Structures of RGL1 RAS-Association Domain in Complex with KRAS and the Oncogenic G12V Mutant.

Ral Guanine Nucleotide Dissociation Stimulator Like 1 (RGL1) is a RAS effector protein that activates Ral GTPase by stimulating nucleotide exchange. Most structures of RAS-effector complexes are for the HRAS isoform; relatively few KRAS-effector structures have been solved, even though KRAS mutations are more frequent in human cancers. We determined crystal structures of KRAS/RGL1-RAS-association (RA) domain complexes and characterized the interaction in solution using nuclear magnetic resonance spectroscopy, size-exclusion chromatography combined with multi-angle light scattering and biolayer interferometry. We report structures of wild-type KRAS and the oncogenic G12V mutant in complex with the RA domain of RGL1 at < 2 Å resolution. KRASWT/RGL1-RA crystallized as a 1:1 heterodimer, whilst KRASG12V/RGL1-RA crystallized as a heterotetrameric structure in which RGL1-RA dimerized via domain-swapping the C-terminal beta-strand. Solution data indicated that KRASWT and KRASG12V in complex with RGL1-RA both exist predominantly as 1:1 dimers, while tetramerization occurs through very slow association. Through detailed structural analyses, the distance and angle between RAS α1 helix and RBD/RA α1 helix were found to differ significantly among RAS and RBD/RA complexes. The KRAS/RGL1-RA structures possess some of the largest α1RAS/α1Effector distances (21.7-22.2 Å), whereas the corresponding distances in previously reported RAS/RAF complexes are significantly shorter (15.2-17.7 Å). Contact map analysis identified unique structural signatures involving contacts between the ß1-ß2 loop of RA and the α1 helix of RAS, clearly distinguishing the KRAS/RGL1-RA (and other RAS/RA complexes) from RAS/RBD complexes. These results demonstrate that RAS effectors employ an assortment of finely-tuned docking surfaces to achieve optimal interactions with RAS.

Elongation rate and average length of amyloid fibrils in solution using isotope-labelled small-angle neutron scattering.

We demonstrate a solution method that allows both elongation rate and average fibril length of assembling amyloid fibrils to be estimated. The approach involves acquisition of real-time neutron scattering data during the initial stages of seeded growth, using contrast matched buffer to make the seeds effectively invisible to neutrons. As deuterated monomers add on to the seeds, the labelled growing ends give rise to scattering patterns that we model as cylinders whose increase in length with time gives an elongation rate. In addition, the absolute intensity of the signal can be used to determine the number of growing ends per unit volume, which in turn provides an estimate of seed length. The number of ends did not change significantly during elongation, demonstrating that any spontaneous or secondary nucleation was not significant compared with growth on the ends of pre-existing fibrils, and in addition providing a method of internal validation for the technique. Our experiments on initial growth of alpha synuclein fibrils using 1.2 mg ml-1 seeds in 2.5 mg ml-1 deuterated monomer at room temperature gave an elongation rate of 6.3 ± 0.5 Å min-1, and an average seed length estimate of 4.2 ± 1.3 µm.

NMR in integrated biophysical drug discovery for RAS: past, present, and future.

Mutations in RAS oncogenes occur in ~ 30% of human cancers, with KRAS being the most frequently altered isoform. RAS proteins comprise a conserved GTPase domain and a C-terminal lipid-modified tail that is unique to each isoform. The GTPase domain is a 'switch' that regulates multiple signaling cascades that drive cell growth and proliferation when activated by binding GTP, and the signal is terminated by GTP hydrolysis. Oncogenic RAS mutations disrupt the GTPase cycle, leading to accumulation of the activated GTP-bound state and promoting proliferation. RAS is a key target in oncology, however it lacks classic druggable pockets and has been extremely challenging to target. RAS signaling has thus been targeted indirectly, by harnessing key downstream effectors as well as upstream regulators, or disrupting the proper membrane localization required for signaling, by inhibiting either lipid modification or 'carrier' proteins. As a small (20 kDa) protein with multiple conformers in dynamic equilibrium, RAS is an excellent candidate for NMR-driven characterization and screening for direct inhibitors. Several molecules have been discovered that bind RAS and stabilize shallow pockets through conformational selection, and recent compounds have achieved substantial improvements in affinity. NMR-derived insight into targeting the RAS-membrane interface has revealed a new strategy to enhance the potency of small molecules, while another approach has been development of peptidyl inhibitors that bind through large interfaces rather than deep pockets. Remarkable progress has been made with mutation-specific covalent inhibitors that target the thiol of a G12C mutant, and these are now in clinical trials. Here we review the history of RAS inhibitor development and highlight the utility of NMR and integrated biophysical approaches in RAS drug discovery.