl) at pH 7.0 or 7.4. Fractions containing Mdm2 peptides were identified and stored at -80 \u0026deg;C for downstream applications.\u003c/p\u003e\n\n\u003cp\u003e\u003cstrong\u003eIle\u003cem\u003e\u0026delta;\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003e1-[\u003csup\u003e13\u003c/sup\u003eCH\u003csub\u003e3\u003c/sub\u003e]- and Met\u003cem\u003e\u0026epsilon;\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003e-[\u003csup\u003e13\u003c/sup\u003eCH\u003csub\u003e3\u003c/sub\u003e]-\u003csup\u003e15\u003c/sup\u003eN-\u003csup\u003e2\u003c/sup\u003eH-labeled Mdm2 NMR analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFor selective \u003csup\u003e13\u003c/sup\u003eCH₃ labeling of isoleucine (\u0026delta;1) and methionine (\u0026epsilon;) methyl groups in a \u003csup\u003e2\u003c/sup\u003eH/\u003csup\u003e12\u003c/sup\u003eC background, \u003cem\u003eE. coli\u003c/em\u003e cells were progressively adapted to D\u003csub\u003e2\u003c/sub\u003eO by growing in LB medium with increasing concentrations of D\u003csub\u003e2\u003c/sub\u003eO (0 %, 30 %, 70 %, 99 %). The final expression was carried out in 99% D\u003csub\u003e2\u003c/sub\u003eO-based M9 medium supplemented with \u003csup\u003e12\u003c/sup\u003eC,\u003csup\u003e2\u003c/sup\u003eD-glucose (2 g/L; Cortecnet), \u003csup\u003e15\u003c/sup\u003eNH\u003csub\u003e4\u003c/sub\u003eCl (0.5 g/L), 2-ketobutyric acid-4-\u003csup\u003e13\u003c/sup\u003eC,3,3-d\u003csub\u003e2\u003c/sub\u003e (70 mg/L), and \u003csup\u003e13\u003c/sup\u003eC-methionine (100 mg/L). The overexpression and purification of the protein were executed using the protocol described above.\u003c/p\u003e\n\u003cp\u003eResonance assignments for Mdm2 were transferred from published data by Watanabe et al. (2024). The initial NMR sample contained 90 \u0026mu;M Ile\u0026delta;1-[\u003csup\u003e13\u003c/sup\u003eCH₃]- and Met\u0026epsilon;-[\u003csup\u003e13\u003c/sup\u003eCH₃]-labeled Mdm2 in PBS (pH 7.4) with 1\u0026times; EDTA-free protease inhibitor cocktail (cOmplete, Roche), 3% D\u003csub\u003e2\u003c/sub\u003eO, and 100 \u0026mu;M DSS. Phosphorylation was then initiated in PBS supplemented with 50 mM HEPES, 4 mM DTT, 10 mM ATP, 20 mM MgCl\u003csub\u003e2\u003c/sub\u003e, 1x EDTA-free protease inhibitor cocktail (Roche), by the addition of 1 \u0026mu;M unlabeled CK1\u0026delta; kinase. An additional sample was prepared under the same conditions and supplemented with 90 \u0026mu;M unlabeled p53(aa14\u0026ndash;29) peptide. \u003c/p\u003e\n\u003cp\u003eNMR experiments were conducted on these three samples at multiple temperatures (283 K, 288 K, 293 K, 298 K, 303 K, 306 K, 308 K, 310 K, and 312 K) using the same 700 MHz Bruker Avance Neo spectrometer equipped with a cryogenically cooled TCI probe. \u003csup\u003e1\u003c/sup\u003eH-\u003csup\u003e13\u003c/sup\u003eC SOFAST Methyl-TROSY spectra were acquired with 2048 (\u003csup\u003e1\u003c/sup\u003eH) \u0026times; 192 (\u003csup\u003e13\u003c/sup\u003eC) complex points, using sweep widths of 14.9 ppm (\u003csup\u003e1\u003c/sup\u003eH) and 14.0 ppm (\u003csup\u003e13\u003c/sup\u003eC). Data processing was performed using TopSpin 4.1.3 (Bruker).\u003c/p\u003e\n\n\u003cp\u003e\u003cstrong\u003eSynthesized peptides\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSynthesized phosphorylated peptides of Mdm2 were all purchased from Proteogenix (Illkirch, France) in al lyophilized form with no TFA, and resuspended in PBS at final millimolar concentrations; pH was adjusted to 7.0. These peptides are Mdm2(aa214-226) Ac-SESTG-T/pT-PpSNPDLDA-NH\u003csub\u003e2\u003c/sub\u003e and Mdm2(aa211-223) Ac-SSSEpSTG-T/pT-P-S/pS-NPD-NH\u003csub\u003e2\u003c/sub\u003e.