Molybdate Pumping Into the Molybdenum Storage Protein Via an ATP-Powered Piercing Mechanism

Molybdate pumping into the molybdenum storage protein via an ATP-powered piercing mechanism

Steffen Brünlea,1,2, Martin L. Eisingera,1, Juliane Poppea, Deryck J. Millsb, Julian D. Langera, Janet Vonckb, and Ulrich Ermlera,2

aDepartment of Molecular Membrane Biology, Max Planck Institute of Biophysics, 60438 Frankfurt am Main, Germany; and bDepartment of Structural Biology, Max Planck Institute of Biophysics, 60438 Frankfurt am Main, Germany

Edited by Robert Huber, Max Planck Institute of Biochemistry, Planegg-Martinsried, Germany, and approved November 6, 2019 (received for review July 29, 2019) The molybdenum storage protein (MoSto) deposits large amounts that are covalently or noncovalently linked with the polypep- of molybdenum as polyoxomolybdate clusters in a heterohexameric tide (8, 10) vary between 3 and 14 Mo atoms and are termed (αβ)3 cage-like protein complex under ATP consumption. Here, we Mo3, Mo8, Mo5-7, hexagonal (bi)pyramidal Mo7-8, and Mo8-14 suggest a unique mechanism for the ATP-powered molybdate clusters (Fig. 1). pumping process based on X-ray crystallography, cryoelectron mi- Outside the cage, these POM clusters would be instable. On croscopy, hydrogen-deuterium exchange mass spectrometry, and the other hand, molybdate and other transition metal oxide an- mutational studies of MoSto from Azotobacter vinelandii.First, ions spontaneously polymerize to an enormous variety of POM we show that molybdate, ATP, and Mg2+ consecutively bind into clusters favorably in acidic solutions. Their investigation is an old the open ATP-binding groove of the β-subunit, which thereafter but still productive research field in inorganic chemistry (12, 13). becomes tightly locked by fixing the previously disordered N- The occurrence of POM clusters in the cage is therefore based terminal arm of the α-subunit over the β-ATP. Next, we propose a on an interplay between the inherent property of molybdate of nucleophilic attack of molybdate onto the γ-phosphate of β-ATP, self-assembly and the capability of proteins to bind/template/ analogous to the similar reaction of the structurally related UMP encapsulate them (8). kinase. The formed instable phosphoric-molybdic anhydride becomes Both subunits architecturally belong to the amino acid kinase immediately hydrolyzed and, according to the current data, the re- family, with UMP and acetylglutamate kinases as prominent leased and accelerated molybdate is pressed through the cage wall, members (14, 15) and host binding sites for ATP termed α- and BIOCHEMISTRY presumably by turning aside the Metβ149 side chain. A structural β-ATP, respectively. Previous studies have indicated that α-ATP comparison between MoSto and UMP kinase provides valuable insight into how an enzyme is converted into a molecular machine Significance during evolution. The postulated direct conversion of chemical energy into kinetic energy via an activating molybdate kinase and an This study on the cage-like molybdenum storage protein (MoSto) exothermic pyrophosphatase reaction to overcome a proteinous bar- provides detailed insight into how nature realizes molybdenum rier represents a novelty in ATP-fueled biochemistry, because nor- biomineralization. Our data support the occurrence of molybdate mally, ATP hydrolysis initiates large-scale conformational changes to kinase and pyrophosphatase reactions in MoSto to pump mo- drive a distant process. lybdate into the locked inner protein cage against a molybdate gradient. The high molybdate concentration in the cage causes ATP | soluble molybdate pump | molybdate kinase | polyoxometalate a protein-assisted self-assembly process of molybdate to poly- cluster | protein structure oxomolybdate clusters by which approximately 130 Mo are deposited in a compact manner. We believe that this molyb- ature uses ATP binding/hydrolysis and subsequent ADP/ date pumping expands the known mechanistic repertoire of Nphosphate release for driving manifold biochemical processes, ATP-powered processes, since the chemical energy of hy- including those in energy metabolism, active transport, DNA rep- drolysis of the phosphoric-molybdic anhydride intermediate lication and maintenance, translation of genetic information, mo- would be conveyed onto the molybdate for penetration of tility, and protein (un)folding. An unusual ATP-powered process is the cage wall and not onto the protein for pore opening via accomplished by the molybdenum storage protein (MoSto) offer- conformational changes. ing some N2-fixing bacteria a pronounced selection advantage against competitors for Mo (1–3), which is only variably available in Author contributions: U.E. initiated the project; S.B. designed expression constructs and their habitats. N -fixing bacteria continuously demand Mo in form variants; S.B. overproduced, crystallized, and determined X-ray structures; M.L.E. and 2 J.D.L. designed HDX-MS experiments; M.L.E. performed HDX-MS experiments; J.P. was of molybdate for synthesizing the FeMo cofactor of nitrogenases involved in some X-ray structural studies; S.B., D.J.M., and J.V. performed cryo-EM sample (4, 5). MoSto use ATP hydrolysis to deposit approximately 130 Mo preparation, data collection, image processing, and model building; and S.B., M.L.E, J.V., over longer periods in a compact and polypeptide-fixed manner and U.E. wrote the paper. as discrete, structurally diverse, and rather instable polynuclear The authors declare no competing interest. Mo(VI)-O or polyoxometalate (POM) clusters (6–8). In com- This article is a PNAS Direct Submission. parison, Fe is biomineralized by precipitating a large and highly Published under the PNAS license. stable but less defined iron-oxygen adduct inside the ferritin Data deposition: The X-ray models reported in this paper have been deposited in the cavity by oxidizing Fe(II) to Fe(III) (9). Research Collaboratory for Structural Bioinformatics Protein Data Bank, https://www.rcsb. β The MoSto of Azotobacter vinelandii is a heterohexameric org/ (PDB ID codes 6RIS [K 42S], 6RKE [P212121], and 6RIJ [P6422]). The cryo-EM map and αβ α the corresponding model have been deposited in the Electron Microscopy Data Bank ( )3 cage-like structure (7, 8). The 3 -subunits, related by a 3-fold (accession no. EMD-4907) and the Protein Data Bank (PDB ID code 6RKD). axis, form one-half of the cage, and the architecturally similar 1S.B. and M.L.E. contributed equally to this work. β -subunits form the other half in an equivalent manner (Fig. 1). 2To whom correspondence may be addressed. Email: [email protected] or ulrich. The interior of the cage serves as a container for up to approxi- [email protected]. mately 12 POM clusters, whose structures are determined by This article contains supporting information online at https://www.pnas.org/lookup/suppl/ specific pockets inside the cage, the 3-fold symmetry, and the doi:10.1073/pnas.1913031116/-/DCSupplemental. preparation conditions (10, 11). The polynuclear Mo-O aggregates First published December 6, 2019.

