Mechanistic Insights into the Wilson Disease Protein MBD6 and Its Interaction with the Chaperone Atox1 underlying the G626A Mutation

Mechanistic Insights into the Wilson Disease Protein MBD6 and Its Interaction with the Chaperone Atox1 underlying the G626A Mutation | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Mechanistic Insights into the Wilson Disease Protein MBD6 and Its Interaction with the Chaperone Atox1 underlying the G626A Mutation Mingwei Li, Ting Cheng, Zhihong Rao, Yulong Yang, Peng Huang, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8691483/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract The human transporter ATP7B plays a critical role in maintaining hepatic copper homeostasis, a process mediated by the specific interaction between its metal binding domain (MBD) and copper chaperone Atox1. The G626A mutation in MBD are known to cause the fatal hepatoneurological disorder Wilson disease (WD). However, the interaction mode between MBD and Atox1, as well as the molecular mechanism underlying WD-associated mutations impair copper transport, remains poorly understood. To bridge this gap, we conducted molecular dynamics simulations and free energy calculations to explore the dynamic properties of Atox1-MBD complex. Our results indicate that Atox1-Cu(I) binding to MBD triggers spontaneous protonation of C575 and C578, markedly enhancing the dynamic stability of Atox1–MBD complex. Furthermore, we have identified a critical interacting network mediated by hydrogen bonds and electrostatic interactions, and delineate how G626A mutation disrupts the key hydrogen bond between G626 and R21. Our study provides mechanistic insights into the dynamics of Atox1-MBD complex during Cu(I) transfer, establishing a link between WD-associated mutation and the functional deficit. Biological sciences/Biochemistry Biological sciences/Biophysics Biological sciences/Computational biology and bioinformatics Biological sciences/Drug discovery Biological sciences/Structural biology Wilson disease G626A mutation ATP7B metal binding domain molecular dynamics simulations free energy calculations Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Introduction The human ATP-dependent copper exporter ATP7B are essential for cellular copper homeostasis[ 1 , 2 ]. It uses the energy of ATP hydrolysis to transport copper from the cytosol into the secretory pathways, making it available for various copper-dependent enzymes[ 3 , 4 ]. Consequently, dysfunction of ATP7B directly causes Wilson diseases (WD), an autosomal recessive disorder of cooper metabolism involving copper accumulation in the liver, brain and other tissues[ 5 , 6 ]. Over the past decades, more than 700 pathogenic variants in ATP7B have been described as the underlying cause of WD[ 7 , 8 ]. This disease has been associated with a stable risk of increased mortality with 6%-7%[ 9 ], drawing more and more attention. The N-terminal domain (residues 1-633) of ATP7B (1465 amino acids) consist of six repetitive sequences of ~ 70 amino acids that are characterized by identical CxxC motifs (x stands for any amino acid)[ 10 ]. Each of these sequences folds into individual metal binding domain (MBD) and binds a single copper ion in the reduced Cu(I) form by cysteine residues. Each MBD has a ferredoxin-like fold with a compact βαββαβ structure with a conserved metal-binding motif 575CxxC578 located in α1 helix and β1–α1 loop, which binds Cu(I) ions (Fig. 1 A). In vivo, MBDs receive Cu(I) from the copper chaperones Atox1. Atox1 is a small soluble cytosolic Cu(I) receptor (68 amino acids), structured in the same βαββαβ motif of MBD. It coordinates one Cu(I) with the cysteine residues of a conserved 12Cxx15C motif (Fig. 1 B). The structure similarity between the chaperone and the target proteins is a hallmark of Cu(I) transporters. Consequently, the MBD of ATP7B receives Cu(I) through its interaction with Atox1, facilitating copper excretion into bile. Based on this, clinical researchers have discovered that numerous residue mutations directly lead to a loss of functions in the ATP7B involving removal of excess Cu(I), thereby causing Wilson disease. Among these, more than 25 mutations have been identified within the N-terminal MBDs[ 10 ]. These mutations are reported to potentially impair the protein's catalytic and transport activities. However, two fundamental questions remain unresolved: First, what dynamic properties and interaction modes of MBD in complex of Atox1 upon Cu(I) binding? Second, how do WD-associated mutation affect the dynamics of the MBD-Atox1 complex and its Cu(I) transport function? Previous studies have established the roles of the chaperones Atox1 and MBD of ATP7B in copper transport. Experimental evidence indicates the fifth MBD (MBD5) and MBD6 with a tight connecting loop are indispensable for the function of ATP7B, whereas MBD1-MBD4 with long and flexible linkers play a regulatory role[ 11 – 13 ]. Nevertheless, the precise regulation mechanisms of copper translocation remain unclear. Karthik Arumugam and Serge Crouzy have revealed that MBD1-MBD5 exhibit large structural fluctuations from initial structures than MBD6 by molecular dynamics (MD) simulations[ 14 ]. Moreover, Pekal et.al have conducted electron paramagnetic resonance measurements to demonstrate that Lys60 plays a critical role in electrostatically stabilizing the Cu(I)-Atox1 dimer[ 15 ]. Furthermore, in vitro experiments have demonstrated that the Wilson disease-associated G626A mutation significantly diminishes copper transport activity[ 16 ]. This mutation is strategically positioned in a α2–β4 loop adjacent to the 575CxxC578 metal-binding motif (Fig. 1 A), a location that is thus poised to critically influence copper translocation. However, the atomistic details of the mechanism by which G626A mutation disrupts function have remained unknown. To address above questions, in this study we employed all-atom molecular dynamics simulations and free energy calculations to explore the dynamic characteristics of MBD6 interacting with Atox1. Furthermore, we identified a key interaction network stabilized by hydrogen bonds and electrostatic contacts, which explains how the G626A mutation impairs the stability of the Atox1-MBD6 complex and compromises copper transport. Our work unveils the molecular mechanisms governing copper transfer and its pathological disruption in Wilson disease. Results The structural dynamics of Atox1-MBD complex The experimental findings indicate that the metallochaperone Atox1 transfers copper ions to ATP7B through its complex with MBD6 (hereinafter referred to as MBD)[ 11 – 13 ]. To investigate how copper binding influences the structural and dynamic properties of the Atox1–MBD heterodimer, we initiated several all-atom molecular dynamics simulations from Atox1–MBD heterodimer in this section. During the trajectory of the apo Atox1-MBD complex without copper binding (Fig. 2 A, black curve), the RMSD reaches a high average value of ~ 8 Å after an initial 150 ns equilibration phase. Structural alignment based on the Atox1 reveals that this high RMSD arises from a major displacement of β1–α1 loop and α1 helix housing the copper-coordinating residues C575 and C578 away from Atox1, accompanied by a relatively minor shift of α2 helix at the Atox1-MBD interface (Fig. 2 B). Consequently, the distance distributions between C12-C575 and C15-C578 throughout the trajectory are predominantly centered around highly 9.3 Å and 6 Å (Fig. 2 C, purple/orange), further confirm structural instability of MBD. When simulating the holo Atox1-MBD complex containing Cu(I), the MD simulations based on predefined empirical force-filed parameters does not allow one to simulate directly bond breaking and formation between Cu(I) and it’s coordinated cysteines during its delivery from Atox1 to MBD. Therefore, in the simulations of holo complex, we needed to assess a priori protonated/deprotonated state of Cu(I) binding sites. According to X-ray crystal structure, the copper-coordinated C12/C15 in Atox1 were modeled in the deprotonated state in simulations. For the MBD, we performed two sets of MD simulations respectively: one with both C575/C578 protonated, and another with both in a deprotonated state. In the first set, where C575/C578 of the MBD were kept protonated (Atox1-MBDpr), the domain exhibited moderate conformational fluctuations. The average RMSD value reaches ~ 7 Å after an initial 150 ns equilibration phase (Fig. 2 A, red curve), smaller than those in the apo complex. This may be attributed to copper binding, which mildly stabilizes the MBD through its interaction with Atox1. Similar to the apo complex, the structural changes in the MBD were primarily driven by displacements of β1–α1 loop, α1 and α2 helix (Fig. 2 D). The distance distributions between the copper ion and the sulfur atoms of C575/C578 were predominantly centered around 8.5 Å (Fig. 2 C, brown/magenta), which is too large to allow Cu(I) binding. This is most likely due to the neutral state of C575/C578, which is unfavorable for establishing strong electrostatic interactions with Cu(I). However, it is likely that C575/C578 will be deprotonate spontaneously or their protonated state will be co-adjusted upon Cu(I) binding in MD simulations. Therefore, to investigate how the deprotonation of MBD cysteines could impact on the stability of the complex, we performed another 1 µs MD simulation from holo Atox1-MBD with deprotonated C575/C578 (Atox1-MBDdepr). When copper-coordinated sites C575/C578 were kept in a deprotonated state, the flexibility of the MBD was markedly lowered, particularly in the β1–α1 loop, α1 and α2 helix (Fig. 2 F). The RMSD value stabilized at ∼3 Å. Although a transient increase to ∼5 Å was observed from 620 ns to 750 ns, primarily due to partial fluctuation of β1–α1, it promptly decreases to and remains at ~ 3 Å for the last 150 ns (Fig. 2 A, green curve). This stability in the global structure was mirrored by the local metal-coordination site, where the distances between Cu(I) and S atoms of C575/C578 maintained a stable value of ~ 2.5 Å throughout the simulation (Fig. 2 C, blue/cyan), as expected for an optimal coordinated distance for Cu(I). Root mean square fluctuations (RMSF) are usually considered as good makers of the global flexibility of the residues. We calculated the RMSF of Atox1-MBD complex during 1 µs MD simulation by C α atom. Overall, per-residue flexibility of Atox1-MBDdepr (Fig. 2 E, green curve) are significantly lower compared to the apo (Fig. 2 E, black curve) and Atox1-MBDpr complex (Fig. 2 E, red curve) except for the N- and C-termini, especially for copper-coordinated C575-C578 loop in MBD and C12-C15 loop in Atox1. As discussed above, for Cu(I) transfer to occur, the copper-coordinated cysteines must be deprotonated and maintain