Characterization of ATP hydrolysis in the Hsp70 BiP nucleotide binding domain

Characterization of ATP hydrolysis in the Hsp70 BiP nucleotide binding domain | 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 Characterization of ATP hydrolysis in the Hsp70 BiP nucleotide binding domain Sebastian Hiller, Guillaume Mas This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4017836/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 01 Jun, 2025 Read the published version in Nature Communications → Version 1 posted You are reading this latest preprint version Abstract The 70 kDa heat shock protein (Hsp70) family of molecular chaperones is crucial for protein biogenesis and homeostasis in all kingdoms of life. Hsp70 activity is driven by ATP hydrolysis in the nucleotide binding domain (NBD). Here, we report an experimental setup to resolve the functional cycle of Hsp70 in unprecedented spatial and temporal resolution. The method combines high-resolution NMR spectroscopy with embedded kinetic measurements to simultaneously resolve kinetic rates and structural information of the individual states of an Hsp70 functional cycle. We benchmark the method on the example of the NBD of the human Hsp70 chaperone BiP. Precision measurements connect the ATP hydrolysis rate ( k cat ) and the ADP lifetime ( k off ) to conventional bulk experiments and thus reveal that ADP-Pi release and not ATP hydrolysis is the limiting step of the cycle. Unlike commonly thought, the phosphate generated from ATP hydrolysis locks the ADP-Pi into the NBD, and thus decouples the ADP release rate from the effect of external factors such as the bulk phosphate and calcium concentration. The method will serve as a platform for studies of the Hsp70 protein family and their co-chaperones, including full-length constructs that have key roles in biogenesis and disease. Biological sciences/Biochemistry/Structural biology/NMR spectroscopy/Solution-state NMR Biological sciences/Biophysics/Bioenergetics Molecular chaperones NMR spectroscopy allostery Hsp70 real-time NMR molecular machines endoplasmic reticulum ATPase Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Introduction The 70 kDa heat shock protein (Hsp70) family of molecular chaperones is crucial for biogenesis and protein homeostasis 1–4 . The Hsp70s account for up to 4% of total cellular protein mass, making them one of the most abundant proteins in the cell 5–7 . Hsp70s are involved in diverse cellular processes 3,8–12 , including de-novo protein folding at the ribosome 13,14 , protein translocation through pores 15,16 and solubilization of protein aggregates 1,17,18 . Consistently, Hsp70s are connected to multiple pathophysiological conditions including cancer and neurodegenerative diseases 19–21 . In the endoplasmic reticulum (ER), BiP (Binding Immunoglobulin Protein) is the sole Hsp70 isoform in all eukaryotes 22,23 and the most abundant ER chaperone 7,24 . BiP is the central functional hub of the ER chaperone network that ensure protein folding homeostasis in the “folding factory of the cell”. It consequently binds to most of the proteins that are processed in the ER, promoting their folding and preventing their aggregation 25,26 . Additionally, it also acts as the central regulator of the unfolded protein response (UPR) by binding to the UPR sensors in a stress-dependent manner 27–29 . Furthermore, BiP targets unfolded protein to the degradation machinery associated with the ER-associated degradation pathway (ERAD) 30–32 . Moreover, BiP is overexpressed in many human cancers, making it a major therapeutic target 33–36 . Hsp70 chaperones comprise two distinct domains, the nucleotide-binding domain (NBD) and the substrate-binding domain (SBD) that are connected by a flexible linker 37,38 . The SBD is sub-divided into the subdomains SBDα and SBDβ enclosing the client binding site 12 . The NBD has a clamp-like shape with two lobes I and II 12 . Each lobe is further subdivided into two subdomains A and B. A nucleotide binding site is located in the center of the domain in the cleft between lobes I and II. Hsp70 chaperones go through a functional cycle encompassing ATP-binding, ATP-hydrolysis, and ADP-Pi release, all of which takes place in the NBD 39 . ATP binding leads to a rearrangement of lobe I, which triggers the SBD to open the client binding site 40–42 . Following ATP hydrolysis, the NBD is in an ADP-bound state that leads to the closure of the client binding site. From there, ADP is released at one point, resulting in the apo form, to which a new ATP molecule binds to restart the cycle. The overall Hsp70 chaperone activity resulting from this fundamental cycle dependents on the cellular context and manifests into a diverse set of effective functions such as a foldase, holdase, translocase, unfoldase or disaggregase 11,12,43 . Importantly, these functions are fundamentally regulated by the timing originating from nucleotide processing in the NBD and understanding the Hsp70 functional cycle thus requires elucidating the nucleotide reaction steps and their modulation by environmental change. Interestingly, despite exhaustive biochemical characterization of Hsp70 proteins, measurements of the functional cycle kinetic parameters have so far been possible only by isolating individual steps and not at atomic resolution 44–46 . In this study, we develop a method that combines the power of methyl NMR spectroscopy to resolve atomic siteswith a temporal dimension to resolve the reaction kinetics of the cycle individual steps. We benchmark the method on the example of the NBD of the human Hsp70 chaperone BiP from the endoplasmic reticulum (ER) and compare the results from the in cyclo experiments with classical single turn-over and ADP release experiments. We find that the functional cycle is completely independent from the bulk concentration of free inorganic phosphate (Pi) and characterize absence of any significant effect of the concentration of calcium. The method established here provides a technology platform for a fundamental understanding of the Hsp70 functional cycle at the atomic level, also in the context of the full-length Hsp70s and their regulation by co-chaperones. NMR resonance assignment of NBD methyl groups As a first step towards observing individual atomic sites during the functional cycle of BiP NBD, we established a highly pure and homogenous sample preparation. Since we want to be able to detect conformational sub-states of the protein with potentially low populations, the preparation needs to be free of any, specifically or non-specifically bound nucleotides and other contaminants. Such contaminants have been reported as a major cause of concern in the literature 47 . We thus purified the protein using well-established protocols and then added an affinity column purification step under denaturing conditions of 8 M Urea. The protein is entirely unfolded under these conditions and thus loses the affinity for bound impurities, which are washed through the column. After elution from the column, the protein was slowly refolded via dialysis. The refolded BiP NBD was analyzed by SEC-MALS showing a perfect overlap with BiP NBD purified without unfolding/refolding step (Supplementary Fig. 1a,b). Its NMR spectrum was 100% homogenous, evidencing the absence of any significant amounts of ligands. The proper conformation of the refolded protein was assessed by a comparison of the NMR spectra of the BiP NBD with and without denaturation (Supplementary Fig. 1c). The two spectra overlapped perfectly, demonstrating that the BiP NBD reaches its native state after denaturation and refolding. This high purification standard was kept in all subsequent experiments. In a next step, we isotope-labelled the methyl groups of three amino acids, Ile-[ 13 C 1 H] δ1 , Met-[ 13 C 1 H] ε and Val-[ 13 C 1 H] g 1/ g 2 , on an otherwise deuterated background. This is a well-established technique to allow atomic resolution NMR studies even at large molecular sizes up to several 100 kDa 48 . 2D [ 13 C, 1 H]-methyl-TROSY spectra 49 with high sensitivity can be recorded in times as short as 5 minutes at protein concentrations of 100 μM. This high sensitivity is key to detect also minor sub-states of the cycle when longer experiment times are used. With the purification and preparation steps, NMR spectra of the protein in thermodynamic equilibrium were recorded in the apo form or in presence of ADP. The resulting equilibrium spectra serve as references for the respective conformational states of the protein. In presence of 5 mM ADP-Pi, we observed a homogeneous spectrum with a single set of 102 resonances, precisely matching the 102 resonances expected from the chemical structure of the molecule (Fig. 1a). We established sequence-specific assignments of these resonances using a strategy that combines single point-mutagenesis and NOESY experiments. As a first step, we established the assignments of all 6 methionine residues by single point mutagenesis (M148L, M153L, M196L, M263L, M332L, M339L). Then, using these anchor points, we expanded the assignment using a 3D 13 C, 13 C-resolved [ 1 H, 1 H]-NOESY experiments that we manually curated against a published crystal structure of the BiP NBD (PDB 5EVZ) (Fig. 1b and Supplementary Fig. 2a). The methyl groups of Met, Val and Ile have unambiguously separated chemical shift ranges for their NMR signals, permitting a direct identification of the amino acid type for a given signal and thus to easily distinguish different NOESY network. We resolved around 4 NOE contacts per residue, guaranteeing unambiguous assignments as a network effect. We additionally exploited the good correlation between the NOE cross peaks intensities and the calculated distance in the BiP NBD structure to confirm the correctness of these network (Supplementary Fig. 2b). Overall, we could resolve 100 NOESY contacts up to 5 Å, 255 NOESY contacts in the distance range 5–8 Å and 91 NOESY contacts in the range 8–10 Å, leading to a high-confidence assignment (Fig. 1c). As a final validation step, we selected 7 individual residues at the core of large NOESY networks and confirmed the correctness of their assignment by single-point mutagenesis. In total, this approach led to the stereospecific assignment of 60 residues, thereof 32/35 valines, 22/25 isoleucine, 6/6 methionine. Among the assigned 32 valines, 24 had both methyl Cg1 and Cg2 assigned and 8 one of them, resulting in a total of 84 observable NMR signals (Fig. 1a and d, Supplementary Fig. 3). For the subsequent experiments, we thus have 84 atomic level reporters that we can observe simultaneously and with high sensitivity for local structural changes. Setup of the functional cycle with the ATP regeneration system Based on these prerequisites of a highly pure preparation and near-complete resonance assignments, we set up the experiment to monitor the BiP NBD functional cycle. We selected a buffer composition corresponding to optimal Hsp70 activity (25 mM HEPES pH 7.5, 150 mM KCl and 10 mM MgCl 2 ), and the physiological temperature of 37°C. Magnesium is required for the Hsp70 ATP hydrolysis as it coordinates the ATP b and g phosphate 50 . The potassium concentration was chosen to match previous reports that it acts as a cofactor of the Hsp70 hydrolysis of ATP increasing the activity by 5-fold in the optimal range of concentration between 100 and 150 mM 51 . Notably, while it is possible to prepare pure apo and pure ADP-bound state, it is not possible to prepare a stable ATP-bound state, due to the catalytic activity of the protein. For example, the addition of 5 mM ATP to 100 μM of the BiP NBD leads to the significant accumulation of ADP already in the first few minutes and results in a non-equilibrium situation with rapidly changing ADP/ATP concentration ratio (Supplementary Fig. 4). In order to create a stable steady state condition, we implemented an ATP regeneration system inside the NMR tube that steadily converts ADP into ATP 52 . This system exploits the activity of pyruvate kinase, at catalytic amounts, to combine phosphoenol pyruvate (PEP) and ADP to form a novel ATP molecule (Fig. 2a). ATP and ADP molecules can be unambiguously distinguished by their H8 adenine proton in 1D 1 H NMR experiments, PEP by its methylene protons and pyruvate by its methyl protons (ATP: 8.554 ppm, ADP: 8.558 ppm, PEP: 5.424 ppm, pyruvate: 2.424 ppm). Monitoring of ATP, ADP, PEP and pyruvate concentrations by 1D 1 H NMR spectra shows that the ATP regeneration system keeps the concentrations of ATP and ADP effectively constant with no detectable signal for the ADP ([ATP] = 5 mM and [ADP] < 5 μM) (Fig. 2b,c), while the PEP concentration is linearly decreasing and the pyruvate concentration linearly increasing with the same rate (Fig. 2d,e). Thereby, the linear increase of pyruvate corresponds stoichiometrically to the ATP consumption and thus directly allows to calculate the ATP hydrolysis rate of the BiP NBD (Fig. 2e). To distinguish this experiment from equilibrium experiments, we refer to this setup with ATP regeneration as the “in cyclo” NMR experiment. The ATP consumption in our setup was 0.087 ± 0.003 mM min -1 , in a sample with 100 μM BiP NBD (Fig. 2f), which corresponds to a molecular hydrolysis rate of k hydr = 0.95 ± 0.03 min -1 . Mechanistically, this number is the sum of the ATP hydrolysis rate k cat and the ADP release k off (ADP). The inverse of this rate, t = k hydr -1 = 63 ± 2 s is the length of the functional cycle of BiP NBD. Notably, the hydrolysis rate determined in the in cyclo experiment matches classical experiments. A typical assay is the NADH-coupled ATPase assay 53 . This assay exploits the enzymatic activity of lactate dehydrogenase to turn pyruvate into lactate by coupled NADH oxidation, which can be monitored by photospectrometry (A 340 ) to measure the ATP consumption. We determined the rate with this assay and found perfect correspondence within error under the steady state conditions of our in cyclo NMR experiment ( k hydr = 0.92 ± 0.11 min -1 ) (Fig. 2f). Our assay thus faithfully reproduces established properties of the cycle, while simultaneously allowing atomic resolution observations. Our experimental setup shows an excellent match in terms of its consumption rate with conventional ATP hydrolysis assay. The crucial advantage is however that it now allows the simultaneous observation of conformational states at atomic resolution. Direct observation of the ATP-bound state In a next step, we wanted to exploit these properties to get atomic level insights. 