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Browsing by Author "Molt, Robert W., Jr."
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Item A GAP‐GTPase‐GDP‐Pi Intermediate Crystal Structure Analyzed by DFT Shows GTP Hydrolysis Involves Serial Proton Transfers(Wiley, 2019) Jin, Yi; Molt, Robert W., Jr.; Pellegrini, Erika; Biochemistry and Molecular Biology, School of MedicineCell signaling by small G proteins uses an ON to OFF signal based on conformational changes following the hydrolysis of GTP to GDP and release of dihydrogen phosphate (Pi). The catalytic mechanism of GTP hydrolysis by RhoA is strongly accelerated by a GAP protein and is now well defined, but timing of inorganic phosphate release and signal change remains unresolved. We have generated a quaternary complex for RhoA‐GAP‐GDP‐Pi. Its 1.75 Å crystal structure shows geometry for ionic and hydrogen bond coordination of GDP and Pi in an intermediate state. It enables the selection of a QM core for DFT exploration of a 20 H‐bonded network. This identifies serial locations of the two mobile protons from the original nucleophilic water molecule, showing how they move in three rational steps to form a stable quaternary complex. It also suggests how two additional proton transfer steps can facilitate Pi release.Item Metal Fluorides: Tools for Structural and Computational Analysis of Phosphoryl Transfer Enzymes(Springer, 2017-04) Jin, Yi; Molt, Robert W., Jr.; Blackburn, G. Michael; Chemistry and Chemical Biology, School of ScienceThe phosphoryl group, PO3-, is the dynamic structural unit in the biological chemistry of phosphorus. Its transfer from a donor to an acceptor atom, with oxygen much more prevalent than nitrogen, carbon, or sulfur, is at the core of a great majority of enzyme-catalyzed reactions involving phosphate esters, anhydrides, amidates, and phosphorothioates. The serendipitous discovery that the phosphoryl group could be labeled by "nuclear mutation," by substitution of PO3- by MgF3- or AlF4-, has underpinned the application of metal fluoride (MF x ) complexes to mimic transition states for enzymatic phosphoryl transfer reactions, with sufficient stability for experimental analysis. Protein crystallography in the solid state and 19F NMR in solution have enabled direct observation of ternary and quaternary protein complexes embracing MF x transition state models with precision. These studies have underpinned a radically new mechanistic approach to enzyme catalysis for a huge range of phosphoryl transfer processes, as varied as kinases, phosphatases, phosphomutases, and phosphohydrolases. The results, without exception, have endorsed trigonal bipyramidal geometry (tbp) for concerted, "in-line" stereochemistry of phosphoryl transfer. QM computations have established the validity of tbp MF x complexes as reliable models for true transition states, delivering similar bond lengths, coordination to essential metal ions, and virtually identical hydrogen bond networks. The emergence of protein control of reactant orbital overlap between bond-forming species within enzyme transition states is a new challenging theme for wider exploration.Item Octahedral Trifluoromagnesate, an Anomalous Metal Fluoride Species, Stabilizes the Transition State in a Biological Motor(American Chemical Society, 2021-03-05) Ge, Mengyu; Molt, Robert W., Jr.; Jenkins, Huw T.; Blackburn, G. Michael; Jin, Yi; Antson, Alfred A.; Biochemistry and Molecular Biology, School of MedicineIsoelectronic metal fluoride transition state analogue (TSA) complexes, MgF3 - and AlF4 -, have proven to be immensely useful in understanding mechanisms of biological motors utilizing phosphoryl transfer. Here we report a previously unobserved octahedral TSA complex, MgF3(H2O)-, in a 1.5 Å resolution Zika virus NS3 helicase crystal structure. 19F NMR provided independent validation and also the direct observation of conformational tightening resulting from ssRNA binding in solution. The TSA stabilizes the two conformations of motif V of the helicase that link ATP hydrolysis with mechanical work. DFT analysis further validated the MgF3(H2O)- species, indicating the significance of this TSA for studies of biological motors.