Cryo‐EM analysis provides new mechanistic insight into ATP binding to Ca 2+ ‐ATPase SERCA2b
2021; Springer Nature; Volume: 40; Issue: 19 Linguagem: Inglês
10.15252/embj.2021108482
ISSN1460-2075
AutoresYuxia Zhang, Satoshi Watanabe, Akihisa Tsutsumi, Hiroshi Kadokura, Masahide Kikkawa, Kenji Inaba,
Tópico(s)Calcium signaling and nucleotide metabolism
ResumoArticle30 August 2021free access Transparent process Cryo-EM analysis provides new mechanistic insight into ATP binding to Ca2+-ATPase SERCA2b Yuxia Zhang Yuxia Zhang orcid.org/0000-0001-9442-8769 Institute of Multidisciplinary Research for Advanced Materials, Tohoku University, Sendai, Japan Search for more papers by this author Satoshi Watanabe Satoshi Watanabe orcid.org/0000-0002-1130-0477 Institute of Multidisciplinary Research for Advanced Materials, Tohoku University, Sendai, Japan Search for more papers by this author Akihisa Tsutsumi Akihisa Tsutsumi Graduate School of Medicine, The University of Tokyo, Tokyo, Japan Search for more papers by this author Hiroshi Kadokura Hiroshi Kadokura orcid.org/0000-0001-7308-7141 Institute of Multidisciplinary Research for Advanced Materials, Tohoku University, Sendai, Japan Search for more papers by this author Masahide Kikkawa Masahide Kikkawa orcid.org/0000-0001-7656-8194 Graduate School of Medicine, The University of Tokyo, Tokyo, Japan Search for more papers by this author Kenji Inaba Corresponding Author Kenji Inaba [email protected] orcid.org/0000-0001-8229-0467 Institute of Multidisciplinary Research for Advanced Materials, Tohoku University, Sendai, Japan Search for more papers by this author Yuxia Zhang Yuxia Zhang orcid.org/0000-0001-9442-8769 Institute of Multidisciplinary Research for Advanced Materials, Tohoku University, Sendai, Japan Search for more papers by this author Satoshi Watanabe Satoshi Watanabe orcid.org/0000-0002-1130-0477 Institute of Multidisciplinary Research for Advanced Materials, Tohoku University, Sendai, Japan Search for more papers by this author Akihisa Tsutsumi Akihisa Tsutsumi Graduate School of Medicine, The University of Tokyo, Tokyo, Japan Search for more papers by this author Hiroshi Kadokura Hiroshi Kadokura orcid.org/0000-0001-7308-7141 Institute of Multidisciplinary Research for Advanced Materials, Tohoku University, Sendai, Japan Search for more papers by this author Masahide Kikkawa Masahide Kikkawa orcid.org/0000-0001-7656-8194 Graduate School of Medicine, The University of Tokyo, Tokyo, Japan Search for more papers by this author Kenji Inaba Corresponding Author Kenji Inaba [email protected] orcid.org/0000-0001-8229-0467 Institute of Multidisciplinary Research for Advanced Materials, Tohoku University, Sendai, Japan Search for more papers by this author Author Information Yuxia Zhang1, Satoshi Watanabe1, Akihisa Tsutsumi2, Hiroshi Kadokura1, Masahide Kikkawa2 and Kenji Inaba *,1 1Institute of Multidisciplinary Research for Advanced Materials, Tohoku University, Sendai, Japan 2Graduate School of Medicine, The University of Tokyo, Tokyo, Japan *Corresponding author. Tel: +81 22 217 5604; Fax: +81 22 217 5605; E-mail: [email protected] The EMBO Journal (2021)40:e108482https://doi.org/10.15252/embj.2021108482 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract Sarco/endoplasmic reticulum Ca2+-ATPase (SERCA) 2b is a ubiquitous SERCA family member that conducts Ca2+ uptake from the cytosol to the ER. Herein, we present a 3.3 Å resolution cryo-electron microscopy (cryo-EM) structure of human SERCA2b in the E1·2Ca2+ state, revealing a new conformation for Ca2+-bound SERCA2b with a much closer arrangement of cytosolic domains than in the previously reported crystal structure of Ca2+-bound SERCA1a. Multiple conformations generated by 3D classification of cryo-EM maps reflect the intrinsically dynamic nature of the cytosolic domains in this state. Notably, ATP binding residues of SERCA2b in the E1·2Ca2+ state are located at similar positions to those in the E1·2Ca2+-ATP state; hence, the cryo-EM structure likely represents a preformed state immediately prior to ATP binding. Consistently, a SERCA2b mutant with an interdomain disulfide bridge that locks the closed cytosolic domain arrangement displayed significant autophosphorylation activity in the presence