2D [ 13 C, 1 H]- methyl-TROSY spectra recorded in cyclo with active ATP regeneration show that the resonances split into two distinct sets of NMR signals. The relative amounts of the two signals is very similar for all residues and these signals thus correspond to two conformational states of the BiP NBD under the steady state conditions in cyclo. One of the two states matches perfectly with the NMR signals in 5 mM ADP-Pi in equilibrium experiment, unambiguously identifying this resonance set as the ADP-Pi-bound state (Supplementary Fig. 5a). The second set of NMR signals did neither match apo BiP NBD, ADP-Pi BiP NBD nor ADP BiP NBD (Fig. 3a,b and Supplementary Fig. 5b,c). It was detected only in presence of the ATP regeneration system or in the early phase of non-equilibrium experiments with pure ATP added (Supplementary Fig. 5b,c). This set of NMR signals therefore corresponds to the ATP-bound state. We term the BiP NBD ADP-Pi-bound state the D state and BiP NBD ATP-bound state T state. Since we did not yet have sequence-specific resonance assignment for the ATP-bound state, we established them by direct transfer from the ADP-Pi-bound state for all signals in unambiguous spectral regions (Fig. 3a,b) and further established and confirmed assignment by the same 13 single point mutants that had previously been used for the assignment of the ADP-Pi-bound state. In total, 74 unambiguous assignments for the ATP-bound state were established (Fig. 3a and Supplementary Fig. 6). These assignments now give us valuable information about the ATP-bound state. On the one hand, we note that for every single residue in the entire BiP NBD, we observe a distinct signal for the ADP-Pi-bound state and the ATP-bound state. This leads to the conclusion that the structural rearrangements of the BiP NBD to adapt between the ATP and ADP-Pi molecules involve the entire BiP NBD. Second, we can interpret the chemical shift differences between the two states in a structure-specific manner. The chemical shift differences were largest for residues located in the vicinity of the bound nucleotide, as expected (Fig. 3c and Supplementary Fig. 7a). Additional large chemical shift differences were however also observed in the lobe IA. These reflect the rotation of the lobe IA resulting from ATP binding, which in the full-length protein result in docking of the SBD 12 . The availability of assignments of the ATP-bound state allowed us also to assess the effects of slow-hydrolysable ATP analogs AMPPNP (adenylyl imidodiphosphate), AMPPCP (adenylyl methylenediphosphate) and ATP-γ-S (adenosine 5’-(gamma-thiotriphosphate)). These analogs are frequently used to mimic the ATP-bound state 54 . The comparison with the 2D [ 13 C, 1 H]-methyl-TROSY spectra fingerprints for the three commons ATP analogs shows large chemical shift deviations for the entire BiP NBD (Supplementary Fig. 7b). The shift differences for AMPPNP and AMPPCP shows that these two analogs shift the BiP NBD conformation in a state that is more similar to the ADP-Pi-bound state than ATP-bound state, i.e. their NMR fingerprints are closer to the one of the ADP-Pi than ATP-bound state (Supplementary Fig. 7b). The ATP-γ-S fingerprint spectrum features three NMR signals per residue, two major states that resemble the ADP-Pi-bound state, and one minor state that is closer to the ATP-bound state but does not overlap with it (Supplementary Fig. 7b). Therefore, ATP-γ-S leads to the formation of an heterogenous mix of conformations that do not represent the ATP-bound state. This fully explains why neither of these three analogs induces the expected Hsp70 interdomain conformational change that is triggered by ATP binding as it has been reported in the literature 44,55,56 . As a consequence, our in cyclo experiment is the only setup that enables studies of the native ATP-bound state at atomic resolution. Combined measurement of the functional cycle kinetic parameters With the assignments of the two functional states at hand, we could next determine the complete kinetic parameters of the functional cycle. The NMR signal intensities of each of the two states is proportional to their relative population and thus to the kinetic rate constants that connect the two states. We integrated 51 non-overlapping, independent methyl groups to quantify the population ratio p D / p T . Along the entire protein, this ratio showed very little variation, clearly establishing that BiP NBD completely splits into two independent states (Fig. 3d). The relative populations are T: 21 ± 4% and D: 79 ± 4% (Fig. 3d,e). Because the length of a complete cycle is 63 ± 2 s, these population levels correspond to mean-lifetimes t T = 13.4 ± 2 s for the ATP-bound T state and t D = 49.7 ± 3 s for the ADP-Pi-bound D state (Fig. 3f). The inverse of these life times thus correspond to the catalytic ATP hydrolysis rate k cat = t T -1 = 0.075 s -1 and the ADP release rate k off = t D -1 = 0.02 s -1 during the functional cycle of NBD. The ATP lifetime in cyclo is identical to classical single turn-over experiments Conventional approaches do not allow the determination of the ATP hydrolysis rate k cat from a steady-state experiment, but require separate single turn-over experiments. We wanted to benchmark the ATP lifetime obtained in cyclo by comparison with standard single-turn over experiments 44,46 . The single-turn over experiments were performed according to the standard protocol adapted from Theyssen et al. 44 . First, the NBD ATP complex was formed by incubation of an excess of ATP with BiP NBD at 4°C. At this temperature, it is generally assumed that the hydrolysis rate can be neglected. Next, the complex was separated from unbound nucleotide by gel filtration columns and then incubated at 37°C for variable time, until the reaction was stopped by addition of HCl. From the resulting samples, the [ATP]/[ADP] ratio was determined by anion exchange chromatography 57 . The data were fitted to a monoexponential, which corresponds to the ATP half-life time (Fig. 4a). The single turn-over experiments showed an excellent agreement with the measurements in cyclo (t T = 14.3 ± 2 s (single turn-over) vs t T = 13.4 ± 2 s (in cyclo)) (Fig 4b). The in cyclo experiment thus faithfully reports the ATP lifetime of the system in a steady state experiment, while simultaneously also giving atomic level structural information. ADP-Pi release is the limiting step of the BiP NBD functional cycle We next wanted to benchmark also the second kinetic rate obtained with the in cyclo experiment by classical experiments. The standard assay in the Hsp70 field to measure ADP lifetimes is the ADP displacement assay 44 . This assay relies on a fluorescently labelled ADP derivate, N8-(4-N'-methylan-thraniloylaminobutyl)-8-aminoadenosine 5'-diphosphate (MABA-ADP), that shows a substantial increase of fluorescence by 140% upon binding to nucleotide-free Hsp70 44 . With this reagent, nucleotide release is determined by real-time measurements of the fluorescence signal of Hsp70 with bound MABA-ADP in the presence of an excess of non-fluorescent ADP. The MABA-ADP unbinds over time, leading to a decrease in fluorescence intensity, because the non-fluorescent ADP prevents re-binding. Fitting the data with a mono-exponential gives the ADP half-life time. The MABA-ADP release times and the ADP release in the functional cycle should perfectly match and this is what is generally assumed in the literature when interpreting the MABA-ADP results. Direct measurements of ADP release during the functional cycle have so far not been accessible. Strikingly, our measurements show a large and highly significant deviation between the two experiments (t D = 13.6 ± 2 s (MABA-ADP) vs t D = 49.7 ± 3 s (in cyclo)) (Fig 5a,b). To resolve this conundrum, we realized that the functional state encountered in the functional cycle is the ADP-Pi-bound state, while the MABA-ADP experiment is phosphate-free and its lifetime thus corresponds to the ADP-bound state. It is well established that there is a difference in lifetime between the two cases and the presence of Pi in the buffer increases the ADP affinity and lifetime 58,59 . We therefore measured the MABA-ADP release at Pi concentrations from 0.1 mM to 30 mM, leading to an increase of the ADP lifetime from t D = 13.6 ± 2 s in the absence of Pi to t D = 153.2 ± 5 s in the presence of 30 mM Pi (Fig. 5a,b). Thereby, the effect of Pi concentration corresponds to an IC 50 of 5.9 ± 0.2 mM. To provide a structural rationale for this observation, we compared the ADP-bound state and the ADP-Pi-bound states in cyclo of BiP NBD using the NMR chemical shifts of our 84 atomic probes. We identify large chemical shift differences between the two states, which are localized in the nucleotide binding pocket and in the vicinity of the phosphate binding site (lobe IIA) (Supplementary Fig. 8a,b). Because the ADP lifetime in equilibrium experiments is strongly correlated to the bulk phosphate concentration, we wanted to assess the presence of the same effect on the functional cycle. We thus performed the in cyclo experiment at different phosphate concentration in the range from 0.1 mM to 30 mM (Supplementary Fig. 8c,d). Strikingly, the kinetic parameters of the functional cycle, and in particular, the ADP lifetime were completely inert to the bulk Pi concentration (Fig. 5b). This finding leads us to the conclusion that the sole determinant of the long-lived ADP-Pi-bound state during the functional cycle is the phosphate generated upon ATP hydrolysis in the BiP NBD nucleotide binding pocket. This phosphate remains inside the nucleotide binding pocket, “gluing” ADP into the BiP NBD and leaving the pocket only concomitantly. Rebinding of phosphate molecules from the bulk plays no role for the functional cycle of the BiP NBD and the cycle is thus robust to fluctuations of the phosphate concentration. The in cyclo experiment thus overcomes the limitations of static experiments. Ca 2+ does not affect the functional cycle Ca 2+ is a key ER stress marker and plays a fundamental role in regulating the activity of multiple ER proteins 60 . While the role of magnesium ions for the catalytic activity of Hsp70 is well established 50 , the potential role played by calcium on the Hsp70 functional cycle is not yet well understood 61 . This question is of special interest for the BiP as the ER can show large variations in calcium, during protein folding homeostasis and stress, with concentrations ranging from 0.1 mM under homeostatic conditions up to 0.8 mM in stress conditions 62,63 . It has been proposed that the calcium concentration might decrease the ATP hydrolysis rate of BiP by 2-fold at physiological Ca 2+ levels compared to no calcium 50,64 and increase the ADP-bound lifetime in a concentration-dependent manner (4-folds at physiological concentration 61 ). Published crystal structures of the nucleotide bound BiP NBD in presence of calcium show that it binds in the nucleotide binding site 61 , in which the ADP-bound state calcium contacts both phosphate groups (a and b) while magnesium only contact the b phosphate (Supplementary Fig. 9a). This might suggest a mechanism for the variation of the kinetic parameters, if Ca 2+ replaces Mg 2+ ions. Importantly, the Ca 2+ concentration is always lower than the Mg 2+ concentration also in the ER 65,66 . Accordingly, to test the effect of calcium on the kinetic parameters of the BiP NBD in the following experiments, we keep the magnesium concentration at 10 mM and vary the calcium concentration as indicated for each experiment. First, we probed the presence of calcium binding to the ADP-bound state by NMR equilibrium experiments. Upon addition of 3.3 mM Ca 2+ to ADP-bound BiP NBD in presence of 10 mM Mg 2+ ([Mg 2+ ]/[Ca 2+ ]=3) large CSPs were observed, clearly confirming the Ca 2+ binding (Supplementary Fig. 9b). Mapping of these CSPs on the structure showed changes consistent with a calcium binding site in lobes IA and IIA as expected from the published crystal structure of NBD bound to ADP-Ca 2+ (PDB 6ZYH) 61 (Supplementary Fig. 9c). Next, we used MABA-ADP release to measure the ADP mean life as a function of calcium concentration at a fixed concentration of magnesium. We observed a strong dependence of the ADP lifetime on the calcium concentration with an IC 50 of 212+7 μM, in excellent agreement with the literature 61 (Fig. 6a,b and Supplementary Fig. 10a). Since the BiP NBD nucleotide binding pocket is always occupied by an ADP and a phosphate moiety during the functional cycle, we wanted to explore the effect of calcium on the ADP-Pi-bound state and thus repeated the experiment in the presence of 0.5 mM Pi. Strikingly, this experiment showed that the lifetime of bound ADP-Pi is completely independent of the calcium concentration (Fig. 6a,b and Supplementary Fig. 10b). The data thus readily suggest that the presence of calcium should also have no effect on the functional cycle. And indeed, in the presence of 3.3 mM calcium in the in cyclo setup, the ATP consumption is 0.090 ± 0.003 mM min -1 , which corresponds to a molecular hydrolysis rate of k hyd = 0.99 ± 0.03 min -1 (Fig. 5c). This is essentially equivalent to the molecular hydrolysis rate in the absence of calcium k hyd = 0.95 ± 0.03 min -1 . Also the relative populations of the two states, with T: 22 ± 4% and D: 78 ± 4% were the same as in the absence of calcium (T: 21 ± 4% and D: 79 ± 4%) (Fig. 5d,e and Supplementary Fig. 11a). Together, this determined the lifetimes of bound nucleotides to t T = 13.3 ± 3 s and t D = 47.4 ± 3 s, which are similar to the lifetimes in the absence of calcium (t T = 13.4 ± 2 s and and t D = 49.7 ± 3 s) (Fig. 5f-h). These data thus completely exclude any effect of calcium on the native functional cycle, in the cellular concentration range as well as up to several mM. Notably, the in cyclo experiment now allows us also to use our structural probes to probe the effect of calcium binding during the functional cycle. The 2D NMR experiments showed no significant CSP, neither for the ATP nor the ADP-Pi-bound state, evidencing an absence in structural changes, in full agreement with the finding that the kinetics are unchanged. (Supplementary Fig. 11c). The data thus clearly establish that calcium ions do bind to the ADP-bound state of the BiP NBD, but not to the ADP-Pi-bound state, and consequently, since the ADP-bound state is not part of the functional cycle of the BiP NBD, physiological variations of calcium have no effect on the BiP NBD functional cycle. Discussion The experimental approach presented in this work has resolved the kinetics and states encountered during the functional cycle of the Hsp70 BiP NBD at atomic resolution (Fig. 7 a,b). The BiP NBD passes cyclically through a total of three distinct states: the apo state, an ATP-bound state and an ADP-Pi-bound state. Starting from the apo state, the BiP NBD binds an ATP molecule to reach the ATP-bound state. From this state, the BiP NBD hydrolyzes ATP to reach the ADP-Pi-bound state. ADP-Pi release brings the BiP NBD into the apo form, from where a new cycle can start. Importantly, the ATP hydrolysis rate determined in cyclo matches the value determined by single turn-over experiments, while the ADP release rate does not correspond to classical experiments, since the phosphate generated upon ATP hydrolysis is “gluing” the ADP into the nucleotide binding pocket, thus dramatically transforming the second step of the functional cycle. The second step of ADP-Pi release is independent of the bulk phosphate concentration, the NBD-ADP-Pi complex decays in a monomolecular reaction. Formation of the ADP-Pi post-hydrolysis state has major consequences for the regulation of the functional cycle in response to environmental change. The phosphate concentration in the cytoplasm of the eukaryotic call is known to vary between at least 1 and 10 mM 67 , and can be expected to have similar or even large fluctuations in the ER. Our experimental setup has shown that the functional cycle is inert to such fluctuations, whereas previous interpretations based on equilibrium experiments that came to opposite conclusions need to be revised. This formation of the ADP-Pi post-hydrolysis state also excludes any effect of Ca 2+ in calcium concentrations on the BiP NBD functional cycle. While the activity of several other ER chaperones is strongly affected by variations in calcium concentrations (Grp94 68 , Calreticulin 69 and