www.pnas.org/cgi/doi/10.1073/pnas.1913031116 PNAS | December 26, 2019 | vol. 116 | no. 52 | 26497–26504 Downloaded by guest on September 29, 2021 Fig. 1. The (αβ)3 hexameric MoSto structure. (A) MoSto is characterized by a cage-like architecture with a completely locked cavity, formed by 3 α-subunits (green) and 3 β-subunits (blue). (B) Removing 1 α- and β-subunit at the front side of MoSto reveals the inner cage, filled with POM clusters. Mo atoms of each POM cluster are depicted as different-colored spheres (Mo3, yellow; hexagonal bipyramidal Mo8, blue; Mo5, wheat; covalent and noncovalent Mo8, cyanand orange; Mo14, pink). The topologies of 4 POM clusters are also depicted as polyhedra (in the same color) formed by oxygens at the vertices that link the metals in a corner- and edge-sharing manner. Each subunit hosts a binding site for ATP, termed α-ATP or β-ATP (sticks), accessible from the outside of the cage.

is more strongly bound and β-ATP significantly more weakly bound homogeneously bound ATP in the β-ATP–binding groove (Fig. 2) to MoSto, and that the hydrolysis of ATP to ADP and phosphate is with no undefined surrounding electron density as is found in most strictly coupled to POM cluster assembly inside the cage (16). of the previously reported MoSto structures (16). The absence of + Mg2 was detectable only in the α-ATP–binding site but never in the Lysβ42 side chain implicates a shorter hydrogen bond distance the β-ATP–binding site. Consequently, due to the strict depen- between Lysβ189 and the β-phosphate oxygen and, concomitantly, 2+ dency on Mg for ATP hydrolysis, the α-ATP–binding site has a 0.5-Å shift of the entire β-ATP away from the cage (Fig. 2). This been considered as the motor for molybdate pumping. The MoSto result definitively proves a pivotal function of the β-ATP–binding A. vinelandii of occurs in the MoStozero,MoStobasal, and MoStofunct site in Mo pumping. states containing neither ATP/ADP nor POM clusters, only ATP/ADP and both ATP/ADP and POM clusters, respectively The Cryoelectron Microscopy Structure of MoSto. Cryo-grids were (16). In the MoStofunct state, the cell can be supplied with mo- prepared with a freshly purified MoSto solution supplemented + lybdate on request. with molybdate and ATP/Mg2 to adjust turnover conditions such Several lines of evidence indicate that POM cluster storage that the obtained MoSto structure reflects a functionally active is separated into a rapid ATP hydrolysis-dependent molybdate state. This was uncertain for the P6322 crystal structure, because transport across the proteinous cage wall and a slow protein- molybdate loading in the crystalline state is infeasible. From the assisted self-assembly of the POM clusters promoted by the high 1,238 micrographs recorded on a JEOL 3200 FSC microscope, molybdate concentrations inside the cage (16). Despite establish- ing MoSto as a soluble, ATP-driven molybdate pump, the role of the 2 different ATP-binding sites, their potential communication, the entry site of molybdate, and in particular, the coupling mechanism of ATP hydrolysis with molybdate translocation remain unknown and are largely answered in this work. Results Functional Analyses of the Kα45S and Kβ42S Variants. The variants Kα45S and Kβ42S are attractive candidates for exploring the unknown purpose of the 2 ATP-binding sites, since both lysine residues are placed adjacent to the β- and γ-phosphates of ATP in their respective binding sites and may influence ATP binding and/or hydrolysis and, consequently, molybdate pumping as well. Unexpectedly, variant Kα45S could not be expressed in significant amounts. We explained this finding by a drastically reduced affinity for α-ATP on lysine exchange, resulting in denaturation of the MoSto complex. This interpretation is in line with previous studies indicating that the strongly bound α-ATP can be removed only under denaturating conditions or enzymatically when the obtained MoStozero is stabilized by high phosphate concentrations (16). In contrast, the variant Kβ42S can be prepared as a stable protein Fig. 2. β-ATP–binding site in the structurally characterized variant Kβ42S. complex. However, it showed neither ATP hydrolysis activity nor Variant Kβ42S (orange) has lost the capability for ATP hydrolysis and POM β POM cluster formation capability in the cage, according to the cluster formation, as reflected in a homogeneous, highly occupied -ATP (carbon in green). The 2Fo − Fc electron density is drawn in gray. Com- malachite green assay (phosphate detection), the dithiooxamide β β β SI pared with -ATP of native MoSto (yellow), -ATP of variant K 42S is shifted assay (molybdate detection) (16), and a 2.1-Å X-ray structure ( slightly away from the cage wall to form a strong hydrogen bond with Appendix,TableS1). The variant Kβ42S structure contains the Lysβ189 in the absence of Lysβ42. The introduced serine at position 42 is completely occupied α-ATP–binding site as well as a strongly and linked by 2 water molecules with the γ-phosphate of ATP.

