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Not AvailableOur previously proposed Ras dimerization model is consistent with recent details observed by NMR in that Raf activation is centered on the Ras/Raf dimer, distinct from one in which Ras activates Raf as a monomer with the Raf cysteine rich domain inserted in the membrane. We review mechanistic understanding of Raf activation within nanoclusters of Ras on the membrane, with a shift to dimers upon binding Raf. This sets the stage for a signaling platform composed of Ras/Raf and Galectin dimers that facilitates the release of Raf autoinhibition and folding of the Raf intrinsically disordered region between the Ras-binding domains and the kinase bound to 14-3-3 and MEK. This platform could provide synchronized units for signal amplification and is consistent with a Ras stationary phase observed in cells. 

Abstract RAS GTPases are proto‐oncoproteins that regulate cell growth, proliferation, and differentiation in response to extracellular signals. The signaling functions of RAS, and other small GTPases, are dependent on their ability to cycle between GDP‐bound and GTP‐bound states. Structural analyses suggest that GTP hydrolysis catalyzed by HRAS can be regulated by an allosteric site located between helices 3, 4, and loop 7. Here we explore the relationship between intrinsic GTP hydrolysis on HRAS and the position of helix 3 and loop 7 through manipulation of the allosteric site, showing that the two sites are functionally connected. We generated several hydrophobic mutations in the allosteric site of HRAS to promote shifts in helix 3 relative to helix 4. By combining crystallography and enzymology to study these mutants, we show that closure of the allosteric site correlates with increased hydrolysis of GTP on HRAS in solution. Interestingly, binding to the RAS binding domain of RAF kinase (RAF‐RBD) inhibits GTP hydrolysis in the mutants. This behavior may be representative of a cluster of mutations found in human tumors, which potentially cooperate with RAF complex formation to stabilize the GTP‐bound state of RAS. 

The scattering of neutrons can be used to provide information on the structure and dynamics of biological systems on multiple length and time scales. Pursuant to a National Science Foundation-funded workshop in February 2018, recent developments in this field are reviewed here, as well as future prospects that can be expected given recent advances in sources, instrumentation and computational power and methods. Crystallography, solution scattering, dynamics, membranes, labeling and imaging are examined. For the extraction of maximum information, the incorporation of judicious specific deuterium labeling, the integration of several types of experiment, and interpretation using high-performance computer simulation models are often found to be particularly powerful.