\u003c/p\u003e\n\u003cp\u003eThe peptide p53(aa14-29) Ac-LSQETFSDLWKLLPEN-NH\u003csub\u003e2\u003c/sub\u003e was purchased and resuspended exactly in the same way.\u003c/p\u003e\n\n\u003cp\u003e\u003cstrong\u003eNMR assignments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e- Synthesized peptides: the assignments were achieved using \u003csup\u003e1\u003c/sup\u003eH-\u003csup\u003e1\u003c/sup\u003eH-TOCSY and \u003csup\u003e1\u003c/sup\u003eH-\u003csup\u003e1\u003c/sup\u003eH-ROESY experiments to obtain the \u003csup\u003e1\u003c/sup\u003eH\u003csub\u003eN\u003c/sub\u003e amide assignments, which was later correlated to the \u003csup\u003e1\u003c/sup\u003eH-\u003csup\u003e15\u003c/sup\u003eN crosspeaks from two-dimensional \u003csup\u003e1\u003c/sup\u003eH-\u003csup\u003e15\u003c/sup\u003eN HSQC spectra recorded at natural abundance. All experiments were recorded at pH 7.0 and 283 K in 3 mm diameter tubes, using a 600 MHz spectrometer equipped with a cryoprobe. The peptides being dissolved at ~ 3 mM concentrations in PBS in presence of protease inhibitors and DSS at 100 mM. \u003csup\u003e1\u003c/sup\u003eH-\u003csup\u003e1\u003c/sup\u003eH-TOCSY (mlevgpph19) and \u003csup\u003e1\u003c/sup\u003eH-\u003csup\u003e1\u003c/sup\u003eH-ROESY spectra (roesygpph19.2) were recorded with 2048 (\u003csup\u003e1\u003c/sup\u003eH direct dimension) x 448 (\u003csup\u003e1\u003c/sup\u003eH indirect) complex points and sweep widths of 16 ppm (\u003csup\u003e1\u003c/sup\u003eH direct dimension) and 10 ppm (\u003csup\u003e1\u003c/sup\u003eH indirect dimension), 8 scans and interscan delays of 1.5 s, and processed with a cosine apodization in the direct dimension.\u003csup\u003e 1\u003c/sup\u003eH-\u003csup\u003e15\u003c/sup\u003eN HSQC (hsqcetfpf3gpsi2) spectra were recorded with 2048 (\u003csup\u003e1\u003c/sup\u003eH) x 192 (\u003csup\u003e15\u003c/sup\u003eN) complex points and sweep widths of 16 ppm (\u003csup\u003e1\u003c/sup\u003eH) and 32 ppm (\u003csup\u003e15\u003c/sup\u003eN), 128 scans and interscan delays of 1.25 s, and processed with a cosine apodization in both the direct and indirect dimensions, respectively.\u003c/p\u003e\n\u003cp\u003e- \u003cem\u003eu\u003c/em\u003e-\u003csup\u003e15\u003c/sup\u003eN/\u003csup\u003e13\u003c/sup\u003eC-labeled Mdm2 and phosphorylated Mdm2: NMR assignments of backbone amide resonances of uniformly-labeled peptides (\u003csup\u003e13\u003c/sup\u003eC/\u003csup\u003e15\u003c/sup\u003eN) was achieved via HNCO, HN(CA)CO, HNCACB, (H)N(CA)NH and HNCA 3D experiments. [\u003cem\u003eu\u003c/em\u003e-\u003csup\u003e13\u003c/sup\u003eC/\u003csup\u003e15\u003c/sup\u003eN]-Mdm2(aa111-293) was assigned at 600 mM in a buffer containing HEPES at 10 mM, sodium chloride at 50 mM, TCEP at 4 mM, 3% D\u003csub\u003e2\u003c/sub\u003eO, protease inhibitors (cOmplete EDTA-free, Roche) and DSS at 0.1 mM, at pH 6.2 and 283 K using a Bruker Avance III 700 MHz spectrometer equipped with a cryoprobe. CK1d-phosphorylated [\u003cem\u003eu\u003c/em\u003e-\u003csup\u003e13\u003c/sup\u003eC/\u003csup\u003e15\u003c/sup\u003eN]-Mdm2(aa111-333_C127A-C200A-C206A-C207A) was assigned at 180 mM in a buffer containing sodium phosphate at 20 mM, sodium chloride at 150 mM, 3% D\u003csub\u003e2\u003c/sub\u003eO, protease inhibitors (cOmplete EDTA-free, Roche), DSS at 0.1 mM at pH 7.4 and 283K, using a Bruker Avance III 950 MHz spectrometer equipped with a cryoprobe.