@article{schwabe_holzapfel_mattos_2020, title={Exploring the Rap1A Active Site Through Accelerated Molecular Dynamic Simulations}, volume={34}, ISSN={["1530-6860"]}, DOI={10.1096/fasebj.2020.34.s1.02775}, abstractNote={GTPases are hydrolytic proteins that convert GTP to GDP and serve as bivalent switches, where the GTP‐bound state is active, and the GDP‐bound state is inactive. Although Ras is the archetypal GTPase, Rap1A is closely related with 50% sequence homology and similar tertiary structure; a key difference occurs at position 61. In Ras Q61 participates in the hydrolysis mechanism and mutation leads to oncogenesis, while T61 in Rap1A is not thought to be involved in catalysis. The goal of this project is to explore the Rap1A active site and method of intrinsic hydrolysis. Two crystal structures were obtained with differing conformations of T61 in the Rap1A active site. Accelerated molecular dynamic simulations were run for 200 ns to sample the variety of motion present in Rap1A. Results indicate coordinated movements between the switch I, switch II, and helix 3 regions. Further, switch II appears less mobile than simulations with Ras isoforms.}, journal={FASEB JOURNAL}, author={Schwabe, Michael and Holzapfel, Genevieve and Mattos, Carla}, year={2020}, month={Apr} }
 @article{knihtila_holzapfel_weiss_meilleur_mattos_2015, title={Neutron Crystal Structure of RAS GTPase Puts in Question the Protonation State of the GTP gamma-Phosphate}, volume={290}, ISSN={["1083-351X"]}, DOI={10.1074/jbc.m115.679860}, abstractNote={Background: The GTP nucleotide is thought to be fully deprotonated when bound to RAS. Results: The neutron crystal structure of RAS bound to the GTP analogue GppNHp shows a protonated γ-phosphate. Conclusion: The active site of RAS significantly increases the pKa of the nucleotide. Significance: This work provides insight to the GTP hydrolysis mechanism, with implications to the superfamily of small GTPases. RAS GTPase is a prototype for nucleotide-binding proteins that function by cycling between GTP and GDP, with hydrogen atoms playing an important role in the GTP hydrolysis mechanism. It is one of the most well studied proteins in the superfamily of small GTPases, which has representatives in a wide range of cellular functions. These proteins share a GTP-binding pocket with highly conserved motifs that promote hydrolysis to GDP. The neutron crystal structure of RAS presented here strongly supports a protonated γ-phosphate at physiological pH. This counters the notion that the phosphate groups of GTP are fully deprotonated at the start of the hydrolysis reaction, which has colored the interpretation of experimental and computational data in studies of the hydrolysis mechanism. The neutron crystal structure presented here puts in question our understanding of the pre-catalytic state associated with the hydrolysis reaction central to the function of RAS and other GTPases.}, number={52}, journal={JOURNAL OF BIOLOGICAL CHEMISTRY}, publisher={American Society for Biochemistry & Molecular Biology (ASBMB)}, author={Knihtila, Ryan and Holzapfel, Genevieve and Weiss, Kevin and Meilleur, Flora and Mattos, Carla}, year={2015}, month={Dec}, pages={31025–31036} }
 @article{holzapfel_buhrman_mattos_2012, title={Shift in the Equilibrium between On and Off States of the Allosteric Switch in Ras-GppNHp Affected by Small Molecules and Bulk Solvent Composition}, volume={51}, ISSN={["0006-2960"]}, DOI={10.1021/bi300509j}, abstractNote={Ras GTPase cycles between its active GTP-bound form promoted by GEFs and its inactive GDP-bound form promoted by GAPs to affect the control of various cellular functions. It is becoming increasingly apparent that subtle regulation of the GTP-bound active state may occur through promotion of substates mediated by an allosteric switch mechanism that induces a disorder to order transition in switch II upon ligand binding at an allosteric site. We show with high-resolution structures that calcium acetate and either dithioerythritol (DTE) or dithiothreitol (DTT) soaked into H-Ras-GppNHp crystals in the presence of a moderate amount of poly(ethylene glycol) (PEG) can selectively shift the equilibrium to the "on" state, where the active site appears to be poised for catalysis (calcium acetate), or to what we call the "ordered off" state, which is associated with an anticatalytic conformation (DTE or DTT). We also show that the equilibrium is reversible in our crystals and dependent on the nature of the small molecule present. Calcium acetate binding in the allosteric site stabilizes the conformation observed in the H-Ras-GppNHp/NOR1A complex, and PEG, DTE, and DTT stabilize the anticatalytic conformation observed in the complex between the Ras homologue Ran and Importin-β. The small molecules are therefore selecting biologically relevant conformations in the crystal that are sampled by the disordered switch II in the uncomplexed GTP-bound form of H-Ras. In the presence of a large amount of PEG, the ordered off conformation predominates, whereas in solution, in the absence of PEG, switch regions appear to remain disordered in what we call the off state, unable to bind DTE.}, number={31}, journal={BIOCHEMISTRY}, author={Holzapfel, Genevieve and Buhrman, Greg and Mattos, Carla}, year={2012}, month={Aug}, pages={6114–6126} }
 @article{buhrman_holzapfel_fetics_mattos_2010, title={Allosteric modulation of Ras positions Q61 for a direct role in catalysis}, volume={107}, ISSN={["0027-8424"]}, DOI={10.1073/pnas.0912226107}, abstractNote={
            Ras and its effector Raf are key mediators of the Ras/Raf/MEK/ERK signal transduction pathway. Mutants of residue Q61 impair the GTPase activity of Ras and are found prominently in human cancers. Yet the mechanism through which Q61 contributes to catalysis has been elusive. It is thought to position the catalytic water molecule for nucleophilic attack on the γ-phosphate of GTP. However, we previously solved the structure of Ras from crystals with symmetry of the space group R32 in which switch II is disordered and found that the catalytic water molecule is present. Here we present a structure of wild-type Ras with calcium acetate from the crystallization mother liquor bound at a site remote from the active site and likely near the membrane. This results in a shift in helix 3/loop 7 and a network of H-bonding interactions that propagates across the molecule, culminating in the ordering of switch II and placement of Q61 in the active site in a previously unobserved conformation. This structure suggests a direct catalytic role for Q61 where it interacts with a water molecule that bridges one of the γ-phosphate oxygen atoms to the hydroxyl group of Y32 to stabilize the transition state of the hydrolysis reaction. We propose that Raf together with the binding of Ca
            2+
            and a negatively charged group mimicked in our structure by the acetate molecule induces the ordering of switch I and switch II to complete the active site of Ras.
          }, number={11}, journal={PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA}, author={Buhrman, Greg and Holzapfel, Genevieve and Fetics, Susan and Mattos, Carla}, year={2010}, month={Mar}, pages={4931–4936} }