stable electronic interactions with Cu(I). Consistent with this requirement, the RMSF values of C575 and C578 in the Atox1-MBDdepr decrease to 0.82 Å and 0.62 Å, respectively—compared to 1.44 Å and 1.13 Å in the apo, and 1.34 Å and 1.03 Å in Atox1-MBDpr complex. Similarly, the RMSF values of C12 and C15 in Atox1 show a relatively modest reduction to 0.80 Å and 0.71 Å in the MBDdepr state, compared to their respective values in the apo state (1.38 Å/1.04 Å) and MBDpr state (1.30 Å/0.99 Å). This observation is readily explained, as the stable coordination between the deprotonated MBD residues C575/C578 and Cu(I) likely reinforces metal binding to C12/C15 in Atox1, thereby enhance their stability. The binding free energy between Atox1 and MBD Having established that deprotonated Cu(I)-coordinated cysteines markedly enhance the structural stability of Atox1-MBD complex, we will further calculate binding free energy between Atox1 and MBD to quantify the thermodynamic driving force. For simplicity, the distance of center of mass (COM) between Atox1 and MBD were chosen as a collective variable to quantify the binding free energy of complex. First, we performed metadynamics simulations to qualitatively trace the dissociation pathway of the Atox1-MBD complex. Subsequently, representative configurations spanning a range of COM distances were extracted from the trajectories and used as initial structures for umbrella sampling simulations. These configurations systematically sampled key states along the dissociation pathway: structures with COM distances around 2.0–2.3 Å corresponded to closely bound complexes, a distance around 2.3–2.7 Å reflected a moderately dissociated intermediate, and a separation around 2.7–3.2 Å represented a nearly fully dissociated state (Fig. 3 C). Second, we calculated the dissociation free energy as a function of the COM distance between Atox1 and MBD. The system was shown to be effectively equilibrated across all sampling windows, as evidenced by the stable distribution of the COM distance and sufficient overlap of the potential energy between adjacent windows (Fig. 3 A). These confirm that the simulations have converged, providing a reliable basis for constructing the potential of mean force (PMF) and deriving the binding free energy. When Atox1 forms a complex with MBD in the absence of Cu(I), the MBD tends to dissociate, exhibiting a binding free energy of 13.2 kJ/mol (Fig. 3 B, black curve). When MBD binds to copper-loaded Atox1 with protonated copper-coordinated residues C575/C578, the binding free energy does not increase significantly, reaching only 19.5 kJ/mol (Fig. 3 B, red curve). In contrast, deprotonation of C575/C578 in copper-bound state markedly enhances the binding free energy to 34.5 kJ/mol. The above results clearly demonstrate that deprotonation of the copper-coordinated cysteines substantially strengthens the interaction between Atox1 and MBD, which is consistent with the previously observed conformational stabilization of MBD mediated by C575 and C578 deprotonation (Fig. 2 ). The hydrogen bonds and electrostatic interactions between Atox1 and MBD contribute to the complex stability To elucidate the key residue interactions that maintain the stability of the Atox1-MBD complex, we statistically analyzed the interchain interactions between Atox1 and MBD throughout the MD simulation trajectories. From each simulation, a snapshot was extracted every 100 ps, resulting in a trajectory of 10,000 conformations for contacts analysis. Interchain interactions were categorized and calculated separately as hydrogen bonds and electrostatic contacts. A hydrogen bond was defined by a donor-acceptor distance ≤ 3.5 Å and a donor-hydrogen-acceptor angle ≥ 150°. An electrostatic contact was defined as occurring between oppositely charged residues within a closest-atom distance of 4 Å. We then calculated the population distribution of frames based on their counts of hydrogen bonds or electrostatic contacts. In the apo complex, interactions between Atox1 and MBD exhibit a limited phenomenon. The number of hydrogen-bonded pairs observed in trajectory was centered at 3 (Fig. 4 A, black), and the population of frames with number of 2–4 hydrogen bonds accounted for 68% of the trajectory. In additions, the dominant population of electrostatic contacts was 2–3 pairs, observed in 58% of the frames (Fig. 4 B, black). When the complex was bound to Cu(I) with C575/C578 protonated, the interaction distributions relatively shifted. The population of frames with 3–5 hydrogen bonds increased to 62%, and the probability of observing higher numbers of hydrogen bonds (6, 7 or 8 pairs) was slightly enhanced compared to the apo complex (Fig. 4 A, red). Although the frames with 4 electrostatic pairs increased, the propensity to form a larger number of electrostatic interactions (Fig. 4 B, red) did not surpass that of the apo state. A dramatic enhancement in interfacial interactions between Atox1 and MBD was observed when C575 and C578 were deprotonated upon Cu(I) binding. Both hydrogen bonds and electrostatic contacts increased markedly. The population of trajectory frames containing 4–7 hydrogen bonds reached 51%, with a significant increased 12% of frames even forming 8–12 hydrogen bonds (Fig. 4 A, green). Moreover, the population for 5–7 electrostatic pairs rose substantially to 30%, compared to only 15% and 17% in the apo and Atox1-MBDpr complexes, respectively. In summary, the Atox1-MBDdepr complex exhibits the increased number of stabilizing intermolecular interactions containing hydrogen bonds and electrostatic contacts, comparing to the apo and Atox1-MBDpr complex. This finding is consistent with aforementioned analysis (Fig. 2 – 3 ), indicating that the Atox1-MBD complex achieves stability only when coupled with deprotonation of the coordinating cysteines C575 and C578 upon Cu(I) binding. Furthermore, we classified interactions by residues type to identify the key residue pairs. Figure 5 displays the top three residue pairs with the highest population frequency. In the apo complex, the top three hydrogen-bonding pairs were T574-K60, N581-K57 and G626-R21, with populations of 53%, 52%, and 40%, respectively. All three pairs exhibited two distinct peaks in their distance distributions: one near 3 Å and another at a larger distance of approximately 5.2 Å (Fig. 5 A). The dominant electrostatic pairs were K57/K25-E624 and E623-R21, with population of 52%, 45%, and 34%, respectively. The first two pairs showed a primary peak around 3 Å and a secondary peak near 4.6 Å, while R21-E623 displayed peaks at approximately 3.5 Å and 4.4 Å (Fig. 5 B). Figure 5 C shows the predominant hydrogen-bonding and electrostatic residues pairs in representative structure. In the Atox1–MBDpr complex, the top three hydrogen-bonding pairs were N581-K57, G626-R21 and G620-S16, with populations of 57%, 55%, and 43%, respectively (Fig. 5 D). The leading electrostatic pairs are K57-E624, K25/R21-E623 with populations of 58%, 49%, and 42% (Fig. 5 E). Figure 5 F shows the predominant N581-K57 and E624-K57 residues pairs in representative structure. Similar to the apo complex, the Atox1-MBDpr interactions also exhibited a bimodal distance distribution, indicating that both the hydrogen bonds and electrostatic contacts are dynamic in rather than stably maintained. In the sharp contrast, there are more stable residues pairs of hydrogen bonds and electrostatic contacts in the Atox1-MBDdepr complex. The top three hydrogen-bonding residues pairs are formed by G626 and R21, then N581 and T58/G59, with populations of 65%, 56%, and 54%, respectively (Fig. 5 G). In additions, A multi-residue electrostatic hub involving R21, K25 and E623 where both K25/R21 interact with the E623 simultaneously and K57-E624 with populations of 69%, 64%, and 51%, respectively (Fig. 5 H). The electrostatic interactions between R21, K25, E623 and E624 are remain in apo complex and Atox1-MBDpr/depr, which indicate that these interactions serve as a foundational scaffold for basal Atox1-MBD complex stability. In additions, both hydrogen bonds and electrostatic interactions in Atox1-MBDdepr showed a greater population concentrated near 3 Å, reflecting enhanced complex stability. In summary, there are primarily two regions’ interactions for Atox1-MBD upon Cu(I) binding (Fig. 5 I): (i) hydrogen bonds between G626-R21, and N581-T58/G59. (ii) electrostatic interactions between E623-R21/K25 and E624-K57. The WD-associated G626A destabilizes the Atox1-MBD complex by disrupting a key hydrogen bond with R21 Previous in vivo studies have reported that the G626A mutation in human ATP7B leads to markedly reduced copper transport activity[ 16 ], yet the underlying molecular mechanism remains unclear. To address this gap, we performed computational mutagenesis G626A and conducted 1 µs MD simulation of the mutant to elucidate how this mutation impairs Cu(I) trafficking from a structural-dynamic perspective. The RMSD of the mutant increased significantly relative to the WT (Fig. 6 A, green) with an average value of ~ 4.8 Å (Fig. 6 A, red). Similarly, RMSF analysis revealed enhanced residue flexibility throughout the mutant, particularly in structural regions adjacent to the mutant site A626 (Fig. 6 B). Structural alignment based on Atox1 further revealed a pronounced conformational change, primarily stemming from a major displacement of the α2 helix (adjacent to A626 in MBD) away from Atox1 (Fig. 6 C). Given these conformational changes, we next sought to quantify their impact on the Atox1-MBD interaction by calculating the binding free energy. The free energy plot shows that the G626A substantially reduces the binding affinity between Atox1-MBD, from 34.5 kJ/mol in WT to 17.7 kcal/mol in mutant complex. Therefore, the above results clearly indicate that the G626A mutation destroy the binding affinity between Atox1 and MBD, thereby significantly compromising the stability of the Atox1-MBD complex. To decipher the residues-specific determinants of the impaired Atox1-MBD binding, we mapped the interaction landscape at the complex interface, focusing on how the G626A substitution undermines complex stability. We calculated the number of intermolecular contacts over every frame among mutant MD trajectory, categorizing them into hydrogen bonds and electrostatic interactions. Contacts analysis reveal a marked reduction of intermolecular hydrogen bonds in the mutant system compared to WT, with a population of frames with number of 2–3 hydrogen bonds accounted for highly 72% of the trajectory (Fig. 7 A, red curve). Furthermore, the maximum number of hydrogen bonds observed in any frame was limited to 4–5, representing only ~ 10% of the sampled conformations. In contrast, the overall distributions of electrostatic interaction show no significant