Engineering and Structural Analysis of a Covalent HRas Dimer

Abstract

Ras is part of the major signaling cascade Ras/Raf/MEK/ERK which regulates cell proliferation. The dysregulation of this pathway through the mutation of Ras is highly implicated in up to 30% of human cancers. Pathway activation is dependent on the binding of Ras to Raf‐RBD and the subsequent dimerization of the Ras/Raf complex. This complex, however, is only robust in the presence of the membrane. In order to create a form of the Ras/Raf‐RBD dimer that is more stable in solution, we engineered mutations to promote covalent bonding at the dimer interface. Our data reveal the structure of a covalent HRas dimer in the absence of Raf‐RBD, and we present an analysis of the disulfide bonded dimer interface and its impact on allosteric connections to the active site. Going forward we plan to use crosslinking as a means to characterize the Ras/Raf‐RBD dimer biochemically, including affinity of the Ras dimer for Raf‐RBD and hydrolysis rate constants in the presence and absence of ligand binding at an allosteric site near the dimer interface.Support or Funding InformationNSFCHE‐1757078