@article{johnson_fetics_davis_rodrigues_mattos_2023, title={Allosteric site variants affect GTP hydrolysis on Ras}, volume={32}, ISSN={["1469-896X"]}, DOI={10.1002/pro.4767}, abstractNote={AbstractRAS GTPases are proto‐oncoproteins that regulate cell growth, proliferation, and differentiation in response to extracellular signals. The signaling functions of RAS, and other small GTPases, are dependent on their ability to cycle between GDP‐bound and GTP‐bound states. Structural analyses suggest that GTP hydrolysis catalyzed by HRAS can be regulated by an allosteric site located between helices 3, 4, and loop 7. Here we explore the relationship between intrinsic GTP hydrolysis on HRAS and the position of helix 3 and loop 7 through manipulation of the allosteric site, showing that the two sites are functionally connected. We generated several hydrophobic mutations in the allosteric site of HRAS to promote shifts in helix 3 relative to helix 4. By combining crystallography and enzymology to study these mutants, we show that closure of the allosteric site correlates with increased hydrolysis of GTP on HRAS in solution. Interestingly, binding to the RAS binding domain of RAF kinase (RAF‐RBD) inhibits GTP hydrolysis in the mutants. This behavior may be representative of a cluster of mutations found in human tumors, which potentially cooperate with RAF complex formation to stabilize the GTP‐bound state of RAS.}, number={10}, journal={PROTEIN SCIENCE}, author={Johnson, Christian W. and Fetics, Susan K. and Davis, Kathleen P. and Rodrigues, Jose A. and Mattos, Carla}, year={2023}, month={Oct} }
 @article{fetics_guterres_kearney_buhrman_ma_nussinov_mattos_2015, title={Allosteric Effects of the Oncogenic RasQ61L Mutant on Raf-RBD}, volume={23}, ISSN={["1878-4186"]}, DOI={10.1016/j.str.2014.12.017}, abstractNote={The Ras/Raf/MEK/ERK signal transduction pathway is a major regulator of cell proliferation activated by Ras-guanosine triphosphate (GTP). The oncogenic mutant RasQ61L is not able to hydrolyze GTP in the presence of Raf and thus is a constitutive activator of this mitogenic pathway. The Ras/Raf interaction is essential for the activation of the Raf kinase domain through a currently unknown mechanism. We present the crystal structures of the Ras-GppNHp/Raf-RBD and RasQ61L-GppNHp/Raf-RBD complexes, which, in combination with MD simulations, reveal differences in allosteric interactions leading from the Ras/Raf interface to the Ras calcium-binding site and to the remote Raf-RBD loop L4. In the presence of Raf, the RasQ61L mutant has a rigid switch II relative to the wild-type and increased flexibility at the interface with switch I, which propagates across Raf-RBD. We show that in addition to local perturbations on Ras, RasQ61L has substantial long-range effects on the Ras allosteric lobe and on Raf-RBD.}, number={3}, journal={STRUCTURE}, author={Fetics, Susan K. and Guterres, Hugo and Kearney, Bradley M. and Buhrman, Greg and Ma, Buyong and Nussinov, Ruth and Mattos, Carla}, year={2015}, month={Mar}, pages={505–516} }
 @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} }