Nucleotide and metalloid-driven conformational changes in the arsenite efflux ATPase ArsA

Nucleotide and metalloid-driven conformational changes in the arsenite efflux ATPase ArsA | bioRxiv /* */ /* */ <!-- <!-- /*! * yepnope1.5.4 * (c) WTFPL, GPLv2 */ (function(a,b,c){function d(a){return"[object Function]"==o.call(a)}function e(a){return"string"==typeof a}function f(){}function g(a){return!a||"loaded"==a||"complete"==a||"uninitialized"==a}function h(){var a=p.shift();q=1,a?a.t?m(function(){("c"==a.t?B.injectCss:B.injectJs)(a.s,0,a.a,a.x,a.e,1)},0):(a(),h()):q=0}function i(a,c,d,e,f,i,j){function k(b){if(!o&&g(l.readyState)&&(u.r=o=1,!q&&h(),l.onload=l.onreadystatechange=null,b)){"img"!=a&&m(function(){t.removeChild(l)},50);for(var d in y[c])y[c].hasOwnProperty(d)&&y[c][d].onload()}}var j=j||B.errorTimeout,l=b.createElement(a),o=0,r=0,u={t:d,s:c,e:f,a:i,x:j};1===y[c]&&(r=1,y[c]=[]),"object"==a?l.data=c:(l.src=c,l.type=a),l.width=l.height="0",l.onerror=l.onload=l.onreadystatechange=function(){k.call(this,r)},p.splice(e,0,u),"img"!=a&&(r||2===y[c]?(t.insertBefore(l,s?null:n),m(k,j)):y[c].push(l))}function j(a,b,c,d,f){return q=0,b=b||"j",e(a)?i("c"==b?v:u,a,b,this.i++,c,d,f):(p.splice(this.i++,0,a),1==p.length&&h()),this}function k(){var a=B;return a.loader={load:j,i:0},a}var l=b.documentElement,m=a.setTimeout,n=b.getElementsByTagName("script")[0],o={}.toString,p=[],q=0,r="MozAppearance"in l.style,s=r&&!!b.createRange().compareNode,t=s?l:n.parentNode,l=a.opera&&"[object Opera]"==o.call(a.opera),l=!!b.attachEvent&&!l,u=r?"object":l?"script":"img",v=l?"script":u,w=Array.isArray||function(a){return"[object Array]"==o.call(a)},x=[],y={},z={timeout:function(a,b){return b.length&&(a.timeout=b[0]),a}},A,B;B=function(a){function b(a){var a=a.split("!"),b=x.length,c=a.pop(),d=a.length,c={url:c,origUrl:c,prefixes:a},e,f,g;for(f=0;f<d;f++)g=a[f].split("="),(e=z[g.shift()])&&(c=e(c,g));for(f=0;f<b;f++)c=x[f](c);return c}function g(a,e,f,g,h){var i=b(a),j=i.autoCallback;i.url.split(".").pop().split("?").shift(),i.bypass||(e&&(e=d(e)?e:e[a]||e[g]||e[a.split("/").pop().split("?")[0]]),i.instead?i.instead(a,e,f,g,h):(y[i.url]?i.noexec=!0:y[i.url]=1,f.load(i.url,i.forceCSS||!i.forceJS&&"css"==i.url.split(".").pop().split("?").shift()?"c":c,i.noexec,i.attrs,i.timeout),(d(e)||d(j))&&f.load(function(){k(),e&&e(i.origUrl,h,g),j&&j(i.origUrl,h,g),y[i.url]=2})))}function h(a,b){function c(a,c){if(a){if(e(a))c||(j=function(){var a=[].slice.call(arguments);k.apply(this,a),l()}),g(a,j,b,0,h);else if(Object(a)===a)for(n in m=function(){var b=0,c;for(c in a)a.hasOwnProperty(c)&&b++;return b}(),a)a.hasOwnProperty(n)&&(!c&&!--m&&(d(j)?j=function(){var a=[].slice.call(arguments);k.apply(this,a),l()}:j[n]=function(a){return function(){var b=[].slice.call(arguments);a&&a.apply(this,b),l()}}(k[n])),g(a[n],j,b,n,h))}else!c&&l()}var h=!!a.test,i=a.load||a.both,j=a.callback||f,k=j,l=a.complete||f,m,n;c(h?a.yep:a.nope,!!i),i&&c(i)}var i,j,l=this.yepnope.loader;if(e(a))g(a,0,l,0);else if(w(a))for(i=0;i (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];var j=d.createElement(s);var dl=l!='dataLayer'?'&l='+l:'';j.src='//www.googletagmanager.com/gtm.js?id='+i+dl;j.type='text/javascript';j.async=true;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-M677548'); Skip to main content Home About Submit ALERTS / RSS Search for this keyword Advanced Search New Results Nucleotide and metalloid-driven conformational changes in the arsenite efflux ATPase ArsA View ORCID Profile Shivansh Mahajan , View ORCID Profile Ashley E. Pall , View ORCID Profile Yancheng E. Li , View ORCID Profile Timothy L. Stemmler , View ORCID Profile Douglas C. Rees , View ORCID Profile William M. Clemons Jr. doi: https://doi.org/10.1101/2025.03.21.644500 Shivansh Mahajan a Division of Chemistry and Chemical Engineering, California Institute of Technology , Pasadena, CA 91125 Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Shivansh Mahajan Ashley E. Pall b Department of Pharmaceutical Sciences, Wayne State University , Detroit, MI Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Ashley E. Pall Yancheng E. Li a Division of Chemistry and Chemical Engineering, California Institute of Technology , Pasadena, CA 91125 Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Yancheng E. Li Timothy L. Stemmler b Department of Pharmaceutical Sciences, Wayne State University , Detroit, MI Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Timothy L. Stemmler Douglas C. Rees a Division of Chemistry and Chemical Engineering, California Institute of Technology , Pasadena, CA 91125 c Howard Hughes Medical Institute Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Douglas C. Rees For correspondence: dcrees{at}caltech.edu clemons{at}caltech.edu William M. Clemons Jr. a Division of Chemistry and Chemical Engineering, California Institute of Technology , Pasadena, CA 91125 d Chan Zuckerberg Initiative Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for William M. Clemons Jr. For correspondence: dcrees{at}caltech.edu clemons{at}caltech.edu Abstract Full Text Info/History Metrics Supplementary material Preview PDF Abstract A common mechanism of arsenic detoxification in bacteria is arsenite (As III ) efflux facilitated by the ArsAB pump that couples metalloid transport to ATP hydrolysis. The cytoplasmic ATPase component, ArsA, binds and hydrolyzes ATP and facilitates the transfer of As III to the integral membrane transporter, ArsB. The underlying molecular mechanism of As III efflux by ArsAB remains unclear. ArsA is a member of the Intradimeric Walker A (IWA) family of ATPases that undergo dramatic nucleotide-dependent conformational changes to facilitate their respective biological functions. Similar conformational transitions in ArsA have been postulated to drive As III binding and transport via ArsB but have not been demonstrated. Here, we report multiple structures of ArsA determined by single-particle cryogenic electron microscopy in an open MgADP-bound state, open MgATP-bound state, and a distinct closed MgATP-bound state liganded to As III . Using X-ray absorption spectroscopy, we confirmed that As III coordinates three conserved cysteines at the metalloid-binding site of the closed state in a three-coordinate fashion. Coupled with biochemical characterization, our cryo-EM structures reveal key conformational changes in the ArsA catalytic cycle consistent with other members of the IWA family and provide the structural basis for allosteric activation of nucleotide hydrolysis by As III . This work enhances our understanding of how the ArsA catalytic cycle regulates metalloid efflux by ArsB. Introduction With a natural abundance of 1.5-2 ppm, arsenic is a toxic metalloid that contaminates soil and groundwater, posing a widespread public health risk across the globe 1 , 2 . Several bacteria and archaea resist trivalent arsenite (As III ) toxicity by using the arsenite efflux machinery encoded in the ars operon 3 – 6 . A major component of the ars operon is an integral membrane transporter, ArsB, that exports As III out of the cell. Though ArsB can function alone, it often associates with a cytoplasmic ATPase, ArsA, that sequesters As III , and enhances the efficiency of As III efflux via ArsB by coupling this process to ATP hydrolysis 7 , 8 . Intriguingly, properties of this ArsAB system are distinct from other ATP-dependent transporters, including ABC transporters and P-type ATPases, both in terms of the protein fold of the ATPase domain and that the ATPase activity is only required to improve the efficiency of transport 9 – 11 . In addition to the questions concerning the mechanism of toxic metalloid detoxification, the ArsAB transporter has garnered interest due to its biotechnological potential for arsenic bioremediation of contaminated environments 12 , 13 . ArsA is a 63 kDa pseudodimeric ATPase that consists of two homologous nucleotide-binding domains covalently linked by a flexible ∼25-residue linker 14 , 15 (Fig. S1A). The metalloid-binding site, composed of three conserved cysteines 16 , is located at the pseudodimer interface and can ligand As III or antimonite (Sb III ). Both domains of ArsA, namely the N- and C-domains, can support the binding and hydrolysis of ATP; however, the N-domain does so at a significantly higher rate than the C-domain 17 . ArsA belongs to a family of dimeric ATPases of diverse biological functions, known as the ‘Intradimeric Walker A (IWA) ATPases’ that are characterized by a conserved X-K-G-G-X-G-K-[T/S] Walker A or phosphate-binding loop (P-loop) motif 18 , 19 (Fig. S1B). The first conserved Lys residue, referred to as the ‘IWA lysine’, is critical for facilitating nucleotide hydrolysis in these enzymes 20 , 21 . Nucleotide-dependent conformational changes at the dimer interface are conserved across the IWA family 18 . The conformational landscape of other IWA ATPases such as the tail-anchored (TA) membrane protein targeting factor Get3 as well as the nitrogenase Fe-protein NifH have been extensively characterized, and their respective catalytic cycles involve conformational switching of the dimer between a ‘closed’ state that is catalytically competent for ATP hydrolysis and an ‘open’ state that is ATPase inactive 20 – 25 . The open conformation is reported for either the apo or post-hydrolysis MgADP-bound states of the ATPase cycle and is characterized by P-loops that are far apart 24 , 26 , whereas the closed state is reported for the pre- or mid-hydrolysis states where the two P-loops are adjacent 20 , 21 , mediating the position of the IWA lysines and thereby regulating nucleotide hydrolysis across the dimer interface. Two additional loop motifs, Switch I and II (Fig. S1A-B), are conserved across IWA ATPases and adopt discrete conformations depending on the nucleotide state. These nucleotide-dependent conformational transitions are critical for the function of IWA ATPases as ATP binding and hydrolysis is functionally coupled to the targeting of a substrate specific to each enzyme 18 . Biochemical and spectroscopic characterization of ArsA suggests that the ATPase undergoes a series of conformational changes through its catalytic cycle in the presence of nucleotide and metalloid 27 , 28 . Four X-ray crystal structures have been reported for E. coli ArsA ( Ec ArsA) in multiple nucleotide-bound states 14 , 29 . In each of these structures, MgADP is bound at the nucleotide-binding site on the N-domain, whereas MgADP (PDB:1F48), MgATP (PDB: 1II0), MgAMP•PNP (PDB: 1II9), or MgADP•AlF 3 (PDB: 1IHU) are bound at the analogous site on the C-domain. Despite the distinct nucleotide states, only subtle conformational changes are seen between the various structures with a root mean square deviation (r.m.s.d.) of 0.38 Å over 536 residues (Fig. S1C), supporting that crystallization has likely restricted the range of motion in the protein. How ArsA undergoes nucleotide-dependent conformational transitions through its catalytic cycle to facilitate As III efflux remains unclear. The presence of metalloid substrate activates ArsA by stimulating ATP hydrolysis, which is the proposed rate-limiting step of the enzyme 27 . The underlying mechanism for this enhancement is poorly understood. Biochemical studies with Sb III suggest that the metalloid substrate binds at a single high-affinity site in ArsA 30 . Of the three conserved cysteines, Cys172, located on a flexible loop between helices 7 and 8 of the N-domain of Ec ArsA, has been proposed to control the metalloid affinity of this site 30 , 31 . As the crystal structures are not consistent with these results, the mechanism of metalloid binding and ArsA activation requires further investigation. In this article, we report single-particle cryogenic electron microscopy (cryo-EM) structures of ArsA from a thermotolerant and acidophilic bacteria, Leptospirillum ferriphilum , in nucleotide-bound open and closed conformations. In the MgADP-bound open state, the ArsA pseudodimer adopts the conformation of the previously reported ArsA crystal structures, all representing an ATPase inactive open state. MgATP alone is unable to stabilize a major conformational change from this open state. In the presence of MgATP and As III , a distinct closed conformation is adopted that is analogous to the closed conformations observed for other IWA ATPases. These structures provide views of the conformational transitions in the ArsA catalytic cycle. Combined with X-ray absorption spectroscopy (XAS) analysis and supporting biochemical studies, our cryo-EM structures suggest the molecular mechanism of As III -activated ATP hydrolysis in ArsA. Altogether, this work provides a structural and biochemical foundation for understanding the mechanism of As III efflux by the ArsAB system. Results ArsA ATPase from Leptospirillum ferriphilum strain ML-04 Following a bioinformatic analysis of ars operons resembling the well-studied E. coli R773 plasmid ars operon 3 , we identified an ArsA homolog from Leptospirillum ferriphilum strain ML-04 ( Lf ArsA), an acidophilic and moderately thermophilic bacterial species, where some strains are found in arsenic-rich acid mine drainage ecosystems (Fig. S2A) 32 . The ArsA gene has been shown to express under arsenic stress conditions in this strain, and the operon confers arsenic resistance in closely related L. ferriphilum strains 32 – 34 . Lf ArsA has 69% sequence identity to Ec ArsA, retaining all the characteristic features of ArsA (Fig. S1B). We cloned, overexpressed, and purified Lf ArsA with a C-terminal 6x-His tag. Purified Lf ArsA hydrolyzes ATP in vitro with a pseudo first-order rate constant of 0.5 ± 0.1 min −1 , and saturating concentrations of As III stimulate the ATPase activity ∼15-fold above the basal activity (Fig. S2B, black plot). To elucidate the conformational landscape of the ArsA catalytic cycle, we sought to determine nucleotide-bound Lf ArsA structures both in the presence and absence of its substrate As III using cryo-EM. Structure of the open conformation of ArsA bound to MgADP To determine the conformation of ArsA in an MgADP and As III -bound state, ArsA was incubated with 2 mM MgCl 2 , 2 mM ADP, and 5 mM sodium arsenite, and grids were prepared for single-particle analysis. From a dataset of 4,732 movies collected on a 300 kV Titan Krios Transmission Electron Microscope (TEM) and several rounds of 2D and 3D classification (Fig. S3), we obtained a 103,258-particle reconstruction of the ArsA•MgADP state ( Fig. 1A ). The final Coulomb potential map was refined to a 3.4 Å gold-standard Fourier shell correlation (FSC) resolution and sharpened using a uniform B-factor of −165 Å 2 to better visualize high-resolution features. AlphaFold2 35 predictions for the N- and C-domains of Lf ArsA were docked into the sharpened map, followed by model building and refinement to obtain the ArsA•MgADP structure ( Fig. 1B ). The average Q-score for the structure was 0.64 (Fig. S4A), indicating good resolvability of the cryo-EM map 36 . About 95% of the protein chain was modeled into the map, with missing residues including part of the linker region between the N- and C-domains (residues 295-307), residues 