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Copper binding leads to increased dynamics in the regulatory N-terminal domain of full-length human copper transporter ATP7B [1]

['Fredrik Orädd', 'Department Of Chemistry', 'Umeå University', 'Umeå', 'Jonas Hyld Steffen', 'Department Of Biomedical Sciences', 'University Of Copenhagen', 'Copenhagen', 'Pontus Gourdon', 'Department Of Experimental Medical Science']

Date: 2022-09

ATP7B is a human copper-transporting P 1B -type ATPase that is involved in copper homeostasis and resistance to platinum drugs in cancer cells. ATP7B consists of a copper-transporting core and a regulatory N-terminal tail that contains six metal-binding domains (MBD1-6) connected by linker regions. The MBDs can bind copper, which changes the dynamics of the regulatory domain and activates the protein, but the underlying mechanism remains unknown. To identify possible copper-specific structural dynamics involved in transport regulation, we constructed a model of ATP7B spanning the N-terminal tail and core catalytic domains and performed molecular dynamics (MD) simulations with (holo) and without (apo) copper ions bound to the MBDs. In the holo protein, MBD2, MBD3 and MBD5 showed enhanced mobilities, which resulted in a more extended N-terminal regulatory region. The observed separation of MBD2 and MBD3 from the core protein supports a mechanism where copper binding activates the ATP7B protein by reducing interactions among MBD1-3 and between MBD1-3 and the core protein. We also observed an increased interaction between MBD5 and the core protein that brought the copper-binding site of MBD5 closer to the high-affinity internal copper-binding site in the core protein. The simulation results assign specific, mechanistic roles to the metal-binding domains involved in ATP7B regulation that are testable in experimental settings.

Living organisms depend upon active transport against gradients across biological membranes for survival. Such transport can be accomplished by ATP-dependent membrane protein transporters for which the activity must be regulated to maintain optimal concentrations in the cellular compartments. The regulatory mechanisms often involve structural responses inherent to the protein structure, which because of their dynamic nature can be hard to assess experimentally. A prime example is regulation of cellular copper levels by a copper-binding tail in the human copper transporter ATP7B. Dysregulation can cause severe diseases, for example the copper metabolism disorder Wilson’s disease, which is caused by mutations in ATP7B regulation machinery. Due to the practical difficulties in working with membrane proteins, most studies of ATP7B have been conducted in the absence of the membrane-bound protein core. Here, we used computer simulations of full-length ATP7B to study how structural dynamics in the regulatory tail differ between copper-bound and copper-free states. Copper induced increased dynamics in the tail, resulting in an overall movement towards the ion-binding site in the protein core. The simulations identified several, hitherto not reported, interactions between the regulatory tail and the protein core that can be targeted experimentally to enhance our understanding of this medically relevant regulatory mechanism.

Introduction

P-type ATPase proteins transport ions across biological membranes to provide critical cofactors and uphold gradients [1]. To ensure cellular homeostasis, these ion transporters have evolved intricate regulation mechanisms that involve small peptides, lipid components in the membrane and internal protein domains [2–4]. A prime example of P-type ATPase regulation is maintenance of cellular copper levels. Copper is an essential metal that is under tight control by chaperones and membrane transporters due to its high toxicity in the free form [5,6]. The copper-transporting ATPase 2 (ATP7B) plays a key role in the cellular copper homeostasis in humans and is primarily expressed in the liver and brain [7]. At normal copper concentrations, ATP7B resides in the trans-golgi network and transports copper into the secretory pathway for incorporation into proteins, but at elevated cellular copper levels ATP7B moves to the cell membrane to export copper from the cell [7,8]. Mutations in ATP7B lead to Wilson’s disease, which is characterized by copper accumulation in the liver and brain that can result in potentially fatal hepatoneurological symptoms if left untreated [9]. ATP7B is also upregulated in several cancer forms and is involved in platinum chemotherapy resistance [10]. An internal copper-binding domain is central to the regulation of ATP7B activity, but the molecular basis of regulation is still elusive.

