Thermodynamic basis for CFTR activity potentiation

Thermodynamic basis for CFTR activity potentiation | 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PDF Research Article Thermodynamic basis for CFTR activity potentiation Guangyu Wang This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7339733/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 05 Jan, 2026 Read the published version in Biomedicine & Pharmacotherapy → Version 1 posted You are reading this latest preprint version Abstract Trikafta modulators can correct the thermal and gating defects of the most common cystic fibrosis mutant F508del of the human cystic fibrosis transmembrane conductance regulator (hCFTR). While folding correctors VX-445 and VX-809 are sufficient to restore the Mg/ATP-dependent dimerization between the two nucleotide binding domains (NBD1 and NBD2) for channel opening, the thermodynamic basis for the activity potentiation by VX-770 in Trikafta remains unknown. Here, the thermoring structures and interdomain interactions of NBD2 were examined and compared with the counterparts of NBD1 with or without F508 in response to ligand binding. The results demonstrated that comparable thermostability between dimerized NBD1 and NBD2 was required to stabilize an activated intermediate for the channel activity potentiation by VX-770. Thus, a global induced fit across the interdomain interfaces upon ligand binding may optimize cooperative ligand-mediated NBD dimerization and improve the treatment of cystic fibrosis. General Biochemistry Biophysics Structural Biology Biochemical Research Methods Bioinformatics Computational Biology Biotechnology and Bioengineering Systems Biology Chemical Biology Mathematical and Theoretical Biology Drug Discovery, Design, & Development Physiology Applied Mathematics Computational Chemistry Biological Chemistry allosteric coupling digital biology interdomain interaction ligand modulation thermodynamic signature least-stable interaction melting threshold thermoring protein stability Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Introduction Induced fit is a term used to describe the process in which an enzyme's shape changes to fit its substrate during a reaction. An example of this can be seen in a metallo-dependent or class II Fructose-1,6-bisphosphate (FBP) aldolase from the extreme thermophile, Thermus aquaticus (Taq). This enzyme is a tetramer composed of two dimers. The loop region of each subunit can be in an open or closed conformation near the active site, facilitating the relocation of metal for keto bond polarization during catalysis ( 1 ). Another example is the multiple inositol polyphosphate phosphatases (MINPP) from the Gram-positive bacterium Bifidobacterium longum (BlMINPP). This enzyme has an α-domain polypeptide insertion called the U-loop, which is responsible for large ligand-driven conformational changes during the catalytic cycle, thermal stability, recovery of activity after heating, and kinetic parameters for hydrolysis of phytate ( 2 ). Furthermore, a ligand-induced conformational change in a dimerization loop is crucial for epidermal growth factor (EGF) receptor dimerization and activation. An abnormal orientation between two loop-related domains can cause autoinhibition ( 3 ) Induced fit has also been proposed to explain the gating mechanisms of the cystic fibrosis (CF) transmembrane conductance regulator (CFTR). The allosteric nature of this ligand-gated channel is combined with the enzymatic activity of an ATP-binding Cassette (ABC) transporter ( 4 – 7 ). Although the Mg/ATP-mediated dimerization between two nucleotide binding domains (NBD1 and NBD2) upon regulatory (R) domain phosphorylation by protein kinase A (PKA) is critical for coupling two transmembrane domains (TMD1 and TMD2) via intracellular loops (ICLs) for CFTR activation ( 8 – 12 ), the specific thermodynamic basis for the induced fit across the interdomain interfaces is still missing. Both NBD1 and NBD2 in hCFTR have highly conserved sequences and motifs at a dimerization interface ( 13 – 14 ). In an ATP-binding “head” subdomain, the aromatic ring of ATP is packed against W401 in NBD1 or Y1219 in NBD2 via a π−π interaction, and the remaining phosphate moiety is anchored by several parts via H-bonds. These parts include the Walker A motif ( 458 GSTGAGKTS 466 in NBD1 and 1244 GRTGSGKST 1252 in NBD2), the Walker B motif (from R560 to D572 and S573 in NBD1 or from R1358 to D1370 and E1371 in NBD2), and the switch regions (Q-loop involving Q493 in NBD1 or Q1291 in NBD2 while H-loop containing S605 in NBD1 or H1402 in NBD2). In the α-helical (or “tail”) subdomain, the conserved ATP-binding cassette (ABC) signature ( 548 LSGGQ 552 in NBD1 but 1346 LSHGH 1350 in NBD2), together with the Walker, is also needed to sandwich ATP. Based on this head-to-tail dimerization configuration, the two ATP-binding sites are inherently asymmetric because only E1371 and H1402 at site 2 rather than S573 and S605 at site 1 can hydrolyze ATP ( 15 – 19 ). The cryo-electron microscopy (cryo-EM) structure of human CFTR revealed that NBD1 and NBD2 are separated by the R domain in the dephosphorylated closed state (PDB, 5UAK). NBD1 contains a disordered regulatory insertion (RI) (spanning residues 405–436) and an unstructured regulatory extension (RE) (spanning residues 647–678), which are unique to CFTR compared to other ABC transporters. On the other hand, NBD2 maintains its entire structure within a peptide range from 1207 to 1436 ( 8 ). Upon R domain phosphorylation and Mg/ATP-binding (PDB, 6MSM), NBD1 and NBD2 dimerize. In this state, the unstructured RI in NBD1 shortens to a segment from 410 to 434, while the disordered RE in NBD1 begins at residue 638. Meanwhile, structured NBD2 expands from 1202 to 1451 ( 9 ). On the other hand, when the most common cystic fibrosis-causing F508 deletion destabilizes NBD1 ( 20 – 25 ), the ATP-dependent NBD dimerization is compromised ( 26 ). Although a combination of folding correctors elexacaftor/VX-445 and lumacaftor/VX-809 is enough to restore the NBD dimerization ( 27 ), the activity potentiator ivacaftor/VX-770 in Trikafta is still necessary to maximize the F508del activity ( 28 , 29 ). Given that the additional binding of VX-770 to F508del-CFTR with VX-445, VX-661 bound fail to further enhance the ICL4-NBD1-NBD2 and TMD1-TMD2 interactions ( 30 , 31 ), how VX-770 potentiates the channel activity is still unknown. In this study, a highly-sensitive grid thermodynamic model, which was recently developed and examined ( 30 – 39 ), was used to test a hypothesis that comparable thermostability between Mg/ATP-dimerized NBD1 and NBD2 is required for maximal channel activity. To confirm this hypothesis, the VX-770-induced changes in the thermoring structures of NBD2 and the ICL2-NBD2 interactions of hCFTR/E1371Q and (F508del)hCFTR/E1371Q were further analyzed and compared with the counterparts of NBD1 in tightly dimerized or partially dimerized states. The results showed that the binding of VX-770 to hCFTR with or without F508 significantly induced comparable thermostability between Mg/ATP-dimerized NBD1 and NBD2, stabilizing an activated intermediate for maximal channel activity with a minimal activation energy barrier. Therefore, a global induced fit across the interdomain interfaces was still necessary to rescue the gating defect of the F508del mutant. Results Melting threshold of the dimerized NBD2 in phosphorylated (E1371Q)hCFTR with Mg/ATP bound matched the thermal inactivation temperaqture of 45°C When (E1371Q)hCFTR is phosphorylated for Mg/ATP-dependent NBD1-NBD2 dimerization, the NBD2 (PDB, 6MSM) is structured from D1202 to P1451 but becomes disordered after P1451 ( 9 ). In this case, it is worth examining if NBD2 has a melting threshold (T m,th ) that is comparable to that of NBD1. Similar to the normal Mg/ATP binding site in NBD1 ( 30 ), a stable and rigid triangle was formed by S1251, Q1291 and D1370 via Mg 2+ in NBD2. Meanwhile, ATP connected Y1219, T1246, K1250 and T1252 (Fig. 1 a-b, Table S1). Notably, in addition to the H1348-H1375 H-bond between α and β subdomains, I1226-N1419-E1228 and T1246-I1416-S1248 H-bonds appeared between N- and C-termini (Fig. 1 a). When the total numbers of noncovalent interactions and grid sizes were 45 and 76 (Table S1), respectively, the systematic thermal instability (T i ) of NBD2 was 1.69 in 6MSM (Table 1 ), lower than the NBD1’s 1.88 in the same construct ( 30 ). Table 1 Grid thermodynamic model-based new parameters of NBD2. Construct hCFTR/E1371Q PDB ID 6MSM 6O2P 8EIG 8EIO 8EIQ F508 + + - - - Mg/ATP + + + + + Phosphorylation + + + + + Sampling temperature, °C 4 4 4 4 4 Tight NBD dimerization + + - + + NBDi 2 2 2 2 2 Normal Mg 2+ site + - - - - Name of the biggest grid Grid 12 Grid 10 Grid 12’ Grid 13 Grid 10’ Grid size (s) 12 10 12 13 10 # of energetically equivalent basic H-bonds (n) controlled by Grid s 1.5 1.5 1.8 1.5 1.5 Total non-covalent interactions (N) 45 42 46 52 51 Total grid sizes (S), a.a. 76 53 103 75 91 Systematic thermal instability (T i ) 1.69 1.26 2.24 1.44 1.78 Calculated T m,th, °C 45 49 48 43 49 Measured inactivation temperature, °C 45 Ref. for measured T inact ( 40 ) At the NBD2-ICL2 interface, in addition to the E264-S1297 H-bond, seven π interactions such as R1358-Y1307-W277-F1296 and N1303-F1296-F1294-W277-Y275 emerged. Similar to the triangle formed by F508, Y1073 and F1074 at the NBD1-ICL4 interface ( 30 ), F1296, W277, and F1294 also formed the smallest triangle to stabilize or rigidize the NBD2-ICL2 interface in this activated intermediate (Fig. 1 b). Consequently, although the weakest Q525-E585 H-bond between the α and β subdomains finalizes the posttranslational folding of NBD1 ( 31 ), the least-stable H-bond between the side chain of T1396 in the β-subdomain and the backbone C = O of G1208 in the N-terminal subdomain was responsible for the final posttranslational folding of NBD2 (Fig. 1 a). It was controlled by the biggest Grid 12 via a thermoring from L1367, D1370, Mg 2+ , Q1291, F1286, W1282, W1204, G1208, T1396, F1392, and back to L1367 (Figs. 1 c-d). For 1.5 equivalent basic H-bonds to seal it, the calculated T m,th of NBD2 in the 6MSM structure was approximately 45°C (Table 1 ), lower than the T m,th of 50°C for NBD1 in the same structure (30). Therefore, the thermostability between the dimerized NBD1 and NBD2 in hCFTR was not comparable upon Mg/ATP binding. Given that the predicted T m,th of 45°C of NBD2 was in excellent agreement with the experimental inactivation temperature of WT hCFTR (Table 1 ) ( 40 ), the thermal inactivation may result from the heat unfolding of the weakest G1208-T1396 H-bond of NBD2. Comparable thermostability between the dimerized NBD1 and NBD2 in phosphorylated (E1371Q)hCFTR with Mg/ATP/VX770 bound Along with additional VX-770 binding to the TMD1/TMD2 interface to enhance CFTR activity ( 41 – 45 ), the same NBD2-ICL2 interactions were also associated with the weakest G1208-T1396 H-bond (Fig. 2 a-b, Table S2). However, this H-bond was controlled by the biggest Grid 10 via a thermoring from L1367 to D1370, S1251, Q1291, F1286, W1282, W1274, D1270, G1208, T1396, F1392 and back to L1367 (Fig. 2 c-d). When this H-bond was energetically equivalent to 1.5 basic H-bonds (1.5 kcal/mol), the calculated T m,th of NBD2 in 6O2P was about 49°C (Table 1 ), similar to the T m,th of 50°C for NBD1 in 6O2P ( 31 ). Therefore, the comparable thermostability between NBD1 and NBD2 was observed with their dimerization induced by Mg/ATP/VX770 binding in the activated intermediate to maximize channel activity. Meanwhile, along with a decrease in the totals of noncovalent interactions and grid sizes from 45 and 76 in 6MSM to 42 and 53 in 6O2P, the systematic thermal instability (Ti) of NBD2 also declined from 1.69 to 1.26 (Table 1 ). Given that Trikafta contains the potentiator VX-770 and significantly enhances the activity of (F508del)hCFTR ( 28 – 29 ), it is intriguing to explore whether tight NBD dimerization induced by Mg/ATP/Trikafta also corresponds to balanced thermostability for both NBD1 and NBD2 even in the presence of the destabilizing F508 deletion. Matched thermostability between the dimerized NBD1 and NBD2 in phosphorylated (E1371Q/F508del)hCFTR with Mg/ATP/Trikafta bound When F508 was deleted, both NBD1 and NBD2 changed their thermoring structures to maintain Mg/ATP-dependent dimerization in the presence of Trikafta (Figs. 3 a; Table S3) ( 30 ). In addition to the intact ATP site and the compromised S1251-Mg 2+ -Q1291 bridge along with the broken R1358-Y1307 CH-π interaction at the NBD2-ICL2 interface (Fig. 3 a-b), only a small fraction of 37 noncovalent interactions were conserved in NBD2. For example, the R1259-W1274-W1282-F1286, F1294-F1296-N1303, V1327-H1350, F1331-F1337 and L1367-F1392 π interactions, together with the Y1219-T1252 and S1373-E1401 H-bonds. Despite these changes, the G1208-T1396 H-bond between N-terminal and β- subdomains was still the weakest to finalize the posttranslational NBD2 folding (Fig. 3 a). However, it was governed by another biggest Grid 10’ via a thermoring from S1251, Mg 2+ , Q1291, F1286, W1282, W1274, D1270, G1207, G1208, T1396, C1400, D1370, and back to S1251 (Fig. 3 c-d). Since the weakest G1208-T1396 H-bond was still energetically equivalent to 1.5 basic H-bonds, the calculated melting threshold (T m,th ) was 49°C (Table 1 ), which was exactly the same as the 49°C of NBD1 in the same construct ( 30 ). On the other hand, when the total numbers of noncovalent interactions and grid sizes increased from 42 and 53 to 51 and 91, respectively, the systematic thermal instability (T i ) of NBD2 also increased from 1.26 to 1.78 (Table 1 ). In contrast, when Trikafta was replaced with VX-445 and VX-809 in 8EIO, the weakest G1208-T1396 H-bond in dimerized NBD2 was governed by the biggest Grid 13 via a thermoring from L1367 to D1370, S1251, Mg 2+ , Q1291, F1286, W1282, W1274, I1267, I1269, W1204, G1208, T1396, F1392, and back to L1367 (Fig. 4 ; Table S4). Since