Structure of the MRAS-SHOC2-PP1C phosphatase complex.

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.

A strategy to assess the cellular activity of E3 ligase components against neo-substrates using electrophilic probes.

While there are hundreds of predicted E3 ligases, characterizing their applications for targeted protein degradation has proved challenging. Here, we report a chemical biology approach to evaluate the ability of modified recombinant E3 ligase components to support neo-substrate degradation. Bypassing the need for specific E3 ligase binders, we use maleimide-thiol chemistry for covalent functionalization followed by E3 electroporation (COFFEE) in live cells. We demonstrate that electroporated recombinant von Hippel-Lindau (VHL) protein, covalently functionalized at its ligandable cysteine with JQ1 or dasatinib, induces degradation of BRD4 or tyrosine kinases, respectively. Furthermore, by applying COFFEE to SPSB2, a Cullin-RING ligase 5 receptor, as well as to SKP1, the adaptor protein for Cullin-RING ligase 1 F box (SCF) complexes, we validate this method as a powerful approach to define the activity of previously uncharacterized ubiquitin ligase components, and provide further evidence that not only E3 ligase receptors but also adaptors can be directly hijacked for neo-substrate degradation.

Targeting a helix-in-groove interaction between E1 and E2 blocks ubiquitin transfer.

The ubiquitin-proteasome system (UPS) is a highly regulated protein disposal process critical to cell survival. Inhibiting the pathway induces proteotoxic stress and can be an effective cancer treatment. The therapeutic window observed upon proteasomal blockade has motivated multiple UPS-targeting strategies, including preventing ubiquitination altogether. E1 initiates the cascade by transferring ubiquitin to E2 enzymes. A small molecule that engages the E1 ATP-binding site and derivatizes ubiquitin disrupts enzymatic activity and kills cancer cells. However, binding-site mutations cause resistance, motivating alternative approaches to block this promising target. We identified an interaction between the E2 N-terminal alpha-1 helix and a pocket within the E1 ubiquitin-fold domain as a potentially druggable site. Stapled peptides modeled after the E2 alpha-1 helix bound to the E1 groove, induced a consequential conformational change and inhibited E1 ubiquitin thiotransfer, disrupting E2 ubiquitin charging and ubiquitination of cellular proteins. Thus, we provide a blueprint for a distinct E1-targeting strategy to treat cancer.

Homogeneous Oligomers of Pro-apoptotic BAX Reveal Structural Determinants of Mitochondrial Membrane Permeabilization.

BAX is a pro-apoptotic protein that transforms from a cytosolic monomer into a toxic oligomer that permeabilizes the mitochondrial outer membrane. How BAX monomers assemble into a higher-order conformation, and the structural determinants essential to membrane permeabilization, remain a mechanistic mystery. A key hurdle has been the inability to generate a homogeneous BAX oligomer (BAXO) for analysis. Here, we report the production and characterization of a full-length BAXO that recapitulates physiologic BAX activation. Multidisciplinary studies revealed striking conformational consequences of oligomerization and insight into the macromolecular structure of oligomeric BAX. Importantly, BAXO enabled the assignment of specific roles to particular residues and α helices that mediate individual steps of the BAX activation pathway, including unexpected functionalities of BAX α6 and α9 in driving membrane disruption. Our results provide the first glimpse of a full-length and functional BAXO, revealing structural requirements for the elusive execution phase of mitochondrial apoptosis.

Identification of a Covalent Molecular Inhibitor of Anti-apoptotic BFL-1 by Disulfide Tethering.

The BCL-2 family is composed of anti- and pro-apoptotic members that respectively protect or disrupt mitochondrial integrity. Anti-apoptotic overexpression can promote oncogenesis by trapping the BCL-2 homology 3 (BH3) "killer domains" of pro-apoptotic proteins in a surface groove, blocking apoptosis. Groove inhibitors, such as the relatively large BCL-2 drug venetoclax (868 Da), have emerged as cancer therapies. BFL-1 remains an undrugged oncogenic protein and can cause venetoclax resistance. Having identified a unique C55 residue in the BFL-1 groove, we performed a disulfide tethering screen to determine if C55 reactivity could enable smaller molecules to block BFL-1's BH3-binding functionality. We found that a disulfide-bearing N-acetyltryptophan analog (304 Da adduct) effectively targeted BFL-1 C55 and reversed BFL-1-mediated suppression of mitochondrial apoptosis. Structural analyses implicated the conserved leucine-binding pocket of BFL-1 as the interaction site, resulting in conformational remodeling. Thus, therapeutic targeting of BFL-1 may be achievable through the design of small, cysteine-reactive drugs.

Identification of a Structural Determinant for Selective Targeting of HDMX.

p53 is a critical tumor-suppressor protein that guards the human genome against mutations by inducing cell-cycle 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.