of Ca2+. We propose a novel mechanism of ATP binding to SERCA2b. SYNOPSIS Sarco/endoplasmic reticulum Ca2+-ATPase 2b (SERCA2b) is a ubiquitous transporter that conducts Ca2+ uptake from the cytosol into the ER. Here, structural analysis reveals an unexpectedly compact conformation of human SERCA2b in the Ca2+-bound but ATP-unbound state, suggesting a novel mechanism of ATP binding to SERCA family members. Cryo-EM analysis of SERCA2b in the E1∙2Ca2+ state at 3.3 Å resolution shows a much closer arrangement of the cytosolic domains than previously reported for SERCA1a. An ATP molecule can bind to the ATP-binding pocket of closed-form SERCA2b without major cytosolic domain rearrangements. A SERCA2b Cys-bridge mutant with locked closed cytosolic domains displays significant autophosphorylation in the presence of Ca2+. The closed form of Ca2+-bound SERCA2b likely represents a preformed state immediately prior to ATP binding. Introduction Sarco/endoplasmic reticulum Ca2+ ATPase (SERCA) proteins are members of the P-type ATPase superfamily that are involved in transporting various cations including protons, calcium, potassium, and sodium ions across cell membranes (Bublitz et al, 2010), and other physiological processes such as lipid flipping (Axelsen & Palmgren, 1998). In terms of structures and mechanisms of action, SERCAs are the best studied members of the superfamily (Møller et al, 2005; Michelangeli & East, 2011). About 20 years ago, the first crystal structure of SERCA1a, an isoform expressed specifically in fast-twitch skeletal-muscle fibers (Zhang et al, 1995), was reported in the E1·2Ca2+ state (Toyoshima et al, 2000). Crystal structures of SERCA1a in different intermediate states have since been determined, providing deep insights into the underlying mechanism of Ca2+ transport by the Ca2+ ATPase (Fig 1A), as well as the general structural and mechanistic features of the P-type ATPase superfamily (Toyoshima, 2009; Bublitz et al, 2013; Dyla et al, 2019b). Figure 1. Cryo-EM structure of SERCA2b WT in the E1∙2Ca2+ state Catalytic cycle for SERCA to transport Ca2+ from the cytosol to the ER lumen through ATP hydrolysis. The intermediate state of which cryo-EM structures have been determined in this work is colored magenta. Cryo-EM map of SERCA2b WT in the E1∙2Ca2+ state (upper) and its cartoon representation (lower). The A, N, and P domains and transmembrane helices (TM1−TM11) are colored yellow, magenta, cyan, and wheat, respectively. Download figure Download PowerPoint Despite remarkable progress in structural and mechanistic studies on SERCA isoforms, their catalytic mechanisms remain contentious. While it is believed that the Ca2+ transport cycle of SERCA is initiated by the coordinated binding of two Ca2+ ions and one ATP molecule to the transmembrane (TM) and nucleotide-binding (N) domains, respectively (Mueller et al, 2004; Inesi et al, 2006; Toyoshima, 2009; Møller et al, 2010), one of the most discussed issues is the functional significance of the widely opened cytosolic domain arrangement observed in the crystal structure of SERCA1a in the E1·2Ca2+ state (Liu & Barth, 2003; Dyla et al, 2019b). Thus, it is still under debate as to whether such a gate opening accompanied by large domain movements actually takes place when the ATP molecule enters the ATP-binding pocket (Ravishankar et al, 2020). Based on the highly mobile nature of the actuator (A) and N domains in this state, and the possible bias caused by crystal packing, the crystal structure may represent only one structural aspect of the calcium-bound but ATP-unbound state. In this context, scientists have been seeking genuine and as-yet-unidentified SERCA intermediates, and visualizing their structures by employing various spectroscopic and computational methods, including fluorescence resonance energy transfer (FRET) (Dyla et al, 2017; Raguimova et al, 2018), molecular dynamics (MD) simulation (Mueller et al, 2004; Huang et al, 2009; Kekenes-Huskey et al, 2012; Das et al, 2014), and time-resolved X-ray solution scattering (TR-XSS) (Ravishankar et al, 2020). Recent TR-XSS analysis combined with MD simulations identified three transient states of SERCA1a during the transition to the ATP-bound state, named the pre-pulse (i.e., pre-ATP-bound), intermediate, and late states, with respect to the cytosolic domain arrangements (Ravishankar et al, 2020), although this approach did not provide high-resolution structures due to its inherent technical limitations. In the present work, we employed cryo-EM single-particle analysis and thereby determined a new intermediate structure of human SERCA2b in the E1·2Ca2+ state at a resolution of 3.3 Å. We revealed that a significant portion of SERCA2b in this state adopts a much more closed cytosolic domain arrangement than observed in previous crystal structures of SERCA1a (Toyoshima et al, 2000). Interestingly, the cryo-EM structure is highly similar to our previously reported cryo-EM structure of SERCA2b in the E1·2Ca2+-AMPPCP state (Zhang et al, 2020), in terms of both the TM helix arrangement and the location and orientation of ATP-binding residues. In this conformation, SERCA2b in the E1·2Ca2+ state possesses a compact headpiece cluster of the cytosolic domains, forming a cavity that appears primed for ATP binding. In line with these structural findings, a SERCA2b mutant with an interdomain disulfide bridge that locks the relative positions of the A and N domains in the closed form displayed significant autophosphorylation activity, suggesting that the newly identified structure represents a preformed state immediately prior to ATP binding. Based on these results, we propose a new mechanism of ATP binding to Ca2+-bound SERCA2b and discuss the ligand-induced conformational transitions in SERCA during its catalytic cycle. Results SERCA2b displays multiple conformations in the E1·2Ca2+ state We expressed and purified SERCA2b essentially as described previously (Inoue et al, 2019; Zhang et al, 2020). The cryo-EM structure of SERCA2b in the E1·2Ca2+ state was determined at a resolution of 3.3 Å (Fig 1 and Table EV1). In the first round of 3D classification based on selected 2D classification images, cryo-EM maps of Ca2+-bound SERCA2b were classified into four different classes (Fig EV1A). The additional round of 3D classification generated two major classes of conformations named "closed form" and "possible open form" in significant abundance (˜20.7 and 37.7%, respectively; Fig EV1A). The "possible-open-form" cryo-EM maps roughly superpose with the crystal structure of SERCA1a in the E1·2Ca2+ state (PDB ID: 1SU4 and 2C9M) (Toyoshima et al, 2000; Jensen et al, 2006) (Fig EV1C), suggesting that SERCA2b actually adopts the previously reported "open form" in the presence of Ca2+. However, the resolution of the "possible-open-form" cryo-EM map was low (˜12 Å resolution), and not improved by additional 3D classifications, probably due to the highly mobile cytosolic domains or the ensemble of their heterogeneous arrangements in this state. Alternatively, it is possible that such low-resolution cryo-EM maps may be ascribed to particle damage during grid preparation and/or data collection, resulting in inaccurate averaged particle densities. Click here to expand this figure. Figure EV1. Workflow of data processing for cryo-EM single-particle analysis and local resolution analysis of SERCA2b in the E1∙2Ca2+ state Multiple classes of density map were generated after the first round of 3D classification, which were divided into two classes during the second round of 3D classification. A workflow of data processing for the cryo-EM single-particle analysis is shown on the left. Local resolution estimation of the "closed-form" cryo-EM map of SERCA2b in the E1∙2Ca2+ state. The panel on the right shows the FSC curve for post-processing of SERCA2b WT in the E1∙2Ca2+ state. Superposition of the crystal structure of SERCA1a in the E1∙2Ca2+ state (PDB ID: 1SU4) onto the present "possible-open-form" class 2 and class 3 cryo-EM maps. Download figure Download PowerPoint Importantly, the closed form identified by the present cryo-EM analysis places three cytosolic domains in much closer proximity than the open form (Figs 1B and 2). The presence of four similar cryo-EM maps (Fig EV1A) suggests that the closed form is less diverse in cytosolic domain arrangement or fluctuates to a lesser extent than the open form. In this context, a pre-ATP-bound form had been identified by unrestrained MD simulations from the "open-form" crystal structure of SERCA1a (Ravishankar et al, 2020), during which the A and N domains were calculated to approach each other, with the distance of 45 Å between Thr171 (A domain) and Lys515 (N domain) decreasing to 29 Å. Eventually, these domains were settled at the intermediate positions between those in the present "closed-form" cryo-EM structure of SERCA2b and those in the "open-form" crystal structure of SERCA1a. Figure 2. Comparison between a "closed-form" cryo-EM structure of SERCA2b and an "open-form" crystal structure of SERCA1a in the E1∙2Ca2+ state Cryo-EM structure of SERCA2b WT in the E1∙2Ca2+ state (left) and the crystal structure of SERCA1a (PDB ID: 1SU4) in the E1∙2Ca2+ state (right). The A, N, and P domains are colored yellow, magenta, and cyan, respectively. TM helices are colored wheat. TM1 in these two structures is highlighted by cartoons on the right of ribbon diagrams. Calcium ions are represented as green spheres. Orange circles indicate Thr171 and Lys514 (Lys515 in SERCA1a), and the double-headed arrows in black represent the distance between these two residues. Top view of the P domain (left), the N and A domains (right) in the cryo-EM structure of SERCA2b (yellow), and the crystal structure of SERCA1a in the E1∙2Ca2+ state (gray). The rearrangements of these cytosolic domains are indicated by black arrows. Top view of the TM helix domain (left) in the cryo-EM structure of SERCA2b (yellow) and the crystal structure of SERCA1a (gray) in the E1∙2Ca2+ state. The right inset highlights the rearrangements of TM1−TM5 between these two structures. Structures are superimposed such that the RMSD of Cα atoms in TM7 to TM10 is minimized. The green circles represent Asp59 and Glu309 located at the kink sites of TM1 and TM4, respectively. Download figure Download PowerPoint Overall cryo-EM structure of the closed form in the E1·2Ca2+ state In the present "closed-form" cryo-EM map of SERCA2b in the E1·2Ca2+ state, all cytosolic domains and TM helices, including TM11 and the luminal extension tail (LE), can be clearly seen (Figs 1B, EV2A and EV5). Similar to E1·2Ca2+-AMPPCP and E2-BeF3− states (Zhang et al, 2020), the LE in the E1·2Ca2+ state is located near the luminal ends of TM10 and TM7, and approaches the short α-helix (Phe866−Ser870) in L7/8 (Fig EV2A, bottom inset). The P domain displayed stronger density and higher resolution than the A and N domains (Fig EV1B), suggesting that it is relatively static in this state. By contrast, the A domain and the upper part of the N domain were not well-defined and yielded fragmented density, resulting in a lower resolution (˜4.5 Å) than other domains (Fig EV1B). The density of two Ca2+ ions bound in the pocket formed by TM4, 5, 6, and 8 is present at almost the same position as in the crystal structure of SERCA1a in the E1·2Ca2+ state (Fig EV2B and D) and the cryo-EM structure of SERCA2b in the E1·2Ca2+-AMPPCP state (Fig EV2C) (Zhang et al, 2020). Click here to expand this figure. Figure EV2. Close-up views of the C-terminal part, Ca2+-binding sites, and ATP-binding pocket of SERCA2b TM11 and the LE of SERCA2b are highlighted in red, while TM1-TM10 are colored wheat. L7/8 is colored blue. The backbone model of TM11 in SERCA2b in the E1∙2Ca2+ state was placed based on the cryo-EM map (left inset). Density is shown at a contour level of 3.5 RMSD. The backbone model of the LE in SERCA2b in the E1∙2Ca2+ state was placed based on the cryo-EM map (bottom inset). Density is shown at a contour level of 2.7 RMSD. Close-up view of the Ca2+-binding sites of SERCA2b (yellow) and SERCA1a (green) in the E1∙2Ca2+ state, in which bound Ca2+ ions are depicted as spheres. Ca2+ binding residues are shown as sticks. Note that the mode of Ca2+ binding is almost identical between these two states. Close-up view of the Ca2+-binding sites of SERCA2b in E1∙2Ca2+ (yellow) and E1∙2Ca2+-AMPPCP (cyan) states, in which bound Ca2+ ions are depicted as spheres. Ca2+ binding residues are shown as sticks. Note that the mode of Ca2+ binding is almost identical between these two states. Close-up view of the Ca2+-binding sites in the E1∙2Ca2+ state, in which bound Ca2+ ions and their density are represented by purple spheres and violet meshes, respectively. Neighboring