The ATP7B transporter belongs to the P IB subclass of P-type ATPases and uses energy from ATP to drive copper transport. In the general Post-Albers reaction cycle, P-type ATPase proteins pass through inward-facing E1 structural states with high substrate affinity and outward-facing E2 states with low substrate affinity to achieve membrane transport [1]. The overall structure of P-type ATPases share a common topology consisting of a transmembrane (M) domain and cytosolic actuator- (A), phosphorylation- (P), and nucleotide-binding- (N) domains [1] (Fig 1A), and this topology has also been observed in crystal structures of a bacterial copper ATPase from Legionella pneumophila (LpCopA) [11,12] and the recent cryo-EM structure of Xenopus tropicalis ATP7B [13]. While there is no high-resolution structure of full-length human ATP7B, the solution structures of the A and N domains have been solved experimentally [14,15], and the crystal structure of LpCopA has enabled homology modeling of the ATP7B core domains [16,17], giving results very similar to the recent X. tropicalis structure [13]. The ATP7B protein also hosts a large regulatory N-terminal tail that contains six independently folded metal-binding domains (MBD1-6) connected by long, unstructured linkers [18] (Fig 1A). The solution structures of the MBDs have revealed that they share the same ferredoxin-like fold with one copper-binding CXXC motif each [19–23]. Nanobody binding, nuclear magnetic resonance (NMR) relaxation, and electron paramagnetic resonance (EPR) spectroscopy have showed that the N-terminal tail is organized into two functional units composed of MBD1-3 and MBD5-6, with MBD4 either belonging to the MBD5-6 complex or serving as a linker between the two groups [19,24,25], and reviewed in [26]. There is also an unstructured N-terminal peptide preceding MBD1 in the sequence that contains a signal peptide for trafficking of ATP7B to the cell membrane [27,28].

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TIFF original image Download: Fig 1. Structure of the ATP7B protein core and metal-binding domains. (A) Schematic and (B) all-atom homology model of the human copper ATPase ATP7B in the E2.P i state. The N-terminal peptide is shown in purple, MBD1-6 in green, the A domain in yellow, the P domain in blue, the N domain in red, and the M domain in brown. The lipid headgroups are shown as light brown spheres (A) and orange surface (B). https://doi.org/10.1371/journal.pcbi.1010074.g001

Positioning of the MBDs relative to the core protein is still uncertain, especially for MBD1-4 which have not been observed in any crystallography or electron microscopy data. Although electron densities have been resolved for MBD5 and MBD6, or corresponding MBDs in homologs with fewer MBDs, their positions vary greatly and the density is often vague. The MBD5 and MBD6 positions were resolved in the cryo-EM structure of a deletion mutant of X. tropicalis ATP7B lacking MBD1-4 (ATP7B-ΔMBD1-4) in the absence of copper, where they primarily contacted the A and P domains, with MBD5 placed above the copper entry site in the M domain [13]. However, a significant fraction of the particles lacked density for MBD5 and MBD6, suggesting that the observed positions were not the only ones occupied in the apo state [13]. Low-resolution electron densities in Archaeoglobus fulgidus CopA and human ATP7B-ΔMBD1-4 suggested that the MBDs closest to the core protein in the sequence are located on the opposite side of the A domain compared to the X. tropicalis structure, and the electron density in human ATP7B placed one MBD closer to the membrane and the copper entry site [29–31]. In all the suggested positions, the MBD(s) would have contact with the A, P and N domains, which is in agreement with co-purification experiments that identified interactions between the ATP7B N-terminal tail and the A domain [32] and the P and N domains [33]. In addition, NMR chemical shift perturbation experiments showed interaction between the N-terminal peptide and the N domain, and it has been suggested that the N-terminal peptide binds at the interface between the A and N domains [34]. Similar interfaces exist in P4- and P5-ATPases, which have autoinhibitory tails that bind at the interface of the A, N and P domains [35,36].

