H-Ras, K-Ras, and N-Ras are small GTPases that are important in the control of cell proliferation, differentiation, and survival, and their mutants occur frequently in human cancers. The G-domain, which catalyzes GTP hydrolysis and mediates downstream signaling, is 95% conserved between the Ras isoforms. Because of their very high sequence identity, biochemical studies done on H-Ras have been considered representative of all three Ras proteins. We show here that this is not a valid assumption. Using enzyme kinetic assays under identical conditions, we observed clear differences between the three isoforms in intrinsic catalysis of GTP by Ras in the absence and presence of the Ras-binding domain (RBD) of the c-Raf kinase protein (Raf-RBD). Given their identical active sites, isoform G-domain differences must be allosteric in origin, due to remote isoform-specific residues that affect conformational states. We present the crystal structure of N-Ras bound to a GTP analogue and interpret the kinetic data in terms of structural features specific for H-, K-, and N-Ras.

Background:The GTP nucleotide is thought to be fully deprotonated when bound to RAS. Results: The neutron crystal structure of RAS bound to the GTP analogue GppNHp shows a protonated ␥-phosphate. Conclusion: The active site of RAS significantly increases the pK a of the nucleotide. Significance: This work provides insight to the GTP hydrolysis mechanism, with implications to the superfamily of small GTPases.

Ras is at the hub of signal transduction pathways controlling cell proliferation and survival. Its mutants, present in about 30% of human cancers, are major drivers of oncogenesis and render tumors unresponsive to standard therapies. Here we report the engineering of a protein scaffold for preferential binding to K-Ras G12D. This is the first reported inhibitor to achieve nanomolar affinity while exhibiting specificity for mutant over wild type (WT) K-Ras. Crystal structures of the protein R11.1.6 in complex with K-Ras WT and K-Ras G12D offer insight into the structural basis for specificity, highlighting differences in the switch I conformation as the major defining element in the higher affinity interaction. R11.1.6 directly blocks interaction with Raf and reduces signaling through the Raf/MEK/ERK pathway. Our results support greater consideration of the state of switch I and provide a novel tool to study Ras biology. Most importantly, this work makes an unprecedented contribution to Ras research in inhibitor development strategy by revealing details of a targetable binding surface. Unlike the polar interfaces found for Ras/effector interactions, the K-Ras/R11.1.6 complex reveals an extensive hydrophobic interface that can serve as a template to advance the development of high affinity, non-covalent inhibitors of K-Ras oncogenic mutants.

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The biopharmaceutical industry is transitioning from currently deployed batch‐mode bioprocessing to a highly efficient and agile next‐generation bioprocessing with the adaptation of continuous bioprocessing, which reduces capital investment and operational costs. Continuous bioprocessing, aligned with FDA's quality‐by‐design platform, is designed to develop robust processes to deliver safe and effective drugs. With the deployment of knowledge‐based operations, product quality can be built into the process to achieve desired critical quality attributes (CQAs) with reduced variability. To facilitate next‐generation continuous bioprocessing, it is essential to embrace a fundamental shift‐in‐paradigm from “quality‐by‐testing” to “quality‐by‐design,” which requires the deployment of process analytical technologies (PAT). With the adaptation of PAT, a systematic approach of process and product understanding and timely process control are feasible. Deployment of PAT tools for real‐time monitoring of CQAs and feedback control is critical for continuous bioprocessing. Given the current deficiency in PAT tools to support continuous bioprocessing, we have integrated Infinity 2D‐LC with a post‐flow‐splitter in conjunction with the SegFlow autosampler to the bioreactors. With this integrated system, we have established a platform for online measurements of titer and CQAs of monoclonal antibodies as well as amino acid analysis of bioreactor cell culture.

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.

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