difference from WT system (Fig. 7 B). The population of trajectory frames forming 2–3 electrostatic pairs in the mutant was 43%, comparable to the value of 44% in the WT. Therefore, these observations demonstrate that the destabilization induced by G626A is mainly caused by a reduction in hydrogen bonds between Atox1 and MBD, with minimal impact on the electrostatic interactions. In G626A mutant, the top three hydrogen-bonding interactions were S577–T11 and N581–P9/K60 with occupancies of 60%, 54%, and 13%, respectively (Fig. 7 C). Notably, the hydrogen bond between G626 and R21, observed in the apo complex and Atox1-MBDpr/depr, was completely absent in the mutant. The distance between G626 and R21 is mostly remain ~ 3.3 Å with stable hydrogen bond, while increased to above 4.5 Å after G626 mutant to A626 (Fig. 7 E). That is to say, the G626A mutation significantly destroyed the hydrogen bond between G626 and R21 in Atox1 α2 helix. In additions, the key electrostatic pairs (R21-E623 and K25/K57-E624) were conserved in the mutant with the same residue types as the WT, demonstrating that the G626A does not significantly perturb electrostatic interactions between Atox1 and MBD. Figure 5 F shows the predominant T11-S577, R21-E623 and K25-E624 residues pairs in representative structure. In summary, the WD-related G626A mutation primarily destroyed the key hydrogen bond between G626 and R21 in Atox1, which contribute to the significant instability of Atox1-MBD, thus offer a potential mechanism for reduced Cu(I) transition activity. Conclusion and discussion The Wilson disease protein ATP7B possesses a long N-terminal tail featuring six metal-binding domains (MBDs), which receive Cu(I) from the metallochaperone Atox1 and subsequently transport the copper ions to perform its biological functions[ 19 ]. It has been proposed that the copper transfer is facilitated by specific protein-protein interactions[ 20 ]. However, the structural and dynamic properties underlie this interaction remain poorly characterized at the molecular level. To address this gap, we have employed all-atom molecular dynamics (MD) simulations and free energy calculations to investigate the underling molecular mechanism. In the first part of our study, we initiated several 1 µs MD simulations from the Atox1-MBD complex involving with or without Cu(I) and varying protonation states of the coordinating cysteine residues. Since no experimental structure of the Atox1-MBD complex has been resolved, we constructed a model based on the crystal structure of the Cu(I)-bound Atox1 homodimer. In this dimer, the Cu(I) ion is coordinated by four cysteine residues—two from each Atox1 monomer. This coordination geometry is highly consistent with the expected binding mode between MBD and Atox1, according to the four Cu(I)-coordinated cysteines occupying the same binding positions. Given the high structural similarity between MBD and Atox1 as evidenced by a low RMSD ~ 1.3 Å, we replaced one of the Atox1 monomer in the dimer with MBD to generate the Atox1-MBD complex model. The MD simulations of the Atox1-MBD complex revealed that the deprotonation of the copper-coordinating cysteine C575/C578 in MBD upon Cu(I) binding significantly enhances the dynamic stability of the complex and improves the binding free energy, while the Atox1-MBD complex without Cu(I) binding and coordinating C575/C578 protonation could induce significant conformational fluctuation. Furthermore, contact analysis identify two regions’ interactions for maintaining stability of the Atox1-MBDdepr complex: (i) hydrogen bonds between α1 helix R21-α2 helix G626, α1 helix T58/G59-α2 helix N581. (ii) electrostatic interactions between α1 helix R21/K25/K57 and α2 helix E623/E624. Therefore, our results reveal a molecular blueprint whereby the initial binding of Atox1-Cu(I) to MBD triggers the spontaneous deprotonation of C575/C578. This key event stabilizes the complex and facilitates the formation of specific hydrogen bonds and electrostatic contacts networks, which in turn enforce a stable configuration enable for subsequent Cu(I) transfer. Another pivotal finding of this study is the elucidation of the molecular mechanism by which the WD-associated G626A mutation impairs Cu(I) transport activity. While previous experimental studies had established that the G626A mutation leads to reduced copper transport activity[ 16 ], the underlying molecular mechanism remained entirely unknown. To investigate it, we performed in silico mutagenesis, replacing glycine with alanine at position 626, and conducted 1 µs MD simulation initially from mutant system. Our results demonstrated that the G626A mutant exhibits significantly higher RMSD and RMSF fluctuations compared to WT, with the most pronounced structural changes localized to the α2 helix adjacent to G626. Further contacts analysis revealed that the G626A mutation specifically disrupts a key hydrogen bond between residue 626 (now Ala) and R21 located on the α1 helix of Atox1. G626-R21 hydrogen bond is a primary stabilizing force in the WT Atox1-MBD complex. Therefore, we conclude that the disease-associated G626A mutation impairs copper transport by destabilizing the Atox1-MBD complex through the disruption a critical interfacial hydrogen bond with R21 in Atox1. Our findings fill critical knowledge gap by providing a mechanistic link between the genetic mutation and its pathological consequence. Our work not only deciphers the molecular pathology for G626A mutation but also establishes a foundational framework for understanding the impact of other WD-related mutations, potentially offering the theoretical groundwork for future drug design. Methods and Materials Structure preparation The model of Atox1-MBD complex was built based on the X-ray crystal structure of human Atox1 homodimer, which contains a Cu bound between the two monomers (PDB ID: 1FEE). We superimposed the MBD6 (PDB ID: 7XUK; residues 562–633) to one monomer of the Atox1 homodimers to construct the Atox1-MBD complex. Since MBD6 are structurally very similar to Atox1 and share the same CxxC motif, we also adopted the same position of Cu observed in Atox1. In additions, the G626A mutant model was generated in silico by substituting Gly with Ala at position 626. All-Atom molecular dynamics (MD) simulations The proteins were parameterized using the AMBER ff14SB force field[ 21 ]. Each system was solvated in a cubic box under periodic boundary condition, using the TIP3P water molecules[ 22 ]. The minimum distance between the solute and the box boundary was 15 Å. The Na + and Cl − were added into the box to neutralize the overall charge of the system and achieve a salt concentration of 100 mM, consistent with the experimental conditions. The cutoff for van der Waals interactions was 12 Å. The long-range electrostatic interactions were calculated using the Particle Mesh Ewald (PME)[ 23 ] method, with a 12 Å cutoff for the direct space sums, a 0.16 nm FFT grid spacing, and a 4-order interpolation polynomial for the reciprocal space sums. Temperature coupling at 300 K was maintained using the velocity rescaling (V-rescale) thermostat[ 24 ], and the relaxation time was 0.1 ps. The pressure of the system was controlled with isotropic position scaling at 1 bar by Parrinello-Rahman algorithm[ 25 ], with a relaxation time of 2 ps and a compressibility of 4.5 ⋅ 10 − 5 bar − 1 . For each system, equilibration was carried out for 1 ns NVT followed by 1 ns NPT, with positional restraints applied to all heavy atoms. A 2-fs integration step was used. The production MD simulations were conducted for 1 µs by Gromacs-2025[ 26 ]. Free energy calculation Umbrella sampling[ 27 – 29 ] was performed using Gromacs to calculate the free energy of MBD from Atox1. First, we performed well-tempered metadynamics[ 30 ] to trace the dissociation pathway as a function of the center-of-mass (COM) distance between MBD and Atox1 ( D com ). The bias factor was set to 200, and the value of sigma was set to 0.05. Next, the structures along the pathway were saved every 1 ps and used as the initial structure for the subsequent umbrella sampling. For window i in the umbrella sampling centered at \(\:{D}_{com}^{i}\) , the conformation obtained in metadynamics with D com closest to \(\:{D}_{com}^{i}\) was selected as the initial structure. There are a total of 14 windows, with a spacing of 1 Å from D com = 22 Å to D com = 35 Å. We performed 60-ns simulations at each window, which resulted in a total simulation time of 840 ns. The spring constant was set to 10000 kJ mol − 1 nm − 2 . The trajectories were analyzed using the weighted histogram analysis method (WHAM) [ 31 ] to derive potential of mean force (PMF) as a function of the COM distance, and the error was estimated using the bootstrap method[ 32 ]. Declarations Author Contributions Statement M.L. performed the simulations and analyzed the data. T.C., Z.R., and Y.Y. helped with data analysis. The manuscript was written by M.L., P.H. and W.Y. Declaration of Interests The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Funding This study was financially supported by the Science and Technology Research Project of Anhui Province Department of Education (Grant Nos. 2025AHGXZK40547 and 2025AHGXZK40618), the Regional Innovation and Development Joint Fund of NSFC (Grant No. U22A20366), the Anhui Province Traditional Chinese Medicine Science and Technology Research Special Project (Grant No. 202303a07020004), the Anhui Province Clinical Medical Research Translation Special Programme (Grant No. 202204295107020066). Author Contribution M.L. performed the simulations and analyzed the data. T.C., Z.R., and Y.Y. helped with data analysis. The manuscript was written by M.L., P.H. and W.Y. Acknowledgements We thank the funding support from the Science and Technology Research Project of Anhui Province Department of Education (2025AHGXZK40547, 2025AHGXZK40618), the Regional Innovation and Development Joint Fund of NSFC (No. U22A20366), the Anhui Province Traditional Chinese Medicine Science and Technology Research Special Project (202303a07020004), the Anhui Province Clinical Medical Research Translation Special Programme (202204295107020066). Data Availability The data presented during the current study are available from the corresponding author on reasonable request. References Lutsenko, S., Human copper homeostasis: a network of interconnected pathways. Current Opinion in Chemical Biology, 2010. 14 (2): p. 211-217. Harris, E.D., Cellular copper transport and metabolism. Annual Review of Nutrition, 2000. 20 : p. 291-310. Linz, R. and S. Lutsenko, Copper-transporting ATPases ATP7A and ATP7B: Cousins, not twins. Journal of Bioenergetics and Biomembranes, 2007. 39 (5-6): p. 403-407. Inesi, G., R. Pilankatta, and F. Tadini-Buoninsegni, Biochemical characterization of P-type copper ATPases. Biochemical Journal, 2014. 