26498 | www.pnas.org/cgi/doi/10.1073/pnas.1913031116 Brünle et al. Downloaded by guest on September 29, 2021 137,558 particles were selected, resulting in a 3D reconstruction at Crystal Structures of MoSto. In the P6322 X-ray and cryo-EM 3.2-Å resolution (Fig. 3 and SI Appendix, Table S2 and Fig. S1). structures, the αβ dimers of the hexamer are averaged and solely MoSto was found as a heterododecameric oligomer in essentially ATP, but neither ADP nor molybdatewasfoundintheATP-binding all particles of the cryoelectron microscopy (EM) and negative- sites, although ADP was clearly identified by HPLC studies (16). stain EM images (the latter recorded at low protein concentrations Therefore, using X-ray crystallography, we searched for new of 0.01 mg/mL). The heterododecamer is composed of a dimer of states with the aim of trapping intermediates of the multistep Mo storage process. 2(αβ)3 hexamers arranged upside down on each other (β3α3α3β3) with the interface formed by 3 α-subunits each (Fig. 3A). Before Unexpectedly, 2 MoStofunct structures of the P6322 crystal form SI Appendix cryo-EM studies, the oligomerization state of MoSto was not ( ,TableS1) contained a Mo5 cluster (the Mo atoms β – assigned unambiguously, although hints of a dodecamer existed substantiated by anomalous data) instead of ATP in the -ATP SI Appendix (17). The X-ray structure was pragmatically regarded as a hetero- binding groove ( ,Fig.S3). Three molybdate units were found to approximately superimpose with the β-ATP triphos- hexamer (Fig. 1) because the cage, the apparent functional unit, was β – included and the biological benefit of an additional hexamer was not phates and interact with Arg83 and Arg168 of the -ATP binding site. This state (MoSto-Mo5) provided the first hint about a obvious. However, the EM dodecamer is also present in the P6 22 3 molybdate-binding site at the outer cage wall. In addition, a high crystal structure formed with a 2-fold crystallographic axis between electron density at and around the triphosphate moiety was found the 2 hexamers. in multiple MoSto structures (16), suggesting the binding of a The cryo-EM map clearly reflects a MoSto state. Density funct second molecule besides ATP inside the β-ATP–binding groove. is visible for the Mo3 cluster, covalent and noncovalent Mo8 A 1.7-Å resolution structure of MoSto crystallized under clusters, 2 bipyramidal Mo8 aggregates, and a disordered Mo5 funct B 2+ α – molybdate-loading conditions, determined from a new crystal cluster (Fig. 3 ). ATP and presumably Mg bind to the -ATP form of space group P2 2 2 (SI Appendix, Table S1), contained binding site in a virtually identical fashion as found in the X-ray 1 1 1 β – well-formed covalent Mo8 and Mo3 clusters, a rather weakly structure. The -ATP binding site also contains ATP but encloses occupied noncovalent Mo8 cluster, a hexagonal-pyramidal Mo7 γ β a density beyond the -phosphate moiety. In addition, the -ATP cluster, and a tentatively modeled Mo10 cluster. While the 6 β β together with the expanded segment 193 to 225, enveloping the α-ATP–binding sites of the asymmetric unit are completely oc- + adenosine moiety, is shifted toward the cage wall by approximately cupied with ATP/Mg2 , the β-ATP–binding sites, unexpectedly, SI Appendix 2+ 1.5 Å compared with the P6322 X-ray structure ( ,Fig. contain a highly-occupied ADP, a Mg , and a molybdate (Fig. S2). Most interestingly, the N-terminal arm (residues 3 to 36) of 4A). Even more surprisingly, the αN-terminal arm is in a well-

α BIOCHEMISTRY the -subunit, disordered in the P6322 X-ray structure, is well ordered conformation, very close to that of the cryo-EM struc- + ordered in the EM structure. The αN-terminal arm is arranged in ture. Mg2 is coordinated to the α- and β-phosphate of β-ADP a thread-like conformation (with 1 small helical segment) that and 3 to 4 water ligands (Fig. 4A). The water ligands form hy- wraps around the β-subunit and thereby shields the triphosphate drogen bonds to 7 residues surrounding them and, most in- of β-ATP from the only solvent-accessible side (Fig. 3C). The terestingly, also to Thrα19 and Glnα21 from the fixed αN-terminal terminal residues α6toα11 interact with the β-subunit and the α- arm. The mutation of the water ligand Lysβ42 to serine stops + and β-subunits of the adjacent dimer (Fig. 3C). ATP hydrolysis, perhaps by impairing productive Mg2 binding.

Fig. 3. The cryo-EM structure of MoSto at 3.2-Å resolution. (A) The overall architecture. MoSto was found as a heterododecamer with an interface formed

between the 3 α-subunits (light blue, light green, light magenta) of 2 (αβ)3 hexamers perpendicular to the 3-fold axis. Previous gel filtration, native PAGE, and preliminary ultracentrifugation data weakly argued for a heterododecamer but were not unambiguous (17). The 3 rather hydrophobic contact areas consist of the loops preceding strand α42:α47 and strand α73:α79 of the counter-hexamer and vice versa and the loops preceding strands α266:α271 of both hexamers. Except for the slightly displaced β-ATP–binding site and the rigidified αN-terminal arm, no notable structural differences between the cryo-EM and

P6322 X-ray structures exist, as documented in an overall rmsd of 0.49 Å. (B) The POM clusters. The cryo-EM density revealed the Mo3, the covalent and noncovalent Mo8, the hexagonal bipyramidal Mo8, and the disordered Mo5 clusters (yellow and gray). Their densities are significantly greater than that of the polypeptide, which disappears at σ > 13 to 15. The order of their occupancy approximately corresponds to the ranking seen in the X-ray structures. (C) The

fixed αN-terminal arm with density (gray). In contrast to the P6322 X-ray structure, residues α3toα36 of the cryo-EM structure are found in a well-defined conformation above the β-ATP–binding site. Only Thrα19 is in van der Waals contact with the ribose and α-phosphate of β-ATP. Of note, the βN-terminal arm also interacts with the αN-terminal arm of a partner αβ dimer.

Brünle et al. PNAS | December 26, 2019 | vol. 116 | no. 52 | 26499 Downloaded by guest on September 29, 2021 2+ 2+ Fig. 4. The β-ATP–binding groove. (A) The ADP/Mg -molybdate–binding site in the P212121 X-ray structure. Mg (green sphere) is octahedrally ligated with the α-andβ-phosphates, 3 H2O, and a variable binding site that appears to be occupied by a water, the γ-phosphate of a potentially bound ATP or a phosphate oxygen. The molybdate (Mo in cyan) sits at the bottom of the β-ATP–binding groove in contact with the outer face of the cage wall. Hydrogen bonds are formed between the molybdate oxygens and Tyrβ86, Argβ83, Glyβ79, Thrβ169, Argβ168, and 2 Mg2+ ligated waters, suggesting that the binding pocket is more favorable 2- 2- − for MoO4 than for HPO4 (or H2PO4 ). (B) Cryo-EM map. β-ATP is clearly visible in the cryo-EM density, which is slightly prolonged. After determination of the P212121 X-ray structure, the extra density was interpreted as a molybdate (Mo as a yellow sphere) localized in front of Metβ149. A second molybdate (Mo as a cyan sphere) appears to sit inside the cage contacting Metβ149 and is connected to a disordered POM cluster, identified in some X-ray structures as an Mo5 cluster.