this H-bond was energetically equivalent to 1.5 basic H-bonds (1.5 kcal/mol), the calculated T m,th of NBD2 in 8EIO was 43°C (Table 1 ), which was 4°C lower than the T m,th of 47°C for NBD1 in the same construct ( 31 ). Notably, despite a decrease in the T m,th of NBD2, the systematic thermal instability (Ti) of NBD2 reduced from 1.78 in 8EIQ to 1.44 in 8EIO (Table 1 ). Significant imbalance in thermostability between partially dimerized NBD1 and NBD2 in phosphorylated (F508del)hCFTR/E1371Q with only Mg/ATP/elexacaftor bound To further investigate the comparable thermostability between NBD1 and NBD2 as a requirement for Trikafta to maximize the channel activity of the F508del mutation, it is necessary to determine if their comparable thermostability would be significantly compromised in a partially dimerized state. When only elexacaftor and Mg 2+ /ATP are bound to phosphorylated closed (E1371Q/F508del)hCFTR, NBD1 and NBD2 are partially dimerized ( 27 ). In this scenario, the total numbers of noncovalent interactions and grid sizes of NBD2 changed from 52 and 75 in 8EIO to 46 and 103 in 8EIG, respectively (Fig. 5 a; Table S5). Hence, the systematic thermal instability (T i ) of NBD2 significantly increased from 1.44 in 8EIO to 2.24 in 8EIG (Table 1 ). Notably, the least-stable G1208-T1396 H-bond in the biggest Grid 13 was replaced by the least-stable I1226-N1419 H-bond in the biggest Grid 12’ of NBD2 while the R1358-Y275 π interaction was reestablished at the NBD2-ICL2 interface (Fig. 5 a-b). This biggest thermoring cycled from I1226 to S1233, Q1412, N1419, and back to I1226 (Fig. 5 c-d). Since this H-bind was energetically equivalent to 1.8 basic H-bonds, the calculated T m,th of NBD2 in 8EIG was about 48°C, significantly 9°C higher than the T m,th of 39°C in NBD1 ( 30 ). Therefore, comparable thermostability between Mg/ATP-dimerized NBD1 and NBD2 was indeed required for Trikafta to maximize the channel activity of the F508del mutant. Discussion Interdomain interactions play a crucial role in regulating CFTR activity, yet little is known about how each domain responds to different physical, chemical and genetic stimuli during this process. This study revealed that while the apparent secondary structures of dimerized NBD1 and NBD2 remained the same or similar before vs. after the introduction of the F508 deletion along with various folding correctors, their tertiary thermoring structures underwent a global change, rearranging interdomain interactions. Furthermore, although the additional binding of VX770 to hCFTR or F508del with folding correctors VX445 and VX-809 did not enhance interdomain interactions, a global induced fit across domain-domain interfaces significantly triggered a conformational selection step, ensuring comparable thermostability between Mg/ATP-dimerized NBD1 and NBD2 to stabilize the activated intermediate with a minimal activation energy barrier for maximal channel activity of hCFTR, regardless of the presence of F508del. Comparable thermostability between the dimerized NBD1 and NBD2 upon VX-770 binding maximizes hCFTR activity In this study, despite the tight NBD1-ICL4 or NBD2-ICL2 interactions still allowing for intact Mg/ATP binding sites in the phosphorylated activated intermediate of hCFTR/E1371Q (6MSM) (Figs. 1 a-b) ( 30 ), both dimerized NBD1 and NBD2 exhibited different melting thresholds (T m,th ) of 50°C and 45°C, respectively (Fig. 6 ). Given that the WT hCFTR channel starts thermal inactivation at 45°C ( 40 ), the inactivation may be due to the unfolding of least-stable G1208-T1396 bridge in NBD2 at the T m,th of 45°C (Table 1 , Fig. 6 ). When the potentiator VX-770 was bound to the E1371Q mutant increasing channel activity, the dimerized NBD2 increased the T m,th from 45°C to 49°C (Fig. 6 ). Thus, although VX-770 still potentiates the WT, G551D and W1282X-CFTR activity in a phosphorylation-dependent but ATP-independent manner ( 44 – 46 ), VX-770-induced comparable thermostability between dimerized NBD1 and NBD2 favors the maximal activity of hCFTR in an ATP-dependent manner. Matching thermostability between dimerized NBD1 and NBD2 upon Trikafta binding maximizes (F508del)hCFTR activity In the previous study, two identical isolated hNBD1-Δ(RI, RE) or (F508del)hNBD1-Δ(RI,RE) constructs with the same thermostability can form a stable homodimer (2PZE or 2PZF) for crystal capture upon Mg/ATP binding, suggesting that the RI or RE may serve as an important dimerization loop or segment ( 20 , 21 ). In this study, despite the instablity of NBD1 in ΔF508 ( 27 ), tightly dimerized NBD1 and NBD2 upon Trikafta binding to TMD1 and TMD2 still shared a common T m,th of 49°C (Fig. 6 ) ( 30 ). However, in the presence of elexacaftor and lumacaftor, the T m.th of NBD1 was 47°C, slightly higher than the T m.th of 43°C in NBD2 (Fig. 6 ). Further, the T m,th values of the partially-dimerized NBD1 and NBD2 in phosphorylated (F508del)hCFTR/E1371Q with elexacaftor bound were 39°C and 48°C, respectively (Fig. 6 ) ( 30 ). Therefore, matching thermostability between dimerized NBD1 and NBD2 upon Trikafta binding facilitates correcting the thermal and gating defects of the F508del mutation. Asymmetric weakest noncovalent bridges across the dimerization interface Although both tightly dimerized NBD1 and NBD2 exhibited minimal differences in T m,th under two various conditions, they had distinct but asymmetric weakest noncovalent bridges (Fig. 6 ). When the potentiator VX-770 was bound to hCFTR/E1371Q, the biggest Grid 8 in dimerized NBD1 was found to be responsible for the least-stable Q525-E585 H-bond between α and β-subdomains ( 31 ). However, the biggest Grid 10 in dimerized NBD2 was responsible for the least-stable G1208-T1396 H-bond between C- and N-termini (Figs. 2 a, 6 ). After Trikafta modulators are bound to the E1371Q/F508del mutant, the least-stable Y517-D537 H-bond finalized posttranslational NBD1 folding ( 30 ). In contrast, the weakest G1208-T1396 H-bond was still the last step of the posttranslational NBD2 folding (Figs. 3 a, 6 ). Since this weakest link was also the final posttranslational CFTR folding, its stability is essential for optimizing CFTR activity and cystic fibrosis treatment. Taken together, while folding modulators in Trikafta shift the weakest tertiary link to serve as the final posttranslational NBD1 folding event, they do not impact the final posttranslational NBD2 folding. Conversely, the potentiator VX-770 in Trikafta is essential for the stabilized activated intermediate to maximal channel activity by adjusting the size of the biggest thermoring in NBD2 to align with the thermal stability of NBD1. Therefore, these three modulators in Trikafta play distinct roles in addressing thermal and gating abnormalities in (F508del)hCFTR ( 30 , 31 ). In fact, in a cellular environment where Mg 2+ , ATP and PKA are present along with an allosteric drug like Trikafta or ivacaftor, the induced comparable thermostability between the dimerized NBD1 and NBD2 is essential for CFTR folding, stability and effective therapy, particularly when compromised by disease mutations such as F508del and G551D ( 20 – 27 , 47 – 50 ). Conclusions Induced fit plays a significant role in allosteric enzymatic reactions, especially in the case of CFTR. CFTR functions not only as an anion channel but also as an ATPase. In the CFTR gating cycle, induced fit at the dimerization interface between NBD1 and NBD2 is crucial for an “interdependent protein dance”. When two Mg/ATP agonists bind normally at two interfacial sites, they dimerize NBD1 and NBD2 after R domain phosphorylation. A VX-770-inducd conformational selection step leads to an expanded induced fit between dimerized NBD1 and NBD2. As a result, their thermostability closes to each other to stabilize the activated intermediate with minimal activation energy barrier for maximal channel activity of hCFTR with or without F508del and folding correctors. Therefore, the increasing precision of thermostability evaluation permits increasing activation mechanistic detail and allosteric drug design to optimize the cystic fibrosis treatment. Computational Methods Data mining resources Thermoring structures of phosphorylated and Mg/ATP bound hCFTR constructs with or without various modulators in the activated intermediate were analyzed using cryo-EM structures at 4°C. The structures without F508del included hCFTR/1371Q with Mg/ATP bound (PDB ID, 6MSM, model resolution = 3.2 Å) ( 9 ), and with Mg/ATP/VX-770 bound (PDB ID, 6O2P, model resolution = 3.3 Å) ( 40 ). Furthermore, the structures with F508del covered hCFTR/E1371Q/ΔF508 with Mg/ATP/ elexacaftor (VX445) bound (PDB ID, 8EIG, model resolution = 3.7 Å), Mg/ATP/VX445/VX809 bound (PDB ID, 8EIO, model resolution = 2.8 Å), and Mg/ATP/Trikafta bound (PDB ID, 8EIQ, model resolution = 3.2 Å) ( 27 ). Standard methods for filtering tertiary non-covalent interactions Tertiary non-covalent interactions such as salt bridges, H-bonds and π interactions in NBD2 were filtered using standard methods and precise calculations previously employed to ensure accurate and reproducible results ( 30 – 39 ). Detailed cutoff distances and interaction angles (for an H-bond) can be found in the online Supplementary Information (Tables S1, S2, S3, S4 and S5). It shoud be noted that momentary fluctuation-induced perturbations in noncovalent interactions during protein dynamics were not considered in this study. Thus, approximately 42–52 noncovalent interactions were identified along the single peptide chain from D1202 to P1451 in NBD2 of each protomer. Calculations based on the thermoring structures and the grid thermodynamic model The previously established grid thermodynamic model was used to map the systematic fluidic grid-like noncovalent interaction mesh networks of NBD1 and NBD2 ( 30 – 39 ). In these networks, identified noncovalent interactions and linked amino acid residues were represented by edges and nodes, respectively. When a noncovalent interaction had a direct zero-length path between two linked nodes and the shortest reverse path from one node back to the other through other noncovalent interactions and a peptide segment, a thermoring or grid with the tightest network was formed to control this least-stable noncovalent interaction within it. The grid size was defined as the free or silent residues not involved in any noncovalent interactions along the shortest reverse path. In this way, each thermoring acted like a bow and each least-stable noncovalent interaction functioned like a vibrating bowstring. Thus, the strength of each bowstring was determined not only by itself but also regulated by the bow length, the relevant energy relocation and allosteric propagation. Generally, the intensity of a noncovalent interaction or a bowstring was limited to 1–3 kcal/mol. However, the more free or flexible side chains along the thermoring or the bow, or the larger the thermoring size or the longer the bow, the weaker the controlled noncovalent interaction or the bowstring. Once the biggest grid was identified, the least-stable noncovalent interaction within it was typically the weakest one along the entire polypeptide chain. Its heat unfolding could be characterized by a specific melting temperature threshold using the following equation as previously examined ( 30 – 39 ): T m,th (°C) = 34 + (n − 2) × 10 + (20 – s) × 2 ( 1 ) where, n represents the total number of basic H-bonds (each approximately 1 kcal/mol) equivalent to the least-stable noncovalent interaction controlled by the grid; and s is the grid size used to control the least-stable noncovalent interaction within the grid. In this study, although the I1226-N1419 H-bond was highly conserved in NBD2, it was not the weakest or least-thermostable except in 8EIG. For example, because E1228 also H-bonded to N1419 via their side chains in 6MSM, it was actually controlled by the smaller Grid 1 via a smaller thermoring from I1226 to E1228, N1419, and back to I1226 and the T m,th to unfold it was at least 72°C (Figs. 1 a). In addition to the T m,th , the total grid sizes (S) and the total non-covalent interactions (N) along the same polypeptide chain could be utilized to calculate grid-based systematic thermal instability (T i ) using the same equation as examined previously ( 30 – 39 ): T i = S/N ( 2 ) This parameter reflects the peptide’s compact conformational entropy or flexibility. Abbreviations ABC, ATP-binding cassette BlMINPP, Gram-positive bacterium Bifidobacterium longum CF, cystic fibrosis cryo-EM, cryoelectron microscopy CFTR, cystic fibrosis transmembrane conductance regulator DSC, differential scanning calorimetry EGF, epidermal growth factor FBP, Fructose-1,6-bisphosphate hCFTR, human CFTR ICL2, intracellular loop 2 ICL3, intracellular loop 3 ICL4, intracellular loop 4 MINPP, multiple inositol polyphosphate phosphatases NBD1, nucleotide binding domain 1 NBD2, nucleotide binding domain 2 PKA, protein kinase A R, regulatory RE, regulatory extension RI, regulatory insert T i , systematic thermal instability T m,th , melting temperature threshold Taq, Thermus aquaticus TMD1, transmembrane domain 1 TMD2, transmembrane domain 2 WT, wild type Declarations Acknowledgements The author’s own studies cited in this article were supported by the NIDDK Grant (DK45880 to D.C.D.) and the Cystic Fibrosis Foundation grant (DAWSON0210), the NIDDK grant (2R56DK056796-10) and the American Heart Association (AHA) Grant (10SDG4120011 to GW). Author contributions G. W wrote the main manuscript text and prepared Figures. 