Protein disulfide isomerase 70 , 71 ), this is not the case for the BiP NBD. BiP thus provides a chaperone buffering capacity disconnected from the ER stress level. Our work has focused on the BiP NBD as a benchmark for subsequent studies also of full-length Hsp70. It will be exciting to extend our studies in a next step to full-length Hsp70 proteins, such as BiP, where we expect to understand the interplay between the substrate binding domain and the NBD. Additional interesting implications will be to understand how co-chaperones modulate the functional cycle. Nucleotide exchange factors 72 , 73 and nucleotide exchange inhibitors 74 , 75 accelerate and inhibit ADP release, respectively, and it will be interesting to study their effect in the context of a full-length Hsp70. The mode of action of these co-chaperones has been studied mostly by equilibrium experiments, whereas our experimental setup will be key to provide high resolution mapping of the interaction interface while simultaneously resolving the precise kinetics of the nucleotide conversion and release. Similarly, the methods described here will allow us to probe the effect of J-domain proteins on the ATP hydrolysis with unprecedented precision. On the longer run, it will be interesting to see how the diverse cellular functionalities of Hsp70 emerge from coupling ATP hydrolysis to interaction dynamics of the substrate binding domain. Methods Protein expression and purification Cloning, expression and purification of human BiP NBD Human BiP NBD lacking its ER signal sequence (residues 19–406) with an N-terminal His 6 -Tag including a TEV cleavage sequence was generated by introducing a mutation (G407 to stop) using the QuikChange II mutagenesis protocol (Stratagene) into the Human BiP (residues 19–654) gene synthetized by genscript. The gene was inserted through NcoI and XhoI into a pET28a expression vector (Novagen). BL21-(DE3)-Lemo cells (New England Biolabs) were transformed and grown at 37°C in M9 minimal medium prepared with 99.85% D2O (Sigma-Aldrich) containing 15 NH 4 Cl (1 g/liter; Sigma-Aldrich) and D-glucose-d7 (2 g/L; Sigma-Aldrich). BiP NBD expression was induced at an OD 600 of 0.6 by adding 1 mM isopropyl-β-d-thiogalactopyranoside (IPTG) and expression was continued at 25°C for 12 hours. Cells were harvested by centrifugation at 5000g for 20 min. The pellet was resuspended in 20 ml of lysis buffer per liter of culture (25 mM Hepes, pH 7.5, 150 mM NaCl, 10 mM MgCl 2 , 0.02 mg/mL ribonuclease, 0.01 mg/mL deoxyribonuclease and phenylmethylsulfonyl fluoride PMSF (0.2 mg/mL). Cell lysis was performed using a microfluidizer (Microfluidics) for three cycles at 4°C. The soluble bacterial lysate was separated from cell debris and other components by centrifugation at 14000 g for 60 min and loaded onto a Ni-NTA column (Qiagen) equilibrated in buffer A (25 mM Hepes, pH 7.5, 150 mM NaCl, 10 mM MgCl 2 ). BiP eluted at 500 mM imidazole concentration and was dialyzed overnight against buffer (25 mM Hepes, pH 7.5, 300 mM NaCl, 10 mM MgCl 2 ). BiP NBD was denatured with 6 M urea and loaded onto a Ni-NTA column (Cytiva) equilibrated in buffer A + 8 M urea. BiP NBD eluted at 500 mM imidazole concentration in buffer containing 8 M urea. BiP refolding was achieved by dialysis overnight against buffer (25 mM Hepes, pH 7.5, 300 mM NaCl, 10 mM MgCl 2 ). After refolding, the His-tag was cleaved by incubation with 1 mg of TEV per 50 mg of BiP in cleavage buffer (25 mM Hepes, pH 7.5, 300 mM NaCl, 10 mM MgCl 2 , DTT 1 mM and EDTA 0.5 mM) overnight at 4°C. BiP was separated from TEV and uncleaved BiP via a reverse Ni-NTA column (Cytiva) equilibrated in buffer A. BiP was then applied to an anion-exchange column (Cytiva) equilibrated in the buffer QA (25 mM Tris, pH 8.5) and eluted with 250 mM of KCl. Finally, BiP NBD was concentrated by ultrafiltration and subjected to size exclusion chromatography (Superdex-200 16/600 PG, Cytiva) to further purify the proteins and adjust to its final buffer (25 mM Hepes, pH 7.5, 150 mM KCl and 20 mM MgCl 2 ). Afterward, BiP was concentrated by ultrafiltration and stored at − 20°C until use. Final yield of purified protein was 15–20 mg for wild-type BiP NBD per liter of deuterated M9 minimal medium. The BiP NBD not refolded protein was purified according to the same protocol without the unfolding/refolding steps. The BiP NBD expressed in LB (Luria broth) medium was purified following the same purification protocol. Methyl labeling of human BiP NBD Methyl-labeled BiP proteins were obtained by growing the expression cells in M9 minimal media prepared with 99.85% D2O (Sigma-Aldrich) containing 15 NH 4 Cl (1 g/L; Sigma-Aldrich) and D-glucose-d 7 (2 g/L; Sigma-Aldrich). When the optical density (OD) at 600 nm reached 0.8, a solution containing the labeled precursors was added. For [U- 2 H, 15 N, 12 C], Met-[ 13 CH 3 ] ε , Val-[ 13 CH 3/ 13 CH 3 ] γ1/γ2 , Ile-[ 13 CH 3 ] δ 1 : 100 mg of 2-Keto-3-(methyl-13C)-butyric-4- 13 C acid sodium salt (Sigma-Aldrich), 30 mg of L-leucine-d 10 (Sigma-Aldrich), 80 mg of α-ketobutyric acid methyl 13 C (99%) 3,3-D2 (98%) (Sigma-Aldrich) and 100 mg of [ 13 C] ε -L-methionine (Sigma-Aldrich). For [U- 2 H, 15 N, 12 C], Met-[ 13 CH 3 ] ε , Val-[ 13 CH 3 / 12 C 2 H 3 ] cγ1/cγ2 , Ile-[ 13 CH 3 ] δ 1 : 100 mg of 2-Keto-3-(methyl-d3)-butyric acid-4- 13 C,3-d sodium salt (Sigma-Aldrich), 30 mg of L-leucine-d 10 (Sigma-Aldrich), 80 mg of α-ketobutyric acid methyl 13 C (99%) 3,3-D2 (98%) (Sigma-Aldrich) and 100 mg of [ 13 C] ε -L-methionine (Sigma-Aldrich). For [U- 2 H, 15 N, 12 C], Met-[ 13 CH 3 ] ε , Val-[ 13 CH 3 ] cγ 2 , Ile-[ 13 CH 3 ] δ 1 : 240 mg of 2-hydroxy-2-[ 13 C]methyl-3-oxo-4,4,4-tri-[ 2 H]-butanoate (pro-S acetolactate- 13 C, NMR-Bio), 30 mg of L-leucine-d 10 , 80 mg of α-ketobutyric acid methyl 13 C (99%) 3,3-D2 (98%) (Sigma-Aldrich) and 100 mg of [ 13 C] ε -L-methionine (Sigma-Aldrich). One hour after the addition of the precursors, BiP expression was induced by adding 1 mM isopropyl-β-d-thiogalactopyranoside (IPTG) at 25°C for 12 hours. Mutagenesis, expression and purification of the BiP assignment mutants The QuikChange II mutagenesis protocol (Stratagene) was used to introduce the point mutations in the BiP plasmid: I33V, I76V, I145V, M148L, M153L, I190V, M196L, I199V, I207V, M263L, M332L, M339L and V400I. Polymerase chain reaction primers were obtained from Microsynth. The expression and purification of the mutant proteins was performed as described for the wild-type proteins. The final yield of purified mutants was similar to wild-type at the exception of the mutant M148L that express at very low yield and thus could not be analyzed. NMR spectroscopy All NMR experiments for BiP NBD were performed in NMR buffer (25 mM Hepes, pH 7.5, 150 mM KCl and 20 mM MgCl 2 ) at 37°C. The experiments were recorded on Bruker AscendII 700 MHz, Avance 800 MHz or Avance 900 MHz spectrometers running Topspin 3.0 and equipped with a cryogenically cooled triple-resonance probe. NMR data were processed with nmrPipe 76 and ccpnmr 77 . All equilibrium NMR experiments were recorded in NMR buffer at a concentration of BiP NBD 100 µM. Purified ADP (Sigma-Aldrich) and Pi were added to the sample at concentration of 5 mM. 1D 1 H NMR spectra were used to monitor the purity of the ADP and absence of contaminants. Assignment of BiP NBD Met[CH 3 ] ε , Val[CH 3 ] γ1/γ2 and Ile[CH 3 ] δ 1 methyl groups Met[CH 3 ] ε , Val[CH 3 ] γ1/γ2 and Ile[CH 3 ] δ 1 assignment was obtained using a structure-based approach combining mutagenesis and 3D 13 C, 13 C-resolved [ 1 H, 1 H]-NOESY experiments. The following point mutation were used: I33V, I76V, I145V, M148L, M153L, I190V, M196L, I199V, I207V, M263L, M332L, M339L, V400I. Each mutant sample was recorded at 37°C with an adjusted duration depending on their final concentration (experimental time ranging from 120 to 240 min per sample). For each mutant, spectra were recorded for the ADP-Pi-bound state (5 mM) and in cyclo experiment. Analysis and comparison of the library of mutant spectra allowed the assignment of 1/35 valines (3%), 6/25 isoleucine (24%) and 6/6 methionines (100%). The network of assigned residues was used to expand the assignment using 3D 13 C, 13 C-resolved [ 1 H, 1 H]-NOESY experiments recorded with sample concentrations ranging from 0.4 to 0.8 mM with a mixing time of 400 ms and an adjusted duration depending on the final concentration of each constructs (experimental time ranging from 3 to 5 days per sample). Stereospecific assignment of the valine cγ1/cγ2 for residues located in the BiP NBD has been obtained using a sample [U- 2 H, 15 N, 12 C], Met-[ 13 CH 3 ] ε , Val-[ 13 CH 3/ 13 CH 3 ] γ1/γ2 , Ile-[ 13 CH 3 ] δ 1 and a 3D 13 C, 13 C-resolved [ 1 H, 1 H]-NOESY experiments recorded with a short mixing time of 50 ms that directly correlate the two methyl groups within one amino-acid. Analysis of the 3D 13 C, 13 C-resolved [ 1 H, 1 H]-NOESY cross peaks with the structure of the BiP NBD in the ADP-Pi undocked allowed to expand our mutagenesis-based assignment to assign of 32/35 valines, 22/25 isoleucine and 6/6 methionine. Among the 32 valines, 24 had both methyl Cγ1 and Cγ2 assigned and 8 one of them, resulting in a total of 84 observable NMR signals. NMR in cyclo experimental setup Composition of the in cyclo NMR samples All in cyclo NMR experiments were recorded with BiP NBD samples [U- 2 H, 15 N, 12 C], Met-[ 13 CH 3 ] ε , Val-[ 13 CH 3 / 12 C 2 H 3 ] cγ1/cγ2 , Ile-[ 13 CH 3 ] δ 1 in NMR buffer (25 mM Hepes, pH 7.5, 150 mM KCl and 10 mM MgCl 2 ) at a concentration of BiP constructs 100 µM and specified concentration of the co-chaperones or client-protein. The in cyclo ATP regeneration system was composed of 5 units of pyruvate kinase from rabbit muscle (Sigma-Aldrich) (≈ 200 nM) and the concentration of phosphoenolpyruvate (PEP- Sigma-Aldrich) was adjusted to maintain the system active for at least four hours ([PEP] = 10–100 mM). All the experiments were recorded at minimum as experimental triplicate. Recording of the in cyclo NMR experiments The in cyclo experiment was initiated by the addition of 5 mM ATP (Sigma-Aldrich) and allowed to equilibrate at a temperature of 37°C for 2 minutes in the NMR spectrometer. After the equilibration time 2D [ 13 C, 1 H]-HMQC spectra were recorded in interleaved mode with 1D 1 H NMR spectra. The 1D 1 H NMR spectra were used to monitor the pyruvate increase and the constant concentration of ATP using the NMR signal of 0.5 mM sodium 2,2-dimethyl- 2-silapentane-5-sulfonate-d6 (d, 98%) (DSS; Cambridge Isotope Laboratories Inc.) as an internal reference. MABA-ADP release assay Nucleotide release and binding measurements were performed using the fluorescent nucleotide analogues MABA-ADP (8-[(4-Amino)butyl]-amino-ADP-MAN; Jena-bioscience) carrying a MANT moiety whose fluorescence increases upon binding to Hsp70s 44 , 58 . To measure nucleotide release, 1.8 µM fluorescent nucleotide analogues were incubated with 1.8 µM proteins in NMR buffer for at least 45 minutes at 25°C to allow for complex formation (Solution A). Solution B is NMR buffer with 7.5 mM ADP. The final concentrations after mixing were 1.44 µM for each protein, 1.44 µM fluorescent nucleotides, and 1.5 mM ADP. The measurements were started by adding 20 µL of solution B to 80 µl of solution A in a 96-well plate (Corning) and fluorescence (excitation 360 nm, emission 420 nm) was detected over time with a fluorescence spectrometer (Tecan Plate Reader) at 37°C. The dead time between sample mixing and signal recording was ∼ 2 s. All solutions contained 25 mM HEPES, pH 7.5, 150 mM KCl, 10 mM MgCl 2 at 37°C. The solutions A contained CaCl 2 and phosphate where indicated (final concentrations are stated in figure legends). The dissociation rate constants ( k off ) were determined by fitting the data to a one phase exponential function using a python script. ATP analog preparation AMPPNP (adenylyl imidodiphosphate), AMPPCP (adenylyl methylenediphosphate) and ATP-γ-S (adenosine 5’-(gamma-thiotriphosphate)) (Jena-bioscience) were dissolved in the HKM reaction buffer HKM (25 mM HEPES pH 7.5, 150 mM KCl and 10 mM MgCl 2 ) at a concentration of 50 mM directly before to be mixed with the BiP NBD sample. Preparation of the ATP analogs just before usage allows to circumvent the known instability of these analog that undergo slow hydrolysis in solution. NADH ATP assay BiP NBD was diluted to a final concentration of 1 µM in the HKM reaction buffer HKM (25 mM HEPES pH 7.5, 150 mM KCl and 10 mM MgCl 2 ). The reactions buffer also contained 10 mM Phosphoenolpyruvate (Sigma-Aldrich), 0.5 mM NADH (Sigma-Aldrich), pyruvate kinase (Sigma-Aldrich) and lactate dehydrogenase (Sigma-Aldrich) (diluted to 5 U). The reactions were started by addition of 5 mM ATP in a final volume of 160 µl in 96-well microplates (Corning) and NADH absorbance at 340 nm (A 340 ) was measured over time at 37°C with a Tecan Plate Reader. Linear regression analysis was performed and the ATP hydrolysis activity was calculated using the molar extinction coefficient of NADH (ε = 6220 M − 1 cm − 1 ). Single turn-over assay The ATPase activity of BiP NBD was determined under single-turnover conditions as previously described with minor modifications of the protocol 44 . The main-modification came to replace the radioactivity-based detection by a label free anion-exchange based quantification 57 . The BiP NBD-ATP complex was formed by mixing BiP NBD with 10 mM ATP in formation buffer (25 mM HEPES pH 7.5, 50 mM KCl, 10 mM MgCl 2 ) and incubating the mixture for 5 min on ice. The BiP NBD was separated from unbound ATP at 4°C by gel filtration on NICK columns (Cytiva) equilibrated in reaction buffer (25 mM HEPES pH 7.5, 150 mM KCl, 10 mM MgCl 2 ). Fractions containing the BiP NBD ATP complexes were pooled and snap frozen in liquid nitrogen and kept at -80°C. For the ATPase activity determination, BiP NBD ATP complex solutions were thawed and the reaction started by mixing with the reaction buffer prewarmed at 37°C (25 mM HEPES pH 7.5, 150 mM KCl, 10 mM MgCl 2 ). At given time points, samples were withdrawn from the reaction, mixed with an equivalent volume of HCl and snap frozen in liquid nitrogen and kept at -80°C. The relative ATP/ADP ratio was determined using an anion chromatography-based assay. SEC-MALS SEC-MALS measurements of BiP NBD were performed at 25°C in NMR buffer (25 mM Hepes, pH 7.5, 150 mM KCl and 20 mM MgCl 2 ) using a SEC-3 HPLC column (300 Å pore size; Agilent Technologies) on an Agilent 1260 HPLC. Elution was monitored by multiangle light scattering (Heleos II 8+; Wyatt Technology), differential refractive index (Optilab T-rEX; Wyatt Technology) and absorbance at 280 and 254 nm (1260 UV; Agilent Technologies). All the system parameters were calibrated using an injection of 2 mg/ml BSA solution (ThermoPierce) and standard protocols in ASTRA 6. Molar mass (MM) and mass distributions were calculated using the ASTRA 6 software (Wyatt Technology). Data availability All data needed to evaluate the conclusions of the paper are presented in the manuscript. The 84 state-specific methyl resonance assignments of BiP have been submitted to the Biological Magnetic Resonance Data Bank under accession code 51751. Additional data related to this paper will be provided by the authors upon request. Declarations Acknowledgements This work was funded by the Swiss National Science Foundation (grants 185388 and 207755 to S. H.). 