Molybdate binds at the bottom of the β-ATP–binding groove close of a control aliquot composed of 9 μMMoStobasal solution, 1 mM A 2− to the cage wall (Fig. 4 ). This binding site is coated by 7 proton- MgCl2,1mMMoO4 , and a reaction aliquot also containing 1 mM donating groups (2 of which originate from the water ligands of ATP (Fig. 5A). Deuterium uptake was slightly increased in the + Mg2 ) and thus is designed to preferentially bind unprotonated α-ATP–binding pocket, particularly in segment α198 to α250, 2− molybdate (MoO4 ) instead of the partially protonated phos- which dominantly consists of the 2 rather flexible loops, α190 to 2− − A phate (HPO4 or H2PO4 )(Fig.4 ). In addition, the difference α211 and α222 to α229, encapsulating the adenosine moiety (SI Fourier peak around the central atom of 3.4σ or 20.0σ after re- Appendix,Fig.S2). Deuterium uptake was substantially decreased fining with either molybdate or phosphate, respectively, along with in the β-ATP–binding groove under molybdate loading conditions an anomalous difference map based on a dataset (SI Appendix, (Fig. 5A), which can be directly explained by the protection of this Table S1) collected at a wavelength of 1.738 Å, clearly argue for area on the binding of ATP. This interpretation is in line with molybdate (SI Appendix,Fig.S4). structural and HPLC data revealing no β-ATP binding in the Leuα20 fixed by Leuα15, Tyrβ86, Argβ83, and Ileβ164 are in MoStobasal state but binding in diverse MoStofunct states (16). A van der Waals contact with molybdate, which becomes completely marked decrease in HDX was found, particularly in segment β196 α encapsulated in the reaction chamber on fixation of the N- to β228, containing the extended loops β190 to β209 and β219 to β terminal arm. We term this trapped state locked MoSto- -ADP/ β228 that envelop the adenosine moiety of β-ATP (Fig. 5A). 2+ β – Mg -molybdate. Some of the 6 -ATP binding sites appear to Deuterium uptake was also decreased between β36 and β57, most + contain a weakly occupied phosphate in van der Waals contact to strongly in segment β40 to β48, which contains the ATP/Mg2 - ADP that partially overlaps with the molybdate-binding site. This β 2+ binding residue Lys 42, the exchange of which to serine stops Mo state is termed locked MoSto-β-ADP/Mg -phosphate. In the α γ storage. Most strikingly, deuterium uptake in the N-terminal arm light of these findings, the EM density beyond the -phosphate of (α3toα36) was strongly decreased (Fig. 5A). This is interpreted as β-ATP might be interpreted as a result of the averaging of ATP, B its fixation during the molybdate pumping process. ADP plus molybdate, and ATP plus molybdate (Fig. 4 ). Considering the defined conformation of the αN-terminal arm Furthermore, a 1.9-Å resolution X-ray structure of MoSto, + in the EM and P2 2 2 X-ray structures and the disordered con- cocrystallized with ATP/Mg2 and molybdate, was determined 1 1 1 formation in the P6 22 (8) and P6 22 X-ray structures, we iden- from a crystal form adopting the space group P6 22 (SI Appen- 3 4 4 tified both as real states of the reaction cycle. Interestingly, the dix, Table S1). No POM clusters were found in the cage, and the + N-terminal extension also plays a crucial role in the related α-subunits were again completely occupied with ATP/Mg2 .In acetylglutamate kinase by linking dimers to higher oligomers (20). contrast, the content of the 3 β-ATP–binding sites ranged from 2+ Finally, a decreased HDX was identified in segment β115 to β131 nearly empty to a weakly bound ADP/Mg dependent on the β β respective β-ATP–binding groove. At the bottom of all 3 β-ATP– containing the glycine-rich loop 127 to 132, involved in POM cluster binding, which becomes occluded during continuous POM binding sites sits a phosphate/molybdate as found in the P212121 structure (SI Appendix, Fig. S5), thereby supporting the as- cluster assembly (11). Mo storage was then investigated by a re- α versed order of ATP and molybdate addition. Thus, a MoStobasal sumption of a second anion-binding site. The N-terminal arm is μ β – solution was incubated with 1 mM MgCl2 and 50 M ATP (con- not rigidified over the -ATP binding groove but rather is at- 2− tached until Glnα18 to another heterohexamer in the crystal trol aliquot), along with 1 mM MoO4 (reaction aliquot). Sub- B lattice in an obviously artificial manner. The 3 active sites enable sequent HDX-MS analysis (Fig. 5 ) revealed no changes in the α – a view of the Mo storage process after ATP hydrolysis and POM -ATP binding site. No uptake changes were observed at the β – B cluster degradation (SI Appendix, Fig. S5). -ATP binding site (Fig. 5 ), indicating molybdate-independent ATP binding. The deuterium uptake of the αN-terminal arm HDX-MS Analysis of MoSto. HDX-MS (18) was performed to explore (polypeptide fragment α3toα43) decreased markedly (Fig. 5B). the conformational dynamics of MoSto during Mo pumping and the Thus, its fixation is a result of a concerted binding of both ATP/ 2+ 2− roles of the different ligands in this process (19). We systematically Mg and MoO4 , as possibly seen in the EM map. The minor tested different states of MoSto and compared the deuterium up- HDX decrease in the β-strand containing Lysβ42 independent of + take among them, termed reaction and control (Dataset S1). The the substrate order is an indicator of Mg2 binding (Fig. 4A) after continuous molybdate storage process was followed by preparation the binding of both ATP and molybdate. Finally, we again observed

26500 | www.pnas.org/cgi/doi/10.1073/pnas.1913031116 Brünle et al. Downloaded by guest on September 29, 2021 BIOCHEMISTRY

Fig. 5. HDX-MS analysis of MoSto. Deuterium uptake differences between reaction and control samples were determined after 90 s of incubation and

projected onto the P212121 MoSto crystal structure, color-coded by gradients ranging from red (decreased uptake) to white (unchanged) to blue (increased uptake). Gray-colored regions indicate missing sequence coverage. The location of Kβ42 and the gating residue Metβ149 are highlighted by a 2+ 2- sphere. ATP, ADP (C, in yellow), Mg (lime), and MoO4 (Mo, in orange) are in a ball- and-stick representation. (A)MoStofunct was generated from 2− 2+ MoStobasal supplemented with MoO4 and Mg by adding ATP, as shown in overall (α/β-unit; Left) and zoom-in (β-ATP– binding groove; Right)rep- 2+ 2− resentations. (B)MoStofunct was generated from MoStobasal supplemented with ATP and Mg by adding MoO4 . Again, ATP hydrolysis and POM cluster 2- 2+ formation occurred during the incubation. (C and D)ATP(C)orMoO4 (D) was added to MoStobasal supplemented with EDTA to withdraw residual Mg and prevent background hydrolysis.