1, 2, 3, 4, 5, 6 and Table 1, Supplementary Information (Tables S1, S2, S3, S4 and S5) and reviewed the manuscript. Competing interests The author declares no conflict of interest. Data availability Data are provided within the manuscript or supplementary information files Additional information Supplementary Information The online version contains supplementary material available. References Izard T, Sygusch J (2004) Induced fit movements and metal cofactor selectivity of class II aldolases: structure of Thermus aquaticus fructose-1,6-bisphosphate aldolase. J Biol Chem 279:11825–11833 Acquistapace IM, Zi Etek MA, Li AWH, Salmon M, Kühn I, Bedford MR, Brearley CA, Hemmings AM (2020) Snapshots during the catalytic cycle of a histidine acid phytase reveal an induced-fit structural mechanism. J Biol Chem 295:17724–17737 Schlessinger J (2002) Ligand-induced, receptor-mediated dimerization and activation of EGF receptor. Cell. 110, 669 – 72 Riordan JR, Rommens JM, Kerem B, Alon N, Rozmahel R, Grzelczak Z, Zielenski J, Lok S, Plavsic N, Chou JL et al (1989) Identification of the cystic fibrosis gene: cloning and characterization of complementary DNA. Science 245:1066–1073 Hyde SC, Emsley P, Hartshorn MJ, Mimmack MM, Gileadi U, Pearce SR, Gallagher MP, Gill DR, Hubbard RE, Higgins CF (1990) Structural model of ATP-binding proteins associated with cystic fibrosis, multidrug resistance and bacterial transport. Nature 346:362–365 Dean M, Rzhetsky A, Allikmets R (2001) The human ATP-binding cassette (ABC) transporter superfamily. Genome Res 11:1156–1166 Kirk KL, Wang W (2011) A unified view of cystic fibrosis transmembrane conductance regulator (CFTR) gating: combining the allosterism of a ligand-gated channel with the enzymatic activity of an ATP-binding cassette (ABC) transporter. J Biol Chem 286:12813–12819 Liu F, Zhang Z, Csanády L, Gadsby DC, Chen J (2017) Molecular Structure of the Human CFTR Ion Channel. Cell 169:85–95e8 Zhang Z, Liu F, Chen J (2018) Molecular structure of the ATP-bound, phosphorylated human CFTR. Proc Natl Acad Sci U S A 115:12757–12762 Csanády L, Vergani P, Gadsby DC (2019) STRUCTURE, GATING, AND REGULATION OF THE CFTR ANION CHANNEL. Physiol Rev 99:707–738 Vernon RM, Chong PA, Lin H, Yang Z, Zhou Q, Aleksandrov AA, Dawson JE, Riordan JR, Brouillette CG, Thibodeau PH, Forman-Kay JD (2017) Stabilization of a nucleotide-binding domain of the cystic fibrosis transmembrane conductance regulator yields insight into disease-causing mutations. J Biol Chem 292:14147–14164 Levring J, Terry DS, Kilic Z, Fitzgerald G, Blanchard SC, Chen J (2023) CFTR function, pathology and pharmacology at single-molecule resolution. Nature. 616(7957), 606–614 10.1038/s41586-023-05854-7 . Epub 2023 Mar 22. Erratum in: Nature. 617(7961), E11 (2023). doi: 10.1038/s41586-023-06115-3 Lewis HA, Buchanan SG, Conners K, Dickey M, Dorwart M, Fowler R, Gao X, Guggino WB, Hendrickson WA, Hunt JF, Kearins MC, Lorimer D, Maloney PC, Post KW et al (2004) Structure of nucleotide-binding domain 1 of the cystic fibrosis transmembrane conductanceregulator. EMBO J 23:282–293 Lewis HA, Zhao X, Wang C, Sauder JM, Rooney I, Noland BW, Lorimer D, Kearins MC, Conners K, Condon B, Maloney PC, Guggino WB, Hunt JF, Emtage S (2005) Impact of the deltaF508 mutation in first nucleotide-binding domain of human cystic fibrosis transmembrane conductance regulator on domain folding and structure. J Biol Chem 280:1346–1353 Aleksandrov L, Aleksandrov AA, Chang XB, Riordan JR (2002) The First Nucleotide Binding Domain of Cystic Fibrosis Transmembrane Conductance Regulator Is a Site of Stable Nucleotide Interaction, whereas the Second Is a Site of Rapid Turnover. J Biol Chem 277:15419–15425 Basso C, Vergani P, Nairn AC, Gadsby DC (2003) Prolonged nonhydrolytic interaction of nucleotide with CFTR’s NH2-terminal nucleotide binding domain and its role in channel gating. J Gen Physiol 122:333–348 Zaitseva J, Jenewein S, Jumpertz T, Holland IB, Schmitt L (2005) H662 is the linchpin of ATP hydrolysis in the nucleotide-binding domain of the ABC transporter HlyB. EMBO J 24:1901–1910 Zaitseva J, Oswald C, Jumpertz T, Jenewein S, Wiedenmann A, Holland IB, Schmitt L (2006) A structural analysis of asymmetry required for catalytic activity of an ABC-ATPase domain dimer. EMBO J 25:3432–3443 Stratford FL, Ramjeesingh M, Cheung JC, Huan LJ, Bear CE (2007) The Walker B motif of the second nucleotide-binding domain (NBD2) of CFTR plays a key role in ATPase activity by the NBD1-NBD2 heterodimer. Biochem J 401(2):581–586. 10.1042/BJ20060968 Atwell S, Brouillette CG, Conners K, Emtage S, Gheyi T, Guggino WB, Hendle J, Hunt JF, Lewis HA, Lu F, Protasevich II, Rodgers LA, Romero R, Wasserman SR, Weber PC, Wetmore D, Zhang FF, Zhao X (2010) Structures of a minimal human CFTR first nucleotide-binding domain as a monomer, head-to-tail homodimer, and pathogenic mutant. Protein Eng Des Sel 23:375–384 Protasevich I, Yang Z, Wang C, Atwell S, Zhao X, Emtage S, Wetmore D, Hunt JF, Brouillette CG (2010) Thermal unfolding studies show the disease causing F508del mutation in CFTR thermodynamically destabilizes nucleotide-binding domain 1. Protein Sci 19:1917–1931 Wang C, Protasevich I, Yang Z, Seehausen D, Skalak T, Zhao X, Atwell S, Spencer Emtage J, Wetmore DR, Brouillette CG, Hunt JF (2010) Integrated biophysical studies implicate partial unfolding of NBD1 of CFTR in the molecular pathogenesis of F508del cystic fibrosis. Protein Sci 19:1932–1947 Rabeh WM, Bossard F, Xu H, Okiyoneda T, Bagdany M, Mulvihill CM, Du K, di Bernardo S, Liu Y, Konermann L, Roldan A, Lukacs GL (2012) Correction of both NBD1 energetics and domain interface is required to restore ∆F508 CFTR folding and function. Cell 148:150–163 He L, Aleksandrov AA, An J, Cui L, Yang Z, Brouillette CG, Riordan JR (2015) Restoration of NBD1 thermal stability is necessary and sufficient to correct ∆F508 CFTR folding and assembly. J Mol Biol 427:106–120 Soya N, Roldan A, Lukacs GL (2019) Differential Scanning Fluorimetry and Hydrogen Deuterium Exchange Mass Spectrometry to Monitor the Conformational Dynamics of NBD1 in Cystic Fibrosis. Methods Mol Biol 1873:53–67 Jih KY, Li M, Hwang TC, Bompadre SG (2011) The most common cystic fibrosis-associated mutation destabilizes the dimeric state of the nucleotide-binding domains of CFTR. J Physiol 589(Pt 11):2719–2731 Fiedorczuk K, Chen J (2022) Molecular structures reveal synergistic rescue of ∆508 CFTR by Trikafta modulators. Science 378(6617):284–290. 10.1126/science.ade2216 Veit G, Roldan A, Hancock MA, Da Fonte DF, Xu H, Hussein M, Frenkiel S, Matouk E, Velkov T, Lukacs GL (2020) Allosteric folding correction of F508del and rare CFTR mutants by elexacaftor-tezacaftor-ivacaftor (Trikafta) combination. JCI Insight 5:e139983 Veit G, Vaccarin C, Lukacs GL (2021) Elexacaftor co-potentiates the activity of F508del and gating mutants of CFTR. J Cyst Fibros 20:895–898 Wang G (2025) Trikafta rescues F508del-CFTR by tightening specific phosphorylation-dependent interdomain interactions. Nat Sci 6:e70009. https://doi.org/10.1002/ntls.70009 Wang G Thermodynamic coupling between folding correctors and the first of dimerized nucleotide binding domains in CFTR. ACS Bio. Med.Au (in press). https://doi.org/10.1021/acsbiomedchemau.5c00014 Wang G (2023) The network basis for the structural thermostability and the functional thermoactivity of aldolase B. Molecules 28:1850 Wang G (2023) Network basis for the heat-adapted structural thermostability of bacterial class II fructose bisphosphate aldolase. ACS Omega 8:17731–17739 Wang G (2023) Thermal ring-based heat switches in hyperthermophilic class II bacterial fructose aldolase. ACS Omega 8:24624–24634 Wang G (2023) Thermoring-Based Heat Activation Switches in the TRPV1 Biothermometer. Int J Biol Macromol 248:125915 Wang G (2023) Thermoring basis for the TRPV3 bio-thermometer. Sci Rep 13:21594 Wang G (2024) Phosphatidylinositol-4,5-biphosphate (PIP 2 )-Dependent Thermoring Basis for Cold-Sensing of the Transient Receptor Potential Melastatin-8 (TRPM8) Biothermometer. Physchem 4:106–119 Wang G (2024) Thermoring basis for heat unfolding-induced inactivation in TRPV1. Nat Sci 4:e20240008 Wang G (2024) ATP-dependent thermoring basis for the heat unfolding of the first nucleotide-binding domain isolated from human CFTR. Res Sq [Preprint]. Nov 21:rs.3.rs-5479740. 10.21203/rs.3.rs-5479740/v1 . Nat Sci. 5, e70007 (2025). https://doi.org/10.1002/ntls.70007 Aleksandrov LA, Jensen TJ, Cui L, Kousouros JN, He L, Aleksandrov AA, Riordan JR (2015) Thermal stability of purified and reconstituted CFTR in a locked open channel conformation. Protein Expr Purif 116:159–166 Liu F, Zhang Z, Levit A, Levring J, Touhara KK, Shoichet BK, Chen J (2019) Structural identification of a hotspot on CFTR for potentiation. Science 364(6446):1184–1188. 10.1126/science.aaw7611 Hadida S, Van Goor F, Zhou J, Arumugam V, McCartney J, Hazlewood A, Decker C, Negulescu P, Grootenhuis PD (2014) Discovery of N-(2,4-di-tert-butyl-5-hydroxyphenyl)-4-oxo-1,4-dihydroquinoline-3-carboxamide (VX-770, ivacaftor), a potent and orally bioavailable CFTR potentiator. J Med Chem 57(23):9776–9795. 10.1021/jm5012808 Van Goor F, Hadida S, Grootenhuis PD, Burton B, Cao D, Neuberger T, Turnbull A, Singh A, Joubran J, Hazlewood A, Zhou J, McCartney J, Arumugam V, Decker C, Yang J, Young C, Olson ER, Wine JJ, Frizzell RA, Ashlock M, Negulescu P (2009) Rescue of CF airway epithelial cell function in vitro by a CFTR potentiator, VX-770. Proc Natl Acad Sci U S A 106(44):18825–18830. 10.1073/pnas.0904709106 Jih KY, Hwang TC (2013) Vx-770 potentiates CFTR function by promoting decoupling between the gating cycle and ATP hydrolysis cycle. Proc Natl Acad Sci U S A 110(11):4404–4409. 10.1073/pnas.1215982110 Eckford PD, Li C, Ramjeesingh M, Bear CE (2012) Cystic fibrosis transmembrane conductance regulator (CFTR) potentiator VX-770 (ivacaftor) opens the defective channel gate of mutant CFTR in a phosphorylation-dependent but ATP-independent manner. J Biol Chem 287(44):36639–36649. 10.1074/jbc.M112.393637 Wang W, Hong JS, Rab A, Sorscher EJ, Kirk KL (2016) Robust Stimulation of W1282X-CFTR Channel Activity by a Combination of Allosteric Modulators. PLoS ONE 11(3):e0152232. 10.1371/journal.pone.0152232 Yu H, Burton B, Huang CJ, Worley J, Cao D, Johnson JP Jr., Urrutia A, Joubran J, Seepersaud S, Sussky K et al (2012) Ivacaftor potentiation of multiple CFTR channels with gating mutations. J Cyst Fibros 11:237–245. https://doi.org/10.1016/j.jcf.2011.12.005 Liu X, Dawson DC (2014) Cystic fibrosis transmembrane conductance regulator (CFTR) potentiators protect G551D but not ∆F508 CFTR from thermal instability. Biochemistry 53:5613–5618 Soya N, Xu H, Roldan A, Yang Z, Ye H, Jiang F, Premchandar A, Veit G, Cole SPC, Kappes J, Hegedüs T, Lukacs GL (2023) Folding correctors can restore CFTR posttranslational folding landscape by allosteric domain-domain coupling. Nat Commun 14:6868 Wang C et al (2022) Mechanism of dual pharmacological correction and potentiation of human CFTR. bioRxiv https://doi.org/10.1101/2022.10.10.510913 Additional Declarations The authors declare no competing interests. Supplementary Files SupplementaryInformationCFTRpotentiationmechanismv1.6.pdf Supplemntal Tables S1-S5 Cite Share Download PDF Status: Published Journal Publication published 05 Jan, 2026 Read the published version in Biomedicine & Pharmacotherapy → Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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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-7339733","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":498329234,"identity":"848322a7-6095-486a-88bb-515a622438d4","order_by":0,"name":"Guangyu Wang","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA60lEQVRIiWNgGAWjYFADZsbGB0CKh494LezMhw1AWtiI18LPliYBoglqkXc/+0yCsc0uT96Zx6zya46dDBsD88NHN/BoMTyTbgbUklxseJjH7LbstmSgw9iMjXPwaWlIY5Ng3MacuLEZqEVyGzNQCw+bNF4t/c9AWurBWoolt9UT1iIvAbblcOJ8ZrY0xo/bDhPWYiDxjNki8d/xxA3MzIelGbcd52FjJuAX+f40xhsfzlQnzu8/2Pjx57Zqe3725oeP8dpygIFFIgHCYGDmAQkx41EOtqWBgfkDlMHA+IOA6lEwCkbBKBiZAACt70H8iuWZ5QAAAABJRU5ErkJggg==","orcid":"","institution":"","correspondingAuthor":true,"prefix":"","firstName":"Guangyu","middleName":"","lastName":"Wang","suffix":""}],"badges":[],"createdAt":"2025-08-10 15:37:33","currentVersionCode":1,"declarations":{"humanSubjects":false,"vertebrateSubjects":false,"conflictsOfInterestStatement":false,"humanSubjectEthicalGuidelines":false,"humanSubjectConsent":false,"humanSubjectClinicalTrial":false,"humanSubjectCaseReport":false,"vertebrateSubjectEthicalGuidelines":false},"doi":"10.21203/rs.3.rs-7339733/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-7339733/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1016/j.biopha.2025.118936","type":"published","date":"2026-01-06T00:00:00+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":88866453,"identity":"c6b3f80e-eb19-43c0-abec-9860666f518b","added_by":"auto","created_at":"2025-08-12 08:34:22","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":261267,"visible":true,"origin":"","legend":"\u003cp\u003eThe thermoring structures of NBD2 in phosphorylated hCFTR/E1371Q with F508 in an activated intermediate at 4 °C. (\u003cstrong\u003ea\u003c/strong\u003e) The grid-like noncovalently interacting mesh network of NBD2 based on the cryo-EM structure of hCFTR/E1371Q with F508 in the presence of Mg/ATP/PKA at 4 °C (PDB ID, 6MSM, 3.2 Å). No software was used to create the image. \u0026nbsp;Salt bridges, H-bonds and pi interactions are colored purple, orange, and green, respectively. The constrained grid sizes required to control the least-stable noncovalent interactions in the grids are labeled with black numbers. The least-stable G1208-T1396 H-bond in the biggest Grid\u003csub\u003e12\u003c/sub\u003e is highlighted.\u0026nbsp; The total grid sizes and the total grid size-controlled noncovalent interactions along the single peptide chain of NBD2 from D1202 to P1451 are shown in cyan and black circles, respectively. (\u003cstrong\u003eb\u003c/strong\u003e) \u0026nbsp;The NBD2-ICL2 interactions. (\u003cstrong\u003ec\u003c/strong\u003e) The structure of the biggest Grid\u003csub\u003e12 \u003c/sub\u003ewith a 12-residue size to control the least-stable G1208-T1396 H-bond. The grid size and the equivalent basic H-bonds for the least-stable noncovalent interaction are shown in and near a red circle. \u0026nbsp;(\u003cstrong\u003ed\u003c/strong\u003e) The sequence of the biggest Grid\u003csub\u003e12 \u003c/sub\u003eto control the least-stable G1208-T1396 H-bond in the blue box.