\u003c/p\u003e\n\u003cp\u003eAssignments of unmodified and phosphorylated human Mdm2(aa111-293), Mdm2(aa111-230_C127A-C200A-C206A-C207A) and CK1d-phosphorylated Mdm2(aa111-333_C127A-C200A-C206A-C207A), as well as details of NMR experiments used to derive them have been deposited in the Biological Magnetic Resonance Data Bank (BMRB, accession numbers 52016, 52028 and 52289, respectively). As reported in the literature \u003csup\u003e10\u003c/sup\u003e, we observed two stable conformations of the Zn-finger corresponding to all-trans- and all-cis-prolines. The relative peak intensities of the two species were the same in the isolated Zn-finger and in the longer constructs aa111-333 or aa1-333 (trans:cis~2:1). Secondary structure propensities were obtained with the neighbor-corrected structural propensity calculator ncSCP \u003csup\u003e44\u003c/sup\u003e (https://www.jku.at/en/institute-of-biochemistry/protein-nmrorg/ , https://st-protein02.chem.au.dk/ncSPC/) using DSS referenced Ca and Cb chemical shifts as input, and the random coil values of phospho-residues reported by Kragelund and colleagues \u003csup\u003e82\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003eAssignments of synthesized peptides have also been deposited in the BMRB: Mdm2(aa214-226_pT218-pS220) (BMRB 52251), Mdm2(aa211-223_pS215-pT218) (BMRB 52255), Mdm2(aa214-226_pS220) (BMRB 52256), Mdm2(aa211-223_pS215) (BMRB 52257).\u003c/p\u003e\n\n\u003cp\u003e\u003cstrong\u003eTitration \u003csup\u003e15\u003c/sup\u003eN-Mdm2(aa111-333) vs \u003csup\u003e14\u003c/sup\u003eN-Mdm2(aa1-112) \u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNMR samples were prepared by spiking 150 mL of \u003csup\u003e15\u003c/sup\u003eN-labeled Mdm2(aa111-333) at 100 mM with\u003csup\u003e \u003c/sup\u003enon-labeled Mdm2(aa1-112) at 475 mM, both in PBS at pH7.0 supplemented with 1x ETDA-Free protease inhibitors (cOmplete, Roche), 3% D₂O, and 100 \u0026mu;M DSS. The pH was adjusted to 7.00 very carefully for every sample. At the final point of the titration, the concentrations were 54 and 219 mM for \u003csup\u003e15\u003c/sup\u003eN-labeled Mdm2(aa111-333) and for\u003csup\u003e \u003c/sup\u003enon-labeled Mdm2(aa1-112), respectively. All NMR experiments were conducted on a 600 MHz Bruker Avance III-HD spectrometer equipped with a cryogenically cooled triple-resonance TCI probe. \u003csup\u003e1\u003c/sup\u003eH-\u003csup\u003e15\u003c/sup\u003eN HSQC (hsqcetfpf3gpsi2) experiments were recorded at 283 K with 2048 (\u003csup\u003e1\u003c/sup\u003eH) x 256 (\u003csup\u003e15\u003c/sup\u003eN) complex points and sweep widths of 16 ppm (\u003csup\u003e1\u003c/sup\u003eH) and 26 ppm (\u003csup\u003e15\u003c/sup\u003eN), 4 to 32 scans and interscan delays of 0.8 s, and processed in Topspin 4.5 with a cosine apodization in the direct dimension and no apodization in the indirect dimension, respectively. The chemical shift analysis was carried out using CcpNmr 3.1 \u003csup\u003e83\u003c/sup\u003e. The peak intensities were normalized according to the dilution factors and plotted using Kaleidagraph5. The chemical shift perturbations were calculated from the combination of \u003csup\u003e1\u003c/sup\u003eH and \u003csup\u003e15\u003c/sup\u003eN chemical shift changes Dd=\u0026radic;((d\u003csub\u003e1H\u003c/sub\u003e\u003csup\u003e2\u003c/sup\u003e+0.2*d\u003csub\u003e15N\u003c/sub\u003e\u003csup\u003e2\u003c/sup\u003e)/2), and plotted using Kaleidagraph5.\u003c/p\u003e\n\n\u003cp\u003e\u003cstrong\u003eIn Vitro CK1\u0026delta;\u003c/strong\u003e\u003cstrong\u003ephosphorylation assays by NMR\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNMR samples were prepared by combining 450 \u0026mu;L of \u003cem\u003eu\u003c/em\u003e-\u003csup\u003e15\u003c/sup\u003eN/\u003csup\u003e13\u003c/sup\u003eC-labeled Mdm2 at a final concentration of 100 \u0026mu;M in PBS supplemented with 50 mM HEPES, 4 mM DTT, 10 mM ATP, 20 mM MgCl\u003csub\u003e2\u003c/sub\u003e, 1x ETDA-Free protease inhibitors (cOmplete, Roche), 3% D₂O, and 100 \u0026mu;M DSS. The pH was adjusted to 7.0. Phosphorylation reaction was initiated by the addition of 2 \u0026mu;M unlabeled CK1\u0026delta; kinase. Reaction progress was monitored using NMR spectroscopy.