2+ decreased deuterium uptake for segment β127 to β142, indicating instead of 1 mM Mg to MoStobasal in the absence and presence continuous POM cluster assembly (11). of ATP. Strong β-ATP binding, according to an HDX decrease, + To further deconvolute the effects of the individual compo- was detected even at low micromolar concentrations without Mg2 2+ nents, the specific effects of ATP, Mg , and molybdate were (Fig. 5C). The αN-terminal segment displayed no decreased HDX + explored. Accordingly, a MoStobasal solution supplemented with 2 + in the absence of Mg , however. 1mMMg2 prepared without (control aliquot) or with 50 μM 2− To investigate the effect of MoO4 binding, a MoStobasal solu- α – + ATP (reaction aliquot) revealed no HDX in the -ATP binding tion with 1 mM Mg2 (SI Appendix,Fig.S6B)or1mMEDTA(Fig. site, reflecting its complete occupancy with ATP. The decreased 2− 5D) was prepared with MoO4 (reaction aliquot) and without HDX in the β-ATP–binding groove, again visible in segments − MoO 2 (control aliquot) in the absence of ATP. Only minor up- β190 to β209, β219 to β228, and β40 to β48, confirms the binding 4 SI Appendix A take differences in restricted segments of the protein were observed, of ATP ( ,Fig.S6 ). The observed decrease in deuterium α uptake of the αN-terminal segment on ATP addition in the ab- whereas the vast majority of the complex, including the N-terminal 2− 2− arm, was unaffected by adding MoO 2-. These results demonstrate sence of MoO4 was interpreted by the small MoO4 amounts 4 ubiquitous in aqueous solutions, which might induce ATP hydro- that both the ordered and disordered αN-terminal arms reflect real lysis analogous to that seen in the background hydrolysis activity of states of molybdate pumping, and that the binding of all 3 ligands— 2− 2+ 2−— α MoSto before the addition of MoO4 (16). To suppress back- ATP, Mg ,andMoO4 is necessary to fixate the N-terminal + ground hydrolysis, Mg2 was sequestered by adding 1 mM EDTA arm over the β-ATP–binding groove.

Brünle et al. PNAS | December 26, 2019 | vol. 116 | no. 52 | 26501 Downloaded by guest on September 29, 2021 Discussion structural integrity. Likewise, in ATP synthases, only β-ATP and Before this work, the α-ATP–binding site of MoSto was considered not α-ATP is catalytically competent (21). the site of ATP cleavage, mainly due to the finding that the func- Our experimental results allow us to outline a comprehensive + tionally essential Mg2 was exclusively present in the α-ATP–binding mechanistic proposal for molybdate binding-induced β-ATP cleavage site but not in the β-ATP–binding site (16). The presented site- and the subsequent molybdate translocation into the cage on the directed mutagenesis, cryo-EM, X-ray, and HDX-MS data com- basis of indirect evidence (Fig. 6A): 1) Molybdate in form of 2- pletely changed our view. The importance of the β-ATP–binding MoO4 or a Mo5 cluster binds to the positively charged bottom groove was demonstrated by the incapability of the Kβ42S variant to of the β-ATP–binding groove (Fig. 4). 2) β-ATP binds afterward + cleave ATP (Fig. 2), by the newly found Mg2 -andmolybdate- and in case of a Mo5 cluster prebound one of the molybdate binding sites therein (Fig. 4A), as well by the rigidification of the units of the thereby destroyed POM cluster is placed into the αN-terminal arm (Fig. 3C). Significant structural changes during established binding site (Fig. 4). 3) The binding of both ATP and + molybdate pumping, monitored by HDX measurements (Fig. 5), molybdate enables Mg2 binding (Fig. 4A). 4) The αN-terminal + were essentially restricted to the β-ATP–binding groove. The arm becomes fixed (Fig. 3C), and the Mg2 ligation shell, the α-ATP–binding site is exclusively filled with ATP (never with ADP molybdate, and the triphosphate of ATP are thereby completely or without nucleotide), whose γ-phosphate is shielded from a nu- packed inside the protein matrix. Based on the MoSto-β-ATP β 2+ cleophilic attack by H2O. Moreover, the removal of the strongly structure (PDB ID code 6GUJ) and the MoSto- -ADP/Mg - attached α-ATP is only feasible by enzymatic cleavage, leading to molybdate structure (PDB ID code 6RKE), we modeled a cat- + unfolding in the absence of phosphate (16), and the Kα45S variant alytically competent MoSto-β-ATP/Mg2 -molybdate state by + is presumably unstable due to its inability to bind ATP. Taken which 1 γ-phosphate oxygen occupies 1 coordination site of Mg2 together, our results strongly argue that ATP-fueled molybdate (Fig. 6B). 5) The encapsulation is accompanied by several con- pumping occurs in the β-ATP–binding groove, acting as a re- formational changes in the β-subunit, such as the swinging of the + action chamber, while α-ATP has a passive role, essential for Arg83 side chain toward molybdate and the Mg2 -water ligands. A

Fig. 6. Mechanism of molybdate pumping. (A) Scheme of the reaction cycle (cage wall in green; β-ATP–binding site in grey and lightbrown). Molybdate is both + the substrate for the activation reaction and the metabolite to be transported. (B) Modeling of the locked MoSto-ATP/Mg2 -molybdate structure. The modeled + γ-phosphate oxygens interact with Mg2 ,Thrβ169, Glnβ46, Glyβ77, Alaβ78, and Argβ83. The distance between the γ-phosphate phosphorous and the closest and potentially attacking molybdate oxygen is 3.0 Å. No space for a water molecule is available between molybdate and ATP to perform ATP hydrolysis. (C)The molybdate entrance site. The modeled (m) anhydride is shown in orange, and the modeled molybdates (green) are shown in a surface representation to highlight 2- the exit pathway. MoO4 enters the cage adjacent to the noncovalent Mo8 cluster. Metβ149 is shown in the closed (o) and modeled open (m) conformations. The space for penetration is essentially created by a conformational change of the Metβ149 side chain. This process is denoted by black arrows.

