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 in- sight into how an enzyme is converted into a molecular machine Significance during evolution. The postulated direct conversion of chemical en- ergy 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 mech- anism of ATP hydrolysis with molybdate translocation remain unknown and are largely answered in this work.