463 : p. 167-176. Scheiber, I.F., R. Bruha, and P. Dusek, Pathogenesis of Wilson disease. Handbook of clinical neurology, 2017. 142 : p. 43-55. Czlonkowska, A., et al., Wilson disease. Nature Reviews Disease Primers, 2018. 4 . Gao, J., S. Brackley, and J.P. Mann, The global prevalence of Wilson disease from next-generation sequencing data. Genetics in Medicine, 2019. 21 (5): p. 1155-1163. Zhang, S., et al., Clinical and genetic characterization of a large cohort of patients with Wilson's disease in China. Translational Neurodegeneration, 2022. 11 (1). Aberg, F., et al., Four-fold increased mortality rate in patients with Wilson's disease: A population-based cohort study of 151 patients. United European Gastroenterology Journal, 2023. 11 (9): p. 852-860. Arioz, C., Y. Li, and P. Wittung-Stafshede, The six metal binding domains in human copper transporter, ATP7B: molecular biophysics and disease-causing mutations. Biometals, 2017. 30 (6): p. 823-840. Huster, D. and S. Lutsenko, The distinct roles of the N-terminal copper-binding sites in regulation of catalytic activity of the Wilson's disease protein. Journal of Biological Chemistry, 2003. 278 (34): p. 32212-32218. Cater, M.A., et al., Intracellular trafficking of the human Wilson protein: the role of the six N-terminal metal-binding sites. Biochemical Journal, 2004. 380 : p. 805-813. Guo, Y., et al., NH2-terminal signals in ATP7B Cu-ATPase mediate its Cu-dependent anterograde traffic in polarized hepatic cells. American Journal of Physiology-Gastrointestinal and Liver Physiology, 2005. 289 (5): p. G904-G916. Arumugam, K. and S. Crouzy, Dynamics and Stability of the Metal Binding Domains of the Menkes ATPase and Their Interaction with Metallochaperone HAH1. Biochemistry, 2012. 51 (44): p. 8885-8906. Perkal, O., et al., Cu(I) Controls Conformational States in Human Atox1 Metallochaperone: An EPR and Multiscale Simulation Study . Journal of Physical Chemistry B, 2020. 124 (22): p. 4399-4411. Huster, D., et al., Diverse Functional Properties of Wilson Disease ATP7B Variants. Gastroenterology, 2012. 142 (4): p. 947-U429. Wernimont, A.K., et al., Structural basis for copper transfer by the metallochaperone for the Menkes/Wilson disease proteins . Nature Structural Biology, 2000. 7 (9): p. 766-771. Yang, G.-M., et al., Structures of the human Wilson disease copper transporter ATP7B . Cell Reports, 2023. 42 (5). Banci, L., et al., Copper(I)-mediated protein-protein interactions result from suboptimal interaction surfaces. Biochemical Journal, 2009. 422 : p. 37-42. Larin, D., et al., Characterization of the interaction between the Wilson and Menkes disease proteins and the cytoplasmic copper chaperone, HAH1p. Journal of Biological Chemistry, 1999. 274 (40): p. 28497-28504. Maier, J.A., et al., ff14SB: Improving the Accuracy of Protein Side Chain and Backbone Parameters from ff99SB. Journal of Chemical Theory and Computation, 2015. 11 (8): p. 3696-3713. Mark, P. and L. Nilsson, Structure and dynamics of the TIP3P, SPC, and SPC/E water models at 298 K. Journal of Physical Chemistry A, 2001. 105 (43): p. 9954-9960. Essmann, U., et al., A SMOOTH PARTICLE MESH EWALD METHOD. Journal of Chemical Physics, 1995. 103 (19): p. 8577-8593. Bussi, G., D. Donadio, and M. Parrinello, Canonical sampling through velocity rescaling. Journal of Chemical Physics, 2007. 126 (1). Berendsen, H.J.C., et al., MOLECULAR-DYNAMICS WITH COUPLING TO AN EXTERNAL BATH. Journal of Chemical Physics, 1984. 81 (8): p. 3684-3690. Abraham, M.J., et al., GROMACS: high performance molecular simulations through multi-level parallelism from laptops to supercomputers. SoftwareX, 2015. 1-2 : p. 19-25. Patey, G.N. and J.P. Valleau, FREE-ENERGY OF SPHERES WITH DIPOLES - MONTE-CARLO WITH MULTISTAGE SAMPLING. Chemical Physics Letters, 1973. 21 (2): p. 297-300. Torrie, G.M. and J.P. Valleau, MONTE-CARLO FREE-ENERGY ESTIMATES USING NON-BOLTZMANN SAMPLING - APPLICATION TO SUBCRITICAL LENNARD-JONES FLUID. Chemical Physics Letters, 1974. 28 (4): p. 578-581. Torrie, G.M. and J.P. Valleau, NON-PHYSICAL SAMPLING DISTRIBUTIONS IN MONTE-CARLO FREE-ENERGY ESTIMATION - UMBRELLA SAMPLING. Journal of Computational Physics, 1977. 23 (2): p. 187-199. Barducci, A., G. Bussi, and M. Parrinello, Well-tempered metadynamics: A smoothly converging and tunable free-energy method. Physical Review Letters, 2008. 100 (2). Kumar, S., et al., The weighted histogram analysis method for free-energy calculations on biomolecules. I. The method. J. Comput. Chem., 1992. 13 (8): p. 1011-1021. Hub, J.S. and B.L. de Groot, Does CO2 permeate through aquaporin-1? Biophys. J., 2006. 91 (3): p. 842-848. Additional Declarations No competing interests reported. Cite Share Download PDF Status: Posted 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. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. 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-8691483","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":587893704,"identity":"c32ba93e-cd5d-4968-9bba-6ca389befa1c","order_by":0,"name":"Mingwei Li","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAq0lEQVRIiWNgGAWjYFAC5oYDH6BMCSK1MDYcnAFVTbwWZh6StOjOSGw8bNtWV8ffwHzwNg+DXR5BLWY3EhsO57YdlpA4wJZszcOQXExYy22wlgMSBgw8ZtI8DAcSG4jSYtlWB9TC/40ELYxtzCBb2IjUcv9hw8Gec4clZxxmM7acY5BMhJYzhw9/+FFWx8/f3vzwxpsKO8JaEIAZRBgQr34UjIJRMApGAR4AAPCoOmrTbTZ0AAAAAElFTkSuQmCC","orcid":"","institution":"The First Affiliated Hospital of Anhui University of Chinese Medicine","correspondingAuthor":true,"prefix":"","firstName":"Mingwei","middleName":"","lastName":"Li","suffix":""},{"id":587893705,"identity":"d4c78029-e395-4aa5-8a8c-8fd256263988","order_by":1,"name":"Ting Cheng","email":"","orcid":"","institution":"The First Affiliated Hospital of Anhui University of Chinese Medicine","correspondingAuthor":false,"prefix":"","firstName":"Ting","middleName":"","lastName":"Cheng","suffix":""},{"id":587893706,"identity":"21a89fa5-d295-43c0-bad1-47ae3115fc47","order_by":2,"name":"Zhihong Rao","email":"","orcid":"","institution":"The First Affiliated Hospital of Anhui University of Chinese Medicine","correspondingAuthor":false,"prefix":"","firstName":"Zhihong","middleName":"","lastName":"Rao","suffix":""},{"id":587893707,"identity":"8cb2bb62-fa8f-47ec-8829-9e02e5cbdece","order_by":3,"name":"Yulong Yang","email":"","orcid":"","institution":"The First Affiliated Hospital of Anhui University of Chinese Medicine","correspondingAuthor":false,"prefix":"","firstName":"Yulong","middleName":"","lastName":"Yang","suffix":""},{"id":587893708,"identity":"98bb253b-d261-401f-b798-7db1d59ccd6c","order_by":4,"name":"Peng Huang","email":"","orcid":"","institution":"The First Affiliated Hospital of Anhui University of Chinese Medicine","correspondingAuthor":false,"prefix":"","firstName":"Peng","middleName":"","lastName":"Huang","suffix":""},{"id":587893709,"identity":"d63aca5a-7134-43b0-98f1-27fc7a7aac99","order_by":5,"name":"Wenming Yang","email":"","orcid":"","institution":"The First Affiliated Hospital of Anhui University of Chinese Medicine","correspondingAuthor":false,"prefix":"","firstName":"Wenming","middleName":"","lastName":"Yang","suffix":""}],"badges":[],"createdAt":"2026-01-25 09:38:47","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-8691483/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-8691483/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":102233925,"identity":"c9581036-4b02-4354-8e44-88772ee83f93","added_by":"auto","created_at":"2026-02-09 15:58:38","extension":"jpeg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":433680,"visible":true,"origin":"","legend":"\u003cp\u003eThe experimental structure of MBD6 (A) (PDB ID: 7XUK, residues 562-633)[17] and Atox1 (B) (PDB ID: 1FEE)[18].\u003c/p\u003e","description":"","filename":"floatimage1.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-8691483/v1/21aec44b64f13d7564edd909.jpeg"},{"id":102233912,"identity":"de716ea1-6753-449b-9a23-376f4bf687be","added_by":"auto","created_at":"2026-02-09 15:58:37","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":362634,"visible":true,"origin":"","legend":"\u003cp\u003eConformational dynamics of the Atox1-MBD complex. (A) Time revolution of the RMSD for the apo Atox1-MBD (black), holo Atox1-MBDpr (red) and holo Atox1-MBDdepr (green) during 1 ms MD simulations. (B, D, F) Structural superposition of representative MD conformations with the initial structure, aligned on Atox1. Key interfacial elements (b1-a1 loop, a1 helix, a2 helix) are highlighted in red, green and orange respectively; the direction of their conformational movement is indicated by purple arrows. Copper-coordinated cysteines C12/C15 and C575/C578 are shown in CPK model, and the Cu(I) is depicted as a cyan sphere. Distance measured in (C) were indicated with black dash lines. (C) Distance distributions between Cu(I) and C575/C578 (pr/depr), and between C12–C575 and C15–C578 in the apo Atox1-MBD complex (no Cu(I) bound). (E) Average RMSF values of MBD and Atox1 calculated using C\u003csub\u003eα \u003c/sub\u003eatoms over the last 750 ns MD simulations. The Cu(I)-coordinated loop (C575-C578 in MBD; C12-C15 in Atox1) are highlighted with blue dashed boxes.\u003c/p\u003e","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-8691483/v1/71702df9a39758025e46d759.png"},{"id":102233914,"identity":"0a853fd6-19ca-4e43-aa48-497fe7252586","added_by":"auto","created_at":"2026-02-09 15:58:37","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":244814,"visible":true,"origin":"","legend":"\u003cp\u003eMBD release from Atox1. (A) Distribution of COM distance between MBD and Atox1 of apo, Atox1-MBDpr/depr complex. (B) PMF profiles as a function of the COM distance between MBD and Atox1. The black curve corresponds the apo Atox1-MBD complex, while the red and green curves represent the protonated C575/C578 and deprotonated C575/C578 Atox1-MBD complex, respectively. Binding free energy values for the three systems are labeled in the graph. (C) Representative conformations along the dissociation pathway of the Atox1-MBD complex.\u003c/p\u003e","description":"","filename":"floatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-8691483/v1/d849ae48d1b8b3568a8050ec.png"},{"id":102233911,"identity":"c4e5dfb9-aaff-4fff-b5c7-24d4fcbbd9de","added_by":"auto","created_at":"2026-02-09 15:58:37","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":64518,"visible":true,"origin":"","legend":"\u003cp\u003eThe interfacial contact analysis between Atox1 and MBD. The frames distributions of the contact number of hydrogen bonds (A) and electrostatic contact (B).