\u003c/p\u003e\n\u003cp\u003eAll NMR experiments were conducted on a 700 MHz Bruker Avance Neo spectrometer equipped with a cryogenically cooled triple-resonance TCI probe. \u003csup\u003e1\u003c/sup\u003eH-\u003csup\u003e15\u003c/sup\u003eN HSQC experiments were recorded before and after phosphorylation at 283 K, 290 K, and 298 K to normalize the final phosphorylation levels and analyze temperature-dependent chemical shift variations. A time-resolved series of \u003csup\u003e1\u003c/sup\u003eH-\u003csup\u003e15\u003c/sup\u003eN SOFAST-HMQC spectra was recorded at 298 K until phosphorylation reached completion.\u003c/p\u003e\n\u003cp\u003eAfter phosphorylation, the sample was purified using ion-exchange chromatography on a 6 mL Resource Q column (Cytiva) with a NaCl gradient, followed by size-exclusion chromatography on a Superdex 16/600 200 pg column (GE Healthcare) equilibrated with PBS (pH 7.4).\u003c/p\u003e\n\u003cp\u003eTwo-dimensional \u003csup\u003e1\u003c/sup\u003eH-\u003csup\u003e15\u003c/sup\u003eN HSQC experiments were recorded with 1536 (\u003csup\u003e1\u003c/sup\u003eH) \u0026times; 192 (\u003csup\u003e15\u003c/sup\u003eN) complex points, sweep widths of 16.23 ppm (\u003csup\u003e1\u003c/sup\u003eH) and 28 ppm (\u003csup\u003e15\u003c/sup\u003eN), 8 scans (4 dummy scans), and an interscan delay of 1 s. Two-dimensional \u003csup\u003e1\u003c/sup\u003eH-\u003csup\u003e15\u003c/sup\u003eN SOFAST-HMQC spectra were recorded with 1536 (\u003csup\u003e1\u003c/sup\u003eH) \u0026times; 192 (\u003csup\u003e15\u003c/sup\u003eN) complex points, sweep widths of 16.23 ppm (\u003csup\u003e1\u003c/sup\u003eH) and 28 ppm (\u003csup\u003e15\u003c/sup\u003eN), 16 scans (32 dummy scans), and a 0.2 s interscan delays, resulting in a total acquisition time of 15 minutes. Data were processed using TopSpin 4.1.3 (Bruker), and peak intensities were analyzed using CcpNmr Analysis 2.4. Peak positions were tracked to monitor chemical shift perturbations, and phosphorylation kinetics were normalized using relative intensity increase changes.\u003c/p\u003e\n\u003cp\u003eUsing Mdm2(aa111-237 WT/T218A/S220A), Mdm2(aa111-333 S215A), and unlabeled phosphorylated peptides of Mdm2(aa214-226): Ac-SESTG-T/pT-PpSNPDLDA-NH₂, and Mdm2(aa211-223): Ac-SSSEpSTG-T/pT-P-S/pS-NPD-NH₂, phosphorylation peaks corresponding of pS215, pT218, pS220, pS229, and pS232 were identified using 2D \u003csup\u003e1\u003c/sup\u003eH-\u003csup\u003e15\u003c/sup\u003eN SOFAST-HMQC and HSQC spectra.\u003c/p\u003e\n\u003cp\u003eSimilarly, phosphorylation peaks for pS253 and pS256 were identified using Mdm2(aa230-333 S253A); for pS240, pS242, and pS246 using Mdm2(aa111-333 S246A); and for pS260 and pS262 using Mdm2(aa111-333 S260A/S262A).\u003c/p\u003e\n\u003cp\u003eIn the case of Mdm2(aa111-333 WT), phosphorylation of residues pT218, pS240, pS242, pS256, pS260, and pT279 was monitored, along with adjacent residues including pG217 (for pS215), pD225 (for pS220), pV228 (for pS229), pG233, pD234 and pS232 (for pS232), pE248 and pS246 (for pS246), pV251, pD252 and pS253 (for pS253), pG265 (for pS262), and pQ277 and pT279 (for pT279). The intensities of these neighboring residues increased proportionally with the phosphorylation of the corresponding sites. To enhance the signal-to-noise ratio (S/N), the intensities of peaks corresponding to each phosphosite, along with those of adjacent residues were aggregated and used to trace phosphorylation kinetics.