Copper is delivered to the ATP7B N-terminal tail by the copper chaperone Atox1 [7,37], which has also been suggested to deliver copper to the intramembrane copper binding site [38,39]. Copper binding to the N-terminal tail leads to both limited local structural changes in individual MBDs [18] and changes in the dynamics of the entire N-terminal tail [34,40,41]. For instance, MD simulations showed that copper binding leads to reduced root mean square fluctuations (RMSF) and subtle movements in amino acid side chains in single MBDs [42]. In MD simulations of two-domain MBD constructs, copper binding to one MBD induced correlated changes in Cα fluctuations in the other MBD [43]. Moreover, mutations introduced in the CXXC motif, most notably in MBD2 and MBD3, were observed to change the ability of other MBDs to bind copper, likely via changes to hydrogen bonding networks involving the cysteine thiol groups [44]. The subtle local effects of copper binding alters the dynamics of the entire N-terminal tail and results in protein activation [23]. Such changes in structural dynamics are difficult to monitor, but copper binding has been reported to cause increased mobility in the MBD1-3 part of a MBD1-6 construct [24,34], and result in a more compact formation in a MBD1-4 construct [41]. However, the underlying mechanism of copper-dependent changes in structural dynamics is currently unknown.

The N-terminal tail is generally believed to be autoinhibitory, with copper binding leading to a release of this inhibition [45,46]. Several inhibition mechanisms have been proposed, involving both the MBD1-3 and MBD5-6 groups. The MBD5-6 complex is important for copper transport, and deletion or mutation of both MBD5 and MBD6 renders ATP7B inactive [4,47–49]. This is similar to prokaryotic CopA proteins with one or two MBDs, where deletion or mutation impairs activity [46]. For single-MBD CopA proteins it has been suggested that the copper-free MBD interacts with the A domain to prevent domain movements [50] or that the MBD blocks access to the membrane ion-binding site [46]. In ATP7B, the situation is more complex due to the presence of six MBDs. The MBD5-6 complex has been proposed to play a similar role as in single-MBD prokaryotes [46], but there is also evidence suggesting that MBD5 or MBD6 are directly involved in copper transfer to the intramembrane copper binding site [47]. The MBD1-3 complex, however, is believed to be purely regulatory since it can be mutated or deleted without much effect on total activity [4,34,47]. One proposed mechanism is that the copper-free MBD1-3 complex is disrupted upon delivery of copper by Atox1 chaperones, which is then followed by enhanced MBD1-3 dynamics [34,47]. The movements of MBD1-3 could then enable the N-terminal peptide to dissociate from the interface between the A and N domains, thereby enabling ATP hydrolysis to trigger ATP7B ion transport [34]. Such a mechanism is in agreement with co-purification data showing that copper binding weakens the interaction between the N-terminal tail and a P-N domain construct and increases the ATP affinity of the P-N construct [33]. The mechanism is also similar to the autoinhibitory regulation mechanisms of P4- and P5-ATPases, where a regulatory terminal tail binds at the interface of the cytosolic domains, and upon a stimulatory signal the tail is removed from its binding site, thereby releasing the autoinhibition [35,36]. Despite recent progress in understanding how the isolated ATP7B N-terminal respond to copper binding, the changes in N-terminal structural dynamics in the presence of the protein core domains are still elusive.

In this work, we constructed a homology model of the human ATP7B core domains and N-terminal tail based on the LpCopA crystal structure, solution structures of the A and N domains, NMR structures of the six MBDs, and current information about domain interactions and performed MD simulations to study copper-induced differences in the dynamics of the protein N-terminal tail. We found the N-terminal tail, in particular MBD2 and MBD3, to show higher mobility and to be more distant to the core domains upon copper binding. We also identified a copper-dependent interaction between MBD5 and the linkers connecting the A- and M-domains that positioned the MBD5 copper-binding motif closer to the ion-entry site in the M domain. Thus, the simulation data are congruent with an activation mechanism where MBD1-3 act as a switch that lose inter-domain interactions upon copper binding and results in priming the structure for copper delivery to the internal transport sites via MBD5/6. The simulation data predict several putative points of regulatory interactions that are testable in experimental settings to further improve understanding of ATP7B regulation.

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[1] Url: https://journals.plos.org/ploscompbiol/article?id=10.1371/journal.pcbi.1010074

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