\u003c/p\u003e","description":"","filename":"Figure1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7339733/v1/6a78ff5761b90e6cb5ef05d8.jpg"},{"id":88867359,"identity":"9249cdde-e9b9-4f2e-b071-cf631e30e07b","added_by":"auto","created_at":"2025-08-12 08:42:22","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":256487,"visible":true,"origin":"","legend":"\u003cp\u003eThe thermoring structures of NBD2 in phosphorylated hCFTR/E1371Q/VX770 with F508 in an activated intermediate at 4 °C. (\u003cstrong\u003ea\u003c/strong\u003e) The grid-like noncovalently interacting mesh network of NBD2 based on the cryo-EM structure of hCFTR/E1371Q with F508 in the presence of Mg/ATP/PKA/VX-770 at 4 °C (PDB ID, 6O2P, 3.3 Å). No software was used to create the image.\u0026nbsp; Salt bridges, H-bonds and pi interactions are colored purple, orange, and green, respectively. The constrained grid sizes required to control the least-stable noncovalent interactions in the grids are labeled with black numbers. The least-stable G1208-T1396 H-bond in the biggest Grid\u003csub\u003e10\u003c/sub\u003e is highlighted.\u0026nbsp; The total grid sizes and the total grid size-controlled noncovalent interactions along the single peptide chain of NBD2 from D1202 to P1451 are shown in cyan and black circles, respectively. (\u003cstrong\u003eb\u003c/strong\u003e) \u0026nbsp;The NBD2-ICL2 interactions. (\u003cstrong\u003ec\u003c/strong\u003e) The structure of the biggest Grid\u003csub\u003e10 \u003c/sub\u003ewith a 10-residue size to control the least-stable G1208-T1396 H-bond. The grid size and the equivalent basic H-bonds for the least-stable noncovalent interaction are shown in and near a red circle. \u0026nbsp;(\u003cstrong\u003ed\u003c/strong\u003e) The sequence of the biggest Grid\u003csub\u003e10 \u003c/sub\u003eto control the least-stable G1208-T1396 H-bond in the blue box.\u003c/p\u003e","description":"","filename":"Figure2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7339733/v1/179daecf7431130294b3a6fc.jpg"},{"id":88866456,"identity":"4e43f518-670c-46ca-b52c-6ad2b2f06191","added_by":"auto","created_at":"2025-08-12 08:34:22","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":271294,"visible":true,"origin":"","legend":"\u003cp\u003eThe thermoring structures of NBD2 in phosphorylated hCFTR/E1371Q/DF508 with Trikafta bound in an activated intermediate at 4 °C. (\u003cstrong\u003ea\u003c/strong\u003e) The grid-like noncovalently interacting mesh network of NBD2 based on the cryo-EM structure of hCFTR/E1371Q/DF508 with Trikafta bound in the presence of Mg/ATP/PKA at 4 °C (PDB ID, 8EIQ, 3.2 Å). No software was used to create the image. \u0026nbsp;Salt bridges, H-bonds and pi interactions are colored purple, orange, and green, respectively. The constrained grid sizes needed to control the least-stable noncovalent interactions in the grids are labeled with black numbers. The least-stable G1208-T1396 H-bond in the biggest Grid\u003csub\u003e10’ \u003c/sub\u003eis highlighted.\u0026nbsp; The total grid sizes and the total grid size-controlled noncovalent interactions along the single peptide chain of NBD2 from I1203 to P1451 are shown in cyan and black circles, respectively. (\u003cstrong\u003eb\u003c/strong\u003e) The NBD2-ICL2 interactions. (\u003cstrong\u003ec\u003c/strong\u003e) The structure of the biggest Grid\u003csub\u003e10’ \u003c/sub\u003ewith a 10-residue size to control the least-stable G1208-T1396 H-bond. \u0026nbsp;The grid size and the equivalent basic H-bonds for the least-stable noncovalent interaction are shown in and near a red circle. (\u003cstrong\u003ed\u003c/strong\u003e) The sequence of the biggest Grid\u003csub\u003e10’ \u003c/sub\u003eto control the least-stable G1208-T1396 H-bond in the blue box.\u003c/p\u003e","description":"","filename":"Figure3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7339733/v1/a1c7d2f088c92c9eb4da0117.jpg"},{"id":88866460,"identity":"b5ff40b6-a343-4152-b4e9-a7c6f8a73c38","added_by":"auto","created_at":"2025-08-12 08:34:22","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":269583,"visible":true,"origin":"","legend":"\u003cp\u003eThe thermoring structures of NBD2 in phosphorylated hCFTR/E1371Q/DF508 with VX-445/VX809 bound in an activated intermediate at 4 °C. (\u003cstrong\u003ea\u003c/strong\u003e) The grid-like noncovalently interacting mesh network of NBD2 based on the cryo-EM structure of hCFTR/E1371Q/DF508 with VX-445/VX809 bound in the presence of Mg/ATP/PKA at 4 °C (PDB ID, 8EIO, 2.8 Å). No software was used to create the image.\u0026nbsp; Salt bridges, H-bonds and pi interactions are colored purple, orange, and green, respectively. The constrained grid sizes needed to control the least-stable noncovalent interactions in the grids are labeled with black numbers. The least-stable G1208-T1396 H-bond in the biggest Grid\u003csub\u003e13\u003c/sub\u003e is highlighted.\u0026nbsp; The total grid sizes and the total grid size-controlled noncovalent interactions along the single peptide chain of NBD2 from I1203 to P1451 are shown in cyan and black circles, respectively. (\u003cstrong\u003eb\u003c/strong\u003e) The NBD2-ICL2 interactions. (\u003cstrong\u003ec\u003c/strong\u003e) The structure of the biggest Grid\u003csub\u003e13 \u003c/sub\u003ewith a 13-residue size to control the least-stable G1208-T1396 H-bond. \u0026nbsp;The grid size and the equivalent basic H-bonds for the least-stable noncovalent interaction are shown in and near a red circle. (\u003cstrong\u003ed\u003c/strong\u003e) The sequence of the biggest Grid\u003csub\u003e13 \u003c/sub\u003eto control the least-stable G1208-T1396 H-bond in the blue box.\u003c/p\u003e","description":"","filename":"Figure4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7339733/v1/c3139e06310c459889be7562.jpg"},{"id":88867364,"identity":"ded6c356-155c-4272-986d-d264aa98c911","added_by":"auto","created_at":"2025-08-12 08:42:22","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":249442,"visible":true,"origin":"","legend":"\u003cp\u003eThe thermoring structures of NBD2 in phosphorylated hCFTR/E1371Q/DF508 with Mg\u003csup\u003e2+\u003c/sup\u003e/ATP/elexacaftor (VX445) bound in the closed state at 4 °C. (\u003cstrong\u003ea\u003c/strong\u003e) The grid-like noncovalently interacting mesh network of NBD2 based on the cryo-EM structure of hCFTR/E1371Q/DF508 with elexacaftor bound in the presence of Mg/ATP/PKA at 4 °C (PDB ID, 8EIG, 3.7 Å). No software was used to create the image.\u0026nbsp; Salt bridges, H-bonds and pi interactions are colored purple, orange, and green, respectively. The constrained grid sizes necessary to control the least-stable noncovalent interactions in the grids are labeled with black numbers. The least-stable I1226-N1419 H-bond in the biggest Grid\u003csub\u003e12‘ \u003c/sub\u003eis highlighted.\u0026nbsp; The total grid sizes and the total grid size-controlled noncovalent interactions along the single peptide chain of NBD2 from I1203 to K1429 are shown in cyan and black circles, respectively. (\u003cstrong\u003eb\u003c/strong\u003e) The NBD2-ICL2 interactions. (\u003cstrong\u003ec\u003c/strong\u003e) The structure of the biggest Grid\u003csub\u003e12’ \u003c/sub\u003ewith a 12-residue size to control the least-stable I1226-N1419 H-bond. The grid size and the equivalent basic H-bonds for the least-stable noncovalent interaction are shown in and near a red circle. (\u003cstrong\u003ed\u003c/strong\u003e) The sequence of the biggest Grid\u003csub\u003e12’ \u003c/sub\u003eto control the least-stable I1226-N1419 H-bond in the blue box.\u003c/p\u003e","description":"","filename":"Figure5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7339733/v1/0108c55d08b28e79a2145627.jpg"},{"id":88867361,"identity":"dee50d24-5e59-4ecb-ad11-dd17ac95cd4f","added_by":"auto","created_at":"2025-08-12 08:42:22","extension":"jpg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":180044,"visible":true,"origin":"","legend":"\u003cp\u003eThermostability comparison between NBD1 and NBD2 upon Mg/ATP-induced dimerization for CFTR activation. \u0026nbsp;Cryo-EM structures of phosphorylated hCFTR/E1371Q with Mg/ATP bound (PDB: 6MSM), phosphorylated hCFTR/E1371Q with Mg/ATP/VX-770 bound (PDB: 6O2P), phosphorylated hCFTR/DF508/E1371Q with Mg/ATP/elexacaftor bound (PDB: 8EIG), phosphorylated hCFTR/DF508/E1371Q with Mg/ATP/VX-445/VX-809 bound (PDB: 8EIO), and phosphorylated hCFTR/DF508/E1371Q with Mg/ATP/Trikafta bound (PDB: 8EIQ) in a closed or activated intermediate are used for the model.\u0026nbsp; The Y577-H1375 pi interaction at the NBD1-NBD2 interface acts as a dimerization hallmark.\u0026nbsp; The calculated melting thresholds (T\u003csub\u003em,th\u003c/sub\u003e) and systematic thermal instabilities (T\u003csub\u003ei\u003c/sub\u003e) of NBD1 and NBD2 and their differences are shown below them.\u003c/p\u003e","description":"","filename":"Figure6.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7339733/v1/6b38f84de4e979b789abf390.jpg"},{"id":99643555,"identity":"331b64aa-5ad4-44a2-98fd-70c434efaddd","added_by":"auto","created_at":"2026-01-06 19:24:51","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2501422,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7339733/v1/65452dd3-f2f9-41ea-8893-fa21c0749f32.pdf"},{"id":88867651,"identity":"25cd8796-cf94-44ca-897c-65fbcd5598df","added_by":"auto","created_at":"2025-08-12 08:50:22","extension":"pdf","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":323575,"visible":true,"origin":"","legend":"\u003cp\u003eSupplemntal Tables S1-S5\u003c/p\u003e","description":"","filename":"SupplementaryInformationCFTRpotentiationmechanismv1.6.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7339733/v1/1f599bb96de6446e66ee370e.pdf"}],"financialInterests":"The authors declare no competing interests.","formattedTitle":"\u003cp\u003e\u003cstrong\u003eThermodynamic basis for CFTR activity potentiation\u003c/strong\u003e\u003c/p\u003e","fulltext":[{"header":"Introduction","content":"\u003cp\u003eInduced fit is a term used to describe the process in which an enzyme's shape changes to fit its substrate during a reaction. An example of this can be seen in a metallo-dependent or class II Fructose-1,6-bisphosphate (FBP) aldolase from the extreme thermophile, \u003cem\u003eThermus aquaticus\u003c/em\u003e (Taq). This enzyme is a tetramer composed of two dimers. The loop region of each subunit can be in an open or closed conformation near the active site, facilitating the relocation of metal for keto bond polarization during catalysis (\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e). Another example is the multiple inositol polyphosphate phosphatases (MINPP) from the Gram-positive bacterium \u003cem\u003eBifidobacterium longum\u003c/em\u003e (BlMINPP). This enzyme has an α-domain polypeptide insertion called the U-loop, which is responsible for large ligand-driven conformational changes during the catalytic cycle, thermal stability, recovery of activity after heating, and kinetic parameters for hydrolysis of phytate (\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eFurthermore, a ligand-induced conformational change in a dimerization loop is crucial for epidermal growth factor (EGF) receptor dimerization and activation. An abnormal orientation between two loop-related domains can cause autoinhibition (\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e)\u003c/p\u003e\u003cp\u003eInduced fit has also been proposed to explain the gating mechanisms of the cystic fibrosis (CF) transmembrane conductance regulator (CFTR). The allosteric nature of this ligand-gated channel is combined with the enzymatic activity of an ATP-binding Cassette (ABC) transporter (\u003cspan additionalcitationids=\"CR5 CR6\" citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e). Although the Mg/ATP-mediated dimerization between two nucleotide binding domains (NBD1 and NBD2) upon regulatory (R) domain phosphorylation by protein kinase A (PKA) is critical for coupling two transmembrane domains (TMD1 and TMD2) via intracellular loops (ICLs) for CFTR activation (\u003cspan additionalcitationids=\"CR9 CR10 CR11\" citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e), the specific thermodynamic basis for the induced fit across the interdomain interfaces is still missing.