167-168 and residues 464-476 ( Fig. 1B ). The N (residues 1-298) and C (residues 315-587) domains are pseudo-symmetric with an r.m.s.d. of 1.0 Å over 139 residues ( Fig. 1C ). At both nucleotide-binding sites, ADP is coordinated to a Mg 2+ ion ( Fig. 1D ). The catalytic Switch I aspartates – Asp46 (N) and Asp364 (C) – are oriented away from the Mg 2+ ion (>4.5 Å). The Switch II aspartates – Asp143 (N) and Asp447 (C) – interact with the Mg 2+ ion within 4 Å and form short hydrogen bonds with Thr23 (N) and Thr341 (C), respectively. Notably, the P-loops are sufficiently separated such that the catalytic IWA lysines (Lys17 and Lys335) are oriented away from the ADP bound at the opposing domain, positioning the terminal amino groups of the lysines over 8 Å away from the nucleotide phosphate groups ( Fig. 1E ). Download figure Open in new tab Figure 1. Cryo-EM structures of Lf ArsA in the nucleotide-bound open conformation. (A) Two views of the sharpened cryo-EM map colored based on local resolution (3.4 Å overall) for Lf ArsA•MgADP open state ( ADP-open ) in the presence of As III . (B) Cartoon representation of the ADP-open state colored in viridis. Unmodeled regions are indicated as dotted lines. (C) Left, surface representation of the ArsA pseudodimer highlighting the N-(blue) and C-(green) domains. Right, cartoon representation of the overlay of the two domains based on the pseudo - 2-fold symmetry. (D) Nucleotide-binding sites of the ADP-open state showing the Coulomb potential map around the MgADP shown as sticks. Switch I & II Asp residues of both N- and C-domains are shown as sticks. (E) The pseudo-dimer interface of the ADP-open state highlighting the distance between the IWA lysines. (F) Structure of Lf ArsA•MgADP open state in the absence of As III . Left, 3.8 Å cryo-EM map; middle, ribbons model (pale green) overlayed with the ADP-open state model from B ; right, the metalloid-binding sites from both structures. (G) Cryo-EM map (6-7 Å overall resolution) of the Lf ArsA non-hydrolyzing variant (D46N/D364N) solved in the presence of MgATP with the ADP-open state model from B docked in the map. (H) Nucleotide-binding sites of the Lf ArsA•MgATP state showing Coulomb potential map likely corresponding to ATP. MgATP was manually fit using Coot. We propose that our ArsA•MgADP structure represents an open form of ArsA, designated as the ‘ADP-open’ state for the rest of the discussion. Based on the general mechanism of the IWA ATPase family, this conformation represents an inactive form of ArsA unable to hydrolyze ATP. Comparison of the cryo-EM structure to the X-ray crystal structures of Ec ArsA reveals the structures adopt similar conformations (Fig. S1C); for example, the cryo-EM structure and the Ec ArsA•MgADP crystal structure (PDB: 1F48) exhibit an r.m.s.d. of 0.95 Å over 475 residues. Despite the presence of millimolar concentrations As III in the samples, there was no evidence of As III at the metalloid-binding site in either our EM or the X-ray structures (Fig. S5) 14 , 29 . Structures of nucleotide-bound ArsA in the absence of As III To clarify the absence of As III at the metalloid-binding site in the ArsA•MgADP structure, we solved a cryo-EM structure using identical experimental conditions but in the absence of As III . The resulting 3.8 Å resolution map of the ArsA•MgADP state is in open conformation ( Fig. 1F , S4B & S6), similar to the structure solved in the presence of As III , with an r.m.s.d. of 0.57 over 545 residues. Importantly, the configuration of the metalloid-binding site remains unchanged ( Fig. 1F ; inset on right), confirming that both states lack As III . This suggests that the ADP-open state does not effectively bind the metalloid. The presence of As III stimulates steady-state ATP hydrolysis by ArsA above the basal activity (Fig. S2B) 28 , 30 . To characterize the ATP-bound ArsA conformation in the absence of metalloid, we generated a non-hydrolyzing variant of Lf ArsA by mutating the catalytic Switch I aspartates – Asp46 and Asp364 of the N- and C-domains, respectively – to asparagines (Fig. S2B, red plot). We used this variant to capture the MgATP-bound state, similar to the approach used to obtain the MgATP-bound Get3 structure (PDB: 7SPY) 21 . For the rest of the discussion, structures solved using the D46N/D364N variant will be simply referred to as ArsA. Under experimental conditions similar to those used to solve the ADP-open structures, we could only obtain a low-resolution (6-7 Å) EM map that revealed an open state of ArsA (Fig. S7). We could confidently fit the model of the ArsA•MgADP structure into the map ( Fig. 1G ). While the density observed at the nucleotide-binding sites could not be unambiguously distinguished at this resolution, it is consistent with MgATP binding ( Fig. 1H ). This is reminiscent of NifH that is refractory to crystallization in the presence of ATP but readily crystallizes in the presence of both ATP and its partner protein, NifDK (MoFe protein) 26 . It is plausible that binding of ATP makes ArsA conformationally flexible, preventing high-resolution cryo-EM reconstruction, and that a partner protein or As III is required for stabilization. Importantly, this structure is consistent with the low basal ATPase activity of ArsA. Together, these As III -free structures support that nucleotide binding alone is unable to stabilize a conformation competent for ATP hydrolysis. Structure of the closed conformation of ArsA bound to MgATP and As III To understand the structural basis of As III activation, we sought to characterize the conformation of ArsA in the presence of both MgATP and As III . We incubated the non-hydrolyzing ArsA variant with 2 mM MgCl 2 , 2 mM ATP, and 2 mM sodium arsenite and then performed single-particle cryo-EM (Fig. S8). The resulting Coulomb potential map was resolved to an overall gold-standard FSC resolution of 3.0 Å and sharpened using a uniform B-factor of −124 Å 2 ( Fig. 2A ). AlphaFold2 models of N- and C-domains of Lf ArsA were docked into the sharpened density map, the model was built and refined to obtain the structure of ArsA•MgATP•As III with clear density for MgATP at both nucleotide-binding sites ( Fig 2B-C ). Significantly, at the metalloid-binding site, we observed density enclosed between the three conserved cysteines – Cys114, Cys173, and Cys422 – into which we modeled an arsenic atom ( Fig. 2D ). The average Q-score for the structure was 0.71 (Fig. S4C), indicating high resolvability of the cryo-EM map. About 97% of the protein was modeled in, while the regions with poor density, such as most of the linker region (residues 299-305) and residues 474-479, were omitted from the final model. Download figure Open in new tab Figure 2. Cryo-EM structure of Lf ArsA closed conformation bound to MgATP and As III . (A) Two views of the sharpened cryo-EM map colored based on local resolution (3.0 Å overall) for Lf ArsA•MgATP•As III closed state ( ATP-closed ) of the non-hydrolyzing variant (D46N/D364N). (B) Cartoon representation of the ATP-closed state colored in viridis. Unmodeled regions are indicated as dotted lines. (C) Nucleotide-binding sites of the ATP-closed state showing the Coulomb potential map around MgATP and Switch I catalytic residues (Asn46 and Asn364) shown as sticks. Switch II Asp residues of both N- and C-sites are shown as sticks. The nucleophilic water molecule ( W cat ) is highlighted. (D) Metalloid-binding site of ATP-closed state showing Coulomb potential map for As III (purple atom) coordinated by Cys114, Cys173 and Cys422. The view is