The Effect of Y71 Mutants on The Allosteric Switch of Ras

Abstract

Ras GTPase is involved in a number of signaling pathways that are responsible for the control of many cellular processes, including cell proliferation. Ras functions like a molecular switch, where its inactive form binds to GDP and its active form binds to GTP. We had shown through crystallography work that in its active form, Ras has two major conformational states, including the T‐state (tardy) and the R‐state (reactive). The R‐state is obtained through an allosteric modulation by the binding of calcium acetate in the crystal structure. Through a wild‐type accelerated molecular dynamics (aMD) simulation, we observed that residue Y71 is involved in the transitioning from R‐ to T‐state. In this study, we aim to elucidate the role of residue Y71 in the allosteric modulation of H‐Ras. We work to obtain the structure of Y71F and Y71A mutants of H‐Ras, and conduct aMD simulations for these mutants. For aMD simulation, we make Y71F and Y71A mutants using homology modeling of wild type H‐Ras structure. In lab, we made mutations using site directed mutagenesis and successfully mutate Tyrosine 71 to Phenylalanine and Alanine. We over‐expressed our mutants in Escherichia coli system, used ion exchange and gel filtration chromatography for protein purification. Our aMD simulation shows that Y71F and Y71A mutants changed the conformational balance between the R‐ and T‐states, favoring the T‐state, relative to wild type H‐Ras. We observe different dynamics in these two mutants, specifically Y71F show higher flexibility near the active site residues, relative to the wild type. Our preliminary crystallization result will also be presented. This study will help elucidate the role of a novel allosteric residue that will help in understanding the allosteric mechanism of H‐Ras.Support or Funding InformationNSF MCB‐1244203.