residues are shown as sticks. Density is shown at a contour level of 5.0 α. Density maps of the residues that constitute the ATP-binding pocket of SERCA2b in the E1∙2Ca2+ state, shown at a contour level of 5.0 α. The residues are shown as sticks. Download figure Download PowerPoint Structural comparison between the closed and open forms in the E1·2Ca2+ state The overall domain arrangement in the "closed-form" cryo-EM structure of SERCA2b differs significantly from that in the "open-form" crystal structure of SERCA1a (Toyoshima et al, 2000), with a root mean square deviation (RMSD) value of 5.9 Å for all Cα atoms (Fig 2A). Meanwhile, the TM helices undergo small positional shifts between the open and closed forms, with an RMSD value of 0.83 Å for TM1−TM10. Thus, the positions and orientations of TM helices seem to be stabilized by bound Ca2+. However, among all TM helices, TM1 and TM2 undergo a remarkable movement (Fig 2A and C). TM2 moves upward and shifts away from TM3 (Fig 2C). Similarly, TM1 moves toward the cytosolic side. Notably, the cytosolic part of TM1 is kinked largely at Asp59 in the closed form, so that it becomes nearly parallel to the membrane surface (Fig 2A and C). The kink of TM1 is believed to function as a "sliding door" allowing Ca2+ entry and thereby facilitating Ca2+ binding in SERCA proteins (Winther et al, 2013). Such a kink in the N-terminal TM helix is commonly seen in P-type ATPases of known structure (Dyla et al, 2017; Focht et al, 2017), including P4-type ATPase (Hiraizumi et al, 2019; Timcenko et al, 2019), and Na+/K+-ATPase (Morth et al, 2007; Nyblom et al, 2013). Concomitant with the movements of TM1 and TM2, the A domain undergoes a significant positional shift (8.8 Å) and rotation (32°) (Fig 2B, right). Compared with TM1 and TM2, TM4 and TM5 seem to undergo smaller positional shifts between the open and closed forms (Fig 2C). However, their cytosolic halves, which are located distant from the Ca2+-binding sites and directly linked to the P domain, are bent toward TM2 by 20.7° and 6.9°, respectively, in the closed form (Fig 2C, inset). Concomitantly, the P domain rotates by 12° and approaches the A domain by 5.7 Å relative to that in the open form (Fig 2B, left). As a result, the side chain of Asn213 in the A domain is hydrogen bonded to the side chain of Thr723 in the P domain (Fig 4A, left inset), stabilizing the closed form in the E1·2Ca2+ state. Of note, the position of the N domain in the closed form is largely different from that in the "open-form" crystal structure of SERCA1a (Figs 2A and B, right); the distance between Cα atoms of Lys515 (N domain) and Thr171 (A domain) is 17 Å in the former, but 45 Å in the latter. In the closed form, the close proximity of the N domain to the A domain seems to be ensured by five side-chain interactions between these two domains: Arg134 (A domain)−Asp426 (N domain), Arg139 (A domain)−Glu435 (N domain), Arg139 (A domain)−Lys436 (N domain), Thr171 (A domain)−Glu486 (N domain), and Lys218 (A domain)−Asp422 (N domain) (Fig 4A, right inset). Thus, although the N domain was previously shown to undergo a large movement upon ATP binding to hold the nucleotide tightly (Toyoshima & Mizutani, 2004), the present cryo-EM analysis suggests that such an extensive domain movement can occur within the E1·2Ca2+ state even without ATP binding, hence the closed form accounts for a significant proportion of the population in this state. Structural comparison between the closed form of the E1·2Ca2+ state and the E1·2Ca2+-AMPPCP state We next compared the "closed-form" cryo-EM structure of SERCA2b in the E1·2Ca2+ state with that of SERCA2b in the E1·2Ca2+-AMPPCP state, in which the cytosolic domains are in even closer contact with each other due to bound AMPPCP (Fig 3) (Zhang et al, 2020). Although both structures possess a closed headpiece cluster of the cytosolic domains, they show significant differences, especially in the position of the cytosolic domains, with an RMSD value of 3.6 Å for all Cα atoms (Fig 3B). By contrast, structural alignment based on TM7−TM10 demonstrated that all TM helices including the Ca2+-binding sites are highly superimposable with each other between E1·2Ca2+ and E1·2Ca2+-AMPPCP states, with an RMSD value of 0.197 