Allosteric effects in the complexes between Ras proteins and Raf

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This project focuses on discerning the molecular mechanisms through which key proteins that regulate cell proliferation, Ras and Raf, attain different biological functions through natural variants that affect details of their interactions with each other. The research is founded on earlier discoveries where the interaction between Ras and Raf promotes dimerization of the complex and small variations in the proteins result in changes in biochemical outcomes. This project challenges current dogma for signaling and regulation associated with cell proliferation, with the potential of transforming fundamental understanding of how the system works at the molecular level. This project will provide training for underrepresented minorities in STEM disciplines. Ras GTPases, HRas, KRas and NRas control cell proliferation via the Ras/Raf/MEK/ERK pathway (MAPK pathway) through a process in which Ras dimerization is required, but not mechanistically understood. The homologous GTPase Rap1A also interacts with Raf, but its biological outcome varies depending on specific Raf isoforms. The Ras Binding Domain of Raf (Raf-RBD) promotes Ras dimerization resulting in strong allosteric linkages between the two extreme ends of the dimer (85 Å apart) and most recently solved the crystal structure of Ras in complex with both RBD and the Cysteine Rich Domain (CRD) of Raf. The surfaces on Ras contacted by the CRD both in the monomer and in the dimer are regions of isoform specific residue differences in the Ras GTPases, suggesting a mechanism through which these differences could impact MAPK signaling. This project will test the hypothesis that residue differences in key regions of allosteric connections in Ras and Rap, across their complexes with Raf lead to refinement of allosteric modulations particular to each GTPase/Raf pair, with implications to regulation and signaling. This hypothesis will be tested using a series of biophysical and biochemical approaches. This project is supported by the Molecular Biophysics Cluster in the Division of Molecular and Cellular Biosciences.This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.