ИСТИНА

The fundamental importance of nucleoside triphosphates, such as ATP and GTP, for biological systems stems from their relative stability and the possibility to dramatically accelerate their hydrolysis by recruiting simple positively charged moieties, such as Mg2+ and K+ ions and/or side groups of Lys or Arg residues, for catalysis, as well as by using a polarized water molecule to act as a nucleophile. Hydrolysis of nucleoside triphosphates (NTPs), such as ATP or GTP, by various nucleoside triphosphatases (NTPases) is a key reaction in the cell. Most widespread are the P-loop NTPases that make up 10-20% gene products in a typical cell [1]. P-loop domains appear to be some of the most ancient protein domains dating back to the Last Universal Cellular Ancestor (LUCA) [2, 3]. They are found in proteins that hydrolyze ATP to perform mechanical work, such as kinesin, myosin and dynein; rotary ATP synthases; DNA and RNA helicases, many GTPases, including α-subunits of signaling G-proteins, and other enzymes. While the expansion of most protein families correlates with expansion of their catalytic repertoire, P-loop NTPases are unusual in being abundant but catalyzing only a few types of hydrolytic reactions [4]. The abundance of NTP-utilizing enzymes encoded in every living cell makes regulating their activity a key problem in cell metabolism. Obviously, uncontrolled NTP hydrolysis would lead to the depletion of cellular ATP/GTP and be detrimental for cell survival. P-loop ATPases are unique enzymes as they are usually activated before (or during) each turnover. The controlled fast hydrolysis takes place upon the interaction with a proper physiological partner, which could be a domain of the same protein, a separate protein or a DNA/RNA molecule. Upon this interaction, an activating moiety (an Arg/Lys residue, or a K+/Na+ ion, or the LSGGQ motif) gets inserted into the binding site and promotes the cleavage of the NTP molecule. It is believed that cleavage of the NTP phosphoanhydride bond results from the nucleophilic attack by a polarized water molecule coordinated by so-called "catalytic/essential" residues. Generally, the exact order of events during the NTP hydrolysis remains obscure. It also remains unclear whether there is some universal catalytic mechanism common for all P-loop NTPases. The experimental investigation of all members of the vast superfamily of P-loop NTPases would not be realistic, which prompted us to apply the tools of evolutionary biophysics. With this approach, the key mechanistic elements are identified in well-studied reference systems and then their conservation is checked, from structure and sequence data, throughout the entire protein family. Since in some P-loop NTPases, the role of activating Arg/Lys residues is played by K+ ions [5-7], we have started from combining structural analyses of diverse P-loop NTPases with molecular dynamics (MD) simulations of the Mg2+-ATP complex in water in the presence of monovalent cations. Monovalent cations were found to occupy two specific positions around the Mg2+-ATP complex. Namely, cations bound between β- and γ-phosphates (the BG site), in the position of the conserved Lys residue of the P-loop, and between α- and γ-phosphates (the AG site), in the position of the activating Arg/Lys residue. Additionally, we observed weak cation binding in the proximity of γ-phosphate (the G-site), which matches the location of the catalytic/essential residues in P-loop NTPases. The catalytically prone conformation of the phosphate chain in the active sites of diverse P-loop NTPases was found to be similar to that observed in water in the presence of K+ and NH4+ ions and more stretched than the one observed in the presence of the smaller Na+ ions. We concluded that the shape of the phosphate chain in the active sites of P-loop NTPases and the catalytically favorable electrostatic environment are defined by certain inherent properties of Mg2+-NTP complexes in the presence of large monovalent cations. As next, we zoomed in on small regulatory GTPases that include H-, N- and K-Ras (rat sarcoma) proteins, which are extremely well studied being responsible for about a half of known cancers. Hhuman Ras GTPases get converted into active oncoproteins by mutations in positions 12, 13, or 61 [8]. NTP hydrolysis is achieved through interaction of the P-loop domain with another protein or domain, which provides an activating moiety, usually a Lys or Arg “finger”, that gets inserted into the active site and promotes catalysis [9-11]. In Ras and related GTPases, the Arg finger, which triggers the hydrolysis and cancels the oncogenic signal, comes from specific GTPase activating proteins (GAPs) [9]. By combining comparative structural analysis of major families of P-loop NTPases with MD simulations of human Ras GTPase in a complex with its GAP, we identified several structural traits that are shared by catalytic sites in diverse families of P-loop NTPases and appear to be inherited from the ancestral form(s) of P-loop proteins. Based on the data obtained, we propose a two-step common mechanism