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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-4017836","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":276810393,"identity":"a5f6c805-c57a-4466-9782-043c51c1ac9d","order_by":0,"name":"Sebastian Hiller","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAABEUlEQVRIiWNgGAWjYFACHhAhAWYceFDAwMAPZB5mgAni1JIA1ZJgwMAg2UCcFigDpMXgAAMDMz4t8u29Bz/+/GEhb87AexBoi02+8Y3cg4cLauoY+Gc3YNVicOZcsjRPgoThzga+BKCWNMttN/ISDs84dphB4s4B7FokcgykgX5h3HCAxwCo5bCB2Y0cg8M8bAeAUgnYHTb/jfHPHwkS9lAt/w2MZ4C0/KvDqYXhBo+ZBNBhiVAtBwxA9h7mbWPGqcXgTF6aNU+aRPKGw2AtyQYSZ94YHJ7Zd5hH4gYOh7WfPXzzh02d7YbjPcYfPlTYGfC35xh/LvhWJ8c/A4fD4IAZjc9DQP0oGAWjYBSMAjwAAFoRXuOlEacyAAAAAElFTkSuQmCC","orcid":"https://orcid.org/0000-0002-6709-4684","institution":"University of Basel","correspondingAuthor":true,"prefix":"","firstName":"Sebastian","middleName":"","lastName":"Hiller","suffix":""},{"id":276810394,"identity":"0cbe01da-acee-44d7-b445-870127fce3f7","order_by":1,"name":"Guillaume Mas","email":"","orcid":"","institution":"University of Basel","correspondingAuthor":false,"prefix":"","firstName":"Guillaume","middleName":"","lastName":"Mas","suffix":""}],"badges":[],"createdAt":"2024-03-05 15:55:07","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4017836/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4017836/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1038/s41467-025-60343-x","type":"published","date":"2025-06-01T04:00:00+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":52187824,"identity":"c0f5e04f-9ecd-4307-9bb5-e49e74e4137f","added_by":"auto","created_at":"2024-03-07 18:57:22","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":465844,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eAssignment of the BiP NBD ADP-Pi-bound state. a, \u003c/strong\u003eSections of a 2D [\u003csup\u003e13\u003c/sup\u003eC,\u003csup\u003e1\u003c/sup\u003eH]-methyl-TROSY spectrum of methyl-labeled BiP in presence of 5 mM ADP-Pi. Sequence-specific resonance assignments are indicated. \u003cstrong\u003eb,\u003c/strong\u003e Graphical representation of the NOESY network around methionine 263 (pink). Observed NOESY cross-peaks between two methyls groups are represented by an arrow. Each arrow tip corresponds to one observed NOE cross peak. \u003cstrong\u003ec, \u003c/strong\u003eCompleteness of the BiP Ile, Met and Val methyl NOE network, as a function of the interproton distance. Grey bars show the percentage of NOESY cross-peaks observed versus expected from the structure (PDB 5EVZ) (left axis)). Crosses show the percentage of number of observed NOESY contacts (righ axis - crosses). \u0026nbsp;\u003cstrong\u003ed\u003c/strong\u003e, Location of the assigned methyl groups (green spheres) in a 3D structure of the BiP NBD in the ADP-Pi-bound state (PDB 5EVZ).\u003c/p\u003e","description":"","filename":"floatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-4017836/v1/df2d9f3b08367a1248cecdfa.png"},{"id":52187828,"identity":"b44c880a-359a-4e8b-84d1-383d536b1ecb","added_by":"auto","created_at":"2024-03-07 18:57:22","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":169353,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eExperimental setup for in cyclo NMR with ATP regeneration system. a, \u003c/strong\u003eReaction scheme of the ATP regeneration system. \u003cstrong\u003eb\u003c/strong\u003e, Series of 1D \u003csup\u003e1\u003c/sup\u003eH NMR spectra of the ATP indole H8, recorded at an interval of 6 min. \u003cstrong\u003ec\u003c/strong\u003e, Quantification of the ATP concentration from the 1D \u003csup\u003e1\u003c/sup\u003eH NMR spectra shown in (b): Red crosses represent three independent experiments. \u003cstrong\u003ed, \u003c/strong\u003eSame as (b) for pyruvate (left) and phospho-enol pyruvate (right). \u003cstrong\u003ee\u003c/strong\u003e, Quantification of the pyruvate concentration from the 1D \u003csup\u003e1\u003c/sup\u003eH NMR spectra shown in (d): Grey crosses represent three independent experiments. The black line shows a linear fit to determine the ATP consumption rate. \u003cstrong\u003ef\u003c/strong\u003e, Molecular hydrolysis rate calculated from the in cyclo data presented in (e) and from a classical NADH ATP assay. Data points represent three independent experiments and the error bar the standard deviation.\u003c/p\u003e","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-4017836/v1/ff65a6e9745860b5e7aa216b.png"},{"id":52187830,"identity":"71be758e-7390-46d3-9475-1e3a295d87df","added_by":"auto","created_at":"2024-03-07 18:57:22","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":430739,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eResonance assignment of the NBD ATP-bound state. a, \u003c/strong\u003eSections of\u003cstrong\u003e \u003c/strong\u003e2D [\u003csup\u003e13\u003c/sup\u003eC,\u003csup\u003e1\u003c/sup\u003eH]-methyl-TROSY spectrum of the methyl-labeled NBD in presence of the ATP regeneration system (in cyclo). Sequence-specific resonance assignments of the Ile-[\u003csup\u003e13\u003c/sup\u003eC\u003csup\u003e1\u003c/sup\u003eH]\u003csup\u003eδ1\u003c/sup\u003e and Met-[\u003csup\u003e13\u003c/sup\u003eC\u003csup\u003e1\u003c/sup\u003eH]\u003csup\u003eε\u003c/sup\u003e methyl groups in the ADP-Pi-bound (black) and ATP-bound (red) states are indicated. \u003cstrong\u003eB\u003c/strong\u003e, Selected sections of 2D [\u003csup\u003e13\u003c/sup\u003eC,\u003csup\u003e1\u003c/sup\u003eH]-methyl-TROSY spectra of residues Met196 and Met263 in cyclo (pink) compared to the equilibrium experiments of NBD apo (grey), NBD with 5 mM ADP (light-blue) and NBD with 5 mM ADP-Pi (blue). \u003cstrong\u003eC\u003c/strong\u003e, Methyl groups with significant chemical shift differences between the ATP-bound and ADP-Pi-bound states displayed on the structure of the ATP-bound state (PDB 3LDL). The CSPs are shown in Supplementary Fig. 7a. \u003cstrong\u003ed\u003c/strong\u003e, Residue-specific populations of the the ADP-Pi-bound (blue) and the ATP-bound state (red). Each bar corresponds to one methyl group. (black line = average population): data points represent the mean for three independent experiments. \u003cstrong\u003eE\u003c/strong\u003e, Populations of the ADP-Pi-bound and ATP-bound states, averaged over all resonances: data points and standard deviation for three independent experiments. Dots represent experimental triplicates. \u003cstrong\u003ef\u003c/strong\u003e, Timing of the NBD two-step functional cycle.\u003cstrong\u003e\u003cbr\u003e\n\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"floatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-4017836/v1/0e82265804c25bd5d3f2ece3.png"},{"id":52187825,"identity":"0cfa87a8-f548-4e36-be80-31f9e9ff66f4","added_by":"auto","created_at":"2024-03-07 18:57:22","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":70684,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSingle turn over experiment matches the ATP lifetime in cyclo. a, \u003c/strong\u003eDecrease of ATP concentration over time for six independent experiments. The black line corresponds to the monoexponential fit of the data average.\u003cstrong\u003eb, \u003c/strong\u003eComparison of\u003cstrong\u003e \u003c/strong\u003ethe mean-lifetime of ATP hydrolysis determined in single turn-over experiments and in cyclo: data points and standard deviation for six (single-turnover) and three (in cyclo) independent experiments.\u003c/p\u003e","description":"","filename":"floatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-4017836/v1/a2e5cb614e7f51d2c3861422.png"},{"id":52190522,"identity":"18544220-985d-4036-9012-ba45cfaf53e3","added_by":"auto","created_at":"2024-03-07 19:13:22","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":169964,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThe lifetime of the ADP-Pi-bound state in cyclo is independent of bulk phosphate concentration. a,\u003c/strong\u003eTime courses of MABA-ADP dissociation from NBD at different concentration of Pi from 0 to 30 mM (gradient orange to dark brown): data points represent the mean for three independent experiments. The red lines correspond to the monoexponential fit of the data. \u003cstrong\u003eb, \u003c/strong\u003eLifetime of the MABA-ADP-NBD complex (grey circles) and lifetime of the ADP-Pi-bound state in cyclo (white circle), as a function of the bulk Pi concentration. Data points and standard deviation for three independent experiments.\u003c/p\u003e","description":"","filename":"floatimage5.png","url":"https://assets-eu.researchsquare.com/files/rs-4017836/v1/476773ce45952c6e9a138ce7.png"},{"id":52187826,"identity":"48f96005-5915-4b31-8be7-7bb30d0fdf52","added_by":"auto","created_at":"2024-03-07 18:57:22","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":211042,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCalcium does not affect the NBD functional cycle. a\u003c/strong\u003e, Time courses of MABA-ADP dissociation from NBD with 0.5 mM Pi (orange), 1 mM Ca\u003csup\u003e2+\u003c/sup\u003e (pink) or 1 mM Pi and Ca\u003csup\u003e2+\u003c/sup\u003e (purple): data points represent the mean for three independent experiments. \u003cstrong\u003eb\u003c/strong\u003e, Mean-lifetime of the MABA-ADP-NBD complex at different Ca\u003csup\u003e2+\u003c/sup\u003e concentrations in the range from 0 to 10 mM, in the absence of Pi (purple circles) and with 0.5 mM Pi (grey circles). data points represent the mean for three independent experiments and the error bar the standard deviation. The half maximal inhibitory concentration (IC50) of Ca was calculated from three independent experiments \u003cstrong\u003ec\u003c/strong\u003e, Molecular hydrolysis rate in the presence of 3.3 mM calcium ([Mg\u003csup\u003e2+\u003c/sup\u003e]/[Ca\u003csup\u003e2+\u003c/sup\u003e]=3) calculated from the data presented in Supplementary Fig. 11a. \u003cstrong\u003ed\u003c/strong\u003e, Residue-specific populations of the isolated NBD ADP D (blue) and ATP-bound state T (red) in the presence of 3.3 mM calcium ([Mg\u003csup\u003e2+\u003c/sup\u003e]/[Ca\u003csup\u003e2+\u003c/sup\u003e]=3). Each bar corresponds to one methyl group. (black line = average population and black dash line). The data represents one data set from a triplicate. \u003cstrong\u003ee,\u003c/strong\u003e Average populations of the ADP- and ATP-bound states including dots for experimental triplicates. \u003cstrong\u003ef\u003c/strong\u003e, Overall timing of the NBD two-step functional cycle in the presence of 3.3 mM calcium ([Mg\u003csup\u003e2+\u003c/sup\u003e]/[Ca\u003csup\u003e2+\u003c/sup\u003e]=3). \u003cstrong\u003eg-h\u003c/strong\u003e, In cyclo lifetime of the ATP-bound (g) and ADP-bound states (h) without and with 3.3 mM calcium ([Mg\u003csup\u003e2+\u003c/sup\u003e]/[Ca\u003csup\u003e2+\u003c/sup\u003e]=3).\u003cbr\u003e\n\u003c/p\u003e","description":"","filename":"floatimage6.png","url":"https://assets-eu.researchsquare.com/files/rs-4017836/v1/55070ccee0c3e250f92a1077.png"},{"id":52187831,"identity":"2dc7a15e-5a03-4404-9ea8-4928f8fe63c4","added_by":"auto","created_at":"2024-03-07 18:57:22","extension":"jpeg","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":313512,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThe three-step functional cycle of BiP NBD and its energy landscape.\u003c/strong\u003e \u003cstrong\u003ea\u003c/strong\u003e, The Hsp70 chaperone BiP NBD cycles through three states (apo state (PDB 3LDN), ATP state (PDB 3LDL) and ADP-Pi state (5EVZ)). The kinetic parameters \u003cem\u003ek\u003c/em\u003e\u003csub\u003eoff\u003c/sub\u003e ADP-Pi and \u003cem\u003ek\u003c/em\u003e\u003csub\u003ecat\u003c/sub\u003e were established in this work. The kinetic parameters for nucleotide ADP \u003cem\u003ek\u003c/em\u003e\u003csub\u003eon\u003c/sub\u003e and ATP \u003cem\u003ek\u003c/em\u003e\u003csub\u003eon\u003c/sub\u003e/\u003cem\u003ek\u003c/em\u003e\u003csub\u003eoff\u003c/sub\u003e were reported previously. \u003cstrong\u003eb\u003c/strong\u003e, Schematic free energy landscape of the functional cycle.\u003c/p\u003e\n\u003cp\u003e78,79\u003c/p\u003e","description":"","filename":"floatimage7.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-4017836/v1/e903da0b3f5f77d66fbe08e5.jpeg"},{"id":83723255,"identity":"709eb4a1-f941-4056-a68a-b05f7161fc5b","added_by":"auto","created_at":"2025-06-01 07:06:08","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":3363556,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4017836/v1/9ca47abb-27b3-4a88-b0fd-7d63c672cb0f.pdf"},{"id":52189908,"identity":"28017cfa-d046-4ec4-a85e-1b393047d629","added_by":"auto","created_at":"2024-03-07 19:05:22","extension":"pdf","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":2965296,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cbr\u003e\u003c/p\u003e","description":"","filename":"SINBDmethodv006.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4017836/v1/5c858a35edc3a8ea4bc01a74.pdf"},{"id":52189907,"identity":"2da34fc2-6af7-42bc-b838-39989d8a7895","added_by":"auto","created_at":"2024-03-07 19:05:22","extension":"pdf","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":321198,"visible":true,"origin":"","legend":"Reporting Summary","description":"","filename":"4921420relaedms8760125s9vsnt.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4017836/v1/4eda9959d1743781e95ea87b.pdf"}],"financialInterests":"There is \u003cb\u003eNO\u003c/b\u003e Competing Interest.","formattedTitle":"Characterization of ATP hydrolysis in the Hsp70 BiP nucleotide binding domain","fulltext":[{"header":"Introduction","content":"\u003cp\u003eThe 70 kDa heat shock protein (Hsp70) family of molecular chaperones is crucial for biogenesis and protein homeostasis\u003csup\u003e1–4\u003c/sup\u003e. The Hsp70s account for up to 4% of total cellular protein mass, making them one of the most abundant proteins in the cell\u003csup\u003e5–7\u003c/sup\u003e. Hsp70s are involved in diverse cellular processes \u003csup\u003e3,8–12\u003c/sup\u003e, including de-novo protein folding at the ribosome\u003csup\u003e13,14\u003c/sup\u003e, protein translocation through pores\u003csup\u003e15,16\u003c/sup\u003e and solubilization of protein aggregates\u003csup\u003e1,17,18\u003c/sup\u003e. Consistently, Hsp70s are connected to multiple pathophysiological conditions including cancer and neurodegenerative diseases\u003csup\u003e19–21\u003c/sup\u003e. In the endoplasmic reticulum (ER), BiP (Binding Immunoglobulin Protein) is the sole Hsp70 isoform in all eukaryotes\u003csup\u003e22,23\u003c/sup\u003e and the most abundant ER chaperone\u003csup\u003e7,24\u003c/sup\u003e. BiP is the central functional hub of the ER chaperone network that ensure protein folding homeostasis in the “folding factory of the cell”. It consequently binds to most of the proteins that are processed in the ER, promoting their folding and preventing their aggregation\u003csup\u003e25,26\u003c/sup\u003e. Additionally, it also acts as the central regulator of the unfolded protein response (UPR) by binding to the UPR sensors in a stress-dependent manner\u003csup\u003e27–29\u003c/sup\u003e. Furthermore, BiP targets unfolded protein to the degradation machinery associated with the ER-associated degradation pathway (ERAD)\u003csup\u003e30–32\u003c/sup\u003e. Moreover, BiP is overexpressed in many human cancers, making it a major therapeutic target\u003csup\u003e33–36\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003eHsp70 chaperones comprise two distinct domains, the nucleotide-binding domain (NBD) and the substrate-binding domain (SBD) that are connected by a flexible linker\u003csup\u003e37,38\u003c/sup\u003e. The SBD is sub-divided into the subdomains SBDα and SBDβ enclosing the client binding site\u003csup\u003e12\u003c/sup\u003e. The NBD has a clamp-like shape with two lobes I and II\u003csup\u003e12\u003c/sup\u003e. Each lobe is further subdivided into two subdomains A and B. A nucleotide binding site is located in the center of the domain in the cleft between lobes I and II. Hsp70 chaperones go through a functional cycle encompassing ATP-binding, ATP-hydrolysis, and ADP-Pi release, all of which takes place in the NBD\u003csup\u003e39\u003c/sup\u003e. ATP binding leads to a rearrangement of lobe I, which triggers the SBD to open the client binding site\u003csup\u003e40–42\u003c/sup\u003e. Following ATP hydrolysis, the NBD is in an ADP-bound state that leads to the closure of the client binding site. From there, ADP is released at one point, resulting in the apo form, to which a new ATP molecule binds to restart the cycle. The overall Hsp70 chaperone activity resulting from this fundamental cycle dependents on the cellular context and manifests into a diverse set of effective functions such as a foldase, holdase, translocase, unfoldase or disaggregase\u003csup\u003e11,12,43\u003c/sup\u003e. Importantly, these functions are fundamentally regulated by the timing originating from nucleotide processing in the NBD and understanding the Hsp70 functional cycle thus requires elucidating the nucleotide reaction steps and their modulation by environmental change.