rotated 110° clockwise relative to B . (E) The pseudodimer interface of the ATP-closed state highlighting the distance between the IWA lysines (Lys17 and Lys335). (F) Top-down view, relative to A , comparing the ADP-open and the ATP-closed states of Lf ArsA colored as in Fig. 1C . showing both states in cartoon and then overlayed in surface representation. The overlay is aligned to the N-domain P-loop (residues 16-23) showing the relative change of the C-domain. (G) Structure of Lf ArsA•MgATP•As III closed state solved using wild-type Lf ArsA. Left, 3.0 Å map cryo-EM map; right, ribbons model (yellow) overlayed with the model from B . (H) Comparison of Switch I conformations between the two ATP-closed structures. Differences in interactions are highlighted. Within both nucleotide-binding sites, ATP is stabilized by interactions of its γ-phosphate group with Mg 2+ and Lys (22 and 340, respectively) by electrostatic interactions and the ⍺- and β-phosphates with the P-loop backbone by hydrogen bonds ( Fig. 2C & Fig. S9A). The ribose sugar and the adenine base are stabilized by hydrogen bonds contributed by sidechains from both domains and backbone hydrogen bonds from the loop region corresponding to the adenine-binding loop (A-loop) described in Get3 structures (Fig. S9A) 24 . Additionally, ATP bound to the N-domain is stabilized by potential cation-π interactions formed between the adenine base and both Arg207 (N) and Arg544 (C) (Fig. S9A, left). However, in the C-domain, the corresponding interaction is only observed between the adenine and Arg256 (N). In this structure, the Switch I aspartates (mutated to asparagines here) – Asn46 (N) and Asn364 (C) – are positioned adjacent to each Mg 2+ ion and anchor the catalytic water molecule ( W cat ) near the γ -phosphate of ATP via a 3.0 Å hydrogen bond for nucleophilic attack, priming the enzyme for hydrolysis ( Fig. 2C ). Switch II aspartates – Asp143 (N) and Asp447 (C) – are positioned at 3.2 and 3.4 Å from the Mg 2+ ion respectively and form short hydrogen bonds with the Thr residue coordinated to the respective Mg 2+ ions, similar to the MgADP-bound structure. This structure adopts a closed pseudodimer conformation previously unobserved for ArsA that we define as the ‘ATP-closed’ state. Notably, the P-loops of the two domains are in close proximity ( Fig. 2E ). The terminal amino group of the conserved IWA lysine of each domain – Lys17 (N) and Lys335 (C) – is positioned ∼3 Å from the bridging oxygen atom between the β- and γ-phosphate groups of the ATP bound to the opposing domain. This enables stabilization of negative charge build-up on the nucleotides in the transition state. Together with the Switch I aspartates, which are positioned to activate W cat for nucleophilic attack on ATP, this closed pseudodimer conformation represents a catalytically competent state of ArsA at both nucleotide-binding sites. The ATP-closed state is analogous to the closed dimer states previously reported for Get3 (PDB: 7SPY) and NifH (PDB: 1M34) (Fig. S10) 21 , 37 . Notably, in our structure, similar to the Get3 closed structure, both IWA lysines appear to form a hydrogen bond with a water molecule found at the pseudodimer interface; these waters may assist with anchoring the terminal amino groups close to the nucleotides in preparation for ATP hydrolysis ( Fig. 2E ). Relative to the ADP-open state, the two domains rotate towards the pseudodimer interface, forming a tighter interface in the ATP-closed state ( Fig. 2F ). This generates new inter-domain contacts not observed in the ADP-open state (Fig. S8B). Glu215 and Glu507, from the N- and C-domains, respectively, form hydrogen bonds with the catalytic water molecule and Switch I residues of the opposing nucleotide-binding site. Electrostatic contacts are formed between Arg214 of the N-domain and Asp371 of the C-domain. This results in a buried surface area of 2,380 Å 2 , ∼500 Å 2 more than the open conformation. We next sought to establish the conformation of ArsA under turnover conditions. Wild-type ArsA ( wt ArsA ) was incubated with 2 mM MgCl 2 , 2 mM ATP, and 2 mM sodium arsenite at room temperature for ∼1.5 min before cryo-EM grid preparation. This resulted in a Coulomb potential map at 3.0 Å overall gold-standard FSC resolution ( Fig. 2G , left; S4D & S11). The final refined structure revealed a closed conformation similar to the ArsA non-hydrolyzing variant (r.m.s.d = 0.53 Å over 565 residues) with MgATP present at both nucleotide-binding sites and arsenic modeled at the metalloid-binding site ( Fig. 2G , right). The IWA lysines in this structure are oriented towards the nucleotides (Fig. S12A), supporting a catalytically competent pre-hydrolysis state consistent with the variant structure. While the Switch I aspartate in each nucleotide-binding site is positioned next to the Mg 2+ ion anchoring the W cat for nucleophilic attack, subtle differences are observed in the conformations of the Switch I loop between the wt ArsA and variant closed structures. In the N-domain, the Switch I loop moves towards the nucleotide in wt ArsA such that Asn50 now interacts with the Mg 2+ ion via an intervening water molecule ( Fig. 2H , left). Likewise, in the C-domain, Switch I loop shifts towards the nucleotide, causing His368 to interact with Mg 2+ via an intervening water molecule ( Fig. 2H , right). Additionally, we see evidence for a hydrogen-bonding network composed of ordered water molecules including W cat , that connects the Switch I aspartates at the N-(Asp46) and C-(Asp364) sites, potentially coupling the two active sites (Fig. S12B). Together, these subtle structural changes observed in the pre-hydrolysis state prime the enzyme to initiate hydrolysis in both N- and C-domains. The As III -binding site of ArsA We observed density for arsenic at the metalloid-binding site in the ATP-closed state, coordinated by the thiolate groups of Cys114, Cys173, and Cys422 in a pyramidal geometry ( Fig. 2D and S12C). Cys114 and Cys422 are homologous residues within the pseudodimer, located at the N-terminal end of helix 6, denoted as H6 N and H6 C , respectively, while Cys173 is located on a flexible loop (residues 155-183) between helix 7 and helix 8 in the N-domain. To confirm the As III coordination environment, we performed solution X-ray absorption spectroscopy (XAS). ArsA non-hydrolyzing variant (1.95 mM) supplemented with 15 mM MgCl 2 , 15 mM ATP, and 1.5 mM sodium arsenite was subjected to XAS analysis. X-ray absorption near edge spectra (XANES) confirmed the presence of trivalent arsenic ( Fig. 3A , black spectra), as the K-edge at 11865.9 eV was consistent with the first inflection edge energies of As III model compounds (11867 eV). Best-fit simulations of the extended X-ray absorption fine structure (EXAFS) spectra supported the presence of As III coordinated by three sulfur ligands (AsS 3 ) at an average As-S bond length of 2.27 Å ( Fig. 3B , top panel; Table S2), consistent with cysteine coordination in the cryo-EM structure. Long-range carbon scattering was also observed in the sample, likely from secondary and tertiary sphere carbon atoms associated with the β- and ⍺-carbon atoms of the cysteines. The oxidation state and coordination environment of the ArsA sample are consistent with that of a control sample consisting of As III mixed with L-cysteine ( Fig. 3A , purple spectra & Fig. 3B , middle panel; Table S2). Similar As III coordination has been reported for the As III -responsive DNA repressor, ArsR, and the As III metallochaperone, ArsD, based on XAS analysis 38 – 40 . We