Å (Fig 3B, bottom inset). Thus, TM helices barely move during the transition from the "closed-form" SERCA2b in the E1·2Ca2+ state to the E1·2Ca2+-ATP state. Figure 3. Conformational transition from the closed form of the E1∙2Ca2+ state to the E1∙2Ca2+-ATP state Top view of the superposition of the A, N, and P domains between E1∙2Ca2+ and E1 ∙2Ca2+-ATP states, in which all domains and TM helices are shown with transparency 0 (i.e., dense) for the E1∙2Ca2+ state and 0.5 (i.e., faint) for the E1∙2Ca2+-ATP state. Cryo-EM structures of SERCA2b in E1∙2Ca2+ and E1∙2Ca2+-ATP states are superimposed with each other such that the RMSD of all Cα atoms are minimized. The black and red arrows indicate ATP-induced movements of three β-strands and three α-helices contained in the C-terminal half of the P domain, respectively. The solid and dashed lines indicate the positions of these secondary structure elements before and after ATP binding, respectively. D351 indicated by spheres is a phosphorylation site. Superimposition of cryo-EM structures of SERCA2b in E1∙2Ca2+ (yellow) and E1∙2Ca2+-ATP (cyan) states, in which TM7−TM10 are aligned with each other. The right inset shows a close-up view of the ATP-binding sites of SERCA2b in E1∙2Ca2+ (yellow) and E1∙2Ca2+-AMPPCP (cyan) states, in which the two structures are superimposed such that the RMSD of their Cα atoms in the N domain is minimized. A Mg2+ ion close to AMPPCP is depicted as a green sphere. The lower inset highlights the side (left) and top (right) views of TM1−TM11 in E1∙2Ca2+ (yellow) and E1∙2Ca2+-AMPPCP (cyan) states. The purple and cyan spheres indicate two bound Ca2+ ions in the E1∙2Ca2+ and E1∙2Ca2+-ATP states, respectively. Download figure Download PowerPoint Among the three cytosolic domains, the N domain moves most prominently during the transition from E1·2Ca2+ to E1·2Ca2+-ATP states, with rotation of 16° (Fig 3A, middle). Consequently, the N domain approaches the P domain, leading to tight ATP binding. Concomitant with the N domain movement, the P domain undergoes ATP-induced conformational changes; the C-terminal half of the P domain, including three β-strands and three α-helices, inclines toward the phosphorylation site (Asp351) upon ATP binding (Fig. 3A, right), forming an even more compact globular fold, as was also seen in crystal structure of SERCA2a in the E2-AMPPCP state (Kabashima et al, 2020). Thus, the P domain folds loosely in the closed form of the E1·2Ca2+ state, which may serve to facilitate the delivery of the γ-phosphate group of ATP into the appropriate site in the P domain. The A domain also undergoes positional shifts upon ATP binding, with rotation of 14° (Fig 3A, left), leading to tighter interactions with the N and P domains, as described in the following paragraph. In brief, the cytosolic headpiece cluster of SERCA2b becomes even more compact upon ATP binding. In support of this, multiple salt bridges and hydrogen bonds are formed at the cytosolic domain interfaces in the E1·2Ca2+-ATP state, including those between the A and N domains: Arg134 (A domain)−Asp426 (N domain), Arg134 (A domain)−Tyr427 (N domain), Arg139 (A domain)−Lys436 (N domain), Thr171 (A domain)−Glu486 (N domain), Lys218 (A domain)−Asp422 (N domain), Thr171 (A domain)−Leu577 (N domain), Arg139 (A domain)−Glu435 (N domain), and Lys169 (A domain)−Thr484 (N domain) (Fig 4B, right inset), and those between the A and P domains: Gly156 (A domain)−Thr723 (P domain), Asn213 (A domain)−Thr728 (P domain), and Asn213 (A domain)−Thr723 (P domain) (Fig 4B, left inset). Figure 4. Interface between the A and N domains in E1∙2Ca2+ and E1∙2Ca2+-AMPPCP states Side (left) and top (right) views of the A, N, and P domains in the cryo-EM structure of SERCA2b in the E1∙2Ca2+ state represented as a surface model. Residues critical for the domain interactions in the E1∙2Ca2+ state are represented by sticks in the left and right insets. Gray mesh in the inset indicates the density of the resides shown at a contour level of 5.0 σ. The circle in the left panel indicates a cavity that may serve as an ATP entry gate in the "closed-form" SERCA2b. Side (left) and top (right) views of the A, N, and P domains in the cryo-EM structure of SERCA2b in the E1∙2Ca2+-AMPPCP state shown in