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eInterestingly, despite exhaustive biochemical characterization of Hsp70 proteins, measurements of the functional cycle kinetic parameters have so far been possible only by isolating individual steps and not at atomic resolution\u003csup\u003e44–46\u003c/sup\u003e. In this study, we develop a method that combines the power of methyl NMR spectroscopy to resolve atomic siteswith a temporal dimension to resolve the reaction kinetics of the cycle individual steps.\u0026nbsp;We benchmark the method on the example of the NBD of the human Hsp70 chaperone BiP from the endoplasmic reticulum (ER) and compare the results from the in cyclo experiments with classical single turn-over and ADP release experiments. We find that the functional cycle is completely independent from the bulk concentration of free inorganic phosphate (Pi) and characterize absence of any significant effect of the concentration of calcium. The method established here provides a technology platform for a fundamental understanding of the Hsp70 functional cycle at the atomic level, also in the context of the full-length Hsp70s and their regulation by co-chaperones.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eNMR resonance assignment of NBD methyl groups\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAs a first step towards observing individual atomic sites during the functional cycle of BiP NBD, we established a highly pure and homogenous sample preparation. Since we want to be able to detect conformational sub-states of the protein with potentially low populations, the preparation needs to be free of any, specifically or non-specifically bound nucleotides and other contaminants. Such contaminants have been reported as a major cause of concern in the literature\u003csup\u003e47\u003c/sup\u003e. We thus purified the protein using well-established protocols and then added an affinity column purification step under denaturing conditions of 8 M Urea. The protein is entirely unfolded under these conditions and thus loses the affinity for bound impurities, which are washed through the column. After elution from the column, the protein was slowly refolded via dialysis. The refolded BiP NBD was analyzed by SEC-MALS showing a perfect overlap with BiP NBD purified without unfolding/refolding step (Supplementary Fig. 1a,b). Its NMR spectrum was 100% homogenous, evidencing the absence of any significant amounts of ligands. The proper conformation of the refolded protein was assessed by a comparison of the NMR spectra of the BiP NBD with and without denaturation (Supplementary Fig. 1c). The two spectra overlapped perfectly, demonstrating that the BiP NBD reaches its native state after denaturation and refolding. This high purification standard was kept in all subsequent experiments.\u003c/p\u003e\n\u003cp\u003eIn a next step, we isotope-labelled the methyl groups of three amino acids, Ile-[\u003csup\u003e13\u003c/sup\u003eC\u003csup\u003e1\u003c/sup\u003eH]\u003csup\u003eδ1\u003c/sup\u003e, Met-[\u003csup\u003e13\u003c/sup\u003eC\u003csup\u003e1\u003c/sup\u003eH]\u003csup\u003eε\u003c/sup\u003e and Val-[\u003csup\u003e13\u003c/sup\u003eC\u003csup\u003e1\u003c/sup\u003eH]\u003csup\u003eg\u003c/sup\u003e\u003csup\u003e1/\u003c/sup\u003e\u003csup\u003eg\u003c/sup\u003e\u003csup\u003e2\u003c/sup\u003e, on an otherwise deuterated background. This is a well-established technique to allow atomic resolution NMR studies even at large molecular sizes up to several 100 kDa\u003csup\u003e48\u003c/sup\u003e. 2D [\u003csup\u003e13\u003c/sup\u003eC,\u003csup\u003e1\u003c/sup\u003eH]-methyl-TROSY spectra\u003csup\u003e49\u003c/sup\u003e with high sensitivity can be recorded in times as short as 5 minutes at protein concentrations of 100 μM. This high sensitivity is key to detect also minor sub-states of the cycle when longer experiment times are used. With the purification and preparation steps, NMR spectra of the protein in thermodynamic equilibrium were recorded in the apo form or in presence of ADP. The resulting equilibrium spectra serve as references for the respective conformational states of the protein. In presence of 5 mM ADP-Pi, we observed a homogeneous spectrum with a single set of 102 resonances, precisely matching the 102 resonances expected from the chemical structure of the molecule (Fig. 1a). We established sequence-specific assignments of these resonances using a strategy that combines single point-mutagenesis and NOESY experiments. As a first step, we established the assignments of all 6 methionine residues by single point mutagenesis (M148L, M153L, M196L, M263L, M332L, M339L). Then, using these anchor points, we expanded the assignment using a 3D \u003csup\u003e13\u003c/sup\u003eC,\u003csup\u003e\u0026nbsp;13\u003c/sup\u003eC-resolved [\u003csup\u003e1\u003c/sup\u003eH,\u003csup\u003e\u0026nbsp;1\u003c/sup\u003eH]-NOESY experiments that we manually curated against a published crystal structure of the BiP NBD (PDB 5EVZ) (Fig. 1b and Supplementary Fig. 2a). The methyl groups of Met, Val and Ile have unambiguously separated chemical shift ranges for their NMR signals, permitting a direct identification of the amino acid type for a given signal and thus to easily distinguish different NOESY network. We resolved around 4 NOE contacts per residue, guaranteeing unambiguous assignments as a network effect. We additionally exploited the good correlation between the NOE cross peaks intensities and the calculated distance in the BiP NBD structure to confirm the correctness of these network (Supplementary Fig. 2b). Overall, we could resolve 100 NOESY contacts up to 5 Å, 255 NOESY contacts in the distance range 5–8 Å and 91 NOESY contacts in the range 8–10 Å, leading to a high-confidence assignment (Fig. 1c). As a final validation step, we selected 7 individual residues at the core of large NOESY networks and confirmed the correctness of their assignment by single-point mutagenesis. In total, this approach led to the stereospecific assignment of 60 residues, thereof 32/35 valines, 22/25 isoleucine, 6/6 methionine. Among the assigned 32 valines, 24 had both methyl Cg1 and Cg2 assigned and 8 one of them, resulting in a total of 84 observable NMR signals (Fig. 1a and d, Supplementary Fig. 3). For the subsequent experiments, we thus have 84 atomic level reporters that we can observe simultaneously and with high sensitivity for local structural changes.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSetup of the functional cycle with the ATP regeneration system\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eBased on these prerequisites of a highly pure preparation and near-complete resonance assignments, we set up the experiment to monitor the BiP NBD functional cycle. We selected a buffer composition corresponding to optimal Hsp70 activity (25 mM HEPES pH 7.5, 150 mM KCl and 10 mM MgCl\u003csub\u003e2\u003c/sub\u003e), and the physiological temperature of 37°C. Magnesium is required for the Hsp70 ATP hydrolysis as it coordinates the ATP\u0026nbsp;b\u0026nbsp;and\u0026nbsp;g\u0026nbsp;phosphate\u003csup\u003e50\u003c/sup\u003e. The potassium concentration was chosen to match previous reports that it acts as a cofactor of the Hsp70 hydrolysis of ATP increasing the activity by 5-fold in the optimal range of concentration between 100 and 150 mM\u003csup\u003e51\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003eNotably, while it is possible to prepare pure apo and pure ADP-bound state, it is not possible to prepare a stable ATP-bound state, due to the catalytic activity of the protein. For example, the addition of 5 mM ATP to 100 μM of the BiP NBD leads to the significant accumulation of ADP already in the first few minutes and results in a non-equilibrium situation with rapidly changing ADP/ATP concentration ratio (Supplementary Fig. 4). In order to create a stable steady state condition, we implemented an ATP regeneration system\u0026nbsp;inside the NMR tube that steadily converts ADP into ATP\u003csup\u003e52\u003c/sup\u003e. This system exploits the activity of pyruvate kinase, at catalytic amounts, to combine phosphoenol pyruvate (PEP) and ADP to form a novel ATP molecule (Fig. 2a).\u0026nbsp;ATP and ADP molecules can be unambiguously distinguished by their\u0026nbsp;H8 adenine proton in\u0026nbsp;1D \u003csup\u003e1\u003c/sup\u003eH NMR experiments, PEP by its methylene protons and pyruvate by its methyl protons\u0026nbsp;(ATP:\u0026nbsp;8.554 ppm, ADP: 8.558 ppm, PEP: 5.424 ppm, pyruvate: 2.424 ppm). Monitoring of ATP, ADP, PEP and pyruvate concentrations by 1D \u003csup\u003e1\u003c/sup\u003eH NMR spectra shows that the ATP regeneration system keeps the\u0026nbsp;concentrations of ATP and ADP effectively constant with no detectable signal for the ADP ([ATP] = 5 mM and [ADP] \u0026lt; 5 μM) (Fig. 2b,c), while the PEP concentration is linearly decreasing and the pyruvate concentration linearly increasing with the same rate (Fig. 2d,e). Thereby, the linear increase of pyruvate corresponds stoichiometrically to the ATP consumption and thus directly allows to calculate the ATP hydrolysis rate of the BiP NBD (Fig. 2e). To distinguish this experiment from equilibrium experiments, we refer to this setup with ATP regeneration as the “in cyclo” NMR experiment. The ATP consumption in our setup was\u0026nbsp;0.087\u0026nbsp;± 0.003\u0026nbsp;mM\u0026nbsp;min\u003csup\u003e-1\u003c/sup\u003e, in a sample with 100 μM\u0026nbsp;BiP NBD (Fig. 2f), which corresponds to a molecular hydrolysis rate of \u003cem\u003ek\u003c/em\u003e\u003csub\u003ehydr\u003c/sub\u003e =\u0026nbsp;0.95 ± 0.03 min\u003csup\u003e-1\u003c/sup\u003e. Mechanistically, this number is the sum of the ATP hydrolysis rate k\u003csub\u003ecat\u003c/sub\u003e and the ADP release k\u003csub\u003eoff\u003c/sub\u003e(ADP).\u0026nbsp;The inverse of this rate,\u0026nbsp;t\u0026nbsp;= \u003cem\u003ek\u003c/em\u003e\u003csub\u003ehydr\u003c/sub\u003e\u003csup\u003e-1\u003c/sup\u003e =\u0026nbsp;63 ± 2 s\u0026nbsp;is the length of the functional cycle of BiP NBD.\u003c/p\u003e\n\u003cp\u003eNotably, the hydrolysis rate determined in the in cyclo experiment matches classical experiments. A typical assay is the\u0026nbsp;NADH-coupled ATPase assay\u003csup\u003e53\u003c/sup\u003e. This assay exploits the enzymatic activity of lactate dehydrogenase to turn pyruvate into lactate by coupled NADH oxidation, which can be monitored by photospectrometry (A\u003csub\u003e340\u003c/sub\u003e) to measure the ATP consumption. We determined the rate with this assay and found perfect correspondence within error under the steady state conditions of our in cyclo NMR experiment (\u003cem\u003ek\u003c/em\u003e\u003csub\u003ehydr\u003c/sub\u003e =\u0026nbsp;0.92 ± 0.11 min\u003csup\u003e-1\u003c/sup\u003e) (Fig. 2f). Our assay thus faithfully reproduces established properties of the cycle, while simultaneously allowing atomic resolution observations. Our experimental setup shows an excellent match in terms of its consumption rate with conventional ATP hydrolysis assay. The crucial advantage is however that it now allows the simultaneous observation of conformational states at atomic resolution.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDirect observation of the ATP-bound state\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eIn a next step, we wanted to exploit these properties to get atomic level insights. 2D [\u003csup\u003e13\u003c/sup\u003eC,\u003csup\u003e1\u003c/sup\u003eH]- methyl-TROSY spectra recorded in cyclo with active ATP regeneration show that the resonances split into\u0026nbsp;two distinct sets of NMR signals. The relative amounts of the two signals is very similar for all residues and these signals thus correspond to two conformational states of the BiP NBD under the steady state conditions in cyclo. One of the two states matches perfectly with the NMR signals in 5 mM ADP-Pi in equilibrium experiment, unambiguously identifying this resonance set as the ADP-Pi-bound state (Supplementary Fig. 5a). The second set of NMR signals did neither match apo\u0026nbsp;BiP NBD, ADP-Pi\u0026nbsp;BiP NBD nor ADP\u0026nbsp;BiP NBD\u0026nbsp;(Fig. 3a,b and Supplementary Fig. 5b,c). It was detected only in presence of the ATP regeneration system or in the early phase of non-equilibrium experiments with pure ATP added (Supplementary Fig. 5b,c). This set of NMR signals therefore corresponds to the ATP-bound state. We term the BiP NBD ADP-Pi-bound state the D state and BiP NBD ATP-bound state T state.\u003c/p\u003e\n\u003cp\u003eSince we did not yet have sequence-specific resonance assignment for the ATP-bound state, we established them by direct transfer from the ADP-Pi-bound state for all signals in unambiguous spectral regions (Fig. 3a,b) and further established and confirmed assignment by the same 13 single point mutants that had previously been used for the assignment of the ADP-Pi-bound state. In total, 74 unambiguous assignments for the ATP-bound state were established (Fig. 3a and Supplementary Fig. 6).\u003c/p\u003e\n\u003cp\u003eThese assignments now give us valuable information about the ATP-bound state. On the one hand, we note that for every single residue in the entire BiP NBD, we observe a distinct signal for the ADP-Pi-bound state and the ATP-bound state. This leads to the conclusion that the structural rearrangements of the BiP NBD to adapt between the ATP and ADP-Pi molecules involve the entire BiP NBD. Second, we can interpret the chemical shift differences between the two states in a structure-specific manner. The chemical shift differences were largest for residues located in the vicinity of the bound nucleotide, as expected (Fig. 3c and Supplementary Fig. 7a). Additional large chemical shift differences were however also observed in the lobe IA. These reflect the rotation of the lobe IA resulting from ATP binding, which in the full-length protein result in docking of the SBD\u003csup\u003e12\u003c/sup\u003e.