quantified the amount of As III bound to ArsA under conditions resembling those used in cryo-EM sample preparation by inductively coupled plasma mass spectrometry (ICP-MS). In the presence of MgATP, ArsA binds As III with 1:1 stoichiometry ( Fig. 3C , black plot). Taken together, cryo-EM, XAS, and ICP-MS analysis support the binding of one three-coordinate As III at the ArsA pseudodimer interface in the presence of MgATP. Download figure Open in new tab Figure 3. Characterization of the As III binding site of ArsA. (A) X-ray absorption near edge spectra (XANES) for Lf ArsA•MgATP•As III (‘As III -ArsA’; black), As III -L-cysteine control (‘As III -Cys’; purple), and Lf ArsA(C173A)•MgATP•As III (‘As III -ArsA C173A’; teal). (B) Left column, extended X-ray absorption fine structure (EXAFS) spectra (black) with best-fit simulations (green) for each complex. Right column, corresponding Fourier-transforms. A replicate of each XAS sample was individually prepared and analyzed (Fig. S13). (C) ICP-MS analysis for As III quantification in nucleotide-bound Lf ArsA samples (ArsA•MgATP, blac k ; ArsA C173A•MgATP, teal; ArsA•MgADP, gray; ArsA C114A/C173A/C422A•MgADP, yellow). Bars represent mean As/ArsA molar ratio for n = 3 individually prepared samples with error bars representing standard deviation. (D) Steady-state ATPase activity of Lf ArsA (nmol min −1 mg −1 ) plotted against varying As III concentrations (mM) in the presence of 5 mM MgCl 2 and 5 mM ATP at 37°C (wild-type, black; C173A, teal; C114A/C173A/C422A, yellow). Data points represent mean of n = 3 and error bars represent standard deviation. The data was fit to the Michaelis-Menten equation. (E) Comparison of metalloid-binding sites of ADP-open (left) and ATP-closed (right) highlighting the relative positions of Cys114, Cys173 and Cys422 that enable As III (purpl e ) binding. The As III -coordinating cysteines are conserved among ArsA homologs (Fig. S14). Alanine mutations of these residues ablate As III activation of steady-state ATP hydrolysis ( Fig. 3D , yellow plot). By contrast, mutation of Cys173 located on the flexible loop region of N-domain alone appears to only decrease the binding of As III to ArsA, as the C173A variant requires higher As III concentrations than the wild-type enzyme to approach half-maximal activation of ATPase activity ( Fig. 3D , teal plot). XANES and EXAFS spectra for the C173A variant incubated with MgATP and As III suggest AsO 2 S coordination, indicating that As III is no longer coordinated to three cysteines ( Fig. 3A , teal spectra & Fig. 3B , bottom panel; Table S2). Moreover, ICP-MS analysis of this variant shows over 50% decrease in As III -ArsA molar ratio ( Fig. 3C , teal plot). As previously proposed 30 , Cys173 regulates the binding affinity of As III and is not critical for the stimulation of nucleotide hydrolysis. Based on our ICP-MS analysis, ArsA•MgADP binds As III far less tightly than ArsA•MgATP ( Fig. 3C , black and gray plots), corroborating the absence of As III density in the ADP-open structure. Mutating the As III -coordinating cysteines completely disrupts As III binding to ArsA•MgADP ( Fig. 3C , yellow plot). How do the conformational changes between open and closed states modulate the affinity for As III ? Our cryo-EM structures reveal that switching from open to closed state causes reorientation of helix 6 of both domains such that Cys114 on H6 N and Cys422 on H6 C shift ∼9 Å away from the P-loops ( Fig. 3E , top panels). This makes the metalloid-binding site more accessible to the solvent for As III binding. Additionally, a change in the relative positions of the sidechains of Cys114 and Cys442 accompanies this conformational rearrangement. As shown in Fig. 3E (bottom panels), the distance between C β atoms of Cys114 and Cys422 reduces from 6 Å in the open state to 4 Å in the closed state, facilitating As III binding. Therefore, by controlling the relative positions of the three cysteines at the metalloid-binding site, conformational changes in ArsA modulate the binding affinity for As III . Intra-domain conformational rearrangements between open and closed states In addition to the nucleotide-dependent conformational changes at the pseudodimer interface, large-scale structural rearrangements are observed within each domain between the ADP-open and the ATP-closed states ( Fig. 4A-B ). These changes are primarily linked to the Switch I and II motifs. Compared to the ADP-open state, the Switch I loop of both the N- and C-domains rearranges to shift towards the nucleotide in the ATP-closed state ( Fig. 4C-D ). This enables the positioning of Asp46 and Asp364 to activate W cat and initiate ATP hydrolysis. Differences in the Switch I conformation between the two domains allow Ser49 to additionally stabilize the catalytic water in the N-domain ( Fig. 4C & G). While the carboxylate group of Asp364 shifts about 5 Å towards the active site from open to closed state, the carboxylate of Asp46 shifts only 3 Å ( Fig. 4C-D ). Download figure Open in new tab Figure 4. Intra-domain conformational changes between ADP-open and ATP-closed states. (A,B) Cartoon representations of overall structural rearrangements within N- and C-domains. Arrows indicate direction of motion of the corresponding motif from the open to the closed state. This and subsequent panels are colored in viridis. Highlighted residues are shown as sticks. All overlayed structures are aligned to the respective P-loops (residues 16-23 (N) and 334-341 (C)) and the transparent structures represent the ADP-open state. (C, D) Conformational changes in Switch I. (E) Conformational changes in Switch II and H7 N of the N-domain. The ‘metalloid affinity loop’ connecting H7 N and H8 N is highlighted. In both panels E and F, helix 6 has been omitted for visual clarity. (F) Conformational changes in Switch II and H7 C of the C-domain. (G) Comparison of Switch I and II conformations of the N- and C-domains in the ATP-closed state. Switch II, acting as a link between the nucleotide-binding site and the metalloid-binding site, modulates a series of dramatic conformational changes within each domain between the open and closed states. Upon As III binding to the metalloid-binding site of the ATP-closed state, both H6 N and H6 C undergo rigid body rearrangement such that the N-terminal end of each, bearing the As III ligands – Cys114 and Cys422, respectively – rotates away from the nucleotide-binding sites ( Fig. 4A-B ). Reorientation of helix 6 leads to restructuring of the downstream Switch II motif in the ATP-closed state. Although the Switch II aspartates (Asp143 and Asp447) of the respective domains remain anchored near the Mg 2+ ion in both states, downstream residues 144-147 of the N-domain (Switch II N ), residues 448-451 of the C-domain (Switch II C ), and both helix 7s shift towards the nucleotide in the ATP-closed state ( Fig. 4E-F ). Switch II N shifts towards the nucleotide to a greater extent than Switch II C ( Fig. 4G ). Hydrogen-bonding interactions between Thr147 (Switch II N ) and Glu117 (H6 N ) and between Thr451 (Switch II C ) and Glu425 (H6 C ), in the ADP-open state, are destabilized in the ATP-closed state to facilitate the conformational change coordinated by helix 6 and Switch II (Fig. S15). In the wt ArsA ATP-closed state, Switch II C further shifts towards the nucleotide, adopting the same conformation as Switch II N (Fig. S12D). This symmetric conformation of Switch II results in an additional contact between the two domains where His149 (H7 N ) and His453 (H7 C ) form a π-π stacking interaction (Fig. S12E). A striking difference is seen between the N- and C-domains in the conformation of helix 7 following the Switch II loop. In the ADP-open state, H7 N is composed of residues Gly148-Leu154. In the ATP-closed state, this helix is extended by incorporation of formerly loop residues Gln155-Ala166 into the C-terminal end of H7 N transition, resulting in a twisted helix composed of residues Thr147-Ala166 ( Fig. 4E ). By contrast, H7 C transitions from residues Gly452-Ala460 in the ADP-open state to a discontinuous helical segment between Thr451 and Gln471, connected by a loop formed by residues Asp459-Gly462 in the ATP-closed state ( Fig. 4F ). Complete helical rearrangement of H7 N arises by virtue of Cys173 on the loop region between H7 N and H8 N , that serves as the third ligand for As III binding in the ATP-closed state. This loop, designated as the ‘metalloid affinity loop’, undergoes a considerable rearrangement between the open and closed states to stabilize three-coordinate As III binding ( Fig. 4E ). An analogous residue is not found on the C-domain; in fact, the corresponding loop region in the C-domain is poorly resolved in both states. Notably, such helical transition of helix 7 is also observed upon substrate (TA protein) binding at the homologous site in Get3 (PDB: 7SQ0) 21 . Taken together, the conformational changes associated with As III binding in the ATP-closed state demonstrate how three-coordinate As III binding at the metalloid-binding site of ArsA is allosterically coupled to the nucleotide-binding sites via Switch II. Discussion ArsA enhances the efficiency of ArsB by coupling ATP hydrolysis to toxic metalloid (As III or Sb III ) efflux. Based on pre-steady state kinetic analysis, the pseudodimeric ATPase undergoes nucleotide-dependent conformational changes throughout its catalytic cycle 27 , 28 , 41 , that are critical for its interaction with ArsB. MgATP-bound ArsA has been reported to alternate between two conformations that differ in their affinities for the metalloid substrate 27 . Metalloid binding stabilizes one of these conformations, followed by hydrolysis and phosphate release. Our high-resolution cryo-EM efforts provide the structural basis for the conformational states proposed in this mechanism. We have characterized open and closed conformations of ArsA. The open state can exist bound to either ADP or ATP ( Fig 1B , F-G), has low metalloid affinity, and is ATPase inactive. The closed state is stabilized as a ternary complex when As III binds ArsA•MgATP ( Fig. 2B ) and is competent for ATP hydrolysis. The structures enable us to propose a catalytic mechanism for ArsA ( Fig. 5 ). ATP binds both N- and C-sites of ArsA where the enzyme exists in equilibrium between open and closed states (states 1 and 2). ATP alone is unable to drive the equilibrium to favor the closed state; as a result, ArsA hydrolyzes nucleotide at a basal rate. When As III is coordinated by three cysteines at the metalloid-binding site ( Fig. 2D ), the open conformation transitions into the catalytically competent closed conformation (state 3). Coordination of As III to Cys114 and Cys422 allosterically triggers the transition of the pseudodimer from open to closed state via helix 6 and Switch II ( Fig. 4A-B ). Cys173 on the ‘metalloid affinity loop’ acts as a switch controlling the binding affinity of As III for ArsA and stabilizes the ternary complex in the closed state. As III binding by this motif is coupled to the nucleotide-binding site of the N-domain via H7 N and Switch II N ( Fig. 4E ). This mechanism reveals how three-coordinate As III binding allosterically stimulates ATP hydrolysis, which is the proposed rate-limiting step of ArsA 27 . Following ATP hydrolysis, the enzyme loses affinity for As III , releasing the metalloid and phosphate, and returns to the open state (state 4). Nucleotide exchange can then reset the enzyme for another catalytic cycle. Download figure Open in new tab Figure 5. Mechanistic model for ArsA catalytic cycle. ATP-bound ArsA alternates between open (state 1) and closed (state 2). As III binding stabilizes the catalytically competent closed conformation (state 3). Following ATP hydrolysis and release of As III and phosphate (P i ), ArsA switches to the ADP-bound open conformation (state 4). Exchange of the nucleotide resets the enzyme for another catalytic cycle. States 1, 3 and 4 were characterized in this study. ArsA is colored by N (blue) and C (green) domains. Nucleotides are shown as space-filling models. As III (As(OH) 3 at physiological pH) and P i are shown as ball & sticks. The conformational changes of ArsA reported here are consistent with the general structural and mechanistic features of IWA ATPases. Despite each IWA ATPase binding a unique substrate at the (pseudo)dimer interface, the structural changes corresponding to the substrate, e.g., As III binding to ArsA, remain conserved. In the case of Get3, TA protein binding induces a conformational change in helix 6 (substrate helix) and Switch II, thus regulating the dimer dynamics 21 , 25 . Likewise in NifH, association with NifDK affects the positioning of the [4Fe:4S] cluster for electron transport, which then induces conformational changes in the substrate helix and Switch II, stabilizing the closed dimer conformation 20 . Notably, the pseudodimeric architecture of ArsA is distinct from other ATPases in the family. While both N- and C-domains have been shown to be competent for ATP hydrolysis 17 , structural differences between the domains in the ATP-closed structures highlight the functional asymmetry within ArsA. Both Switch I and II loops adopt distinct conformations relative to the nucleotide in the N- and C-domains in the closed state ( Fig. 4G & S16A), reflecting the difference in ATPase activities, where the N-site is more competent for hydrolysis than the C-site (Fig. S16B). These structural differences become less obvious in the wt ArsA closed state under turnover conditions where a potential hydrogen-bonding network linking the two active sites may coordinate hydrolysis at the two sites (Fig. S12B & D). A key gap in our understanding of the ArsAB efflux pump is how the conformational landscape of the ArsA catalytic cycle modulates interactions with ArsB to facilitate As III efflux. ArsB is sufficient to transport the metalloid and coupling the process with ATP hydrolysis enhances the efficiency of efflux 7 . A mutation in the N-domain P-loop of Ec ArsA (G20S) has been shown to abolish ATPase activity and inhibit the ability of ArsB to remove As III from the cell 42 . This suggests that ArsA catalytic cycle is required for interaction with ArsB. In this light, nucleotide-dependent conformational changes between the high As III -affinity closed state and low As III -affinity open state uncovered in this study ensure efficient metalloid transfer to ArsB. This mechanism enables the survival of the bacteria under high arsenic stress conditions. Methods The Lf ArsA gene was designed and purchased from Twist Biosciences (San Francisco, CA). Primers for amplification were designed using the New England Biolabs (NEB) (Ipswich, MA) primer design tool and obtained from Integrated DNA Technologies (Newark, NJ). Cloned constructs were sequenced by whole plasmid sequencing through Plasmidsaurus (Eugene, OR). Cloning, Expression, and Purification of LfArsA and its variants Lf ArsA (Uniprot Accession Number: J9ZFA3) was cloned into the multiple cloning site-1 of pETDuet-1 expression vector encoding a 6x-His tag on the 3’-end, using Hi-Fi DNA assembly