\u003c/p\u003e\n\u003cp\u003eThe percentage of phosphorylation (%phospho) was calculated using the equation %phospho\u003csub\u003efinal\u003c/sub\u003e = 1 - I\u003csub\u003efinal\u003c/sub\u003e/I\u003csub\u003e0\u003c/sub\u003e from unphospho-peaks, where intensities were extracted from 2D \u003csup\u003e1\u003c/sup\u003eH-\u003csup\u003e15\u003c/sup\u003eN HSQC spectra of Mdm2(aa111-333) and pMdm2(aa111-333) at 283 K. Normalization was achieved by combining %phospho values with peak intensities from SOFAST-HMQC spectra. Phosphorylation build-up curves were fitted using the equation %phospho(t)= 1 - (I/ (I\u003csub\u003efinal\u003c/sub\u003e/%phospho\u003csub\u003efinal\u003c/sub\u003e)*exp(-k*t)).\u003c/p\u003e\n\n\u003cp\u003e\u003cstrong\u003eRelaxation Experiments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNMR relaxation experiments were recorded using the same 700 MHz Bruker Avance Neo spectrometer equipped with a cryoprobe. Data acquisition, processing, and spectral analysis were carried out with TopSpin 4.1.3 (Bruker) and CcpNmr Analysis 2.4. Two-dimensional \u003csup\u003e1\u003c/sup\u003eH-\u003csup\u003e15\u003c/sup\u003eN correlation spectra of \u003cem\u003eu\u003c/em\u003e-\u003csup\u003e15\u003c/sup\u003eN-labeled Mdm2 were acquired using a standard HSQC pulse sequence at 283 K with protein concentrations ranging from 200 to 300 \u0026mu;M in PBS (pH 7.4). Duplicate experiments were acquired at each time point to estimate experimental error.\u003c/p\u003e\n\u003cp\u003e- T₁ and T₂ Relaxation Measurements: \u003csup\u003e15\u003c/sup\u003eN amide-backbone relaxation rates, including longitudinal (T₁, 1/R₁) and transverse (T₂, 1/R₂), were determined using standard Bruker pulse sequences. T₁ values were measured at relaxation delay times of 40, 100, 200, 300, 400, 500, 600, 800, and 1200 ms and intensities were fitted to the exponential delay function: I(t)=I\u003csub\u003e0\u003c/sub\u003e*(1-e\u003csup\u003e-t/T1\u003c/sup\u003e). T₂ values were determined using a Carr-Purcell-Meiboom-Gill (CPMG) sequence with delays of 15.7, 31.5, 47, 62.5, 78.5, 110, 141, 188, and 376 ms. Apparent R\u003csub\u003e2\u003c/sub\u003e rates were derived via multi-exponential fitting to account for hydrogen exchange (HX) effects, following methodology described in Alik et al. (2020) \u003csup\u003e10\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003e- Hydrogen Exchange (HX) Rate Measurements: Hydrogen exchange rates (k\u003csub\u003eHX\u003c/sub\u003e) were determined using the CLEANEX-PM experiment (Hwang et al., 1998) \u003csup\u003e84\u003c/sup\u003e with \u003csup\u003e1\u003c/sup\u003eH-\u003csup\u003e15\u003c/sup\u003eN HSQC detection. The fhsqccxf3gpph pulse sequence from Bruker was used with mixing times of 0.005 s, 0.01 s, and 0.02 s at pH 7.4. fhsqcf3gpph \u003csup\u003e1\u003c/sup\u003eH-\u003csup\u003e15\u003c/sup\u003eN HSQC spectra served as reference (V\u003csub\u003e0\u003c/sub\u003e) in the analysis, and k\u003csub\u003eHX\u003c/sub\u003e were calculated using the equation described by Hwang et al. (1998) \u003csup\u003e84\u003c/sup\u003e. Data were acquired with 2048 complex points in the t\u003csub\u003e2\u003c/sub\u003e dimension and 256 complex points in t\u003csub\u003e1\u003c/sub\u003e, using a 2-seconds recycle delay. \u003c/p\u003e\n\u003cp\u003e- Heteronuclear NOE Measurements: Heteronuclear NOEs (hetNOE) values were obtained by comparing peak intensities from spectra acquired with and without a 4-seconds proton saturation during a 5-seconds recycle delay. Residue-specific signal intensity ratios (I/I\u003csub\u003e0\u003c/sub\u003e), corresponding to hetNOE values, were calculated for Mdm2. \u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eNMR Diffusion