\u003c/p\u003e\u003cp\u003eBoth NBD1 and NBD2 in hCFTR have highly conserved sequences and motifs at a dimerization interface (\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e). In an ATP-binding \u0026ldquo;head\u0026rdquo; subdomain, the aromatic ring of ATP is packed against W401 in NBD1 or Y1219 in NBD2 via a π\u0026minus;π interaction, and the remaining phosphate moiety is anchored by several parts via H-bonds. These parts include the Walker A motif (\u003csup\u003e458\u003c/sup\u003eGSTGAGKTS\u003csup\u003e466\u003c/sup\u003e in NBD1 and \u003csup\u003e1244\u003c/sup\u003eGRTGSGKST\u003csup\u003e1252\u003c/sup\u003e in NBD2), the Walker B motif (from R560 to D572 and S573 in NBD1 or from R1358 to D1370 and E1371 in NBD2), and the switch regions (Q-loop involving Q493 in NBD1 or Q1291 in NBD2 while H-loop containing S605 in NBD1 or H1402 in NBD2). In the α-helical (or \u0026ldquo;tail\u0026rdquo;) subdomain, the conserved ATP-binding cassette (ABC) signature (\u003csup\u003e548\u003c/sup\u003eLSGGQ\u003csup\u003e552\u003c/sup\u003e in NBD1 but \u003csup\u003e1346\u003c/sup\u003eLSHGH\u003csup\u003e1350\u003c/sup\u003e in NBD2), together with the Walker, is also needed to sandwich ATP. Based on this head-to-tail dimerization configuration, the two ATP-binding sites are inherently asymmetric because only E1371 and H1402 at site 2 rather than S573 and S605 at site 1 can hydrolyze ATP (\u003cspan additionalcitationids=\"CR16 CR17 CR18\" citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eThe cryo-electron microscopy (cryo-EM) structure of human CFTR revealed that NBD1 and NBD2 are separated by the R domain in the dephosphorylated closed state (PDB, 5UAK). NBD1 contains a disordered regulatory insertion (RI) (spanning residues 405\u0026ndash;436) and an unstructured regulatory extension (RE) (spanning residues 647\u0026ndash;678), which are unique to CFTR compared to other ABC transporters. On the other hand, NBD2 maintains its entire structure within a peptide range from 1207 to 1436 (\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e). Upon R domain phosphorylation and Mg/ATP-binding (PDB, 6MSM), NBD1 and NBD2 dimerize. In this state, the unstructured RI in NBD1 shortens to a segment from 410 to 434, while the disordered RE in NBD1 begins at residue 638. Meanwhile, structured NBD2 expands from 1202 to 1451 (\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eOn the other hand, when the most common cystic fibrosis-causing F508 deletion destabilizes NBD1 (\u003cspan additionalcitationids=\"CR21 CR22 CR23 CR24\" citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e), the ATP-dependent NBD dimerization is compromised (\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e). Although a combination of folding correctors elexacaftor/VX-445 and lumacaftor/VX-809 is enough to restore the NBD dimerization (\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e), the activity potentiator ivacaftor/VX-770 in Trikafta is still necessary to maximize the F508del activity (\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e, \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e). Given that the additional binding of VX-770 to F508del-CFTR with VX-445, VX-661 bound fail to further enhance the ICL4-NBD1-NBD2 and TMD1-TMD2 interactions (\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e, \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e), how VX-770 potentiates the channel activity is still unknown.\u003c/p\u003e\u003cp\u003eIn this study, a highly-sensitive grid thermodynamic model, which was recently developed and examined (\u003cspan additionalcitationids=\"CR31 CR32 CR33 CR34 CR35 CR36 CR37 CR38\" citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e), was used to test a hypothesis that comparable thermostability between Mg/ATP-dimerized NBD1 and NBD2 is required for maximal channel activity. To confirm this hypothesis, the VX-770-induced changes in the thermoring structures of NBD2 and the ICL2-NBD2 interactions of hCFTR/E1371Q and (F508del)hCFTR/E1371Q were further analyzed and compared with the counterparts of NBD1 in tightly dimerized or partially dimerized states. The results showed that the binding of VX-770 to hCFTR with or without F508 significantly induced comparable thermostability between Mg/ATP-dimerized NBD1 and NBD2, stabilizing an activated intermediate for maximal channel activity with a minimal activation energy barrier. Therefore, a global induced fit across the interdomain interfaces was still necessary to rescue the gating defect of the F508del mutant.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cb\u003eMelting threshold of the dimerized NBD2 in phosphorylated (E1371Q)hCFTR with Mg/ATP bound matched the thermal inactivation temperaqture of 45\u0026deg;C\u003c/b\u003e\u003c/p\u003e\u003cp\u003eWhen (E1371Q)hCFTR is phosphorylated for Mg/ATP-dependent NBD1-NBD2 dimerization, the NBD2 (PDB, 6MSM) is structured from D1202 to P1451 but becomes disordered after P1451 (\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e). In this case, it is worth examining if NBD2 has a melting threshold (T\u003csub\u003em,th\u003c/sub\u003e) that is comparable to that of NBD1.\u003c/p\u003e\u003cp\u003eSimilar to the normal Mg/ATP binding site in NBD1 (\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e), a stable and rigid triangle was formed by S1251, Q1291 and D1370 via Mg\u003csup\u003e2+\u003c/sup\u003e in NBD2. Meanwhile, ATP connected Y1219, T1246, K1250 and T1252 (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea-b, Table S1). Notably, in addition to the H1348-H1375 H-bond between α and β subdomains, I1226-N1419-E1228 and T1246-I1416-S1248 H-bonds appeared between N- and C-termini (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea). When the total numbers of noncovalent interactions and grid sizes were 45 and 76 (Table S1), respectively, the systematic thermal instability (T\u003csub\u003ei\u003c/sub\u003e) of NBD2 was 1.69 in 6MSM (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e), lower than the NBD1\u0026rsquo;s 1.88 in the same construct (\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eGrid thermodynamic model-based new parameters of NBD2.\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"6\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eConstruct\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colspan=\"5\" nameend=\"c6\" namest=\"c2\"\u003e\u003cp\u003ehCFTR/E1371Q\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003ePDB ID\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e6MSM\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e6O2P\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e8EIG\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e8EIO\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e8EIQ\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eF508\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e+\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e+\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eMg/ATP\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e+\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e+\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e+\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e+\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e+\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003ePhosphorylation\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e+\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e+\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e+\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e+\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e+\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eSampling temperature, \u0026deg;C\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e4\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e4\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e4\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e4\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e4\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eTight NBD dimerization\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e+\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e+\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e+\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e+\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eNBDi\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e2\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eNormal Mg\u003csup\u003e2+\u003c/sup\u003e site\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e+\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eName of the biggest grid\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eGrid\u003csub\u003e12\u003c/sub\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eGrid\u003csub\u003e10\u003c/sub\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eGrid\u003csub\u003e12\u0026rsquo;\u003c/sub\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eGrid\u003csub\u003e13\u003c/sub\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eGrid\u003csub\u003e10\u0026rsquo;\u003c/sub\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eGrid size (s)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e12\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e10\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e12\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e13\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e10\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e# of energetically equivalent basic H-bonds (n) controlled by Grid\u003csub\u003es\u003c/sub\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e1.5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e1.5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e1.8\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e1.5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e1.5\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eTotal non-covalent interactions (N)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e45\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e42\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e46\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e52\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e51\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eTotal grid sizes (S), a.a.\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e76\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e53\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e103\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e75\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e91\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eSystematic thermal instability (T\u003csub\u003ei\u003c/sub\u003e)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e1.69\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e1.26\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e2.24\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e1.44\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e1.78\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eCalculated T\u003csub\u003em,th,\u003c/sub\u003e \u0026deg;C\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cb\u003e45\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e49\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e48\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e43\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e49\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eMeasured inactivation temperature, \u0026deg;C\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cb\u003e45\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eRef. for measured T\u003csub\u003einact\u003c/sub\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e(\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003eAt the NBD2-ICL2 interface, in addition to the E264-S1297 H-bond, seven π interactions such as R1358-Y1307-W277-F1296 and N1303-F1296-F1294-W277-Y275 emerged. Similar to the triangle formed by F508, Y1073 and F1074 at the NBD1-ICL4 interface (\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e), F1296, W277, and F1294 also formed the smallest triangle to stabilize or rigidize the NBD2-ICL2 interface in this activated intermediate (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eb).\u003c/p\u003e\u003cp\u003eConsequently, although the weakest Q525-E585 H-bond between the α and β subdomains finalizes the posttranslational folding of NBD1 (\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e), the least-stable H-bond between the side chain of T1396 in the β-subdomain and the backbone C\u0026thinsp;=\u0026thinsp;O of G1208 in the N-terminal subdomain was responsible for the final posttranslational folding of NBD2 (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea). It was controlled by the biggest Grid\u003csub\u003e12\u003c/sub\u003e via a thermoring from L1367, D1370, Mg\u003csup\u003e2+\u003c/sup\u003e, Q1291, F1286, W1282, W1204, G1208, T1396, F1392, and back to L1367 (Figs.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ec-d). For 1.5 equivalent basic H-bonds to seal it, the calculated T\u003csub\u003em,th\u003c/sub\u003e of NBD2 in the 6MSM structure was approximately 45\u0026deg;C (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e), lower than the T\u003csub\u003em,th\u003c/sub\u003e of 50\u0026deg;C for NBD1 in the same structure (30). Therefore, the thermostability between the dimerized NBD1 and NBD2 in hCFTR was not comparable upon Mg/ATP binding. Given that the predicted T\u003csub\u003em,th\u003c/sub\u003e of 45\u0026deg;C of NBD2 was in excellent agreement with the experimental inactivation temperature of WT hCFTR (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e) (\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e), the thermal inactivation may result from the heat unfolding of the weakest G1208-T1396 H-bond of NBD2.\u003c/p\u003e\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003eComparable thermostability between the dimerized NBD1 and NBD2 in phosphorylated (E1371Q)hCFTR with Mg/ATP/VX770 bound\u003c/h2\u003e\u003cp\u003eAlong with additional VX-770 binding to the TMD1/TMD2 interface to enhance CFTR activity (\u003cspan additionalcitationids=\"CR42 CR43 CR44\" citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e), the same NBD2-ICL2 interactions were also associated with the weakest G1208-T1396 H-bond (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea-b, Table S2). However, this H-bond was controlled by the biggest Grid\u003csub\u003e10\u003c/sub\u003e via a thermoring from L1367 to D1370, S1251, Q1291, F1286, W1282, W1274, D1270, G1208, T1396, F1392 and back to L1367 (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ec-d). When this H-bond was energetically equivalent to 1.5 basic H-bonds (1.5 kcal/mol), the calculated T\u003csub\u003em,th\u003c/sub\u003e of NBD2 in 6O2P was about 49\u0026deg;C (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e), similar to the T\u003csub\u003em,th\u003c/sub\u003e of 50\u0026deg;C for NBD1 in 6O2P (\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e). Therefore, the comparable thermostability between NBD1 and NBD2 was observed with their dimerization induced by Mg/ATP/VX770 binding in the activated intermediate to maximize channel activity. Meanwhile, along with a decrease in the totals of noncovalent interactions and grid sizes from 45 and 76 in 6MSM to 42 and 53 in 6O2P, the systematic thermal instability (Ti) of NBD2 also declined from 1.69 to 1.26 (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eGiven that Trikafta contains the potentiator VX-770 and significantly enhances the activity of (F508del)hCFTR (\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e), it is intriguing to explore whether tight NBD dimerization induced by Mg/ATP/Trikafta also corresponds to balanced thermostability for both NBD1 and NBD2 even in the presence of the destabilizing F508 deletion.