Elucidating differences in the conformational dynamics of K-Ras isoforms
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Humans ubiquitously express three genes (HRAS, KRAS, and NRAS) that encode four different isoforms of Ras, an integral p21 GTPase acting as an effector of cell surface receptors and regulating cell survival, proliferation, and differentiation. Mutations in KRAS account for the majority of all RAS-mediated cancers, and are amongst the most frequently observed in epithelial carcinomas. Nearly all observed Ras isoform mutations are found at codons 12, 13 and 61, and affect both splice variants of KRAS, K-Ras4A and K-Ras4B. The K-Ras isoforms share significant sequence identity, and differ only in their 23 residue C- terminal hypervariable region (HVR) and in four catalytic domain residues. However, little is known about how these residue differences correlate to K-Ras conformational dynamics, and consequently, their effects on Ras functional behavior and effector interactions. In this investigation, the functional consequences of residue differences between K-Ras4A and K- Ras4B were probed through all-atom molecular dynamics (MD) simulations to elucidate the effects of isoform-specific differences on K-Ras conformational dynamics. These simulations, utilizing the CHARMM programming package and NAMD code, aimed to (1) study the conformational fluctuations of the K-Ras4A catalytic domain, (2) study the dynamics of the full-length K-Ras4A isoform, and (3) characterize potential HVR interactions with the catalytic domain of the protein. These studies suggest that: (1) GDP-bound K-Ras4A exhibits a more exposed nucleotide binding pocket than K-Ras4B, which may facilitate enhanced nucleotide dissociation in this isoform, (2) GDP-bound K-Ras4A exhibits dynamic fluctuations in Switch I and II that differ from GDP-bound K-Ras4B, implying additional conformational states that may influence its interactions with guanine- nucleotide exchange factors (GEFs), (3) residues Q165 and Y166 in K-Ras4A may allosterically modulate the observed dynamic Switch region fluctuations, (4) full-length K-Ras4A exhibits nucleotide-dependent HVR fluctuations, and (5) the HVR of GDP and GTP-bound K-Ras4A confers additional flexibility to the intrinsic fluctuations of its Switch I and Switch II regions. These results provide a basis for further studies examining the functional consequences of K-Ras4A dynamics.