26502 | www.pnas.org/cgi/doi/10.1073/pnas.1913031116 Brünle et al. Downloaded by guest on September 29, 2021 postulated shift of the loops β190 to β209 and β219 to β228 to- In ATP-fueled cellular processes performed by proteins, ATP gether with β-ATP of approximately 1 to 2 Å (SI Appendix,Fig. binding/hydrolysis or ADP/phosphate release generally induces S2) toward the bottom of the groove presses the negatively local conformational changes, which are often transformed into charged β-ATP against molybdate, which may transfer the active rigid-body movements that are transmitted over long distances to site into an activated state. 6) The obtained active site geometry trigger energy-requiring protein docking/undocking, ligand binding/ strongly suggests a nucleophilic attack of the molybdate onto the release, or active transport events (30). For example, ABC trans- γ-phosphate of ATP (Fig. 6 A and B) by which ATP and molyb- porters use wide-reaching polypeptide rearrangements to create a date are converted into ADP and a mixed phosphoric-molybdic passage for ions/solutes (e.g., molybdate) across the cell membrane anhydride. This kinase reaction likely takes place close to the (31). Formerly, for MoSto we considered a similar scenario, in- thermodynamic equilibrium. 7) The instabile phosphoric-molybdic cluding allosterically modulated subunit rearrangements, as used + anhydride might be hydrolyzed by a water ligand of Mg2 ,as by UMP kinase for regulation purposes (32). However, MoSto found for inorganic pyrophosphatases (22). We postulate that the operates by a fundamentally different mechanism which to our released chemical energy is directly transformed into the kinetic knowledge is unique in biochemistry. The chemical energy of ATP energy of molybdate, which penetrates the cage wall at the adja- cleavage is transmitted via a kinase reaction onto a reactive an- cent Metβ149, considered the sole point for molybdate in the hydride intermediate and, after its hydrolysis, is directly converted firmly locked reaction chamber, to release the built-up pressure via a pyrophosphatase reaction into kinetic energy of the molyb- (Fig. 6 A and C). We imagine that the electrostatic and steric date to be pierced through the cage wall. This action mode has repulsion between phosphate and molybdate press the latter striking analogies to the firing of a gun. A cartridge (molybdate) is outward, while phosphate and ADP have no space to move. Be- put into a box (ATP-binding groove) and locked (fixation of the hind the Metβ149 side chain, the rather thin wall is already pierced αN-terminal arm). The reaction is started by pushing the bolt through, and the molybdate migrates along, for example, residues (ATP) toward the cartridge, and the resulting chemical reaction Glyβ103, Serβ104, Alaβ107, Aspβ108, and Serβ147 (some of them (phosphoric-molybdic anhydride formation) induces an explosion conformationally mobile) into the cage. In the EM map, the pu- (anhydride hydrolysis) and an acceleration of the bullet (molyb- tative density for a molybdate inside the cage, 4.5 Å apart from date) through the barrel to, for example, penetrate an object (cage Metβ149, may indicate the endpoint of the route across the wall. wall). This gunshot-like mechanism requires a locked reaction This molybdate is directly linked with a highly disordered Mo5 chamber to ensure the directional movement of molybdate across cluster (Fig. 4B). 8) After molybdate has passed Metβ149, its the cage wall and thus prevent dissipation of the released energy into strained side chain immediately springs back into the original heat. For the same reason, the distance between the energy source BIOCHEMISTRY position and closes the gap in the cage wall to prevent the efflux of and the energy-consuming event must be short (Fig. 6A), in contrast molybdates. 9) After losing the interaction to the channeling to the separation found in many other ATP-cleaving proteins. + molybdate, Mg2 is released, and the αN-terminal arm detaches and becomes disordered. 10) ADP is liberated. In each cycle, 1 Materials and Methods molybdate is pumped per 1 ATP hydrolyzed. Recombinant MoSto and MoSto Variant Production. The production of recombi- The annotation of MoSto as a molybdate kinase was inspired nant MoSto of A. vinelandii was performed in the Escherichia coli strain BL21(DE3) CC5, in which a pET21a-based vector containing the genes encoding by the architecturally related amino acid kinase family, which for the MoSto α-andβ-subunits and a strepII-tag were incorporated (11). uses a highly similar active site, involving conserved residues to MoSto was purified using strep-tag affinity, anion exchange, and size exclu- SI Appendix catalyze the same reaction ( , Fig. S7) (14). In UMP sion chromatography and stored in the MoStofunct state in 50 mM MOPS/NaOH kinases, ATP is attacked by the phosphate group of UMP instead pH 6.5 and 50 mM NaCl at −20 °C. The POM cluster-free MoStobasal was pre- of molybdate in MoSto. This leads to the formation of ADP in both pared as described previously (6). Site-directed mutagenesis was performed reactions but forms UDP in UMP kinase instead of a phosphoric- using the Agilent QuikChange Lightning Site-Directed Mutagenesis Kit. Am- molybdic anhydride in MoSto. While anhydride formation is shared plification of the mutagenesis product was done in E. coli DH5α cells, and by all amino acid kinase family members, the formation of a extraction of the amplified plasmids ere extracted using the Qiagen QIAprep phosphoric-molybdic anhydride is unusual in chemistry and bi- Spin Miniprep Kit. ology but not without precedents. It is plausibly postulated as a Mo Content and Kinetic Analysis. The Mo content in the cage was determined short-lived intermediate for the molybdate-induced increase of by chemical analysis (6, 33). ATP hydrolysis rates were determined by phos- ATP hydrolysis in aqueous solution by a factor of 150 (23, 24), in phate detection with a colorimetric malachite green assay (16, 34). the nucleotide-assisted molybdenum insertion into molybdopterin (25), and in the molybdolysis of ATP sulfurylase (26). The as- Single-Particle Cryo-Electron Microscopy. Negative staining experiments with sumed adenylyl molybdate formation instead of adenylyl sulfate 1% uranyl acetate revealed clearly separable particles. For single particle formation in ATP sulfurylase convincingly demonstrates the fea- cryo-EM, holey carbon grids (CF-MH-4C multi C-flat; ProtoChips) were in- sibility of transforming a pyrophosphate into a mixed phosphoric- cubated for 2 h with chloroform and then glow-discharged. After being loaded with 3 μL of 4 mg/mL MoStofunct solution supplemented with 1 mM molybdic anhydride in a suitable polypeptide environment. The 2+ rapid dissociation of phosphoric-molybdic anhydrides has pre- molybdate and 1 mM ATP/Mg , they were plunge-frozen in liquid ethane in a FEI Vitrobot Mark IV at 10 °C and 70% humidity, after blotting for 11 s. vented their detection; however, a related anhydride between Images were collected with a JEOL 3200 FSC electron microscope at 300 kV. vanadate and pyrophosphate has been identified (27). Moreover, Beam-induced motion was corrected by MotionCor2 with correction for a the estimated free energy of phosphoric-molybdic anhydride hy- magnification distortion (35), which resulted in a pixel size of 1.11 Å. A total drolysis of 5 to 7 kcal/mol is considered sufficiently exothermic to of 1,238 EM micrographs remained, from which 174,681 particles were au- turn aside the Metβ149 side chain (28). However, further site- tomatically picked using Relion 2.0 (36, 37). After 2D and 3D classification, a directed mutagenesis and theoretical studies are needed to final dataset of 137,558 particles was refined in Relion 2.0, applying D3 2 understand, in detail, the translocation of molybdate into the symmetry. Ten frames with an accumulated dose of 16 e/Å were selected cage. Remarkably, the design of the molybdate entry site from for particle ensemble movements and resolution-dependent weighting. an uridine-binding site in UMP kinase is essentially achieved by restructuring the linker between strand β143 to β146 and helix Crystal Structure Analysis. MoSto was crystallized under 3 different conditions; those of the P6 22 crystal form were reported previously (8, 10). The β169 to β179. Overall, MoSto is an impressive example of how a 3 crystallization conditions for the 2 new crystal forms, P212121 and P6422, are molybdate pump is developed from an ATP-consuming enzyme given in SI Appendix, Table S1. Crystals of the latter forms were obtained that underpins the postulated direction of evolution from binding after the removal of molybdate from MoSto and subsequent reloading of + proteins to enzymes and finally to complex machineries (29). MoSto with molybdate by the addition of ATP/Mg2 and molybdate. Data