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe availability of assignments of the ATP-bound state allowed us also to assess the effects of slow-hydrolysable ATP analogs AMPPNP (adenylyl imidodiphosphate), AMPPCP (adenylyl methylenediphosphate) and ATP-γ-S (adenosine 5’-(gamma-thiotriphosphate)). These analogs are frequently used to mimic the ATP-bound state\u003csup\u003e54\u003c/sup\u003e. The comparison with the 2D [\u003csup\u003e13\u003c/sup\u003eC,\u003csup\u003e1\u003c/sup\u003eH]-methyl-TROSY spectra fingerprints for the three commons ATP analogs shows large chemical shift deviations for the entire BiP NBD (Supplementary Fig. 7b). The shift differences for AMPPNP and AMPPCP shows that these two analogs shift the BiP NBD conformation in a state that is more similar to the ADP-Pi-bound state than ATP-bound state, i.e. their NMR fingerprints are closer to the one of the ADP-Pi than ATP-bound state (Supplementary Fig. 7b). The ATP-γ-S fingerprint spectrum features three NMR signals per residue, two major states that resemble the ADP-Pi-bound state, and one minor state that is closer to the ATP-bound state but does not overlap with it (Supplementary Fig. 7b). Therefore, ATP-γ-S leads to the formation of an heterogenous mix of conformations that do not represent the ATP-bound state. This fully explains why neither of these three analogs induces the expected Hsp70 interdomain conformational change that is triggered by ATP binding as it has been reported in the literature\u003csup\u003e44,55,56\u003c/sup\u003e. As a consequence, our in cyclo experiment is the only setup that enables studies of the native ATP-bound state at atomic resolution.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCombined measurement of the functional cycle kinetic parameters\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWith the assignments of the two functional states at hand, we could next determine the complete kinetic parameters of the functional cycle. The NMR signal intensities of each of the two states is proportional to their relative population and thus to the kinetic rate constants that connect the two states. We integrated 51 non-overlapping, independent methyl groups to quantify the population ratio \u003cem\u003ep\u003c/em\u003e\u003csub\u003eD\u003c/sub\u003e/\u003cem\u003ep\u003c/em\u003e\u003csub\u003eT\u003c/sub\u003e. Along the entire protein, this ratio showed very little variation, clearly establishing that BiP NBD completely splits into two independent states (Fig. 3d). The relative populations are T: 21 ± 4% and D: 79 ± 4% (Fig. 3d,e). Because the length of a complete cycle is 63 ± 2 s, these population levels correspond to mean-lifetimes\u0026nbsp;t\u003csub\u003eT\u003c/sub\u003e = 13.4 ± 2 s for the ATP-bound T state and\u0026nbsp;t\u003csub\u003eD\u003c/sub\u003e = 49.7 ± 3 s for the ADP-Pi-bound D state (Fig. 3f). The inverse of these life times thus correspond to the catalytic ATP hydrolysis rate k\u003csub\u003ecat\u003c/sub\u003e = \u0026nbsp;t\u003csub\u003eT\u003c/sub\u003e\u003csup\u003e-1\u003c/sup\u003e = 0.075 s\u003csup\u003e-1\u003c/sup\u003e and the ADP release rate k\u003csub\u003eoff\u003c/sub\u003e\u0026nbsp; = \u0026nbsp;t\u003csub\u003eD\u003c/sub\u003e\u003csup\u003e-1\u003c/sup\u003e = 0.02 s\u003csup\u003e-1\u003c/sup\u003e during the functional cycle of NBD.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eThe ATP lifetime in cyclo is identical to classical single turn-over experiments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eConventional approaches do not allow the determination of the ATP hydrolysis rate \u003cem\u003ek\u003c/em\u003e\u003csub\u003ecat\u003c/sub\u003e from a steady-state experiment, but require separate single turn-over experiments. We wanted to benchmark the ATP lifetime obtained in cyclo by comparison with standard single-turn over experiments\u003csup\u003e44,46\u003c/sup\u003e. The single-turn over experiments were performed according to the standard protocol adapted from Theyssen et al.\u003csup\u003e44\u003c/sup\u003e. First, the NBD ATP complex was formed by incubation of an excess of ATP with BiP NBD at 4°C. At this temperature, it is generally assumed that the hydrolysis rate can be neglected. Next, the complex was separated from unbound nucleotide by gel filtration columns and then incubated at 37°C for variable time, until the reaction was stopped by addition of HCl. From the resulting samples, the [ATP]/[ADP] ratio was determined by anion exchange chromatography\u003csup\u003e57\u003c/sup\u003e. The data were fitted to a monoexponential, which corresponds to the ATP half-life time (Fig. 4a). The single turn-over experiments showed an excellent agreement with the measurements in cyclo (t\u003csub\u003eT\u003c/sub\u003e = 14.3 ± 2 s (single turn-over) vs\u0026nbsp;t\u003csub\u003eT\u003c/sub\u003e = 13.4 ± 2 s (in cyclo)) (Fig 4b). The in cyclo experiment thus faithfully reports the ATP lifetime of the system in a steady state experiment, while simultaneously also giving atomic level structural information.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eADP-Pi release is the limiting step of the BiP NBD functional cycle\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe next wanted to benchmark also the second kinetic rate obtained with the in cyclo experiment by classical experiments. The standard assay in the Hsp70 field to measure ADP lifetimes is the ADP displacement assay\u003csup\u003e44\u003c/sup\u003e. This assay relies on a fluorescently labelled ADP derivate, N8-(4-N'-methylan-thraniloylaminobutyl)-8-aminoadenosine 5'-diphosphate (MABA-ADP), that shows a substantial increase of fluorescence by 140% upon binding to nucleotide-free Hsp70\u003csup\u003e44\u003c/sup\u003e. With this reagent, nucleotide release is determined by real-time measurements of the fluorescence signal of Hsp70 with bound MABA-ADP in the presence of an excess of non-fluorescent ADP. The MABA-ADP unbinds over time, leading to a decrease in fluorescence intensity, because the non-fluorescent ADP prevents re-binding. Fitting the data with a mono-exponential gives the ADP half-life time. The MABA-ADP release times and the ADP release in the functional cycle should perfectly match and this is what is generally assumed in the literature when interpreting the MABA-ADP results. Direct measurements of ADP release during the functional cycle have so far not been accessible. Strikingly, our measurements show a large and highly significant deviation between the two experiments (t\u003csub\u003eD\u003c/sub\u003e = 13.6 ± 2 s (MABA-ADP) vs\u0026nbsp;t\u003csub\u003eD\u003c/sub\u003e = 49.7 ± 3 s (in cyclo)) (Fig 5a,b).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eTo resolve this conundrum, we realized that the functional state encountered in the functional cycle is the ADP-Pi-bound state, while the MABA-ADP experiment is phosphate-free and its lifetime thus corresponds to the ADP-bound state. It is well established that there is a difference in lifetime between the two cases and the presence of Pi in the buffer increases the ADP affinity and lifetime\u003csup\u003e58,59\u003c/sup\u003e. We therefore measured the MABA-ADP release at Pi concentrations from 0.1 mM to 30 mM, leading to an increase of the ADP lifetime from\u0026nbsp;t\u003csub\u003eD\u003c/sub\u003e = 13.6 ± 2 s in the absence of Pi to\u0026nbsp;t\u003csub\u003eD\u003c/sub\u003e = 153.2 ± 5 s in the presence of 30 mM Pi (Fig. 5a,b). Thereby, the effect of Pi concentration corresponds to an IC\u003csub\u003e50\u003c/sub\u003e of 5.9 ± 0.2 mM. To provide a structural rationale for this observation, we compared the ADP-bound state and the ADP-Pi-bound states in cyclo of BiP NBD using the NMR chemical shifts of our 84 atomic probes. We identify large chemical shift differences between the two states, which are localized in the nucleotide binding pocket and in the vicinity of the phosphate binding site (lobe IIA)\u0026nbsp;(Supplementary Fig. 8a,b).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eBecause the ADP lifetime in equilibrium experiments is strongly correlated to the bulk phosphate concentration, we wanted to assess the presence of the same effect on the functional cycle. We thus performed the in cyclo experiment at different phosphate concentration in the range from 0.1 mM to 30 mM (Supplementary Fig. 8c,d). Strikingly, the kinetic parameters of the functional cycle, and in particular, the ADP lifetime were completely inert to the bulk Pi concentration (Fig. 5b). This finding leads us to the conclusion that the sole determinant of the long-lived ADP-Pi-bound state during the functional cycle is the phosphate generated upon ATP hydrolysis in the BiP NBD nucleotide binding pocket. This phosphate remains inside the nucleotide binding pocket, “gluing” ADP into the BiP NBD and leaving the pocket only concomitantly. Rebinding of phosphate molecules from the bulk plays no role for the functional cycle of the BiP NBD and the cycle is thus robust to fluctuations of the phosphate concentration. The in cyclo experiment thus overcomes the limitations of static experiments.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCa\u003csup\u003e2+\u0026nbsp;\u003c/sup\u003e\u003c/strong\u003e\u003cstrong\u003edoes not affect the functional cycle\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCa\u003csup\u003e2+\u0026nbsp;\u003c/sup\u003eis a key ER stress marker and plays a fundamental role in regulating the activity of multiple ER proteins\u003csup\u003e60\u003c/sup\u003e. While the role of magnesium ions for the catalytic activity of Hsp70 is well established\u003csup\u003e50\u003c/sup\u003e, the potential role played by calcium on the Hsp70 functional cycle is not yet well understood\u003csup\u003e61\u003c/sup\u003e. This question is of special interest for the BiP as the ER can show large variations in calcium, during protein folding homeostasis and stress, with concentrations ranging from 0.1 mM under homeostatic conditions up to 0.8 mM in stress conditions\u003csup\u003e62,63\u003c/sup\u003e. It has been proposed that the calcium concentration might decrease the ATP hydrolysis rate of BiP by 2-fold at physiological Ca\u003csup\u003e2+\u003c/sup\u003e levels compared to no calcium\u003csup\u003e50,64\u003c/sup\u003e and increase the ADP-bound lifetime in a concentration-dependent manner (4-folds at physiological concentration\u003csup\u003e61\u003c/sup\u003e). Published crystal structures of the nucleotide bound BiP NBD in presence of calcium show that it binds in the nucleotide binding site\u003csup\u003e61\u003c/sup\u003e, in which the ADP-bound state calcium contacts both phosphate groups (a\u0026nbsp;and\u0026nbsp;b) while magnesium only contact the\u0026nbsp;b\u0026nbsp;phosphate (Supplementary Fig. 9a). This might suggest a mechanism for the variation of the kinetic parameters, if Ca\u003csup\u003e2+\u003c/sup\u003e replaces Mg\u003csup\u003e2+\u003c/sup\u003e ions. Importantly, the Ca\u003csup\u003e2+\u003c/sup\u003e concentration is always lower than the Mg\u003csup\u003e2+\u003c/sup\u003e concentration also in the ER\u003csup\u003e65,66\u003c/sup\u003e. Accordingly, to test the effect of calcium on the kinetic parameters of the BiP NBD in the following experiments, we keep the magnesium concentration at 10 mM and vary the calcium concentration as indicated for each experiment.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eFirst, we probed the presence of calcium binding to the ADP-bound state by NMR equilibrium experiments. Upon addition of 3.3 mM Ca\u003csup\u003e2+\u003c/sup\u003e to ADP-bound BiP NBD in presence of 10 mM Mg\u003csup\u003e2+\u003c/sup\u003e ([Mg\u003csup\u003e2+\u003c/sup\u003e]/[Ca\u003csup\u003e2+\u003c/sup\u003e]=3) large CSPs were observed, clearly confirming the Ca\u003csup\u003e2+\u003c/sup\u003e binding (Supplementary Fig. 9b). \u0026nbsp;Mapping of these CSPs on the structure showed changes consistent with a calcium binding site in lobes IA and IIA as expected from the published crystal structure of NBD bound to ADP-Ca\u003csup\u003e2+\u003c/sup\u003e (PDB 6ZYH)\u003csup\u003e61\u003c/sup\u003e\u0026nbsp; (Supplementary Fig. 9c). \u0026nbsp;Next, we used MABA-ADP release to measure the ADP mean life as a function of calcium concentration at a fixed concentration of magnesium. We observed a strong dependence of the ADP lifetime on the calcium concentration with an IC\u003csub\u003e50\u003c/sub\u003e of 212+7 μM, in excellent agreement with the literature\u003csup\u003e61\u003c/sup\u003e (Fig. 6a,b and Supplementary Fig. 10a). Since the BiP NBD nucleotide binding pocket is always occupied by an ADP and a phosphate moiety during the functional cycle, we wanted to explore the effect of calcium on the ADP-Pi-bound state and thus repeated the experiment in the presence of 0.5 mM Pi. Strikingly, this experiment showed that the lifetime of bound ADP-Pi is completely independent of the calcium concentration (Fig. 6a,b and Supplementary Fig. 10b).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe data thus readily suggest that the presence of calcium should also have no effect on the functional cycle. And indeed, in the presence of 3.3 mM calcium in the in cyclo setup, the ATP consumption is 0.090\u0026nbsp;± 0.003\u0026nbsp;mM min\u003csup\u003e-1\u003c/sup\u003e, which corresponds to a molecular hydrolysis rate of \u003cem\u003ek\u003c/em\u003e\u003csub\u003ehyd\u003c/sub\u003e = 0.99\u0026nbsp;± 0.03\u0026nbsp;min\u003csup\u003e-1\u003c/sup\u003e (Fig. 5c).\u0026nbsp;This is essentially equivalent to the\u0026nbsp;molecular hydrolysis rate in the absence of calcium \u003cem\u003ek\u003c/em\u003e\u003csub\u003ehyd\u003c/sub\u003e =\u0026nbsp;0.95\u0026nbsp;± 0.03 min\u003csup\u003e-1\u003c/sup\u003e. Also the relative populations of the two states, with\u0026nbsp;T: 22 ± 4% and D: 78 ± 4% were the same as in the absence of calcium (T: 21 ± 4% and D: 79 ± 4%) (Fig. 5d,e and Supplementary Fig. 11a). Together, this determined the lifetimes of bound nucleotides to\u0026nbsp;t\u003csub\u003eT\u003c/sub\u003e = 13.3 ± 3 s and\u0026nbsp;t\u003csub\u003eD\u003c/sub\u003e = 47.4 ± 3 s, which are similar to the lifetimes in the absence of calcium (t\u003csub\u003eT\u003c/sub\u003e = 13.4 ± 2 s and and\u0026nbsp;t\u003csub\u003eD\u003c/sub\u003e = 49.7 ± 3 s) (Fig. 5f-h). These data thus completely exclude any effect of calcium on the native functional cycle, in the cellular concentration range as well as up to several mM.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eNotably, the in cyclo experiment now allows us also to use our structural probes to probe the effect of calcium binding during the functional cycle. The 2D NMR experiments showed no significant CSP, neither for the ATP nor the ADP-Pi-bound state, evidencing an absence in structural changes, in full agreement with the finding that the kinetics are unchanged. \u0026nbsp;(Supplementary Fig. 11c). The data thus clearly establish that calcium ions do bind to the ADP-bound state of the BiP NBD, but not to the ADP-Pi-bound state, and consequently, since the ADP-bound state is not part of the functional cycle of the BiP NBD, physiological variations of calcium have no effect on the BiP NBD functional cycle.