for the whole superfamily of P-loop NTPases. (1) Upon NTP binding to the P-loop, the energy of binding is used to bring the NTP molecule into a catalytically prone conformation with eclipsed β- and γ-phosphates via coordination of the Mg2+ ion and phosphate groups by multiple amino acids that are strictly conserved among all P-loop NTPases; (2) NTP hydrolysis is promoted by a specific activating interaction of the P-loop NTPase with another protein or a separate domain of the same protein or a RNA/DNA molecule. This interaction leads to the insertion of a positively charged moiety next to the phosphate chain. In most cases, this moiety is an Arg/Lys residue or a monovalent cation. This interaction, via rotation/pulling of the γ-phosphate group, yields a near-eclipsed conformation of the whole phosphate chain, which causes a further weakening of the PB-O3B-PG bond and a more planar state of γ-phosphate. The MD simulation data assign a key role to the rotation of γ-phosphate and suggests that the backbone nitrogen atom of a specific residue of the P-loop (Gly13 in Ras, whose mutations often lead to cancer) provides an H-bond to stabilize γ-phosphate after its rotation. The reshuffling of the H-bond network around γ-phosphate brings the catalytic water molecule into the attacking position where it is typically supported by the "catalytic"/"essential" residues poorly conserved among different families of P-loop NTPases. The cleavage of the PB-O3B-PG bond proceeds by a mechanism where weakening of this bond and the nucleophilic attack are coupled via the motion of γ-phosphate.   The proposed universal mechanism of NTP hydrolysis by P-loop NTPases, the most widespread catalytic mechanism in living nature, appears to be pretty simple and robust. In a way, removal of γ-phosphate from an NTP molecule resembles plucking an apple from a tree – the "fingers" are used to forcibly rotate the γ-phosphate and then pull it off. The free energy for twisting/pulling the γ-phosphate (ca. 20-25 kJ/mol in case of Ras/RasGAP) could be provided by the interaction between the P-loop protein domain and the activating protein domain. Hence, the rate of hydrolysis by P-loop NTPases could be fine-tuned by affecting protein-protein interactions. We suggest that this easily tunable and versatile mechanism of using the free energy of protein-protein interaction for catalysis provides unlimited possibilities to control NTP hydrolysis, which might explain why P-loop ATPases and GTPases, despite their limited catalytic repertoire, represent the most common protein fold. Acknowledgements Very useful discussions with Drs. A.V. Golovin, A. Gorfe, Y. Kalaidzidis, J. Klare, E.V. Koonin, V.P. Skulachev and H.-J. Steinhoff are greatly appreciated. We are thankful to Dr. D. Dibrova and A. Mulkidzhanyan for their help during the launching phase of this project. This study was supported by the Deutsche Forschungsgemeinschaft, Federal Ministry of Education and Research of Germany (A.Y.M.), the German Academic Exchange Service (D.N.S.) a grant from the Russian Science Foundation (14-50-00029, D.N.S., D.A.C), and the Lomonosov Moscow State University (Supercomputer Facility, D.A.C). M.Y.G. is supported by the Intramural Research Program of the NIH at the National Library of Medicine. References 1. Leipe,D.D. Wolf, Y.I., Koonin, E.V., Aravind, L. 2002. Classification and evolution of P-loop GTPases and related ATPases // J Mol Biol, Vol. 317. P. 41-72. 2. Lupas, A.N. et al. 2001. On the evolution of protein folds: are similar motifs in different protein folds the result of convergence, insertion, or relics of an ancient peptide world?, // J. Struct Biol, Vol. 134 P. 191-203. 3. Alva, V. et al. 2015. A vocabulary of ancient peptides at the origin of folded proteins. // eLife, Vol. 4. e09410. 4. Anantharaman, V., Aravind, L., Koonin, E.V. 2003. Emergence of diverse biochemical activities in evolutionarily conserved structural scaffolds of proteins. // Curr. Opin. Chem. Biol., Vol. 7. P. 12-20. 5. Scrima, A., Wittinghofer, A. 2006. Dimerisation-dependent GTPase reaction of MnmE: how potassium acts as GTPase-activating element // EMBO J. Vol. 25. P. 2940-2951. 6. Bohme, S., Meyer, S., Kruger, S., Steinhoff, H.-J., Wittinghofer, A., Klare, J.P. 2010. Stabilization of G domain conformations in the tRNA-modifying MnmE-GidA complex observed with double electron electron resonance spectroscopy // J. Biol. Chem. Vol. 285. P. 16991-17000. 7. Ash, M.-R. et al. 2012. The cation-dependent G-proteins: in a class of their own // FEBS Lett. V. 586. P. 2218-2224. 8. Bos, J.L. et al. 1987. Prevalence of ras gene mutations in human colorectal cancers // Nature. V. 327. P. 293-297. 9. Wittinghofer, A., Vetter, I.R. 2011. Structure-function relationships of the G domain, a canonical switch motif // Annu. Rev. Biochem. V. 80. P. 943-971. 10. Kamerlin, S.C. et al. 2013. Why nature really chose phosphate // Q. Rev. Biophys. Vol. 46. P. 1-132. 11. Jin, Y. et al. 2017. Metal fluorides: Tools for structural and computational analysis of phosphoryl transfer enzymes // Top. Curr. Chem. (Cham). V. 375. N. 36.