protocol (NEB). This resulted in an Lf ArsA construct with a C-terminal 6x-His tag. The vector backbone and gene fragment were amplified using the Q5 High-Fidelity 2X Master Mix protocol (NEB). ArsA variants were prepared by point mutagenesis, using the Q5 High-Fidelity 2X Master Mix protocol as well. Lf ArsA plasmid was transformed into BL21 Gold (DE3) competent cells (Invitrogen). Starter overnight cultures (5-mL) were prepared by inoculated single colony in Luria broth media in the presence of 100 μg/mL ampicillin and grown for ∼18 h. Sterile 2xYT media (1 L) supplemented with 100 μg/mL ampicillin was inoculated with the overnight culture and grown at 37°C until OD reached ∼0.6, when the cells were induced with 0.4 mM isopropyl β-D-1-thiogalactopyranoside (IPTG), and grown at 18 °C for ∼16-18 h. Cells were harvested, flash frozen in liquid nitrogen and stored at −80°C. For purification, thawed cells were resuspended into ‘buffer A’ (50 mM HEPES pH 7.5, 300 mM NaCl, 10% glycerol, 20 mM imidazole, 5 mM β-mercaptoethanol (β-ME) and 0.2 mM phenylmethylsulfonyl fluoride (PMSF)), and lysed using an M-110L microfluidizer (Microfluidics). The debris was spun down at 24,000 xg for 30 min and the supernatant was incubated with Ni-NTA affinity resin (Qiagen) at 4°C for 1 h. Unbound material was removed by batch method using a bench-top centrifuge at 700 xg for 5 min, followed by 2x washes with 50 column-volumes of buffer A. At this point, the resin was transferred to an Econo-Pac chromatography column (Bio-Rad), and four fractions (1 column-volume each) were eluted with ‘buffer B’ (buffer A + 200 mM imidazole). After SDS-PAGE analysis, fractions containing ArsA were pooled, concentrated to 5 mL in an Amicon 30k concentration filter, and loaded onto a 120-mL HiLoad Superdex-200 column pre-equilibrated with 50 mM HEPES pH 7.5, 100 mM NaCl, 10% glycerol and 5 mM dithiothreitol (DTT). Alternatively, 5 mM tris(2-carboxyethyl)phosphine (TCEP) was used as reducing agent for storage buffer. After SDS-PAGE analysis, pure fractions were stored at −80°C. Protein concentration was determined by measuring the absorbance at 280 nm with a NanoDrop 2000 spectrophotometer (Thermo Fisher), using molar extinction coefficient of 25,400 M −1 cm −1 and molecular weight of 63.8 kDa. X-ray absorption spectroscopy of ArsA ‘As III -ArsA’ sample was prepared by incubating 1.95 mM of Lf ArsA D46N/D364N (non-hydrolyzing variant) with 15 mM MgCl 2 , 15 mM ATP (Sigma), and 1.5 mM of sodium arsenite (Sigma) and then buffer exchanged into 50 mM HEPES pH 7.5, 100 mM NaCl, 30% glycerol and 5 mM TCEP using Micro Bio-Spin P-6 desalting column ( Bio-Rad ) to remove excess unbound arsenite. ‘As III -ArsA C173A’ sample was prepared similarly with 2.5 mM of Lf ArsA (non-hydrolyzing variant) C173A incubated with 20 mM MgCl 2 , 20 mM ATP, and 20 mM sodium arsenite. The ‘As III -cysteine’ sample (control) was prepared by adding 1.2 mM sodium arsenite to 6 mM L-cysteine in 50 mM HEPES pH 7.5, 100 mM NaCl, 30% glycerol, and 5 mM TCEP. A replicate was prepared and analyzed for each sample (Fig. S13 & Table S3). As III -ArsA replicate was supplemented with 15 mM sodium arsenite instead. Samples (∼150 µL each) were injected into leucite cells using a Hamilton syringe, avoiding air bubbles, and immediately frozen in liquid nitrogen. XAS data were collected at the Stanford Synchrotron Radiation Laboratory (SSRL) on beamline 7-3, equipped with Si[220] double-crystal monochromator with a harmonic rejection mirror. Samples were maintained at 10 K using an Oxford Instruments continuous-flow liquid helium cryostat. Protein fluorescence excitation spectra were recorded using a 30-element Ge solid-state array detector. A germanium filter (0.6 mm in width) and solar slits were placed between the cryostat and detector to filter scattering fluorescence not associated with protein-bound arsenic signals. XAS spectra were recorded in 5 eV steps in the pre-edge region (11625– 11825 eV), 0.25 eV steps in the edge region (11850–11900 eV), and 0.05 Å −1 increments in the extended X-ray absorption fine structure (EXAFS) region out to a k range of 14 Å −1 . The data were integrated from 2 to 25 s in a k -weighted manner in the EXAFS region for a total scan length of 45 min. X-ray energies were calibrated using an arsenic foil absorption spectrum collected simultaneously with the protein data. The first inflection point for the arsenic foil edge was assigned to 11867 eV. Each fluorescence channel of each scan was examined for spectral anomalies prior to averaging. The data represent an average of five to six scans for each sample. Data were processed using the Macintosh OS X version of the EXAFSPAK software suite integrated with Feff version 7 for theoretical model generation. Data, collected out to k = 14.0 Å − 1 , corresponds to a spectral resolution of 0.121 Å −1 for all metal–ligand interactions; therefore, only independent scattering environments at distances >0.121 Å were considered resolvable in the EXAFS fitting analysis. The final EXAFS fitting analysis was performed on raw/unfiltered data. Protein EXAFS data were fit using single-scattering F eff theoretical models calculated for carbon, oxygen, and sulfur coordination to simulate arsenic-ligand environments, with values for the scale factors (0.98) and E 0 (−10) following a previously published fitting protocol. All spectra were fit using identical protocols, first by distinguishing the best single-shell fit to the data and then by progressively adding extra scattering environments to the fit. Best fit selection criteria were identified by having the lowest mean square deviation between experimental data and the theoretical fit ( F ’ value), along with an acceptable absorber-scatterer bond disorder value (Debye-Waller factor) of < 6.0 × 10 3 Å 2 . Cryo-EM sample preparation For the ADP-open structure, the Lf ArsA sample was buffer-exchanged into 50 mM HEPES pH 7.5, 100 mM NaCl, and 4 mM TCEP using Micro Bio-Spin P-6 desalting columns (Bio-Rad). The resulting sample was diluted to 10 mg/mL and incubated with 2 mM each of MgCl 2 , ADP (Sigma), and 5 mM sodium arsenite for ∼3 hours. Samples for As III -free structures were prepared similarly. For the ATP-closed structure, Lf ArsA D46N/D364N was buffer-exchanged into 50 mM HEPES pH 7.5, 100 mM NaCl, and 5 mM DTT. The resulting sample was diluted to 10 mg/mL and incubated with 2 mM each of MgCl 2 , ATP, and sodium arsenite for ∼30 min. The sample for wt ArsA ATP-closed structure was prepared similarly but incubated with ligands for 1.5 min prior to grid preparation. For cryo-EM grid preparation, 3 μL of the sample supplemented with 0.05% CHAPSO was applied to glow-discharged Quantifoil holey carbon R1.2/1.3 300 Mesh, Copper (Quantifoil, Micro Tools GmbH) grids using a Vitrobot (FEI Vitrobot Mark v4 x2, Mark v3). Grids were blotted at 100% humidity and 4°C using a blot time of 4-5 seconds and blot force of 7, and immediately followed by plunge-freezing into liquid ethane. Cryo-EM data acquisition and processing The grids were screened for ice thickness and sample quality using a 200 kV Talos Arctica TEM equipped with a Gatan K3 detector. Data collection was performed using a 300 kV Titan Krios TEM equipped with a Gatan K3 direct electron detector and Gatan Energy Filter (slit width 20eV) in super-resolution mode using SerialEM 43 . Each dataset was acquired at a nominal magnification of 130,000x with a raw pixel size of 0.325 Å/pixel, electron exposure of 70 e − /Å 2 over 40 frames (exposure rate of 1.75 e − /Å 2 /frame), and a