Brünle et al. PNAS | December 26, 2019 | vol. 116 | no. 52 | 26503 Downloaded by guest on September 29, 2021 were processed with XDS (38), and the structure was determined by mo- Peptides identified from undeuterated measurements (PLGS 3.0.2; Waters) lecular replacement with PHASER (39) using MoSto with PDB ID code 4NDO were imported into DynamX 3.0 (Waters), and the assigned uptake spectra as a model. Structural refinement was performed with PHENIX (40) and were curated. Statistical analysis was performed in R using an unpaired t test manual model building was done with Coot (41). No POM clusters were (P ≤ 0.05, 2-sided) as described previously (43). Deuterium uptake was found in the P6422 structure, presumably due to the high pH of the crys- tracked with 240 to 340 peptides (depending on the experiment), covering tallization conditions. Figures were generated with PyMOL (Schrödinger) approximately 95% of the protein sequence. Subsequently, significant HDX and Chimera 1.13.X (42). differences observed between reaction and control were plotted onto the MoSto structure (PDB ID code 6RKE) to visualize changes in structural dy- + Hydrogen-Deuterium Exchange MS. To monitor the binding of ATP/Mg2 and namics and solvent accessibility (43). 2− MoO4 and thereby the conformational rearrangements involved, a 9 pmol/μL solution of MoStobasal (50 mM MOPS and 50 mM NaCl, pH 6.5), was split Data Availability. The structure factors and coordinates of the MoSto variant + into 2 identical aliquots. The reaction aliquot was supplemented with a Kβ42S, the MoSto–β-ADP/Mg2 -molybdate complex (P212121), and the specific reactant, and the control aliquot was supplemented with an equal MoSto–β-ADP/Mg2+ complex (P6422) are deposited under PDB ID codes 6RIS, amount of buffer. HDX-MS analysis was performed on a Waters HDX setup 6RKE, and 6RIJ. The cryo-EM map is deposited in the Electron Microscopy as described previously (43). In brief, samples were 15-fold diluted with the Data Bank under the accession number EMD-4907, and the corresponding + corresponding deuterated buffer at 20 °C for defined times (0, 15, 30, 90, coordinates of the MoSto–β-ATP/Mg2 -molybdate complex under the PDB ID 2- and 240 s). Because high MoO4 concentrations inhibit pepsin (44), it was code 6RKD. added only to the samples but not to the labeling buffers. The exchange reaction was rapidly quenched by 1:1 dilution with cooled (2 °C) quench ACKNOWLEDGMENTS. We thank the International Max Planck Research buffer (75 mM KH2PO4 and 75 mM K2PO4, pH 2.5), and 18 pmol of protein School for the scholarship for S.B., Hartmut Michel for financially supporting was subjected to online peptic digestion. Peptides were trapped and washed J.P., Werner Kühlbrandt for general support, Barbara Rathmann and Yvonne for 3 min before chromatographic separation. Eluting peptides were ana- Thielmann (Core Center, Max Planck Institute of Biophysics) for performing lyzed on a quadrupole time-of-flight mass spectrometer. All experiments crystallization screenings, and the staff of the Swiss-Light Source, Villigen for were conducted in technical quadruplicates of 2 purification batches. help with data collection.

1. P. T. Pienkos, W. J. Brill, Molybdenum accumulation and storage in Klebsiella pneu- 21. J. P. Abrahams, A. G. Leslie, R. Lutter, J. E. Walker, Structure at 2.8 Å resolution of moniae and Azotobacter vinelandii. J. Bacteriol. 145, 743–751 (1981). F1-ATPase from bovine heart mitochondria. Nature 370, 621–628 (1994). 2. K. Schneider, A. Müller, K. U. Johannes, E. Diemann, J. Kottmann, Selective removal of 22. T. Kajander, J. Kellosalo, A. Goldman, Inorganic pyrophosphatases: One substrate,