\u0026nbsp;\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eThe experimental approach presented in this work has resolved the kinetics and states encountered during the functional cycle of the Hsp70 BiP NBD at atomic resolution (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003ea,b). The BiP NBD passes cyclically through a total of three distinct states: the apo state, an ATP-bound state and an ADP-Pi-bound state. Starting from the apo state, the BiP NBD binds an ATP molecule to reach the ATP-bound state. From this state, the BiP NBD hydrolyzes ATP to reach the ADP-Pi-bound state. ADP-Pi release brings the BiP NBD into the apo form, from where a new cycle can start. Importantly, the ATP hydrolysis rate determined in cyclo matches the value determined by single turn-over experiments, while the ADP release rate does not correspond to classical experiments, since the phosphate generated upon ATP hydrolysis is \u0026ldquo;gluing\u0026rdquo; the ADP into the nucleotide binding pocket, thus dramatically transforming the second step of the functional cycle. The second step of ADP-Pi release is independent of the bulk phosphate concentration, the NBD-ADP-Pi complex decays in a monomolecular reaction.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eFormation of the ADP-Pi post-hydrolysis state has major consequences for the regulation of the functional cycle in response to environmental change. The phosphate concentration in the cytoplasm of the eukaryotic call is known to vary between at least 1 and 10 mM\u003csup\u003e\u003cspan citationid=\"CR67\" class=\"CitationRef\"\u003e67\u003c/span\u003e\u003c/sup\u003e, and can be expected to have similar or even large fluctuations in the ER. Our experimental setup has shown that the functional cycle is inert to such fluctuations, whereas previous interpretations based on equilibrium experiments that came to opposite conclusions need to be revised. This formation of the ADP-Pi post-hydrolysis state also excludes any effect of Ca\u003csup\u003e2+\u003c/sup\u003e in calcium concentrations on the BiP NBD functional cycle. While the activity of several other ER chaperones is strongly affected by variations in calcium concentrations (Grp94\u003csup\u003e68\u003c/sup\u003e, Calreticulin\u003csup\u003e\u003cspan citationid=\"CR69\" class=\"CitationRef\"\u003e69\u003c/span\u003e\u003c/sup\u003e and Protein disulfide isomerase\u003csup\u003e\u003cspan citationid=\"CR70\" class=\"CitationRef\"\u003e70\u003c/span\u003e,\u003cspan citationid=\"CR71\" class=\"CitationRef\"\u003e71\u003c/span\u003e\u003c/sup\u003e), this is not the case for the BiP NBD. BiP thus provides a chaperone buffering capacity disconnected from the ER stress level.\u003c/p\u003e \u003cp\u003eOur work has focused on the BiP NBD as a benchmark for subsequent studies also of full-length Hsp70. It will be exciting to extend our studies in a next step to full-length Hsp70 proteins, such as BiP, where we expect to understand the interplay between the substrate binding domain and the NBD. Additional interesting implications will be to understand how co-chaperones modulate the functional cycle. Nucleotide exchange factors\u003csup\u003e\u003cspan citationid=\"CR72\" class=\"CitationRef\"\u003e72\u003c/span\u003e,\u003cspan citationid=\"CR73\" class=\"CitationRef\"\u003e73\u003c/span\u003e\u003c/sup\u003e and nucleotide exchange inhibitors\u003csup\u003e\u003cspan citationid=\"CR74\" class=\"CitationRef\"\u003e74\u003c/span\u003e,\u003cspan citationid=\"CR75\" class=\"CitationRef\"\u003e75\u003c/span\u003e\u003c/sup\u003e accelerate and inhibit ADP release, respectively, and it will be interesting to study their effect in the context of a full-length Hsp70. The mode of action of these co-chaperones has been studied mostly by equilibrium experiments, whereas our experimental setup will be key to provide high resolution mapping of the interaction interface while simultaneously resolving the precise kinetics of the nucleotide conversion and release. Similarly, the methods described here will allow us to probe the effect of J-domain proteins on the ATP hydrolysis with unprecedented precision. On the longer run, it will be interesting to see how the diverse cellular functionalities of Hsp70 emerge from coupling ATP hydrolysis to interaction dynamics of the substrate binding domain.\u003c/p\u003e"},{"header":"Methods","content":"\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eProtein expression and purification\u003c/h2\u003e \u003cdiv id=\"Sec12\" class=\"Section3\"\u003e \u003ch2\u003eCloning, expression and purification of human BiP NBD\u003c/h2\u003e \u003cp\u003eHuman BiP NBD lacking its ER signal sequence (residues 19\u0026ndash;406) with an N-terminal His\u003csub\u003e6\u003c/sub\u003e-Tag including a TEV cleavage sequence was generated by introducing a mutation (G407 to stop) using the QuikChange II mutagenesis protocol (Stratagene) into the Human BiP (residues 19\u0026ndash;654) gene synthetized by genscript. The gene was inserted through NcoI and XhoI into a pET28a expression vector (Novagen).\u003c/p\u003e \u003cp\u003eBL21-(DE3)-Lemo cells (New England Biolabs) were transformed and grown at 37\u0026deg;C in M9 minimal medium prepared with 99.85% D2O (Sigma-Aldrich) containing \u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003eNH\u003csub\u003e4\u003c/sub\u003eCl (1 g/liter; Sigma-Aldrich) and D-glucose-d7 (2 g/L; Sigma-Aldrich). BiP NBD expression was induced at an OD\u003csub\u003e600\u003c/sub\u003e of 0.6 by adding 1 mM isopropyl-β-d-thiogalactopyranoside (IPTG) and expression was continued at 25\u0026deg;C for 12 hours. Cells were harvested by centrifugation at 5000g for 20 min. The pellet was resuspended in 20 ml of lysis buffer per liter of culture (25 mM Hepes, pH 7.5, 150 mM NaCl, 10 mM MgCl\u003csub\u003e2\u003c/sub\u003e, 0.02 mg/mL ribonuclease, 0.01 mg/mL deoxyribonuclease and phenylmethylsulfonyl fluoride PMSF (0.2 mg/mL). Cell lysis was performed using a microfluidizer (Microfluidics) for three cycles at 4\u0026deg;C. The soluble bacterial lysate was separated from cell debris and other components by centrifugation at 14000 g for 60 min and loaded onto a Ni-NTA column (Qiagen) equilibrated in buffer A (25 mM Hepes, pH 7.5, 150 mM NaCl, 10 mM MgCl\u003csub\u003e2\u003c/sub\u003e). BiP eluted at 500 mM imidazole concentration and was dialyzed overnight against buffer (25 mM Hepes, pH 7.5, 300 mM NaCl, 10 mM MgCl\u003csub\u003e2\u003c/sub\u003e). BiP NBD was denatured with 6 M urea and loaded onto a Ni-NTA column (Cytiva) equilibrated in buffer A\u0026thinsp;+\u0026thinsp;8 M urea. BiP NBD eluted at 500 mM imidazole concentration in buffer containing 8 M urea. BiP refolding was achieved by dialysis overnight against buffer (25 mM Hepes, pH 7.5, 300 mM NaCl, 10 mM MgCl\u003csub\u003e2\u003c/sub\u003e). After refolding, the His-tag was cleaved by incubation with 1 mg of TEV per 50 mg of BiP in cleavage buffer (25 mM Hepes, pH 7.5, 300 mM NaCl, 10 mM MgCl\u003csub\u003e2\u003c/sub\u003e, DTT 1 mM and EDTA 0.5 mM) overnight at 4\u0026deg;C. BiP was separated from TEV and uncleaved BiP via a reverse Ni-NTA column (Cytiva) equilibrated in buffer A. BiP was then applied to an anion-exchange column (Cytiva) equilibrated in the buffer QA (25 mM Tris, pH 8.5) and eluted with 250 mM of KCl. Finally, BiP NBD was concentrated by ultrafiltration and subjected to size exclusion chromatography (Superdex-200 16/600 PG, Cytiva) to further purify the proteins and adjust to its final buffer (25 mM Hepes, pH 7.5, 150 mM KCl and 20 mM MgCl\u003csub\u003e2\u003c/sub\u003e). Afterward, BiP was concentrated by ultrafiltration and stored at \u0026minus;\u0026thinsp;20\u0026deg;C until use. Final yield of purified protein was 15\u0026ndash;20 mg for wild-type BiP NBD per liter of deuterated M9 minimal medium. The BiP NBD not refolded protein was purified according to the same protocol without the unfolding/refolding steps. The BiP NBD expressed in LB (Luria broth) medium was purified following the same purification protocol.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eMethyl labeling of human BiP NBD\u003c/h2\u003e \u003cp\u003eMethyl-labeled BiP proteins were obtained by growing the expression cells in M9 minimal media prepared with 99.85% D2O (Sigma-Aldrich) containing \u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003eNH\u003csub\u003e4\u003c/sub\u003eCl (1 g/L; Sigma-Aldrich) and D-glucose-d\u003csub\u003e7\u003c/sub\u003e (2 g/L; Sigma-Aldrich). When the optical density (OD) at 600 nm reached 0.8, a solution containing the labeled precursors was added. For [U-\u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003eH, \u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003eN, \u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003eC], Met-[\u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003eCH\u003csub\u003e3\u003c/sub\u003e]\u003csup\u003eε\u003c/sup\u003e, Val-[\u003csup\u003e13\u003c/sup\u003eCH\u003csub\u003e3/\u003c/sub\u003e\u003csup\u003e13\u003c/sup\u003eCH\u003csub\u003e3\u003c/sub\u003e]\u003csup\u003eγ1/γ2\u003c/sup\u003e, Ile-[\u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003eCH\u003csub\u003e3\u003c/sub\u003e]\u003csup\u003eδ\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e: 100 mg of 2-Keto-3-(methyl-13C)-butyric-4-\u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003eC acid sodium salt (Sigma-Aldrich), 30 mg of L-leucine-d\u003csub\u003e10\u003c/sub\u003e (Sigma-Aldrich), 80 mg of α-ketobutyric acid methyl \u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003eC (99%) 3,3-D2 (98%) (Sigma-Aldrich) and 100 mg of [\u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003eC]\u003csup\u003eε\u003c/sup\u003e-L-methionine (Sigma-Aldrich). For [U-\u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003eH, \u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003eN, \u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003eC], Met-[\u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003eCH\u003csub\u003e3\u003c/sub\u003e]\u003csup\u003eε\u003c/sup\u003e, Val-[\u003csup\u003e13\u003c/sup\u003eCH\u003csub\u003e3\u003c/sub\u003e/\u003csup\u003e12\u003c/sup\u003eC\u003csup\u003e2\u003c/sup\u003eH\u003csub\u003e3\u003c/sub\u003e]\u003csup\u003ecγ1/cγ2\u003c/sup\u003e, Ile-[\u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003eCH\u003csub\u003e3\u003c/sub\u003e]\u003csup\u003eδ\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e: 100 mg of 2-Keto-3-(methyl-d3)-butyric acid-4-\u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003eC,3-d sodium salt (Sigma-Aldrich), 30 mg of L-leucine-d\u003csub\u003e10\u003c/sub\u003e (Sigma-Aldrich), 80 mg of α-ketobutyric acid methyl \u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003eC (99%) 3,3-D2 (98%) (Sigma-Aldrich) and 100 mg of [\u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003eC]\u003csup\u003eε\u003c/sup\u003e-L-methionine (Sigma-Aldrich). For [U-\u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003eH, \u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003eN, \u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003eC], Met-[\u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003eCH\u003csub\u003e3\u003c/sub\u003e]\u003csup\u003eε\u003c/sup\u003e, Val-[\u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003eCH\u003csub\u003e3\u003c/sub\u003e]\u003csup\u003ecγ\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e, Ile-[\u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003eCH\u003csub\u003e3\u003c/sub\u003e]\u003csup\u003eδ\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e: 240 mg of 2-hydroxy-2-[\u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003eC]methyl-3-oxo-4,4,4-tri-[\u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003eH]-butanoate (pro-S acetolactate-\u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003eC, NMR-Bio), 30 mg of L-leucine-d\u003csub\u003e10\u003c/sub\u003e, 80 mg of α-ketobutyric acid methyl \u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003eC (99%) 3,3-D2 (98%) (Sigma-Aldrich) and 100 mg of [\u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003eC]\u003csup\u003eε\u003c/sup\u003e-L-methionine (Sigma-Aldrich). One hour after the addition of the precursors, BiP expression was induced by adding 1 mM isopropyl-β-d-thiogalactopyranoside (IPTG) at 25\u0026deg;C for 12 hours.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eMutagenesis, expression and purification of the BiP assignment mutants\u003c/h2\u003e \u003cp\u003eThe QuikChange II mutagenesis protocol (Stratagene) was used to introduce the point mutations in the BiP plasmid: I33V, I76V, I145V, M148L, M153L, I190V, M196L, I199V, I207V, M263L, M332L, M339L and V400I. Polymerase chain reaction primers were obtained from Microsynth. The expression and purification of the mutant proteins was performed as described for the wild-type proteins. The final yield of purified mutants was similar to wild-type at the exception of the mutant M148L that express at very low yield and thus could not be analyzed.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eNMR spectroscopy\u003c/h2\u003e \u003cp\u003eAll NMR experiments for BiP NBD were performed in NMR buffer (25 mM Hepes, pH 7.5, 150 mM KCl and 20 mM MgCl\u003csub\u003e2\u003c/sub\u003e) at 37\u0026deg;C. The experiments were recorded on Bruker AscendII 700 MHz, Avance 800 MHz or Avance 900 MHz spectrometers running Topspin 3.0 and equipped with a cryogenically cooled triple-resonance probe. NMR data were processed with nmrPipe\u003csup\u003e\u003cspan citationid=\"CR76\" class=\"CitationRef\"\u003e76\u003c/span\u003e\u003c/sup\u003e and ccpnmr\u003csup\u003e\u003cspan citationid=\"CR77\" class=\"CitationRef\"\u003e77\u003c/span\u003e\u003c/sup\u003e. All equilibrium NMR experiments were recorded in NMR buffer at a concentration of BiP NBD 100 \u0026micro;M. Purified ADP (Sigma-Aldrich) and Pi were added to the sample at concentration of 5 mM. 1D \u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003eH NMR spectra were used to monitor the purity of the ADP and absence of contaminants.