Experiments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eDouble echo PGSTE-WATERGATE experiments (Zheng et al. 2009) \u003csup\u003e52\u003c/sup\u003e was conducted on 200 \u0026mu;M unlabeled Mdm2 dissolved in PBS (pH 7.4) supplemented with 1x EDTA-free protease inhibitor cocktail (Roche), 3% D\u003csub\u003e2\u003c/sub\u003eO, 100 \u0026mu;M DSS, and 0.03% 1,3-dioxane. Measurements were performed at multiple temperatures (283 K, 288 K, 293 K, 298 K, 303 K, 306 K, 308 K, 310 K, and 312 K) using a 600 MHz Bruker Avance Neo spectrometer equipped with a cryogenically cooled TXI probe.\u003c/p\u003e\n\u003cp\u003eA series of 24 one-dimensional \u003csup\u003e1\u003c/sup\u003eH-NMR spectra were acquired with linearly incremented gradient strengths. Signal intensities were measured by integrating peaks from the 1D spectra. The diffusion coefficients of Mdm2 were determined by fitting the exponential decays of signal intensities in the methyl region (1-0.5 ppm) to the Stejskal-Tanner equation.\u003c/p\u003e\n\u003cp\u003eThe diffusion coefficient of 1,3-dioxane was determined independently by analyzing the intensity decay of its specific resonance. Peak integration and analysis were performed using TopSpin 4.1.3 (Bruker).\u003c/p\u003e\n\u003cp\u003eFor each pulsed-field gradient (PFG) NMR experiment, 16 scans were acquired, with the gradient strength varying from 10% to 100%. The gyromagnetic ratio (\u0026gamma;) was set to 2.68 \u0026times; 10⁸ rad.s⁻\u0026sup1;.T⁻\u0026sup1;, with a diffusion time (∆) of 200 ms and gradient pulse duration (\u0026delta;) of 5 or 7 ms.\u003c/p\u003e\n\n\u003cp\u003e\u003cstrong\u003eIsothermal Titration Calorimetry (ITC)\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eITC experiments were performed using both a VP-ITC and a PEAQ-ITC instruments (MicroCal). In a typical VP-ITC experiment, the sample cell was loaded with Mdm2 at 5 or 10 \u0026mu;M (aa1-112, aa18-112, aa1-333, or aa111-333), while the titrating syringe contained p53(aa1-88) at 50 or 100 \u0026mu;M. For PEAQ-ITC experiments, the cell was loaded with 15 or 20 \u0026mu;M phospho-MDM2(aa1-333) or MDM2(aa1-333del189-208), and the syringe contained p53(aa1-88) of p53FL at 150 or 200 \u0026mu;M. Raw data are shown in Supplementary Figure 15.\u003c/p\u003e\n\u003cp\u003eAll titrations were conducted in PBS buffer at pH7.4 and 20 \u0026deg;C. Data were analyzed using MicroCal Analysis software, with the binding isotherms fitted to a single-site binding model.\u003c/p\u003e\n"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by the CNRS and the CEA-Saclay (CEA/PSAC/DPRS/BE/TG/2021-410), by the French Infrastructure for Integrated Structural Biology (https://frisbi.eu/, grant number ANR-10-INSB-05-01, Acronym FRISBI) and by the French National Research Agency (ANR; research grants ANR-14-ACHN-0015 and ANR-20-CE92-0013). This work has been supported by the Fondation ARC pour la recherche sur le cancer (YL, ARCDOC42023120007483). Financial support from the IR INFRANALYTICS FR2054 for conducting the research is gratefully acknowledged, and we value the commitment and expertise of F. Giraud, E. Lescop and N. Morellet. We also thank the Platform PIM from the I2BC.\u003c/p\u003e\n\n\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eY.L., J.P., C.B., A.A. and F.-X.T. produced protein samples, Y.L., J.P., C.B., M.A.-N. and F.-X.T. conducted the ITC experiments, Y.L. and F.-X.T. conducted and analyzed the NMR experiments, Y.L. and F.-X.T. wrote the manuscript, F.-X.T. secured funding, designed and supervised the project.\u003c/p\u003e\n\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e\n\n\u003cp\u003e\u003cstrong\u003eAdditional information\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSupplementary information \u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe online version contains supplementary material.