\u003c/p\u003e","description":"","filename":"floatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-8691483/v1/eda6d105685d19105d222a92.png"},{"id":102233913,"identity":"0efbccb6-e198-4087-878d-5cc0a3bad910","added_by":"auto","created_at":"2026-02-09 15:58:37","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":316507,"visible":true,"origin":"","legend":"\u003cp\u003eThe key residue pairs between Atox1 and MBD. (A-B, D-E, G-H) \u003cstrong\u003eDistance distribution of the top three interacting residue pairs over 1 μs MD simulations. (C, F, I) Structural \u003c/strong\u003evisualization\u003cstrong\u003e of key residue pairs. \u003c/strong\u003eInteracting residues are highlighted by dashed lines, with their distances annotated.\u003c/p\u003e","description":"","filename":"floatimage5.png","url":"https://assets-eu.researchsquare.com/files/rs-8691483/v1/37d9fbc52e200b00084e56f2.png"},{"id":102233916,"identity":"f40a1bb0-54a4-49a8-b936-e42f5d6dc29f","added_by":"auto","created_at":"2026-02-09 15:58:38","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":327994,"visible":true,"origin":"","legend":"\u003cp\u003eThe Conformational changes of the G626A mutant Atox1-MBD complex. (A) RMSD comparison between G626A mutant and WT complexes. (B) RMSF fluctuation of the G626A mutant. The G626A mutation site and its associated α2 helix are highlighted with a triangle and a dashed box, respectively. (C) Representative MD conformations are superimposed on Atox1. Cu(I)-coordinated cysteines and the G626A site were rendered in CPK model. Purple arrows indicate the directions of displacement of a2 helix. (D) PMF profiles as a function of the COM distance between MBD and Atox1.\u003c/p\u003e","description":"","filename":"floatimage6.png","url":"https://assets-eu.researchsquare.com/files/rs-8691483/v1/4e90276489b427eba3db601b.png"},{"id":102233915,"identity":"077dd933-9e98-42e4-8670-0caebe054d76","added_by":"auto","created_at":"2026-02-09 15:58:38","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":241822,"visible":true,"origin":"","legend":"\u003cp\u003eContacts analysis of the G626A mutant Atox1-MBD complex. Populations of contact numbers for \u003cstrong\u003ehydrogen-bonding pairs (A) and electrostatic pairs (B) over 1 μs MD simulation. (C-D) Distance distribution of the top three \u003c/strong\u003einteracting \u003cstrong\u003epairs. (E) Evolution of the distance between G626/A626 and R21 during 1 μs MD simulation. (F) Structural visualization of key interacting pairs\u003c/strong\u003e.\u003c/p\u003e","description":"","filename":"floatimage7.png","url":"https://assets-eu.researchsquare.com/files/rs-8691483/v1/5693108e522459ca78e2c3c2.png"},{"id":106413757,"identity":"552bf6e7-d617-4c3d-a373-9bf2a3b00961","added_by":"auto","created_at":"2026-04-08 10:05:12","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2760049,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8691483/v1/553d94e4-d1f5-4aba-bb48-b57fdd74564b.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Mechanistic Insights into the Wilson Disease Protein MBD6 and Its Interaction with the Chaperone Atox1 underlying the G626A Mutation","fulltext":[{"header":"Introduction","content":"\u003cp\u003eThe human ATP-dependent copper exporter ATP7B are essential for cellular copper homeostasis[\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. It uses the energy of ATP hydrolysis to transport copper from the cytosol into the secretory pathways, making it available for various copper-dependent enzymes[\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. Consequently, dysfunction of ATP7B directly causes Wilson diseases (WD), an autosomal recessive disorder of cooper metabolism involving copper accumulation in the liver, brain and other tissues[\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. Over the past decades, more than 700 pathogenic variants in ATP7B have been described as the underlying cause of WD[\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. This disease has been associated with a stable risk of increased mortality with 6%-7%[\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e], drawing more and more attention.\u003c/p\u003e \u003cp\u003eThe N-terminal domain (residues 1-633) of ATP7B (1465 amino acids) consist of six repetitive sequences of ~\u0026thinsp;70 amino acids that are characterized by identical CxxC motifs (x stands for any amino acid)[\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. Each of these sequences folds into individual metal binding domain (MBD) and binds a single copper ion in the reduced Cu(I) form by cysteine residues. Each MBD has a ferredoxin-like fold with a compact βαββαβ structure with a conserved metal-binding motif 575CxxC578 located in α1 helix and β1\u0026ndash;α1 loop, which binds Cu(I) ions (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). In vivo, MBDs receive Cu(I) from the copper chaperones Atox1. Atox1 is a small soluble cytosolic Cu(I) receptor (68 amino acids), structured in the same βαββαβ motif of MBD. It coordinates one Cu(I) with the cysteine residues of a conserved 12Cxx15C motif (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB). The structure similarity between the chaperone and the target proteins is a hallmark of Cu(I) transporters. Consequently, the MBD of ATP7B receives Cu(I) through its interaction with Atox1, facilitating copper excretion into bile. Based on this, clinical researchers have discovered that numerous residue mutations directly lead to a loss of functions in the ATP7B involving removal of excess Cu(I), thereby causing Wilson disease. Among these, more than 25 mutations have been identified within the N-terminal MBDs[\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. These mutations are reported to potentially impair the protein's catalytic and transport activities. However, two fundamental questions remain unresolved: First, what dynamic properties and interaction modes of MBD in complex of Atox1 upon Cu(I) binding? Second, how do WD-associated mutation affect the dynamics of the MBD-Atox1 complex and its Cu(I) transport function?\u003c/p\u003e \u003cp\u003ePrevious studies have established the roles of the chaperones Atox1 and MBD of ATP7B in copper transport. Experimental evidence indicates the fifth MBD (MBD5) and MBD6 with a tight connecting loop are indispensable for the function of ATP7B, whereas MBD1-MBD4 with long and flexible linkers play a regulatory role[\u003cspan additionalcitationids=\"CR12\" citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. Nevertheless, the precise regulation mechanisms of copper translocation remain unclear. Karthik Arumugam and Serge Crouzy have revealed that MBD1-MBD5 exhibit large structural fluctuations from initial structures than MBD6 by molecular dynamics (MD) simulations[\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. Moreover, Pekal et.al have conducted electron paramagnetic resonance measurements to demonstrate that Lys60 plays a critical role in electrostatically stabilizing the Cu(I)-Atox1 dimer[\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. Furthermore, in vitro experiments have demonstrated that the Wilson disease-associated G626A mutation significantly diminishes copper transport activity[\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. This mutation is strategically positioned in a α2\u0026ndash;β4 loop adjacent to the 575CxxC578 metal-binding motif (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA), a location that is thus poised to critically influence copper translocation. However, the atomistic details of the mechanism by which G626A mutation disrupts function have remained unknown.\u003c/p\u003e \u003cp\u003eTo address above questions, in this study we employed all-atom molecular dynamics simulations and free energy calculations to explore the dynamic characteristics of MBD6 interacting with Atox1. Furthermore, we identified a key interaction network stabilized by hydrogen bonds and electrostatic contacts, which explains how the G626A mutation impairs the stability of the Atox1-MBD6 complex and compromises copper transport. Our work unveils the molecular mechanisms governing copper transfer and its pathological disruption in Wilson disease.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eThe structural dynamics of Atox1-MBD complex\u003c/h2\u003e \u003cp\u003eThe experimental findings indicate that the metallochaperone Atox1 transfers copper ions to ATP7B through its complex with MBD6 (hereinafter referred to as MBD)[\u003cspan additionalcitationids=\"CR12\" citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. To investigate how copper binding influences the structural and dynamic properties of the Atox1\u0026ndash;MBD heterodimer, we initiated several all-atom molecular dynamics simulations from Atox1\u0026ndash;MBD heterodimer in this section.\u003c/p\u003e \u003cp\u003eDuring the trajectory of the apo Atox1-MBD complex without copper binding (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA, black curve), the RMSD reaches a high average value of ~\u0026thinsp;8 \u0026Aring; after an initial 150 ns equilibration phase. Structural alignment based on the Atox1 reveals that this high RMSD arises from a major displacement of β1\u0026ndash;α1 loop and α1 helix housing the copper-coordinating residues C575 and C578 away from Atox1, accompanied by a relatively minor shift of α2 helix at the Atox1-MBD interface (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB). Consequently, the distance distributions between C12-C575 and C15-C578 throughout the trajectory are predominantly centered around highly 9.3 \u0026Aring; and 6 \u0026Aring; (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC, purple/orange), further confirm structural instability of MBD.\u003c/p\u003e \u003cp\u003eWhen simulating the holo Atox1-MBD complex containing Cu(I), the MD simulations based on predefined empirical force-filed parameters does not allow one to simulate directly bond breaking and formation between Cu(I) and it\u0026rsquo;s coordinated cysteines during its delivery from Atox1 to MBD. Therefore, in the simulations of holo complex, we needed to assess a priori protonated/deprotonated state of Cu(I) binding sites. According to X-ray crystal structure, the copper-coordinated C12/C15 in Atox1 were modeled in the deprotonated state in simulations. For the MBD, we performed two sets of MD simulations respectively: one with both C575/C578 protonated, and another with both in a deprotonated state. In the first set, where C575/C578 of the MBD were kept protonated (Atox1-MBDpr), the domain exhibited moderate conformational fluctuations. The average RMSD value reaches\u0026thinsp;~\u0026thinsp;7 \u0026Aring; after an initial 150 ns equilibration phase (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA, red curve), smaller than those in the apo complex. This may be attributed to copper binding, which mildly stabilizes the MBD through its interaction with Atox1. Similar to the apo complex, the structural changes in the MBD were primarily driven by displacements of β1\u0026ndash;α1 loop, α1 and α2 helix (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD). The distance distributions between the copper ion and the sulfur atoms of C575/C578 were predominantly centered around 8.5 \u0026Aring; (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC, brown/magenta), which is too large to allow Cu(I) binding. This is most likely due to the neutral state of C575/C578, which is unfavorable for establishing strong electrostatic interactions with Cu(I).