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eAssignment of BiP NBD Met[CH\u003csub\u003e3\u003c/sub\u003e]\u003csup\u003eε\u003c/sup\u003e, Val[CH\u003csub\u003e3\u003c/sub\u003e]\u003csup\u003eγ1/γ2\u003c/sup\u003e and Ile[CH\u003csub\u003e3\u003c/sub\u003e]\u003csup\u003eδ\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e methyl groups\u003c/h2\u003e \u003cp\u003eMet[CH\u003csub\u003e3\u003c/sub\u003e]\u003csup\u003eε\u003c/sup\u003e, Val[CH\u003csub\u003e3\u003c/sub\u003e]\u003csup\u003eγ1/γ2\u003c/sup\u003e and Ile[CH\u003csub\u003e3\u003c/sub\u003e]\u003csup\u003eδ\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e assignment was obtained using a structure-based approach combining mutagenesis and 3D \u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003eC, \u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003eC-resolved [\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003eH, \u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003eH]-NOESY experiments. The following point mutation were used: I33V, I76V, I145V, M148L, M153L, I190V, M196L, I199V, I207V, M263L, M332L, M339L, V400I. Each mutant sample was recorded at 37\u0026deg;C with an adjusted duration depending on their final concentration (experimental time ranging from 120 to 240 min per sample). For each mutant, spectra were recorded for the ADP-Pi-bound state (5 mM) and in cyclo experiment. Analysis and comparison of the library of mutant spectra allowed the assignment of 1/35 valines (3%), 6/25 isoleucine (24%) and 6/6 methionines (100%). The network of assigned residues was used to expand the assignment using 3D \u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003eC, \u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003eC-resolved [\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003eH, \u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003eH]-NOESY experiments recorded with sample concentrations ranging from 0.4 to 0.8 mM with a mixing time of 400 ms and an adjusted duration depending on the final concentration of each constructs (experimental time ranging from 3 to 5 days per sample). Stereospecific assignment of the valine cγ1/cγ2 for residues located in the BiP NBD has been obtained using a sample [U-\u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003eH, \u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003eN, \u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003eC], Met-[\u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003eCH\u003csub\u003e3\u003c/sub\u003e]\u003csup\u003eε\u003c/sup\u003e, Val-[\u003csup\u003e13\u003c/sup\u003eCH\u003csub\u003e3/\u003c/sub\u003e\u003csup\u003e13\u003c/sup\u003eCH\u003csub\u003e3\u003c/sub\u003e]\u003csup\u003eγ1/γ2\u003c/sup\u003e, Ile-[\u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003eCH\u003csub\u003e3\u003c/sub\u003e]\u003csup\u003eδ\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e and a 3D \u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003eC, \u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003eC-resolved [\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003eH, \u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003eH]-NOESY experiments recorded with a short mixing time of 50 ms that directly correlate the two methyl groups within one amino-acid. Analysis of the 3D \u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003eC, \u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003eC-resolved [\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003eH, \u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003eH]-NOESY cross peaks with the structure of the BiP NBD in the ADP-Pi undocked allowed to expand our mutagenesis-based assignment to assign of 32/35 valines, 22/25 isoleucine and 6/6 methionine. Among the 32 valines, 24 had both methyl Cγ1 and Cγ2 assigned and 8 one of them, resulting in a total of 84 observable NMR signals.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003eNMR in cyclo experimental setup\u003c/h2\u003e \u003cdiv id=\"Sec18\" class=\"Section3\"\u003e \u003ch2\u003eComposition of the in cyclo NMR samples\u003c/h2\u003e \u003cp\u003eAll in cyclo NMR experiments were recorded with BiP NBD samples [U-\u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003eH, \u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003eN, \u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003eC], Met-[\u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003eCH\u003csub\u003e3\u003c/sub\u003e]\u003csup\u003eε\u003c/sup\u003e, Val-[\u003csup\u003e13\u003c/sup\u003eCH\u003csub\u003e3\u003c/sub\u003e/\u003csup\u003e12\u003c/sup\u003eC\u003csup\u003e2\u003c/sup\u003eH\u003csub\u003e3\u003c/sub\u003e]\u003csup\u003ecγ1/cγ2\u003c/sup\u003e, Ile-[\u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003eCH\u003csub\u003e3\u003c/sub\u003e]\u003csup\u003eδ\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e in NMR buffer (25 mM Hepes, pH 7.5, 150 mM KCl and 10 mM MgCl\u003csub\u003e2\u003c/sub\u003e) at a concentration of BiP constructs 100 \u0026micro;M and specified concentration of the co-chaperones or client-protein. The in cyclo ATP regeneration system was composed of 5 units of pyruvate kinase from rabbit muscle (Sigma-Aldrich) (\u0026asymp;\u0026thinsp;200 nM) and the concentration of phosphoenolpyruvate (PEP- Sigma-Aldrich) was adjusted to maintain the system active for at least four hours ([PEP]\u0026thinsp;=\u0026thinsp;10\u0026ndash;100 mM). All the experiments were recorded at minimum as experimental triplicate.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003eRecording of the in cyclo NMR experiments\u003c/h2\u003e \u003cp\u003eThe in cyclo experiment was initiated by the addition of 5 mM ATP (Sigma-Aldrich) and allowed to equilibrate at a temperature of 37\u0026deg;C for 2 minutes in the NMR spectrometer. After the equilibration time 2D [\u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003eC,\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003eH]-HMQC spectra were recorded in interleaved mode with 1D \u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003eH NMR spectra. The 1D \u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003eH NMR spectra were used to monitor the pyruvate increase and the constant concentration of ATP using the NMR signal of 0.5 mM sodium 2,2-dimethyl- 2-silapentane-5-sulfonate-d6 (d, 98%) (DSS; Cambridge Isotope Laboratories Inc.) as an internal reference.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec20\" class=\"Section2\"\u003e \u003ch2\u003eMABA-ADP release assay\u003c/h2\u003e \u003cp\u003eNucleotide release and binding measurements were performed using the fluorescent nucleotide analogues MABA-ADP (8-[(4-Amino)butyl]-amino-ADP-MAN; Jena-bioscience) carrying a MANT moiety whose fluorescence increases upon binding to Hsp70s\u003csup\u003e\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e,\u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e\u003c/sup\u003e. To measure nucleotide release, 1.8 \u0026micro;M fluorescent nucleotide analogues were incubated with 1.8 \u0026micro;M proteins in NMR buffer for at least 45 minutes at 25\u0026deg;C to allow for complex formation (Solution A). Solution B is NMR buffer with 7.5 mM ADP. The final concentrations after mixing were 1.44 \u0026micro;M for each protein, 1.44 \u0026micro;M fluorescent nucleotides, and 1.5 mM ADP. The measurements were started by adding 20 \u0026micro;L of solution B to 80 \u0026micro;l of solution A in a 96-well plate (Corning) and fluorescence (excitation 360 nm, emission 420 nm) was detected over time with a fluorescence spectrometer (Tecan Plate Reader) at 37\u0026deg;C. The dead time between sample mixing and signal recording was \u0026sim; 2 s. All solutions contained 25 mM HEPES, pH 7.5, 150 mM KCl, 10 mM MgCl\u003csub\u003e2\u003c/sub\u003e at 37\u0026deg;C. The solutions A contained CaCl\u003csub\u003e2\u003c/sub\u003e and phosphate where indicated (final concentrations are stated in figure legends). The dissociation rate constants (\u003cem\u003ek\u003c/em\u003e\u003csub\u003eoff\u003c/sub\u003e) were determined by fitting the data to a one phase exponential function using a python script.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec21\" class=\"Section2\"\u003e \u003ch2\u003eATP analog preparation\u003c/h2\u003e \u003cp\u003eAMPPNP (adenylyl imidodiphosphate), AMPPCP (adenylyl methylenediphosphate) and ATP-γ-S (adenosine 5\u0026rsquo;-(gamma-thiotriphosphate)) (Jena-bioscience) were dissolved in the HKM reaction buffer HKM (25 mM HEPES pH 7.5, 150 mM KCl and 10 mM MgCl\u003csub\u003e2\u003c/sub\u003e) at a concentration of 50 mM directly before to be mixed with the BiP NBD sample. Preparation of the ATP analogs just before usage allows to circumvent the known instability of these analog that undergo slow hydrolysis in solution.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec22\" class=\"Section2\"\u003e \u003ch2\u003eNADH ATP assay\u003c/h2\u003e \u003cp\u003eBiP NBD was diluted to a final concentration of 1 \u0026micro;M in the HKM reaction buffer HKM (25 mM HEPES pH 7.5, 150 mM KCl and 10 mM MgCl\u003csub\u003e2\u003c/sub\u003e). The reactions buffer also contained 10 mM Phosphoenolpyruvate (Sigma-Aldrich), 0.5 mM NADH (Sigma-Aldrich), pyruvate kinase (Sigma-Aldrich) and lactate dehydrogenase (Sigma-Aldrich) (diluted to 5 U). The reactions were started by addition of 5 mM ATP in a final volume of 160 \u0026micro;l in 96-well microplates (Corning) and NADH absorbance at 340 nm (A\u003csub\u003e340\u003c/sub\u003e) was measured over time at 37\u0026deg;C with a Tecan Plate Reader. Linear regression analysis was performed and the ATP hydrolysis activity was calculated using the molar extinction coefficient of NADH (ε\u0026thinsp;=\u0026thinsp;6220 M\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e).\u003c/p\u003e \u003cdiv id=\"Sec23\" class=\"Section3\"\u003e \u003ch2\u003eSingle turn-over assay\u003c/h2\u003e \u003cp\u003eThe ATPase activity of BiP NBD was determined under single-turnover conditions as previously described with minor modifications of the protocol\u003csup\u003e\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e\u003c/sup\u003e. The main-modification came to replace the radioactivity-based detection by a label free anion-exchange based quantification\u003csup\u003e\u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e\u003c/sup\u003e. The BiP NBD-ATP complex was formed by mixing BiP NBD with 10 mM ATP in formation buffer (25 mM HEPES pH 7.5, 50 mM KCl, 10 mM MgCl\u003csub\u003e2\u003c/sub\u003e) and incubating the mixture for 5 min on ice. The BiP NBD was separated from unbound ATP at 4\u0026deg;C by gel filtration on NICK columns (Cytiva) equilibrated in reaction buffer (25 mM HEPES pH 7.5, 150 mM KCl, 10 mM MgCl\u003csub\u003e2\u003c/sub\u003e). Fractions containing the BiP NBD ATP complexes were pooled and snap frozen in liquid nitrogen and kept at -80\u0026deg;C. For the ATPase activity determination, BiP NBD ATP complex solutions were thawed and the reaction started by mixing with the reaction buffer prewarmed at 37\u0026deg;C (25 mM HEPES pH 7.5, 150 mM KCl, 10 mM MgCl\u003csub\u003e2\u003c/sub\u003e). At given time points, samples were withdrawn from the reaction, mixed with an equivalent volume of HCl and snap frozen in liquid nitrogen and kept at -80\u0026deg;C. The relative ATP/ADP ratio was determined using an anion chromatography-based assay.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec24\" class=\"Section2\"\u003e \u003ch2\u003eSEC-MALS\u003c/h2\u003e \u003cp\u003eSEC-MALS measurements of BiP NBD were performed at 25\u0026deg;C in NMR buffer (25 mM Hepes, pH 7.5, 150 mM KCl and 20 mM MgCl\u003csub\u003e2\u003c/sub\u003e) using a SEC-3 HPLC column (300 \u0026Aring; pore size; Agilent Technologies) on an Agilent 1260 HPLC. Elution was monitored by multiangle light scattering (Heleos II 8+; Wyatt Technology), differential refractive index (Optilab T-rEX; Wyatt Technology) and absorbance at 280 and 254 nm (1260 UV; Agilent Technologies). All the system parameters were calibrated using an injection of 2 mg/ml BSA solution (ThermoPierce) and standard protocols in ASTRA 6. Molar mass (MM) and mass distributions were calculated using the ASTRA 6 software (Wyatt Technology).\u003c/p\u003e \u003cdiv id=\"Sec25\" class=\"Section3\"\u003e \u003ch2\u003eData availability\u003c/h2\u003e \u003cp\u003eAll data needed to evaluate the conclusions of the paper are presented in the manuscript. The 84 state-specific methyl resonance assignments of BiP have been submitted to the Biological Magnetic Resonance Data Bank under accession code 51751. Additional data related to this paper will be provided by the authors upon request.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was funded by the Swiss National Science Foundation\u0026nbsp;(grants 185388 and 207755 to S. H.). The authors thank Anna Leder for critical reading of the manuscript and\u0026nbsp;Tim Sharpe and Thomas Müntener for technical support.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eG. M. conducted the experiments. G. M. and S. H. designed the study, analyzed the data, discussed the results, and wrote the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eWentink AS et al (2020) Molecular dissection of amyloid disaggregation by human HSP70. Nature 587:483\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSchneider MM et al (2021) The Hsc70 disaggregation machinery removes monomer units directly from α-synuclein fibril ends. 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Protein Sci 21:1489\u0026ndash;1502\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"nature-portfolio","isNatureJournal":true,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"","title":"Nature Portfolio","twitterHandle":"","acdcEnabled":false,"dfaEnabled":false,"editorialSystem":"ejp","reportingPortfolio":"","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Molecular chaperones, NMR spectroscopy, allostery, Hsp70, real-time NMR, molecular machines, endoplasmic reticulum, ATPase","lastPublishedDoi":"10.21203/rs.3.rs-4017836/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4017836/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eThe 70 kDa heat shock protein (Hsp70) family of molecular chaperones is crucial for protein biogenesis and homeostasis in all kingdoms of life. Hsp70 activity is driven by ATP hydrolysis in the nucleotide binding domain (NBD).\u003c/strong\u003e \u003cstrong\u003eHere, we report an experimental setup to resolve the functional cycle of Hsp70 in unprecedented spatial and temporal resolution. The method combines high-resolution NMR spectroscopy with embedded kinetic measurements to simultaneously resolve kinetic rates and structural information of the individual states of an Hsp70 functional cycle. We benchmark the method on the example of the NBD of the human Hsp70 chaperone BiP. Precision measurements connect the ATP hydrolysis rate (\u003c/strong\u003e\u003cem\u003e\u003cstrong\u003ek\u003c/strong\u003e\u003c/em\u003e\u003csub\u003e\u003cstrong\u003ecat\u003c/strong\u003e\u003c/sub\u003e\u003cstrong\u003e) and the ADP lifetime (\u003c/strong\u003e\u003cem\u003e\u003cstrong\u003ek\u003c/strong\u003e\u003c/em\u003e\u003csub\u003e\u003cstrong\u003eoff\u003c/strong\u003e\u003c/sub\u003e\u003cstrong\u003e) to conventional bulk experiments and thus reveal that ADP-Pi release and not ATP hydrolysis is the limiting step of the cycle. Unlike commonly thought, the phosphate generated from ATP hydrolysis locks the ADP-Pi into the NBD, and thus decouples the ADP release rate from the effect of external factors such as the bulk phosphate and calcium concentration. The method will serve as a platform for studies of the Hsp70 protein family and their co-chaperones, including full-length constructs that have key roles in biogenesis and disease.\u003c/strong\u003e\u003c/p\u003e","manuscriptTitle":"Characterization of ATP hydrolysis in the Hsp70 BiP nucleotide binding domain","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-03-07 18:57:17","doi":"10.21203/rs.3.rs-4017836/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":"171cdfde-4345-4a62-aa87-519c3ba03185","owner":[],"postedDate":"March 7th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":29176324,"name":"Biological sciences/Biochemistry/Structural biology/NMR spectroscopy/Solution-state NMR"},{"id":29176325,"name":"Biological sciences/Biophysics/Bioenergetics"}],"tags":[],"updatedAt":"2025-06-01T07:05:57+00:00","versionOfRecord":{"articleIdentity":"rs-4017836","link":"https://doi.org/10.1038/s41467-025-60343-x","journal":{"identity":"nature-communications","isVorOnly":false,"title":"Nature Communications"},"publishedOn":"2025-06-01 04:00:00","publishedOnDateReadable":"June 1st, 2025"},"versionCreatedAt":"2024-03-07 18:57:17","video":"","vorDoi":"10.1038/s41467-025-60343-x","vorDoiUrl":"https://doi.org/10.1038/s41467-025-60343-x","workflowStages":[]},"version":"v1","identity":"rs-4017836","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4017836","identity":"rs-4017836","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}