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCorrespondence\u003c/strong\u003e and requests for materials should be addressed to Francois-Xavier Theillet.\u003c/p\u003e\n\n\n\n"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eGe, Z. \u003cem\u003eet al.\u003c/em\u003e Integrated Genomic Analysis of the Ubiquitin Pathway across Cancer Types. \u003cem\u003eCell Rep.\u003c/em\u003e \u003cstrong\u003e23\u003c/strong\u003e, 213-226.e3 (2018).\u003c/li\u003e\n\u003cli\u003eKlein, A. 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Accurate quantitation of water-amide proton exchange rates using the phase-modulated CLEAN chemical EXchange (CLEANEX-PM) approach with a Fast-HSQC (FHSQC) detection scheme. \u003cem\u003eJ. Biomol. NMR\u003c/em\u003e \u003cstrong\u003e11\u003c/strong\u003e, 221\u0026ndash;226 (1998).\u003c/li\u003e\n\u003c/ol\u003e"},{"header":"Table 1","content":"\u003cp\u003e\u003cimg 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Indeed, producing purified samples for its structural analysis is difficult: Mdm2 is a multidomain protein, alternating folded and disordered regions, the latter being moreover natively hyperphosphorylated. Here, we report the structural study of Mdm2 constructs including its folded p53-binding domain (p53-BD) and Zinc-Finger, and their spacer, a 190-residue long disordered region (IDR1) hyperphosphorylated by CK1. Using NMR spectroscopy, we revealed that IDR1 establish ultraweak intramolecular contacts with p53-BD in the μs timescale, which provokes the partial release of the N-terminal tail from the p53-binding groove of Mdm2. Then, using ITC, we established that these Mdm2 intramolecular interactions decrease the affinity for p53 by up to 25-fold compared to the isolated p53-binding domain. Surprisingly, the identified 15 CK1-established phosphosites between Mdm2-S192 and -S286 do not improve the affinity for p53 despite their binding to the positively charged DNA-binding domain. Structural studies on multidomain proteins are scarce, and even more rare on natively phosphorylated species. This report shows how disordered regions can interfere with folded domains, and markedly affect binding between cognate cellular partners.","manuscriptTitle":"The disordered region 1 of Mdm2 weakens p53-binding in both its unmodified and hyperphospho-forms","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-03-09 07:12:01","doi":"10.21203/rs.3.rs-9014206/v1","editorialEvents":[],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"nature-communications","isNatureJournal":true,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"NCOMMS","sideBox":"Learn more about [Nature Communications](http://www.nature.com/ncomms/)","snPcode":"","submissionUrl":"https://mts-ncomms.nature.com/","title":"Nature Communications","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Nature Communications","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"abd747aa-75cf-428c-83ab-6cb97b91246b","owner":[],"postedDate":"March 9th, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[{"id":63855812,"name":"Biological sciences/Structural biology/NMR spectroscopy/Solution-state NMR"},{"id":63855813,"name":"Biological sciences/Biophysics/Intrinsically disordered proteins"}],"tags":[],"updatedAt":"2026-03-24T11:16:32+00:00","versionOfRecord":[],"versionCreatedAt":"2026-03-09 07:12:01","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-9014206","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-9014206","identity":"rs-9014206","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}