\u003c/p\u003e \u003cp\u003eHowever, it is likely that C575/C578 will be deprotonate spontaneously or their protonated state will be co-adjusted upon Cu(I) binding in MD simulations. Therefore, to investigate how the deprotonation of MBD cysteines could impact on the stability of the complex, we performed another 1 \u0026micro;s MD simulation from holo Atox1-MBD with deprotonated C575/C578 (Atox1-MBDdepr). When copper-coordinated sites C575/C578 were kept in a deprotonated state, the flexibility of the MBD was markedly lowered, particularly in the β1\u0026ndash;α1 loop, α1 and α2 helix (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eF). The RMSD value stabilized at \u0026sim;3 \u0026Aring;. Although a transient increase to \u0026sim;5 \u0026Aring; was observed from 620 ns to 750 ns, primarily due to partial fluctuation of β1\u0026ndash;α1, it promptly decreases to and remains at ~\u0026thinsp;3 \u0026Aring; for the last 150 ns (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA, green curve). This stability in the global structure was mirrored by the local metal-coordination site, where the distances between Cu(I) and S atoms of C575/C578 maintained a stable value of ~\u0026thinsp;2.5 \u0026Aring; throughout the simulation (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC, blue/cyan), as expected for an optimal coordinated distance for Cu(I).\u003c/p\u003e \u003cp\u003eRoot mean square fluctuations (RMSF) are usually considered as good makers of the global flexibility of the residues. We calculated the RMSF of Atox1-MBD complex during 1 \u0026micro;s MD simulation by C\u003csub\u003eα\u003c/sub\u003e atom. Overall, per-residue flexibility of Atox1-MBDdepr (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eE, green curve) are significantly lower compared to the apo (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eE, black curve) and Atox1-MBDpr complex (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eE, red curve) except for the N- and C-termini, especially for copper-coordinated C575-C578 loop in MBD and C12-C15 loop in Atox1. As discussed above, for Cu(I) transfer to occur, the copper-coordinated cysteines must be deprotonated and maintain stable electronic interactions with Cu(I). Consistent with this requirement, the RMSF values of C575 and C578 in the Atox1-MBDdepr decrease to 0.82 \u0026Aring; and 0.62 \u0026Aring;, respectively\u0026mdash;compared to 1.44 \u0026Aring; and 1.13 \u0026Aring; in the apo, and 1.34 \u0026Aring; and 1.03 \u0026Aring; in Atox1-MBDpr complex. Similarly, the RMSF values of C12 and C15 in Atox1 show a relatively modest reduction to 0.80 \u0026Aring; and 0.71 \u0026Aring; in the MBDdepr state, compared to their respective values in the apo state (1.38 \u0026Aring;/1.04 \u0026Aring;) and MBDpr state (1.30 \u0026Aring;/0.99 \u0026Aring;). This observation is readily explained, as the stable coordination between the deprotonated MBD residues C575/C578 and Cu(I) likely reinforces metal binding to C12/C15 in Atox1, thereby enhance their stability.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eThe binding free energy between Atox1 and MBD\u003c/h3\u003e\n\u003cp\u003eHaving established that deprotonated Cu(I)-coordinated cysteines markedly enhance the structural stability of Atox1-MBD complex, we will further calculate binding free energy between Atox1 and MBD to quantify the thermodynamic driving force. For simplicity, the distance of center of mass (COM) between Atox1 and MBD were chosen as a collective variable to quantify the binding free energy of complex. First, we performed metadynamics simulations to qualitatively trace the dissociation pathway of the Atox1-MBD complex. Subsequently, representative configurations spanning a range of COM distances were extracted from the trajectories and used as initial structures for umbrella sampling simulations. These configurations systematically sampled key states along the dissociation pathway: structures with COM distances around 2.0\u0026ndash;2.3 \u0026Aring; corresponded to closely bound complexes, a distance around 2.3\u0026ndash;2.7 \u0026Aring; reflected a moderately dissociated intermediate, and a separation around 2.7\u0026ndash;3.2 \u0026Aring; represented a nearly fully dissociated state (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC).\u003c/p\u003e \u003cp\u003eSecond, we calculated the dissociation free energy as a function of the COM distance between Atox1 and MBD. The system was shown to be effectively equilibrated across all sampling windows, as evidenced by the stable distribution of the COM distance and sufficient overlap of the potential energy between adjacent windows (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA). These confirm that the simulations have converged, providing a reliable basis for constructing the potential of mean force (PMF) and deriving the binding free energy. When Atox1 forms a complex with MBD in the absence of Cu(I), the MBD tends to dissociate, exhibiting a binding free energy of 13.2 kJ/mol (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB, black curve). When MBD binds to copper-loaded Atox1 with protonated copper-coordinated residues C575/C578, the binding free energy does not increase significantly, reaching only 19.5 kJ/mol (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB, red curve). In contrast, deprotonation of C575/C578 in copper-bound state markedly enhances the binding free energy to 34.5 kJ/mol. The above results clearly demonstrate that deprotonation of the copper-coordinated cysteines substantially strengthens the interaction between Atox1 and MBD, which is consistent with the previously observed conformational stabilization of MBD mediated by C575 and C578 deprotonation (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e\n\u003ch3\u003eThe hydrogen bonds and electrostatic interactions between Atox1 and MBD contribute to the complex stability\u003c/h3\u003e\n\u003cp\u003eTo elucidate the key residue interactions that maintain the stability of the Atox1-MBD complex, we statistically analyzed the interchain interactions between Atox1 and MBD throughout the MD simulation trajectories. From each simulation, a snapshot was extracted every 100 ps, resulting in a trajectory of 10,000 conformations for contacts analysis. Interchain interactions were categorized and calculated separately as hydrogen bonds and electrostatic contacts. A hydrogen bond was defined by a donor-acceptor distance\u0026thinsp;\u0026le;\u0026thinsp;3.5 \u0026Aring; and a donor-hydrogen-acceptor angle\u0026thinsp;\u0026ge;\u0026thinsp;150\u0026deg;. An electrostatic contact was defined as occurring between oppositely charged residues within a closest-atom distance of 4 \u0026Aring;. We then calculated the population distribution of frames based on their counts of hydrogen bonds or electrostatic contacts.\u003c/p\u003e \u003cp\u003eIn the apo complex, interactions between Atox1 and MBD exhibit a limited phenomenon. The number of hydrogen-bonded pairs observed in trajectory was centered at 3 (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA, black), and the population of frames with number of 2\u0026ndash;4 hydrogen bonds accounted for 68% of the trajectory. In additions, the dominant population of electrostatic contacts was 2\u0026ndash;3 pairs, observed in 58% of the frames (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB, black). When the complex was bound to Cu(I) with C575/C578 protonated, the interaction distributions relatively shifted. The population of frames with 3\u0026ndash;5 hydrogen bonds increased to 62%, and the probability of observing higher numbers of hydrogen bonds (6, 7 or 8 pairs) was slightly enhanced compared to the apo complex (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA, red). Although the frames with 4 electrostatic pairs increased, the propensity to form a larger number of electrostatic interactions (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB, red) did not surpass that of the apo state. A dramatic enhancement in interfacial interactions between Atox1 and MBD was observed when C575 and C578 were deprotonated upon Cu(I) binding. Both hydrogen bonds and electrostatic contacts increased markedly. The population of trajectory frames containing 4\u0026ndash;7 hydrogen bonds reached 51%, with a significant increased 12% of frames even forming 8\u0026ndash;12 hydrogen bonds (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA, green). Moreover, the population for 5\u0026ndash;7 electrostatic pairs rose substantially to 30%, compared to only 15% and 17% in the apo and Atox1-MBDpr complexes, respectively. In summary, the Atox1-MBDdepr complex exhibits the increased number of stabilizing intermolecular interactions containing hydrogen bonds and electrostatic contacts, comparing to the apo and Atox1-MBDpr complex. This finding is consistent with aforementioned analysis (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e\u0026ndash;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e), indicating that the Atox1-MBD complex achieves stability only when coupled with deprotonation of the coordinating cysteines C575 and C578 upon Cu(I) binding.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eFurthermore, we classified interactions by residues type to identify the key residue pairs. Figure\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e displays the top three residue pairs with the highest population frequency. In the apo complex, the top three hydrogen-bonding pairs were T574-K60, N581-K57 and G626-R21, with populations of 53%, 52%, and 40%, respectively. All three pairs exhibited two distinct peaks in their distance distributions: one near 3 \u0026Aring; and another at a larger distance of approximately 5.2 \u0026Aring; (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA). The dominant electrostatic pairs were K57/K25-E624 and E623-R21, with population of 52%, 45%, and 34%, respectively. The first two pairs showed a primary peak around 3 \u0026Aring; and a secondary peak near 4.6 \u0026Aring;, while R21-E623 displayed peaks at approximately 3.5 \u0026Aring; and 4.4 \u0026Aring; (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB). Figure\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC shows the predominant hydrogen-bonding and electrostatic residues pairs in representative structure. In the Atox1\u0026ndash;MBDpr complex, the top three hydrogen-bonding pairs were N581-K57, G626-R21 and G620-S16, with populations of 57%, 55%, and 43%, respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eD). The leading electrostatic pairs are K57-E624, K25/R21-E623 with populations of 58%, 49%, and 42% (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eE). Figure\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eF shows the predominant N581-K57 and E624-K57 residues pairs in representative structure. Similar to the apo complex, the Atox1-MBDpr interactions also exhibited a bimodal distance distribution, indicating that both the hydrogen bonds and electrostatic contacts are dynamic in rather than stably maintained.