\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eMatched thermostability between the dimerized NBD1 and NBD2 in phosphorylated (E1371Q/F508del)hCFTR with Mg/ATP/Trikafta bound\u003c/h3\u003e\n\u003cp\u003eWhen F508 was deleted, both NBD1 and NBD2 changed their thermoring structures to maintain Mg/ATP-dependent dimerization in the presence of Trikafta (Figs.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea; Table S3) (\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e). In addition to the intact ATP site and the compromised S1251-Mg\u003csup\u003e2+\u003c/sup\u003e-Q1291 bridge along with the broken R1358-Y1307 CH-π interaction at the NBD2-ICL2 interface (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea-b), only a small fraction of 37 noncovalent interactions were conserved in NBD2. For example, the R1259-W1274-W1282-F1286, F1294-F1296-N1303, V1327-H1350, F1331-F1337 and L1367-F1392 π interactions, together with the Y1219-T1252 and S1373-E1401 H-bonds. Despite these changes, the G1208-T1396 H-bond between N-terminal and β- subdomains was still the weakest to finalize the posttranslational NBD2 folding (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea). However, it was governed by another biggest Grid\u003csub\u003e10\u0026rsquo;\u003c/sub\u003e via a thermoring from S1251, Mg\u003csup\u003e2+\u003c/sup\u003e, Q1291, F1286, W1282, W1274, D1270, G1207, G1208, T1396, C1400, D1370, and back to S1251 (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ec-d). Since the weakest G1208-T1396 H-bond was still energetically equivalent to 1.5 basic H-bonds, the calculated melting threshold (T\u003csub\u003em,th\u003c/sub\u003e) was 49\u0026deg;C (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e), which was exactly the same as the 49\u0026deg;C of NBD1 in the same construct (\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e). On the other hand, when the total numbers of noncovalent interactions and grid sizes increased from 42 and 53 to 51 and 91, respectively, the systematic thermal instability (T\u003csub\u003ei\u003c/sub\u003e) of NBD2 also increased from 1.26 to 1.78 (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eIn contrast, when Trikafta was replaced with VX-445 and VX-809 in 8EIO, the weakest G1208-T1396 H-bond in dimerized NBD2 was governed by the biggest Grid\u003csub\u003e13\u003c/sub\u003e via a thermoring from L1367 to D1370, S1251, Mg\u003csup\u003e2+\u003c/sup\u003e, Q1291, F1286, W1282, W1274, I1267, I1269, W1204, G1208, T1396, F1392, and back to L1367 (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e; Table S4). Since this H-bond was energetically equivalent to 1.5 basic H-bonds (1.5 kcal/mol), the calculated T\u003csub\u003em,th\u003c/sub\u003e of NBD2 in 8EIO was 43\u0026deg;C (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e), which was 4\u0026deg;C lower than the T\u003csub\u003em,th\u003c/sub\u003e of 47\u0026deg;C for NBD1 in the same construct (\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e). Notably, despite a decrease in the T\u003csub\u003em,th\u003c/sub\u003e of NBD2, the systematic thermal instability (Ti) of NBD2 reduced from 1.78 in 8EIQ to 1.44 in 8EIO (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003eSignificant imbalance in thermostability between partially dimerized NBD1 and NBD2 in phosphorylated (F508del)hCFTR/E1371Q with only Mg/ATP/elexacaftor bound\u003c/b\u003e\u003c/p\u003e\u003cp\u003eTo further investigate the comparable thermostability between NBD1 and NBD2 as a requirement for Trikafta to maximize the channel activity of the F508del mutation, it is necessary to determine if their comparable thermostability would be significantly compromised in a partially dimerized state.\u003c/p\u003e\u003cp\u003eWhen only elexacaftor and Mg\u003csup\u003e2+\u003c/sup\u003e/ATP are bound to phosphorylated closed (E1371Q/F508del)hCFTR, NBD1 and NBD2 are partially dimerized (\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e). In this scenario, the total numbers of noncovalent interactions and grid sizes of NBD2 changed from 52 and 75 in 8EIO to 46 and 103 in 8EIG, respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ea; Table S5). Hence, the systematic thermal instability (T\u003csub\u003ei\u003c/sub\u003e) of NBD2 significantly increased from 1.44 in 8EIO to 2.24 in 8EIG (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eNotably, the least-stable G1208-T1396 H-bond in the biggest Grid\u003csub\u003e13\u003c/sub\u003e was replaced by the least-stable I1226-N1419 H-bond in the biggest Grid\u003csub\u003e12\u0026rsquo;\u003c/sub\u003e of NBD2 while the R1358-Y275 π interaction was reestablished at the NBD2-ICL2 interface (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ea-b). This biggest thermoring cycled from I1226 to S1233, Q1412, N1419, and back to I1226 (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ec-d). Since this H-bind was energetically equivalent to 1.8 basic H-bonds, the calculated T\u003csub\u003em,th\u003c/sub\u003e of NBD2 in 8EIG was about 48\u0026deg;C, significantly 9\u0026deg;C higher than the T\u003csub\u003em,th\u003c/sub\u003e of 39\u0026deg;C in NBD1 (\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e). Therefore, comparable thermostability between Mg/ATP-dimerized NBD1 and NBD2 was indeed required for Trikafta to maximize the channel activity of the F508del mutant.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eInterdomain interactions play a crucial role in regulating CFTR activity, yet little is known about how each domain responds to different physical, chemical and genetic stimuli during this process. This study revealed that while the apparent secondary structures of dimerized NBD1 and NBD2 remained the same or similar before \u003cem\u003evs.\u003c/em\u003e after the introduction of the F508 deletion along with various folding correctors, their tertiary thermoring structures underwent a global change, rearranging interdomain interactions. Furthermore, although the additional binding of VX770 to hCFTR or F508del with folding correctors VX445 and VX-809 did not enhance interdomain interactions, a global induced fit across domain-domain interfaces significantly triggered a conformational selection step, ensuring comparable thermostability between Mg/ATP-dimerized NBD1 and NBD2 to stabilize the activated intermediate with a minimal activation energy barrier for maximal channel activity of hCFTR, regardless of the presence of F508del.\u003c/p\u003e\n\u003ch3\u003eComparable thermostability between the dimerized NBD1 and NBD2 upon VX-770 binding maximizes hCFTR activity\u003c/h3\u003e\n\u003cp\u003eIn this study, despite the tight NBD1-ICL4 or NBD2-ICL2 interactions still allowing for intact Mg/ATP binding sites in the phosphorylated activated intermediate of hCFTR/E1371Q (6MSM) (Figs.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea-b) (\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e), both dimerized NBD1 and NBD2 exhibited different melting thresholds (T\u003csub\u003em,th\u003c/sub\u003e) of 50\u0026deg;C and 45\u0026deg;C, respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e). Given that the WT hCFTR channel starts thermal inactivation at 45\u0026deg;C (\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e), the inactivation may be due to the unfolding of least-stable G1208-T1396 bridge in NBD2 at the T\u003csub\u003em,th\u003c/sub\u003e of 45\u0026deg;C (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e, Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e). When the potentiator VX-770 was bound to the E1371Q mutant increasing channel activity, the dimerized NBD2 increased the T\u003csub\u003em,th\u003c/sub\u003e from 45\u0026deg;C to 49\u0026deg;C (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e). Thus, although VX-770 still potentiates the WT, G551D and W1282X-CFTR activity in a phosphorylation-dependent but ATP-independent manner (\u003cspan additionalcitationids=\"CR45\" citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e), VX-770-induced comparable thermostability between dimerized NBD1 and NBD2 favors the maximal activity of hCFTR in an ATP-dependent manner.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\n\u003ch3\u003eMatching thermostability between dimerized NBD1 and NBD2 upon Trikafta binding maximizes (F508del)hCFTR activity\u003c/h3\u003e\n\u003cp\u003eIn the previous study, two identical isolated hNBD1-Δ(RI, RE) or (F508del)hNBD1-Δ(RI,RE) constructs with the same thermostability can form a stable homodimer (2PZE or 2PZF) for crystal capture upon Mg/ATP binding, suggesting that the RI or RE may serve as an important dimerization loop or segment (\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e). In this study, despite the instablity of NBD1 in ΔF508 (\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e), tightly dimerized NBD1 and NBD2 upon Trikafta binding to TMD1 and TMD2 still shared a common T\u003csub\u003em,th\u003c/sub\u003e of 49\u0026deg;C (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e) (\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e). However, in the presence of elexacaftor and lumacaftor, the T\u003csub\u003em.th\u003c/sub\u003e of NBD1 was 47\u0026deg;C, slightly higher than the T\u003csub\u003em.th\u003c/sub\u003e of 43\u0026deg;C in NBD2 (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e). Further, the T\u003csub\u003em,th\u003c/sub\u003e values of the partially-dimerized NBD1 and NBD2 in phosphorylated (F508del)hCFTR/E1371Q with elexacaftor bound were 39\u0026deg;C and 48\u0026deg;C, respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e) (\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e). Therefore, matching thermostability between dimerized NBD1 and NBD2 upon Trikafta binding facilitates correcting the thermal and gating defects of the F508del mutation.\u003c/p\u003e\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\u003ch2\u003eAsymmetric weakest noncovalent bridges across the dimerization interface\u003c/h2\u003e\u003cp\u003eAlthough both tightly dimerized NBD1 and NBD2 exhibited minimal differences in T\u003csub\u003em,th\u003c/sub\u003e under two various conditions, they had distinct but asymmetric weakest noncovalent bridges (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e). When the potentiator VX-770 was bound to hCFTR/E1371Q, the biggest Grid\u003csub\u003e8\u003c/sub\u003e in dimerized NBD1 was found to be responsible for the least-stable Q525-E585 H-bond between α and β-subdomains (\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e). However, the biggest Grid\u003csub\u003e10\u003c/sub\u003e in dimerized NBD2 was responsible for the least-stable G1208-T1396 H-bond between C- and N-termini (Figs.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea, \u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e). After Trikafta modulators are bound to the E1371Q/F508del mutant, the least-stable Y517-D537 H-bond finalized posttranslational NBD1 folding (\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e). In contrast, the weakest G1208-T1396 H-bond was still the last step of the posttranslational NBD2 folding (Figs.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea, \u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e). Since this weakest link was also the final posttranslational CFTR folding, its stability is essential for optimizing CFTR activity and cystic fibrosis treatment.\u003c/p\u003e\u003cp\u003eTaken together, while folding modulators in Trikafta shift the weakest tertiary link to serve as the final posttranslational NBD1 folding event, they do not impact the final posttranslational NBD2 folding. Conversely, the potentiator VX-770 in Trikafta is essential for the stabilized activated intermediate to maximal channel activity by adjusting the size of the biggest thermoring in NBD2 to align with the thermal stability of NBD1. Therefore, these three modulators in Trikafta play distinct roles in addressing thermal and gating abnormalities in (F508del)hCFTR (\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e, \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e). In fact, in a cellular environment where Mg\u003csup\u003e2+\u003c/sup\u003e, ATP and PKA are present along with an allosteric drug like Trikafta or ivacaftor, the induced comparable thermostability between the dimerized NBD1 and NBD2 is essential for CFTR folding, stability and effective therapy, particularly when compromised by disease mutations such as F508del and G551D (\u003cspan additionalcitationids=\"CR21 CR22 CR23 CR24 CR25 CR26\" citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e, \u003cspan additionalcitationids=\"CR48 CR49\" citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e).\u003c/p\u003e\u003c/div\u003e"},{"header":"Conclusions","content":"\u003cp\u003eInduced fit plays a significant role in allosteric enzymatic reactions, especially in the case of CFTR. CFTR functions not only as an anion channel but also as an ATPase. In the CFTR gating cycle, induced fit at the dimerization interface between NBD1 and NBD2 is crucial for an \u0026ldquo;interdependent protein dance\u0026rdquo;. When two Mg/ATP agonists bind normally at two interfacial sites, they dimerize NBD1 and NBD2 after R domain phosphorylation. A VX-770-inducd conformational selection step leads to an expanded induced fit between dimerized NBD1 and NBD2. As a result, their thermostability closes to each other to stabilize the activated intermediate with minimal activation energy barrier for maximal channel activity of hCFTR with or without F508del and folding correctors. Therefore, the increasing precision of thermostability evaluation permits increasing activation mechanistic detail and allosteric drug design to optimize the cystic fibrosis treatment.