Structures of wild-type K-Ras from crystals obtained in the presence of guanosine triphosphate (GTP) or its analogs have remained elusive. Of the K-Ras mutants, only K-RasG12D and K-RasQ61H are available in the PDB representing the activated form of the GTPase not in complex with other proteins. We present the crystal structure of wild-type K-Ras bound to the GTP analog GppCHp, with K-Ras in the state 1 conformation. Signatures of conformational states obtained by one-dimensional proton NMR confirm that K-Ras has a more substantial population of state 1 in solution than H-Ras, which predominantly favors state 2. The oncogenic mutant K-RasG12D favors state 2, changing the balance of conformational states in favor of interactions with effector proteins. Differences in the population of conformational states between K-Ras and H-Ras, as well as between K-Ras and its mutants, can provide a structural basis for focused targeting of the K-Ras isoform in cancer-specific strategies.

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Neutron protein crystallography (NPC) reveals the three‐dimensional structures of proteins, including the positions of H atoms. The technique is particularly suited to elucidate ambiguous catalytic steps in complex biochemical reactions. While NPC uniquely complements biochemical assays and X‐ray structural analyses by revealing the protonation states of ionizable groups at and around the active site of enzymes, the technique suffers from a major drawback: large single crystals must be grown to compensate for the relatively low flux of neutron beams. However, in addition to revealing the positions of hydrogens involved in enzyme catalysis, NPC has the advantage over X‐ray crystallography that the crystals do not suffer radiation damage. The lack of radiation damage can be exploited to conduct in crystallo parametric studies. Here, the use of a single crystal of the small GTPase Ras to collect three neutron data sets at pD 8.4, 9.0 and 9.4 is reported, enabling an in crystallo titration study using NPC. In addition to revealing the behavior of titratable groups in the active site, the data sets will allow the analysis of allosteric water‐mediated communication networks across the molecule, particularly regarding Cys118 and three tyrosine residues central to these networks, Tyr32, Tyr96 and Tyr137, with pKa values expected to be in the range sampled in our experiments.

The interaction between Ras and Raf kinase at the membrane promotes cell proliferation through the mitogen activated protein kinase (MAPK) pathway. Ras mutations drive 20% of all human cancers and despite great efforts, there are currently no drugs targeting Ras. Raf interacts with Ras via its two N‐terminal Ras‐binding domains: the Ras‐binding domain (RBD) and the cysteine‐rich domain. Binding of both the Raf‐RBD and CRD are required for Ras‐mediated activation of Raf kinase, however, the mechanism that results in the activation of the C‐terminal Raf kinase domain remains unknown. Here we present the 2.8 Å crystal structure of Ras in complex with a Raf construct containing both the RBD and CRD, revealing the interface for Raf‐CRD binding. In combination with molecular dynamic simulations, we identify allosteric effects induced by Raf‐CRD binding that stabilize the Ras active site to promote a Ras conformation poised for intrinsic hydrolysis. Support or Funding Information NSF, MCB‐1517295. Northeastern Office of Undergraduate Research and Fellowships

Ras is a small GTPase that is involved in up to 30% of cancers. A wealth of evidence suggests that despite high sequence similarity of the Ras isoforms, H‐Ras, K‐Ras, and N‐Ras, there are distinct differences in signaling, localization, mutation position, and outcomes in cancer. Even in the absence of the C‐terminal hypervariable region (HVR), there are differences between the isoforms in the rates of GTP hydrolysis and in the details of Ras structure and dynamics. Here we present accelerated molecular dynamics simulations paired with solution x‐ray scattering that reveal differences in the conformational sampling of H‐Ras, K‐Ras4B, and N‐Ras. Specific interactions within water‐mediated allosteric networks are altered by the presence of isoform specific residues that impact accessibility to the catalytic state. Thus, the isoforms sample the same main three conformational states, but the population of each one is shifted by changes driven by isoform‐specific residues that may be associated with fine‐tuning preferences for signaling through distinct pathways and susceptibility to specific mutations observed in cancers. Support or Funding Information NSF MCB‐1517295

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