\u003c/p\u003e \u003cp\u003eIn the sharp contrast, there are more stable residues pairs of hydrogen bonds and electrostatic contacts in the Atox1-MBDdepr complex. The top three hydrogen-bonding residues pairs are formed by G626 and R21, then N581 and T58/G59, with populations of 65%, 56%, and 54%, respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eG). In additions, A multi-residue electrostatic hub involving R21, K25 and E623 where both K25/R21 interact with the E623 simultaneously and K57-E624 with populations of 69%, 64%, and 51%, respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eH). The electrostatic interactions between R21, K25, E623 and E624 are remain in apo complex and Atox1-MBDpr/depr, which indicate that these interactions serve as a foundational scaffold for basal Atox1-MBD complex stability. In additions, both hydrogen bonds and electrostatic interactions in Atox1-MBDdepr showed a greater population concentrated near 3 \u0026Aring;, reflecting enhanced complex stability. In summary, there are primarily two regions\u0026rsquo; interactions for Atox1-MBD upon Cu(I) binding (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eI): (i) hydrogen bonds between G626-R21, and N581-T58/G59. (ii) electrostatic interactions between E623-R21/K25 and E624-K57.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e\n\u003ch3\u003eThe WD-associated G626A destabilizes the Atox1-MBD complex by disrupting a key hydrogen bond with R21\u003c/h3\u003e\n\u003cp\u003ePrevious in vivo studies have reported that the G626A mutation in human ATP7B leads to markedly reduced copper transport activity[\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e], yet the underlying molecular mechanism remains unclear. To address this gap, we performed computational mutagenesis G626A and conducted 1 \u0026micro;s MD simulation of the mutant to elucidate how this mutation impairs Cu(I) trafficking from a structural-dynamic perspective.\u003c/p\u003e \u003cp\u003eThe RMSD of the mutant increased significantly relative to the WT (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA, green) with an average value of ~\u0026thinsp;4.8 \u0026Aring; (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA, red). Similarly, RMSF analysis revealed enhanced residue flexibility throughout the mutant, particularly in structural regions adjacent to the mutant site A626 (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eB). Structural alignment based on Atox1 further revealed a pronounced conformational change, primarily stemming from a major displacement of the α2 helix (adjacent to A626 in MBD) away from Atox1 (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eC). Given these conformational changes, we next sought to quantify their impact on the Atox1-MBD interaction by calculating the binding free energy. The free energy plot shows that the G626A substantially reduces the binding affinity between Atox1-MBD, from 34.5 kJ/mol in WT to 17.7 kcal/mol in mutant complex. Therefore, the above results clearly indicate that the G626A mutation destroy the binding affinity between Atox1 and MBD, thereby significantly compromising the stability of the Atox1-MBD complex.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTo decipher the residues-specific determinants of the impaired Atox1-MBD binding, we mapped the interaction landscape at the complex interface, focusing on how the G626A substitution undermines complex stability. We calculated the number of intermolecular contacts over every frame among mutant MD trajectory, categorizing them into hydrogen bonds and electrostatic interactions. Contacts analysis reveal a marked reduction of intermolecular hydrogen bonds in the mutant system compared to WT, with a population of frames with number of 2\u0026ndash;3 hydrogen bonds accounted for highly 72% of the trajectory (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eA, red curve). Furthermore, the maximum number of hydrogen bonds observed in any frame was limited to 4\u0026ndash;5, representing only\u0026thinsp;~\u0026thinsp;10% of the sampled conformations. In contrast, the overall distributions of electrostatic interaction show no significant difference from WT system (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eB). The population of trajectory frames forming 2\u0026ndash;3 electrostatic pairs in the mutant was 43%, comparable to the value of 44% in the WT. Therefore, these observations demonstrate that the destabilization induced by G626A is mainly caused by a reduction in hydrogen bonds between Atox1 and MBD, with minimal impact on the electrostatic interactions.\u003c/p\u003e \u003cp\u003eIn G626A mutant, the top three hydrogen-bonding interactions were S577\u0026ndash;T11 and N581\u0026ndash;P9/K60 with occupancies of 60%, 54%, and 13%, respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eC). Notably, the hydrogen bond between G626 and R21, observed in the apo complex and Atox1-MBDpr/depr, was completely absent in the mutant. The distance between G626 and R21 is mostly remain\u0026thinsp;~\u0026thinsp;3.3 \u0026Aring; with stable hydrogen bond, while increased to above 4.5 \u0026Aring; after G626 mutant to A626 (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eE). That is to say, the G626A mutation significantly destroyed the hydrogen bond between G626 and R21 in Atox1 α2 helix. In additions, the key electrostatic pairs (R21-E623 and K25/K57-E624) were conserved in the mutant with the same residue types as the WT, demonstrating that the G626A does not significantly perturb electrostatic interactions between Atox1 and MBD. Figure\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eF shows the predominant T11-S577, R21-E623 and K25-E624 residues pairs in representative structure.\u003c/p\u003e \u003cp\u003eIn summary, the WD-related G626A mutation primarily destroyed the key hydrogen bond between G626 and R21 in Atox1, which contribute to the significant instability of Atox1-MBD, thus offer a potential mechanism for reduced Cu(I) transition activity.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e"},{"header":"Conclusion and discussion","content":"\u003cp\u003eThe Wilson disease protein ATP7B possesses a long N-terminal tail featuring six metal-binding domains (MBDs), which receive Cu(I) from the metallochaperone Atox1 and subsequently transport the copper ions to perform its biological functions[\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. It has been proposed that the copper transfer is facilitated by specific protein-protein interactions[\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. However, the structural and dynamic properties underlie this interaction remain poorly characterized at the molecular level. To address this gap, we have employed all-atom molecular dynamics (MD) simulations and free energy calculations to investigate the underling molecular mechanism.\u003c/p\u003e \u003cp\u003eIn the first part of our study, we initiated several 1 µs MD simulations from the Atox1-MBD complex involving with or without Cu(I) and varying protonation states of the coordinating cysteine residues. Since no experimental structure of the Atox1-MBD complex has been resolved, we constructed a model based on the crystal structure of the Cu(I)-bound Atox1 homodimer. In this dimer, the Cu(I) ion is coordinated by four cysteine residues—two from each Atox1 monomer. This coordination geometry is highly consistent with the expected binding mode between MBD and Atox1, according to the four Cu(I)-coordinated cysteines occupying the same binding positions. Given the high structural similarity between MBD and Atox1 as evidenced by a low RMSD ~ 1.3 Å, we replaced one of the Atox1 monomer in the dimer with MBD to generate the Atox1-MBD complex model. The MD simulations of the Atox1-MBD complex revealed that the deprotonation of the copper-coordinating cysteine C575/C578 in MBD upon Cu(I) binding significantly enhances the dynamic stability of the complex and improves the binding free energy, while the Atox1-MBD complex without Cu(I) binding and coordinating C575/C578 protonation could induce significant conformational fluctuation. Furthermore, contact analysis identify two regions’ interactions for maintaining stability of the Atox1-MBDdepr complex: (i) hydrogen bonds between α1 helix R21-α2 helix G626, α1 helix T58/G59-α2 helix N581. (ii) electrostatic interactions between α1 helix R21/K25/K57 and α2 helix E623/E624. Therefore, our results reveal a molecular blueprint whereby the initial binding of Atox1-Cu(I) to MBD triggers the spontaneous deprotonation of C575/C578. This key event stabilizes the complex and facilitates the formation of specific hydrogen bonds and electrostatic contacts networks, which in turn enforce a stable configuration enable for subsequent Cu(I) transfer.\u003c/p\u003e \u003cp\u003eAnother pivotal finding of this study is the elucidation of the molecular mechanism by which the WD-associated G626A mutation impairs Cu(I) transport activity. While previous experimental studies had established that the G626A mutation leads to reduced copper transport activity[\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e], the underlying molecular mechanism remained entirely unknown. To investigate it, we performed in silico mutagenesis, replacing glycine with alanine at position 626, and conducted 1 µs MD simulation initially from mutant system. Our results demonstrated that the G626A mutant exhibits significantly higher RMSD and RMSF fluctuations compared to WT, with the most pronounced structural changes localized to the α2 helix adjacent to G626. Further contacts analysis revealed that the G626A mutation specifically disrupts a key hydrogen bond between residue 626 (now Ala) and R21 located on the α1 helix of Atox1. G626-R21 hydrogen bond is a primary stabilizing force in the WT Atox1-MBD complex. Therefore, we conclude that the disease-associated G626A mutation impairs copper transport by destabilizing the Atox1-MBD complex through the disruption a critical interfacial hydrogen bond with R21 in Atox1. Our findings fill critical knowledge gap by providing a mechanistic link between the genetic mutation and its pathological consequence. Our work not only deciphers the molecular pathology for G626A mutation but also establishes a foundational framework for understanding the impact of other WD-related mutations, potentially offering the theoretical groundwork for future drug design.