\u003c/p\u003e"},{"header":"Computational Methods","content":"\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\u003ch2\u003eData mining resources\u003c/h2\u003e\u003cp\u003eThermoring structures of phosphorylated and Mg/ATP bound hCFTR constructs with or without various modulators in the activated intermediate were analyzed using cryo-EM structures at 4\u0026deg;C. The structures without F508del included hCFTR/1371Q with Mg/ATP bound (PDB ID, 6MSM, model resolution\u0026thinsp;=\u0026thinsp;3.2 \u0026Aring;) (\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e), and with Mg/ATP/VX-770 bound (PDB ID, 6O2P, model resolution\u0026thinsp;=\u0026thinsp;3.3 \u0026Aring;) (\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e). Furthermore, the structures with F508del covered hCFTR/E1371Q/ΔF508 with Mg/ATP/ elexacaftor (VX445) bound (PDB ID, 8EIG, model resolution\u0026thinsp;=\u0026thinsp;3.7 \u0026Aring;), Mg/ATP/VX445/VX809 bound (PDB ID, 8EIO, model resolution\u0026thinsp;=\u0026thinsp;2.8 \u0026Aring;), and Mg/ATP/Trikafta bound (PDB ID, 8EIQ, model resolution\u0026thinsp;=\u0026thinsp;3.2 \u0026Aring;) (\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\u003ch2\u003eStandard methods for filtering tertiary non-covalent interactions\u003c/h2\u003e\u003cp\u003eTertiary non-covalent interactions such as salt bridges, H-bonds and π interactions in NBD2 were filtered using standard methods and precise calculations previously employed to ensure accurate and reproducible results (\u003cspan additionalcitationids=\"CR31 CR32 CR33 CR34 CR35 CR36 CR37 CR38\" citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e). Detailed cutoff distances and interaction angles (for an H-bond) can be found in the online Supplementary Information (Tables S1, S2, S3, S4 and S5). It shoud be noted that momentary fluctuation-induced perturbations in noncovalent interactions during protein dynamics were not considered in this study. Thus, approximately 42\u0026ndash;52 noncovalent interactions were identified along the single peptide chain from D1202 to P1451 in NBD2 of each protomer.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e\u003ch2\u003eCalculations based on the thermoring structures and the grid thermodynamic model\u003c/h2\u003e\u003cp\u003eThe previously established grid thermodynamic model was used to map the systematic fluidic grid-like noncovalent interaction mesh networks of NBD1 and NBD2 (\u003cspan additionalcitationids=\"CR31 CR32 CR33 CR34 CR35 CR36 CR37 CR38\" citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e). In these networks, identified noncovalent interactions and linked amino acid residues were represented by edges and nodes, respectively. When a noncovalent interaction had a direct zero-length path between two linked nodes and the shortest reverse path from one node back to the other through other noncovalent interactions and a peptide segment, a thermoring or grid with the tightest network was formed to control this least-stable noncovalent interaction within it. The grid size was defined as the free or silent residues not involved in any noncovalent interactions along the shortest reverse path.\u003c/p\u003e\u003cp\u003eIn this way, each thermoring acted like a bow and each least-stable noncovalent interaction functioned like a vibrating bowstring. Thus, the strength of each bowstring was determined not only by itself but also regulated by the bow length, the relevant energy relocation and allosteric propagation. Generally, the intensity of a noncovalent interaction or a bowstring was limited to 1\u0026ndash;3 kcal/mol. However, the more free or flexible side chains along the thermoring or the bow, or the larger the thermoring size or the longer the bow, the weaker the controlled noncovalent interaction or the bowstring.\u003c/p\u003e\u003cp\u003eOnce the biggest grid was identified, the least-stable noncovalent interaction within it was typically the weakest one along the entire polypeptide chain. Its heat unfolding could be characterized by a specific melting temperature threshold using the following equation as previously examined (\u003cspan additionalcitationids=\"CR31 CR32 CR33 CR34 CR35 CR36 CR37 CR38\" citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e):\u003c/p\u003e\u003cp\u003eT\u003csub\u003em,th\u003c/sub\u003e (\u0026deg;C)\u0026thinsp;=\u0026thinsp;34 + (n \u0026minus;\u0026thinsp;2) \u0026times; 10 + (20 \u0026ndash; s) \u0026times; 2 (\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e)\u003c/p\u003e\u003cp\u003ewhere, n represents the total number of basic H-bonds (each approximately 1 kcal/mol) equivalent to the least-stable noncovalent interaction controlled by the grid; and s is the grid size used to control the least-stable noncovalent interaction within the grid. In this study, although the I1226-N1419 H-bond was highly conserved in NBD2, it was not the weakest or least-thermostable except in 8EIG. For example, because E1228 also H-bonded to N1419 via their side chains in 6MSM, it was actually controlled by the smaller Grid\u003csub\u003e1\u003c/sub\u003e via a smaller thermoring from I1226 to E1228, N1419, and back to I1226 and the T\u003csub\u003em,th\u003c/sub\u003e to unfold it was at least 72\u0026deg;C (Figs.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea).\u003c/p\u003e\u003cp\u003eIn addition to the T\u003csub\u003em,th\u003c/sub\u003e, the total grid sizes (S) and the total non-covalent interactions (N) along the same polypeptide chain could be utilized to calculate grid-based systematic thermal instability (T\u003csub\u003ei\u003c/sub\u003e) using the same equation as examined previously (\u003cspan additionalcitationids=\"CR31 CR32 CR33 CR34 CR35 CR36 CR37 CR38\" citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e):\u003c/p\u003e\u003cp\u003eT\u003csub\u003ei\u003c/sub\u003e = S/N (\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e)\u003c/p\u003e\u003cp\u003eThis parameter reflects the peptide\u0026rsquo;s compact conformational entropy or flexibility.\u003c/p\u003e\u003c/div\u003e"},{"header":"Abbreviations","content":"\u003cp\u003eABC, ATP-binding cassette\u003c/p\u003e\n\u003cp\u003eBlMINPP, Gram-positive bacterium \u003cem\u003eBifidobacterium longum\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eCF, cystic fibrosis\u003c/p\u003e\n\u003cp\u003ecryo-EM, cryoelectron microscopy\u003c/p\u003e\n\u003cp\u003eCFTR, cystic fibrosis transmembrane conductance regulator\u003c/p\u003e\n\u003cp\u003eDSC, differential scanning calorimetry\u003c/p\u003e\n\u003cp\u003eEGF, epidermal growth factor\u003c/p\u003e\n\u003cp\u003eFBP, Fructose-1,6-bisphosphate\u003c/p\u003e\n\u003cp\u003ehCFTR, human CFTR\u003c/p\u003e\n\u003cp\u003eICL2,\u0026nbsp;intracellular loop 2\u003c/p\u003e\n\u003cp\u003eICL3,\u0026nbsp;intracellular loop 3\u003c/p\u003e\n\u003cp\u003eICL4, intracellular loop 4\u003c/p\u003e\n\u003cp\u003eMINPP, multiple inositol polyphosphate phosphatases\u003c/p\u003e\n\u003cp\u003eNBD1, nucleotide binding domain 1\u003c/p\u003e\n\u003cp\u003eNBD2, nucleotide binding domain 2\u003c/p\u003e\n\u003cp\u003ePKA, protein kinase A\u003c/p\u003e\n\u003cp\u003eR, regulatory\u003c/p\u003e\n\u003cp\u003eRE, regulatory extension\u003c/p\u003e\n\u003cp\u003eRI, regulatory insert\u003c/p\u003e\n\u003cp\u003eT\u003csub\u003ei\u003c/sub\u003e, systematic thermal instability\u003c/p\u003e\n\u003cp\u003eT\u003csub\u003em,th\u003c/sub\u003e, melting temperature threshold\u003c/p\u003e\n\u003cp\u003eTaq,\u003cem\u003e\u0026nbsp;Thermus aquaticus\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eTMD1, transmembrane domain 1\u003c/p\u003e\n\u003cp\u003eTMD2, transmembrane domain 2\u003c/p\u003e\n\u003cp\u003eWT, wild type\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe author\u0026rsquo;s own studies cited in this article were supported by the NIDDK Grant (DK45880 to D.C.D.) and the Cystic Fibrosis Foundation grant (DAWSON0210), the NIDDK grant (2R56DK056796-10) and the American Heart Association (AHA) Grant (10SDG4120011 to GW).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eG. W wrote the main manuscript text and prepared Figures. 1, 2, 3, 4, 5, 6 and Table 1, Supplementary Information (Tables S1, S2, S3, S4 and S5) and reviewed the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe author declares no conflict of interest.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eData are provided within the manuscript or supplementary information files\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAdditional information\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSupplementary Information\u003c/strong\u003e The online version contains supplementary material available.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eIzard T, Sygusch J (2004) Induced fit movements and metal cofactor selectivity of class II aldolases: structure of Thermus aquaticus fructose-1,6-bisphosphate aldolase. J Biol Chem 279:11825\u0026ndash;11833\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eAcquistapace IM, Zi Etek MA, Li AWH, Salmon M, K\u0026uuml;hn I, Bedford MR, Brearley CA, Hemmings AM (2020) Snapshots during the catalytic cycle of a histidine acid phytase reveal an induced-fit structural mechanism. J Biol Chem 295:17724\u0026ndash;17737\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eSchlessinger J (2002) Ligand-induced, receptor-mediated dimerization and activation of EGF receptor. \u003cem\u003eCell.\u003c/em\u003e 110, 669\u0026thinsp;\u0026ndash;\u0026thinsp;72\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eRiordan JR, Rommens JM, Kerem B, Alon N, Rozmahel R, Grzelczak Z, Zielenski J, Lok S, Plavsic N, Chou JL et al (1989) Identification of the cystic fibrosis gene: cloning and characterization of complementary DNA. Science 245:1066\u0026ndash;1073\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eHyde SC, Emsley P, Hartshorn MJ, Mimmack MM, Gileadi U, Pearce SR, Gallagher MP, Gill DR, Hubbard RE, Higgins CF (1990) Structural model of ATP-binding proteins associated with cystic fibrosis, multidrug resistance and bacterial transport. Nature 346:362\u0026ndash;365\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eDean M, Rzhetsky A, Allikmets R (2001) The human ATP-binding cassette (ABC) transporter superfamily. Genome Res 11:1156\u0026ndash;1166\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eKirk KL, Wang W (2011) A unified view of cystic fibrosis transmembrane conductance regulator (CFTR) gating: combining the allosterism of a ligand-gated channel with the enzymatic activity of an ATP-binding cassette (ABC) transporter. J Biol Chem 286:12813\u0026ndash;12819\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eLiu F, Zhang Z, Csan\u0026aacute;dy L, Gadsby DC, Chen J (2017) Molecular Structure of the Human CFTR Ion Channel. Cell 169:85\u0026ndash;95e8\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eZhang Z, Liu F, Chen J (2018) Molecular structure of the ATP-bound, phosphorylated human CFTR. Proc Natl Acad Sci U S A 115:12757\u0026ndash;12762\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eCsan\u0026aacute;dy L, Vergani P, Gadsby DC (2019) STRUCTURE, GATING, AND REGULATION OF THE CFTR ANION CHANNEL. Physiol Rev 99:707\u0026ndash;738\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eVernon RM, Chong PA, Lin H, Yang Z, Zhou Q, Aleksandrov AA, Dawson JE, Riordan JR, Brouillette CG, Thibodeau PH, Forman-Kay JD (2017) Stabilization of a nucleotide-binding domain of the cystic fibrosis transmembrane conductance regulator yields insight into disease-causing mutations. J Biol Chem 292:14147\u0026ndash;14164\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eLevring J, Terry DS, Kilic Z, Fitzgerald G, Blanchard SC, Chen J (2023) CFTR function, pathology and pharmacology at single-molecule resolution. \u003cem\u003eNature.\u003c/em\u003e 616(7957), 606\u0026ndash;614 \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1038/s41586-023-05854-7\u003c/span\u003e\u003cspan address=\"10.1038/s41586-023-05854-7\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e. Epub 2023 Mar 22. Erratum in: \u003cem\u003eNature.\u003c/em\u003e 617(7961), E11 (2023). doi: 10.1038/s41586-023-06115-3\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eLewis HA, Buchanan SG, Conners K, Dickey M, Dorwart M, Fowler R, Gao X, Guggino WB, Hendrickson WA, Hunt JF, Kearins MC, Lorimer D, Maloney PC, Post KW et al (2004) Structure of nucleotide-binding domain 1 of the cystic fibrosis transmembrane conductanceregulator. EMBO J 23:282\u0026ndash;293\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eLewis HA, Zhao X, Wang C, Sauder JM, Rooney I, Noland BW, Lorimer D, Kearins MC, Conners K, Condon B, Maloney PC, Guggino WB, Hunt JF, Emtage S (2005) Impact of the deltaF508 mutation in first nucleotide-binding domain of human cystic fibrosis transmembrane conductance regulator on domain folding and structure. J Biol Chem 280:1346\u0026ndash;1353\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eAleksandrov L, Aleksandrov AA, Chang XB, Riordan JR (2002) The First Nucleotide Binding Domain of Cystic Fibrosis Transmembrane Conductance Regulator Is a Site of Stable Nucleotide Interaction, whereas the Second Is a Site of Rapid Turnover. J Biol Chem 277:15419\u0026ndash;15425\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eBasso C, Vergani P, Nairn AC, Gadsby DC (2003) Prolonged nonhydrolytic interaction of nucleotide with CFTR\u0026rsquo;s NH2-terminal nucleotide binding domain and its role in channel gating. J Gen Physiol 122:333\u0026ndash;348\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eZaitseva J, Jenewein S, Jumpertz T, Holland IB, Schmitt L (2005) H662 is the linchpin of ATP hydrolysis in the nucleotide-binding domain of the ABC transporter HlyB. EMBO J 24:1901\u0026ndash;1910\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eZaitseva J, Oswald C, Jumpertz T, Jenewein S, Wiedenmann A, Holland IB, Schmitt L (2006) A structural analysis of asymmetry required for catalytic activity of an ABC-ATPase domain dimer. EMBO J 25:3432\u0026ndash;3443\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eStratford FL, Ramjeesingh M, Cheung JC, Huan LJ, Bear CE (2007) The Walker B motif of the second nucleotide-binding domain (NBD2) of CFTR plays a key role in ATPase activity by the NBD1-NBD2 heterodimer. Biochem J 401(2):581\u0026ndash;586. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1042/BJ20060968\u003c/span\u003e\u003cspan address=\"10.1042/BJ20060968\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eAtwell S, Brouillette CG, Conners K, Emtage S, Gheyi T, Guggino WB, Hendle J, Hunt JF, Lewis HA, Lu F, Protasevich II, Rodgers LA, Romero R, Wasserman SR, Weber PC, Wetmore D, Zhang FF, Zhao X (2010) Structures of a minimal human CFTR first nucleotide-binding domain as a monomer, head-to-tail homodimer, and pathogenic mutant. Protein Eng Des Sel 23:375\u0026ndash;384\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eProtasevich I, Yang Z, Wang C, Atwell S, Zhao X, Emtage S, Wetmore D, Hunt JF, Brouillette CG (2010) Thermal unfolding studies show the disease causing F508del mutation in CFTR thermodynamically destabilizes nucleotide-binding domain 1. Protein Sci 19:1917\u0026ndash;1931\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eWang C, Protasevich I, Yang Z, Seehausen D, Skalak T, Zhao X, Atwell S, Spencer Emtage J, Wetmore DR, Brouillette CG, Hunt JF (2010) Integrated biophysical studies implicate partial unfolding of NBD1 of CFTR in the molecular pathogenesis of F508del cystic fibrosis. Protein Sci 19:1932\u0026ndash;1947\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eRabeh WM, Bossard F, Xu H, Okiyoneda T, Bagdany M, Mulvihill CM, Du K, di Bernardo S, Liu Y, Konermann L, Roldan A, Lukacs GL (2012) Correction of both NBD1 energetics and domain interface is required to restore ∆F508 CFTR folding and function. Cell 148:150\u0026ndash;163\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eHe L, Aleksandrov AA, An J, Cui L, Yang Z, Brouillette CG, Riordan JR (2015) Restoration of NBD1 thermal stability is necessary and sufficient to correct ∆F508 CFTR folding and assembly. J Mol Biol 427:106\u0026ndash;120\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eSoya N, Roldan A, Lukacs GL (2019) Differential Scanning Fluorimetry and Hydrogen Deuterium Exchange Mass Spectrometry to Monitor the Conformational Dynamics of NBD1 in Cystic Fibrosis. Methods Mol Biol 1873:53\u0026ndash;67\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eJih KY, Li M, Hwang TC, Bompadre SG (2011) The most common cystic fibrosis-associated mutation destabilizes the dimeric state of the nucleotide-binding domains of CFTR. J Physiol 589(Pt 11):2719\u0026ndash;2731\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eFiedorczuk K, Chen J (2022) Molecular structures reveal synergistic rescue of ∆508 CFTR by Trikafta modulators. Science 378(6617):284\u0026ndash;290. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1126/science.ade2216\u003c/span\u003e\u003cspan address=\"10.1126/science.ade2216\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eVeit G, Roldan A, Hancock MA, Da Fonte DF, Xu H, Hussein M, Frenkiel S, Matouk E, Velkov T, Lukacs GL (2020) Allosteric folding correction of F508del and rare CFTR mutants by elexacaftor-tezacaftor-ivacaftor (Trikafta) combination. JCI Insight 5:e139983\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eVeit G, Vaccarin C, Lukacs GL (2021) Elexacaftor co-potentiates the activity of F508del and gating mutants of CFTR. J Cyst Fibros 20:895\u0026ndash;898\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eWang G (2025) Trikafta rescues F508del-CFTR by tightening specific phosphorylation-dependent interdomain interactions. Nat Sci 6:e70009. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1002/ntls.70009\u003c/span\u003e\u003cspan address=\"10.1002/ntls.70009\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eWang G Thermodynamic coupling between folding correctors and the first of dimerized nucleotide binding domains in CFTR. \u003cem\u003eACS Bio. Med.Au\u003c/em\u003e (in press). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1021/acsbiomedchemau.5c00014\u003c/span\u003e\u003cspan address=\"10.1021/acsbiomedchemau.5c00014\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eWang G (2023) The network basis for the structural thermostability and the functional thermoactivity of aldolase B. Molecules 28:1850\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eWang G (2023) Network basis for the heat-adapted structural thermostability of bacterial class II fructose bisphosphate aldolase. ACS Omega 8:17731\u0026ndash;17739\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eWang G (2023) Thermal ring-based heat switches in hyperthermophilic class II bacterial fructose aldolase. ACS Omega 8:24624\u0026ndash;24634\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eWang G (2023) Thermoring-Based Heat Activation Switches in the TRPV1 Biothermometer. Int J Biol Macromol 248:125915\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eWang G (2023) Thermoring basis for the TRPV3 bio-thermometer. Sci Rep 13:21594\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eWang G (2024) Phosphatidylinositol-4,5-biphosphate (PIP\u003csub\u003e2\u003c/sub\u003e)-Dependent Thermoring Basis for Cold-Sensing of the Transient Receptor Potential Melastatin-8 (TRPM8) Biothermometer. Physchem 4:106\u0026ndash;119\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eWang G (2024) Thermoring basis for heat unfolding-induced inactivation in TRPV1. Nat Sci 4:e20240008\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eWang G (2024) ATP-dependent thermoring basis for the heat unfolding of the first nucleotide-binding domain isolated from human CFTR. \u003cem\u003eRes Sq [Preprint].\u003c/em\u003e Nov 21:rs.3.rs-5479740. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.21203/rs.3.rs-5479740/v1\u003c/span\u003e\u003cspan address=\"10.21203/rs.3.rs-5479740/v1\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e. \u003cem\u003eNat Sci.\u003c/em\u003e 5, e70007 (2025). https://doi.org/10.1002/ntls.70007\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eAleksandrov LA, Jensen TJ, Cui L, Kousouros JN, He L, Aleksandrov AA, Riordan JR (2015) Thermal stability of purified and reconstituted CFTR in a locked open channel conformation. Protein Expr Purif 116:159\u0026ndash;166\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eLiu F, Zhang Z, Levit A, Levring J, Touhara KK, Shoichet BK, Chen J (2019) Structural identification of a hotspot on CFTR for potentiation. Science 364(6446):1184\u0026ndash;1188. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1126/science.aaw7611\u003c/span\u003e\u003cspan address=\"10.1126/science.aaw7611\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eHadida S, Van Goor F, Zhou J, Arumugam V, McCartney J, Hazlewood A, Decker C, Negulescu P, Grootenhuis PD (2014) Discovery of N-(2,4-di-tert-butyl-5-hydroxyphenyl)-4-oxo-1,4-dihydroquinoline-3-carboxamide (VX-770, ivacaftor), a potent and orally bioavailable CFTR potentiator. J Med Chem 57(23):9776\u0026ndash;9795. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1021/jm5012808\u003c/span\u003e\u003cspan address=\"10.1021/jm5012808\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eVan Goor F, Hadida S, Grootenhuis PD, Burton B, Cao D, Neuberger T, Turnbull A, Singh A, Joubran J, Hazlewood A, Zhou J, McCartney J, Arumugam V, Decker C, Yang J, Young C, Olson ER, Wine JJ, Frizzell RA, Ashlock M, Negulescu P (2009) Rescue of CF airway epithelial cell function in vitro by a CFTR potentiator, VX-770. Proc Natl Acad Sci U S A 106(44):18825\u0026ndash;18830. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1073/pnas.0904709106\u003c/span\u003e\u003cspan address=\"10.1073/pnas.0904709106\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eJih KY, Hwang TC (2013) Vx-770 potentiates CFTR function by promoting decoupling between the gating cycle and ATP hydrolysis cycle. Proc Natl Acad Sci U S A 110(11):4404\u0026ndash;4409. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1073/pnas.1215982110\u003c/span\u003e\u003cspan address=\"10.1073/pnas.1215982110\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eEckford PD, Li C, Ramjeesingh M, Bear CE (2012) Cystic fibrosis transmembrane conductance regulator (CFTR) potentiator VX-770 (ivacaftor) opens the defective channel gate of mutant CFTR in a phosphorylation-dependent but ATP-independent manner. J Biol Chem 287(44):36639\u0026ndash;36649. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1074/jbc.M112.393637\u003c/span\u003e\u003cspan address=\"10.1074/jbc.M112.393637\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eWang W, Hong JS, Rab A, Sorscher EJ, Kirk KL (2016) Robust Stimulation of W1282X-CFTR Channel Activity by a Combination of Allosteric Modulators. PLoS ONE 11(3):e0152232. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1371/journal.pone.0152232\u003c/span\u003e\u003cspan address=\"10.1371/journal.pone.0152232\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eYu H, Burton B, Huang CJ, Worley J, Cao D, Johnson JP Jr., Urrutia A, Joubran J, Seepersaud S, Sussky K et al (2012) Ivacaftor potentiation of multiple CFTR channels with gating mutations. J Cyst Fibros 11:237\u0026ndash;245. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.jcf.2011.12.005\u003c/span\u003e\u003cspan address=\"10.1016/j.jcf.2011.12.005\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eLiu X, Dawson DC (2014) Cystic fibrosis transmembrane conductance regulator (CFTR) potentiators protect G551D but not ∆F508 CFTR from thermal instability. Biochemistry 53:5613\u0026ndash;5618\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eSoya N, Xu H, Roldan A, Yang Z, Ye H, Jiang F, Premchandar A, Veit G, Cole SPC, Kappes J, Heged\u0026uuml;s T, Lukacs GL (2023) Folding correctors can restore CFTR posttranslational folding landscape by allosteric domain-domain coupling. Nat Commun 14:6868\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eWang C et al (2022) Mechanism of dual pharmacological correction and potentiation of human CFTR. \u003cem\u003ebioRxiv\u003c/em\u003e \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1101/2022.10.10.510913\u003c/span\u003e\u003cspan address=\"10.1101/2022.10.10.510913\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[{"identity":"4b0f1a38-d3a8-477f-89c4-489c4ee41bf8","identifier":"10.13039/100000968","name":"American Heart Association","awardNumber":"10SDG4120011","order_by":0}],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":false,"highlight":"","institution":"University of California, Davis","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"allosteric coupling, digital biology, interdomain interaction, ligand modulation, thermodynamic signature, least-stable interaction, melting threshold, thermoring, protein stability","lastPublishedDoi":"10.21203/rs.3.rs-7339733/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7339733/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eTrikafta modulators can correct the thermal and gating defects of the most common cystic fibrosis mutant F508del of the human cystic fibrosis transmembrane conductance regulator (hCFTR). While folding correctors VX-445 and VX-809 are sufficient to restore the Mg/ATP-dependent dimerization between the two nucleotide binding domains (NBD1 and NBD2) for channel opening, the thermodynamic basis for the activity potentiation by VX-770 in Trikafta remains unknown. Here, the thermoring structures and interdomain interactions of NBD2 were examined and compared with the counterparts of NBD1 with or without F508 in response to ligand binding. The results demonstrated that comparable thermostability between dimerized NBD1 and NBD2 was required to stabilize an activated intermediate for the channel activity potentiation by VX-770. Thus, a global induced fit across the interdomain interfaces upon ligand binding may optimize cooperative ligand-mediated NBD dimerization and improve the treatment of cystic fibrosis.\u003c/p\u003e","manuscriptTitle":"Thermodynamic basis for CFTR activity potentiation","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-08-12 08:34:17","doi":"10.21203/rs.3.rs-7339733/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"43ee4efc-79ce-40f4-ace3-56b2fcffba09","owner":[],"postedDate":"August 12th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":52930594,"name":"General Biochemistry"},{"id":52930595,"name":"Biophysics"},{"id":52930596,"name":"Structural Biology"},{"id":52930597,"name":"Biochemical Research Methods"},{"id":52930598,"name":"Bioinformatics"},{"id":52930599,"name":"Computational Biology"},{"id":52930600,"name":"Biotechnology and Bioengineering"},{"id":52930601,"name":"Systems Biology"},{"id":52930602,"name":"Chemical Biology"},{"id":52930603,"name":"Mathematical and Theoretical Biology"},{"id":52930604,"name":"Drug Discovery, Design, \u0026 Development"},{"id":52930605,"name":"Physiology"},{"id":52930606,"name":"Applied Mathematics"},{"id":52930607,"name":"Computational Chemistry"},{"id":52930608,"name":"Biological Chemistry"}],"tags":[],"updatedAt":"2026-01-06T19:24:46+00:00","versionOfRecord":{"articleIdentity":"rs-7339733","link":"https://doi.org/10.1016/j.biopha.2025.118936","journal":{"identity":"biomedicine-and-pharmacotherapy","isVorOnly":true,"title":"Biomedicine \u0026 Pharmacotherapy"},"publishedOn":"2026-01-06 00:00:00","publishedOnDateReadable":"January 6th, 2026"},"versionCreatedAt":"2025-08-12 08:34:17","video":"","vorDoi":"10.1016/j.biopha.2025.118936","vorDoiUrl":"https://doi.org/10.1016/j.biopha.2025.118936","workflowStages":[]},"version":"v1","identity":"rs-7339733","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7339733","identity":"rs-7339733","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}