surface model representation. Residues critical for the domain interactions in the E1∙2Ca2+-AMPPCP state are represented by sticks in the left and right insets. Dotted lines in the right inset indicate hydrogen bonds and salt bridges formed between the residues at the domain interface. Download figure Download PowerPoint Notably, many of the above interactions are already formed prior to ATP binding, between the A and N domains and between the A and P domains (Fig 4A, left and right insets). Additionally, hydrogen bonds are newly identified between the side chain of Arg489 (N domain) and the main-chain carbonyl group of Val678 (P domain) and between the side chains of Lys492 (N domain) and Arg677 (P domain) in the E1·2Ca2+ state (Fig 4A, left inset). As a consequence of these interdomain interactions, the ATP binding residues, namely, Asp351, Met494, Glu442, Phe482, Arg489, Lys514, Arg559, Lys683, Arg677, Asp702, and Asn705, in the E1·2Ca2+ state are located at similar positions to those in the E1·2Ca2+-ATP state, and the cavity constituted by these residues appears to shrink upon ATP binding (Fig 3B, right inset, and Fig EV2E). In this regard, the "closed-form" E1·2Ca2+ state newly identified in this study can be interpreted as a preformed state of SERCA immediately prior to ATP binding. ATP enters and phosphorylates the closed form of SERCA2b To examine whether the "closed-form" SERCA2b in the E1·2Ca2+ state does allow ATP to enter the cavity, we carried out biochemical experiments. With the purpose of locking the relative positions of the A and N domains in the closed form (Lopez-Redondo et al, 2018), we focused upon the interface between the A and N domains and found several amino acid pairs that have potential to form an interdomain disulfide bridge. We thus prepared six kinds of SERCA2b mutants by site-directed mutagenesis: Q138C/D426C, V137C/D426C, A154C/G438C, V155C/G438C, T171C/E486C, and T171C/L577C (Fig EV3A). Among them, T171C/L577C showed a slight but significant upward band shift relative to WT after oxidative treatment with potassium ferricyanide (K3Fe(CN)6) (Fig EV3B). Consistently, a Cβ-Cβ distance between T171 and L577 is 4.1 Å in the cryo-EM structure of SERCA2b in the E1·2Ca2+ state (Fig 5A). Additionally, it is predicted that two sulfur atoms of the introduced cysteines are located apart by a distance of 2.0 Å, and that a torsion angle of the Cβ–S–S–Cβ is +110° (Fig 5A, inset). These conditions are likely to meet the criteria for forming a disulfide bond between these two sites (Dombkowski et al, 2014). Click here to expand this figure. Figure EV3. Cytosolic domain interface in the "closed-form" of SERCA2b and critical residues for the cytosolic domain interactions in the E1∙2Ca2+ state Closed-up view of the interface between the A and N domains in the "closed-form" cryo-EM structure of SERCA2b. The amino acid pairs in which Cα atoms are located within a distance of 8.5 Å are represented by sticks, and their cysteine mutants were prepared in this study. Six kinds of SERCA2b mutants including Q138C/D426C, V137C/D426C, A154C/G438C, V155C/G438C, T171C/E486C, or T171C/L577C were treated with no reagent (upper), DTT (middle) or K3Fe(CN)6 (lower). Among them, T171C/L577C showed a significant upward band shift relative to WT after oxidative treatment with K3Fe(CN)6. Close-up views of the interface between the A and P domains in the "open-form" crystal structure of SERCA1a in the E1∙2Ca2+ state (upper) (PDB ID: 1SU4), and the interfaces between the cytosolic A, P, and N domains in the "closed-form" cryo-EM structure of SERCA2b in the E1∙2Ca2+ state (lower). Critical residues for the cytosolic domain interactions are represented by sticks. Dotted lines indicate hydrogen bonds or salt bridges formed between those residues. Magenta spheres represent two bound Ca2+ ions. Download figure Download PowerPoint Figure 5. Functional characterization of the "closed-form" SERCA2b Close-up view of the interface between the A and N domains. The engineered site in the T171C/L577C mutant is highlighted in the inset. SDS–PAGE analysis of purified SERCA2b WT and T171C/L577C (0.5 µg each) after treatment with no reagent (−), 10 mM DTT, or 5 mM K3Fe(CN
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