\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003cdiv id=\"Sec9\" class=\"Section3\"\u003e \u003c/div\u003e \u003c/div\u003e"},{"header":"Methods and Materials","content":"\u003ch2\u003eStructure preparation\u003c/h2\u003e\u003cp\u003eThe model of Atox1-MBD complex was built based on the X-ray crystal structure of human Atox1 homodimer, which contains a Cu bound between the two monomers (PDB ID: 1FEE). We superimposed the MBD6 (PDB ID: 7XUK; residues 562–633) to one monomer of the Atox1 homodimers to construct the Atox1-MBD complex. Since MBD6 are structurally very similar to Atox1 and share the same CxxC motif, we also adopted the same position of Cu observed in Atox1. In additions, the G626A mutant model was generated in silico by substituting Gly with Ala at position 626.\u003c/p\u003e\n\u003ch3\u003eAll-Atom molecular dynamics (MD) simulations\u003c/h3\u003e\n\u003cp\u003eThe proteins were parameterized using the AMBER ff14SB force field[\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. Each system was solvated in a cubic box under periodic boundary condition, using the TIP3P water molecules[\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. The minimum distance between the solute and the box boundary was 15 \u0026Aring;. The Na\u003csup\u003e+\u003c/sup\u003e and Cl\u003csup\u003e\u0026minus;\u003c/sup\u003e were added into the box to neutralize the overall charge of the system and achieve a salt concentration of 100 mM, consistent with the experimental conditions. The cutoff for van der Waals interactions was 12 \u0026Aring;. The long-range electrostatic interactions were calculated using the Particle Mesh Ewald (PME)[\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e] method, with a 12 \u0026Aring; cutoff for the direct space sums, a 0.16 nm FFT grid spacing, and a 4-order interpolation polynomial for the reciprocal space sums. Temperature coupling at 300 K was maintained using the velocity rescaling (V-rescale) thermostat[\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e], and the relaxation time was 0.1 ps. The pressure of the system was controlled with isotropic position scaling at 1 bar by Parrinello-Rahman algorithm[\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e], with a relaxation time of 2 ps and a compressibility of 4.5 \u0026sdot; 10\u003csup\u003e\u0026minus;\u0026thinsp;5\u003c/sup\u003e bar \u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e. For each system, equilibration was carried out for 1 ns NVT followed by 1 ns NPT, with positional restraints applied to all heavy atoms. A 2-fs integration step was used. The production MD simulations were conducted for 1 \u0026micro;s by Gromacs-2025[\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e].\u003c/p\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eFree energy calculation\u003c/h2\u003e \u003cp\u003eUmbrella sampling[\u003cspan additionalcitationids=\"CR28\" citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e] was performed using Gromacs to calculate the free energy of MBD from Atox1. First, we performed well-tempered metadynamics[\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e] to trace the dissociation pathway as a function of the center-of-mass (COM) distance between MBD and Atox1 (\u003cem\u003eD\u003c/em\u003e\u003csub\u003e\u003cem\u003ecom\u003c/em\u003e\u003c/sub\u003e). The bias factor was set to 200, and the value of sigma was set to 0.05. Next, the structures along the pathway were saved every 1 ps and used as the initial structure for the subsequent umbrella sampling. For window \u003cem\u003ei\u003c/em\u003e in the umbrella sampling centered at \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:{D}_{com}^{i}\\)\u003c/span\u003e\u003c/span\u003e, the conformation obtained in metadynamics with \u003cem\u003eD\u003c/em\u003e\u003csub\u003e\u003cem\u003ecom\u003c/em\u003e\u003c/sub\u003e closest to \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:{D}_{com}^{i}\\)\u003c/span\u003e\u003c/span\u003e was selected as the initial structure.\u003c/p\u003e \u003cp\u003eThere are a total of 14 windows, with a spacing of 1 \u0026Aring; from \u003cem\u003eD\u003c/em\u003e\u003csub\u003e\u003cem\u003ecom\u003c/em\u003e\u003c/sub\u003e = 22 \u0026Aring; to \u003cem\u003eD\u003c/em\u003e\u003csub\u003e\u003cem\u003ecom\u003c/em\u003e\u003c/sub\u003e = 35 \u0026Aring;. We performed 60-ns simulations at each window, which resulted in a total simulation time of 840 ns. The spring constant was set to 10000 kJ mol\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e nm\u003csup\u003e\u0026minus;\u0026thinsp;2\u003c/sup\u003e. The trajectories were analyzed using the weighted histogram analysis method (WHAM) [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e] to derive potential of mean force (PMF) as a function of the COM distance, and the error was estimated using the bootstrap method[\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e"},{"header":"Declarations","content":"\u003ch2\u003eAuthor Contributions Statement\u003c/h2\u003e\n\u003cp\u003eM.L. performed the simulations and analyzed the data. T.C., Z.R., and Y.Y. helped with data analysis. The manuscript was written by M.L., P.H. and W.Y.\u003c/p\u003e\n\u003ch2\u003eDeclaration of Interests\u003c/h2\u003e\n\u003cp\u003eThe authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.\u003c/p\u003e\n\u003ch2\u003eFunding\u003c/h2\u003e\n\u003cp\u003eThis study was financially supported by the Science and Technology Research Project of Anhui Province Department of Education (Grant Nos. 2025AHGXZK40547 and 2025AHGXZK40618), the Regional Innovation and Development Joint Fund of NSFC (Grant No. U22A20366), the Anhui Province Traditional Chinese Medicine Science and Technology Research Special Project (Grant No. 202303a07020004), the Anhui Province Clinical Medical Research Translation Special Programme (Grant No. 202204295107020066).\u003c/p\u003e\n\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\n\u003cp\u003eM.L. performed the simulations and analyzed the data. T.C., Z.R., and Y.Y. helped with data analysis. The manuscript was written by M.L., P.H. and W.Y.\u003c/p\u003e\n\u003ch2\u003eAcknowledgements\u003c/h2\u003e\n\u003cp\u003eWe thank the funding support from the Science and Technology Research Project of Anhui Province Department of Education (2025AHGXZK40547, 2025AHGXZK40618), the Regional Innovation and Development Joint Fund of NSFC (No. U22A20366), the Anhui Province Traditional Chinese Medicine Science and Technology Research Special Project (202303a07020004), the Anhui Province Clinical Medical Research Translation Special Programme (202204295107020066).\u003c/p\u003e\n\u003ch2\u003eData Availability\u003c/h2\u003e\n\u003cp\u003eThe data presented during the current study are available from the corresponding author on reasonable request.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eLutsenko, S., \u003cem\u003eHuman copper homeostasis: a network of interconnected pathways.\u003c/em\u003e Current Opinion in Chemical Biology, 2010. \u003cstrong\u003e14\u003c/strong\u003e(2): p. 211-217.\u003c/li\u003e\n\u003cli\u003eHarris, E.D., \u003cem\u003eCellular copper transport and metabolism.\u003c/em\u003e Annual Review of Nutrition, 2000. \u003cstrong\u003e20\u003c/strong\u003e: p. 291-310.\u003c/li\u003e\n\u003cli\u003eLinz, R. and S. 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Chem., 1992. \u003cstrong\u003e13\u003c/strong\u003e(8): p. 1011-1021.\u003c/li\u003e\n\u003cli\u003eHub, J.S. and B.L. de Groot, \u003cem\u003eDoes CO2 permeate through aquaporin-1?\u003c/em\u003e Biophys. J., 2006. \u003cstrong\u003e91\u003c/strong\u003e(3): p. 842-848.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"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":"Wilson disease, G626A mutation, ATP7B metal binding domain, molecular dynamics simulations, free energy calculations","lastPublishedDoi":"10.21203/rs.3.rs-8691483/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8691483/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThe human transporter ATP7B plays a critical role in maintaining hepatic copper homeostasis, a process mediated by the specific interaction between its metal binding domain (MBD) and copper chaperone Atox1. The G626A mutation in MBD are known to cause the fatal hepatoneurological disorder Wilson disease (WD). However, the interaction mode between MBD and Atox1, as well as the molecular mechanism underlying WD-associated mutations impair copper transport, remains poorly understood. To bridge this gap, we conducted molecular dynamics simulations and free energy calculations to explore the dynamic properties of Atox1-MBD complex. Our results indicate that Atox1-Cu(I) binding to MBD triggers spontaneous protonation of C575 and C578, markedly enhancing the dynamic stability of Atox1\u0026ndash;MBD complex. Furthermore, we have identified a critical interacting network mediated by hydrogen bonds and electrostatic interactions, and delineate how G626A mutation disrupts the key hydrogen bond between G626 and R21. Our study provides mechanistic insights into the dynamics of Atox1-MBD complex during Cu(I) transfer, establishing a link between WD-associated mutation and the functional deficit.\u003c/p\u003e","manuscriptTitle":"Mechanistic Insights into the Wilson Disease Protein MBD6 and Its Interaction with the Chaperone Atox1 underlying the G626A Mutation","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-02-09 15:58:32","doi":"10.21203/rs.3.rs-8691483/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":"906467ab-3e98-4898-bd76-8678ccd753da","owner":[],"postedDate":"February 9th, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":62541978,"name":"Biological sciences/Biochemistry"},{"id":62541979,"name":"Biological sciences/Biophysics"},{"id":62541980,"name":"Biological sciences/Computational biology and bioinformatics"},{"id":62541981,"name":"Biological sciences/Drug discovery"},{"id":62541982,"name":"Biological sciences/Structural biology"}],"tags":[],"updatedAt":"2026-04-08T09:49:00+00:00","versionOfRecord":[],"versionCreatedAt":"2026-02-09 15:58:32","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8691483","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8691483","identity":"rs-8691483","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}