Highlights d Fos-12 induces homogeneous BAX oligomers that recapitulate physiologic activation d SAXS, HXMS, and crosslinking analyses reveal conformational features of BAX O d BAX O distinguishes between the structural determinants of activation and poration d BAX O uncovered roles for a6 and a9 in the execution phase of mitochondrial apoptosis
RAS–MAPK signalling is fundamental for cell proliferation and is altered in most human cancers1–3. However, our mechanistic understanding of how RAS signals through RAF is still incomplete. Although studies revealed snapshots for autoinhibited and active RAF–MEK1–14-3-3 complexes4, the intermediate steps that lead to RAF activation remain unclear. The MRAS–SHOC2–PP1C holophosphatase dephosphorylates RAF at serine 259, resulting in the partial displacement of 14-3-3 and RAF–RAS association3,5,6. MRAS, SHOC2 and PP1C are mutated in rasopathies—developmental syndromes caused by aberrant MAPK pathway activation6–14—and SHOC2 itself has emerged as potential target in receptor tyrosine kinase (RTK)–RAS-driven tumours15–18. Despite its importance, structural understanding of the SHOC2 holophosphatase is lacking. Here we determine, using X-ray crystallography, the structure of the MRAS–SHOC2–PP1C complex. SHOC2 bridges PP1C and MRAS through its concave surface and enables reciprocal interactions between all three subunits. Biophysical characterization indicates a cooperative assembly driven by the MRAS GTP-bound active state, an observation that is extendible to other RAS isoforms. Our findings support the concept of a RAS-driven and multi-molecular model for RAF activation in which individual RAS–GTP molecules recruit RAF–14-3-3 and SHOC2–PP1C to produce downstream pathway activation. Importantly, we find that rasopathy and cancer mutations reside at protein–protein interfaces within the holophosphatase, resulting in enhanced affinities and function. Collectively, our findings shed light on a fundamental mechanism of RAS biology and on mechanisms of clinically observed enhanced RAS–MAPK signalling, therefore providing the structural basis for therapeutic interventions.
Highlights d A disulfide tethering screen identifies small molecules that target BFL-1 C55 d Structural analyses reveal the conformational consequences of disulfide formation d Lead molecule 4E14 effectively competes with BH3-binding at the BFL-1 groove d Covalent 4E14 targeting of C55 inhibits BFL-1 suppression of mitochondrial apoptosis
p53 is a critical tumor-suppressor protein that guards the human genome against mutations by inducing cellcycle arrest or apoptosis. Cancer cells subvert p53 by deletion, mutation, or overexpression of the negative regulators HDM2 and HDMX. For tumors that retain wild-type p53, its reactivation by pharmacologic targeting of HDM2 and/or HDMX represents a promising strategy, with a series of selective small-molecule HDM2 inhibitors and a dual HDM2/HDMX stapled-peptide inhibitor being evaluated in clinical trials. Because selective HDM2 targeting can cause hematologic toxicity, selective HDMX inhibitors could provide an alternative p53-reactivation strategy, but clinical candidates remain elusive. Here, we applied a mutation-scanning approach to uncover p53-based stapled peptides that are selective for HDMX. Crystal structures of stapled-peptide/HDMX complexes revealed a molecular mechanism for the observed specificity, which was validated by HDMX mutagenesis. Thus, we provide a blueprint for the development of HDMX-selective inhibitors to dissect and target the p53/HDMX interaction.
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