molybdenum traces from growth media of N2-fixing bacteria. Anal. Biochem. 193, three mechanisms. FEBS Lett. 587, 1863–1869 (2013). 292–298 (1991). 23. H. Weil-Malherbe, R. H. Green, The catalytic effect of molybdate on the hydrolysis of 3. M. Navarro-Rodríguez, J. M. Buesa, L. M. Rubio, Genetic and biochemical analysis of organic phosphate bonds. Biochem. J. 49, 286–292 (1951). the Azotobacter vinelandii molybdenum storage protein. Front. Microbiol. 10, 579 24. W. T. Jenkins, J. S. Patrick, Catalysis of the dephosphorylation of an ATP-molybdate (2019). complex by calcium and magnesium ions. J. Inorg. Biochem. 27, 163–172 (1986). 4. B. M. Hoffman, D. Lukoyanov, Z. Y. Yang, D. R. Dean, L. C. Seefeldt, Mechanism of 25. A. Llamas, T. Otte, G. Multhaup, R. R. Mendel, G. Schwarz, The mechanism of nitrogen fixation by nitrogenase: The next stage. Chem. Rev. 114, 4041–4062 (2014). nucleotide-assisted molybdenum insertion into molybdopterin: A novel route toward 5. T. Spatzal et al., Nitrogenase FeMoco investigated by spatially resolved anomalous metal cofactor assembly. J. Biol. Chem. 281, 18343–18350 (2006). dispersion refinement. Nat. Commun. 7, 10902 (2016). 26. R. S. Bandurski, L. G. Wilson, C. L. Squires, The mechanism of “active sulfate” for- 6. D. Fenske et al., A new type of metalloprotein: The Mo storage protein from Azo- mation. J. Am. Chem. Soc. 78, 6408–6409 (1956). tobacter vinelandii contains a polynuclear molybdenum-oxide cluster. Chembiochem 27. M. J. Gresser, A. S. Tracey, K. M. Parkinson, Vanadium(V) oxyanions: The interaction of – 6, 405 413 (2005). vanadate with pyrophosphate, and arsenate. J. Am. Chem. Soc. 108, 6229–6234 7. J. Schemberg et al., Towards biological supramolecular chemistry: A variety of pocket- (1986). templated, individual metal oxide cluster nucleations in the cavity of a mo/w-storage 28. X. Zhu, P. E. Lopes, J. Shim, A. D. MacKerell, Jr, Intrinsic energy landscapes of amino protein. Angew. Chem. Int. Ed. Engl. 46, 2408–2413 (2007). acid side-chains. J. Chem. Inf. Model. 52, 1559–1572 (2012). 8. B. Kowalewski et al., Nature’s polyoxometalate chemistry: X-ray structure of the Mo 29. B. E. Clifton et al., Evolution of cyclohexadienyl dehydratase from an ancestral solute- storage protein loaded with discrete polynuclear Mo-O clusters. J. Am. Chem. Soc. binding protein. Nat. Chem. Biol. 14, 542–547 (2018). 134, 9768–9774 (2012). 30. A. Wittinghofer, GTP and ATP hydrolysis in biology. Biopolymers 105, 419–421 (2016). 9. K. Zeth, E. Hoiczyk, M. Okuda, Ferroxidase-mediated iron oxide biomineralization: 31. K. P. Locher, Review. Structure and mechanism of ATP-binding cassette transporters. Novel pathways to multifunctional nanoparticles. Trends Biochem. Sci. 41, 190–203 Philos. Trans. R. Soc. Lond. B Biol. Sci. 364, 239–245 (2009). (2016). 32. P. Meyer et al., Structural and functional characterization of Escherichia coli UMP 10. J. Poppe et al., Structural diversity of polyoxomolybdate clusters along the three-fold kinase in complex with its allosteric regulator GTP. J. Biol. Chem. 283, 36011–36018 axis of the molybdenum storage protein. J. Inorg. Biochem. 138, 122–128 (2014). (2008). 11. S. Brünle, J. Poppe, R. Hail, U. Demmer, U. Ermler, The molybdenum storage protein— 33. R. P. Pantaler, A kinetic method for determining traces of tungsten and molybdenum. A bionanolab for creating experimentally alterable polyoxomolybdate clusters. J. J Anal Chem USSR 18, 519–524 (1963). Inorg. Biochem. 189, 172–179 (2018). 34. K. Itaya, M. Ui, A new micromethod for the colorimetric determination of inorganic 12. M. T. Pope, A. Müller, Polyoxometalate chemistry: An old field with new dimensions phosphate. Clin. Chim. Acta 14, 361–366 (1966). in several disciplines. Angew. Chem. Int. Ed. Engl. 30,34–48 (1991). 35. S. Q. Zheng et al., MotionCor2: Anisotropic correction of beam-induced motion for 13. H. N. Miras, D. L. Long, L. Cronin, Advances in Inorganic Chemistry: Polyoxometalate – Chemistry, R. van Eldik, L. Cronin, Eds. (Academic Press, 2017), vol. 69, pp. 1–28. improved cryo-electron microscopy. Nat. Methods 14, 331 332 (2017). 14. C. Marco-Marín, F. Gil-Ortiz, V. Rubio, The crystal structure of Pyrococcus furiosus 36. S. H. Scheres, RELION: Implementation of a Bayesian approach to cryo-EM structure – UMP kinase provides insight into catalysis and regulation in microbial pyrimidine determination. J. Struct. Biol. 180, 519 530 (2012). nucleotide biosynthesis. J. Mol. Biol. 352, 438–454 (2005). 37. D. Kimanius, B. O. Forsberg, S. H. Scheres, E. Lindahl, Accelerated cryo-EM structure 15. S. Ramón-Maiques, A. Marina, F. Gil-Ortiz, I. Fita, V. Rubio, Structure of acetylgluta- determination with parallelisation using GPUs in RELION-2. eLife 5, e18722 (2016). – mate kinase, a key enzyme for arginine biosynthesis and a prototype for the amino 38. W. Kabsch, XDS. Acta Crystallogr. D Biol. Crystallogr. 66, 125 132 (2010). – acid kinase enzyme family, during catalysis. Structure 10, 329–342 (2002). 39. A. J. McCoy et al., Phaser crystallographic software. J. Appl. Cryst. 40, 658 674 (2007). 16. J. Poppe et al., The molybdenum storage protein: A soluble ATP hydrolysis-dependent 40. P. V. Afonine et al., phenix.model_vs_data: A high-level tool for the calculation of – molybdate pump. FEBS J. 285, 4602–4616 (2018). crystallographic model and data statistics. J. Appl. Cryst. 43, 669 676 (2010). 17. J. Schemberg, “Biochemische charakterisierung und untersuchungen der röntgenstruktur 41. P. Emsley, K. Cowtan, Coot: Model-building tools for molecular graphics. Acta Crys- am molybdänspeicherprotein aus Azotobacter vinelandii,” PhD thesis, Universität Bielefeld, tallogr. D Biol. Crystallogr. 60, 2126–2132 (2004). Bielefeld (2006). 42. E. F. Pettersen et al., UCSF Chimera—a visualization system for exploratory research 18. J. R. Engen, Analysis of protein conformation and dynamics by hydrogen/deuterium and analysis. J. Comput. Chem. 25, 1605–1612 (2004). exchange MS. Anal. Chem. 81, 7870–7875 (2009). 43. M. L. Eisinger, L. Nie, A. R. Dörrbaum, J. D. Langer, H. Michel, The xenobiotic extrusion 19. Z. Zhang, D. L. Smith, Determination of amide hydrogen exchange by mass spec- mechanism of the MATE transporter NorM_PS from Pseudomonas stutzeri. J. Mol. trometry: A new tool for protein structure elucidation. Protein Sci. 2, 522–531 (1993). Biol. 430, 1311–1323 (2018). 20. S. de Cima, F. Gil-Ortiz, M. Crabeel, I. Fita, V. Rubio, Insight on an arginine synthesis 44. S. Yenjai, P. Malaikaew, T. Liwporncharoenvong, A. Buranaprapuk, Selective cleavage metabolon from the tetrameric structure of yeast acetylglutamate kinase. PLoS One of pepsin by molybdenum metallopeptidase. Biochem. Biophys. Res. Commun. 419, 7, e34734 (2012). 126–129 (2012).

26504 | www.pnas.org/cgi/doi/10.1073/pnas.1913031116 Brünle et al. Downloaded by guest on September 29, 2021