defocus range of −0.5 to −2.5 μm. Correlated double sampling (CDS) mode was enabled to improve the signal-to-noise ratio of the images 44 . All datasets were processed in cryoSPARC v4.4.1-4.5.1 using the same overall processing workflow 45 . Movie frames were motion corrected using ‘patch motion correction’ with 0.5 F-cropping, resulting in a pixel size of 0.65 Å/pixel. The contrast transfer function (CTF) for the motion-corrected micrographs was estimated using ‘patch CTF estimation’. Micrographs were manually curated, and particles were picked using ‘blob-picking’, extracted with a 2x bin (1.3 Å/pixel), and subjected to multiple rounds of 2D classification to retain good-quality particle picks. Ab-initio reconstruction was performed on this particle set to result in four 3D volumes, followed by ‘heterogenous refinement’ that yielded one good volume revealing secondary structural features of ArsA. Particles were re-extracted from the micrographs with no binning (0.65 Å/pixel) and subjected to multiple rounds of ab-initio reconstruction and heterogeneous refinement followed by ‘reference-based motion correction’, ‘global CTF refinement’, and ‘non-uniform refinement’ 46 , to obtain a final set of particles resulting in a good-quality map. The overall resolution was estimated from the gold-standard Fourier shell correlation (FSC) curve at a cut-off of 0.143 in cryoSPARC. B-factor sharpening values were determined using ‘sharpening tools’ in cryoSPARC. Sharpened maps were exported from cryoSPARC for model building and refinement. The local resolution of each map was calculated using ‘local resolution estimation’ job in cryoSPARC. For the ArsA•MgATP map, iterative rounds of ab-initio reconstruction were performed instead to obtain the final set of particles. The image processing pipeline for each cryo-EM map reported in this work is presented in the SI Appendix, Fig. S3 (ArsA•MgADP in the presence of As III ), Fig. S6 (ArsA•MgADP in the absence of As III ), Fig. S7 (ArsA•MgATP), Fig. S8 (ArsA•MgATP•As III ), and Fig. S11 ( wt ArsA•MgADP•As III ). Model building and refinement All models were built and refined similarly unless stated otherwise. Initial models were obtained by docking the N (residues 1-294) and C (residues 316-587) domains of Lf ArsA AlphaFold2 35 model prediction determined using ColabFold v1.5.2, as distinct model entries into the sharpened EM maps using ‘Dock in map’ in Phenix v1.21.2 47 . The model was adjusted by manual model building into good-quality regions of the map in Coot v0.9.8.7 48 . Ligands (Mg 2+ , nucleotides and/or As III ) and any ordered water molecules in the nucleotide-binding sites were docked in Coot. Models were refined using ‘Real-space refinement’ in Phenix and ‘Real-space refine zone’ in Coot. Custom geometry restraints for As-S bond length were obtained from the XAS data and applied to the As III binding site during refinement cycles in Phenix for the ArsA•MgATP•As III structure. Each As-S bond length was restrained to 2.27 Å with a sigma-value of 0.06 Å derived from the Debye-Waller factor σ 2 of 3.49 × 10 3 Å 2 corresponding to As-S bond distance of ‘As III -ArsA’ sample (Table S2). Average and per-residue Q-scores for each refined model were calculated using the Qscore plugin in ChimeraX v1.7.1. ATPase assays Steady-state ATPase activity of ArsA was measured spectrophotometrically using an NADH-linked coupled assay with an ATP regeneration system that couples ATP hydrolysis to oxidation of NADH 49 . The reaction mixture (100 µL) consisted of 50 mM HEPES pH 7.5, containing 5 mM MgCl 2 , 2 mM phosphoenol pyruvate (Roche), 20 U/mL pyruvate kinase from rabbit muscle (MP Biomedicals), 20 U/mL L-lactate dehydrogenase from rabbit muscle (Sigma), 0.2 mM NADH (Roche), 2-5 µM ArsA or its variant and varying concentrations of sodium arsenite (0, 0.05, 0.1, 0.2, 0.5 and 0.8 mM). The mixture was incubated for 5-10 min at 37 °C. The assay was conducted in a 96-well plate setup using SpectraMax M3 plate reader (Molecular Devices). The reaction was initiated by adding 5 mM ATP into the wells with gentle mixing. This was immediately followed by the measurement of a steady-state decrease in the NADH absorbance at 340 nm for 20 min on the plate reader at 37°C. Slopes were calculated for the steady region of the progress curves in the units of A 340 min −1 . Rates were calculated as nanomoles of ATP hydrolyzed per min per mg of ArsA (nmol min −1 mg −1 ) using the extinction coefficient of NADH at 340 nm (6,220 M −1 cm −1 ). ATPase activity was fitted to the Michaelis-Menten equation as a function of arsenite concentration and plotted using Python 3 in Jupyter notebook with assistance from ChatGPT, OpenAI in writing the code. Inductively coupled plasma mass spectroscopy (ICP-MS) ArsA-bound arsenic was quantified for ArsA•MgATP, ArsA C173A•MgATP, ArsA•MgADP and ArsA C114A/C173A/C422A•MgADP samples. The non-hydrolyzing variant was utilized when trapping complexes with MgATP. Each sample was prepared similar to the cryo-EM samples by incubating 10 mg/mL of Lf ArsA or its variant, with 2 mM MgCl 2, 2 mM ATP/ADP, and 5 mM sodium arsenite at 4°C for 3 hours. Free arsenite was removed from the samples by exchanging the buffer with 50 mM HEPES pH 7.5 using Micro Bio-Spin P-6 desalting column. Subsequently, arsenic was extracted from the protein by incubating 30 µL of the sample with 1.1 mL of 70% (v/v) HNO 3 (ACS grade) in borosilicate glass tubes at ∼50°C for 30 min. Each sample was then diluted to 15 mL with double distilled water in a 50-mL flat bottom tube, resulting in maximum arsenic concentration equivalent to 23.5 ppb or 0.313 µM of ArsA in 5% (v/v) HNO 3 . Arsenic concentrations were determined by ICP-MS using an Agilent 8800. The sample introduction system consisted of a Miramist nebulizer, Scott-type spray chamber, and 2.0 mm fixed injector quartz torch. A guard electrode was used, and the plasma was operated at 1500 W. Arsenic analysis was performed in He mode using MS/MS scan mode. Arsenic standards were prepared in 5% (v/v) HNO 3 from a 10 µg/mL arsenic standard solution (Inorganic Ventures) in the range of 0 to 40 ppb of As. The probe was subjected to 4 rinses (1 flowing, 3 static) between samples to minimize any possibility of cross contamination. Sample to sample carryover has been found to be less than 1% between the highest standard (40 ppb) and the blank for similar analyses. Results were analyzed using ICP Masshunter 4.5 (Agilent Technologies). Data Availability Cryo-EM maps and the corresponding atomic models generated in this study have been deposited into the Protein Data Bank (PDB) and the Electron Microscopy Data Bank (EMDB) for release upon publication. Scripts for the ATPase assay and ICP-MS plots can be accessed at https://github.com/smahajan82/ArsAplots.git . Author contributions S.M., D.C.R. and W.M.C. designed research; S.M., Y.E.L., A.E.P. and T.L.S. performed research; S.M., T.L.S., D.C.R. and W.M.C. analyzed data; S.M., D.C.R. and W.M.C. wrote the manuscript. Competing interests The authors declare no competing interest. Acknowledgements This work was supported by funding from Howard Hughes Medical Institute (D.C.R.), Chan Zuckerberg Initiative (W.M.C.), and the Center for Environmental-Microbial Interactions at Caltech (S.M.). Cryo-EM data were collected at the Caltech Cryo-EM Resource Center supported by the Beckman Institute, and we are grateful to Songye Chen, Tyler J. Brittain, and Victor Garcia for their assistance with data collection and processing. 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