Ubiquitination controls the stability of most cellular proteins, and its deregulation contributes to human diseases including cancer. Deubiquitinases remove ubiquitin from proteins, and their inhibition can induce the degradation of selected proteins, potentially including otherwise 'undruggable' targets. For example, the inhibition of ubiquitin-specific protease 7 (USP7) results in the degradation of the oncogenic E3 ligase MDM2, and leads to re-activation of the tumour suppressor p53 in various cancers. Here we report that two compounds, FT671 and FT827, inhibit USP7 with high affinity and specificity in vitro and within human cells. Co-crystal structures reveal that both compounds target a dynamic pocket near the catalytic centre of the auto-inhibited apo form of USP7, which differs from other USP deubiquitinases. Consistent with USP7 target engagement in cells, FT671 destabilizes USP7 substrates including MDM2, increases levels of p53, and results in the transcription of p53 target genes, induction of the tumour suppressor p21, and inhibition of tumour growth in mice.

Regulator of G protein signaling (RGS) proteins accelerate GTP hydrolysis by G␣ subunits and thus facilitate termination of signaling initiated by G protein-coupled receptors (GPCRs). RGS proteins hold great promise as disease intervention points, given their signature role as negative regulators of GPCRs-receptors to which the largest fraction of approved medications are currently directed. RGS proteins share a hallmark RGS domain that interacts most avidly with G␣ when in its transition state for GTP hydrolysis; by binding and stabilizing switch regions I and II of G␣, RGS domain binding consequently accelerates G␣-mediated GTP hydrolysis. The human genome encodes more than three dozen RGS domaincontaining proteins with varied G␣ substrate specificities. To facilitate their exploitation as drug-discovery targets, we have taken a systematic structural biology approach toward cataloging the structural diversity present among RGS domains and identifying molecular determinants of their differential G␣ selectivities. Here, we determined 14 structures derived from NMR and x-ray crystallography of members of the R4, R7, R12, and RZ subfamilies of RGS proteins, including 10 uncomplexed RGS domains and 4 RGS domain/G␣ complexes. Heterogeneity observed in the structural architecture of the RGS domain, as well as in engagement of switch III and the all-helical domain of the G␣ substrate, suggests that unique structural determinants specific to particular RGS protein/G␣ pairings exist and could be used to achieve selective inhibition by small molecules.GTPase-accelerating proteins ͉ NMR structure ͉ RGS proteins ͉ x-ray crystallography G protein-coupled receptors (GPCRs) are critical for many physiological processes including vision, olfaction, neurotransmission, and the actions of many hormones (1). As such, GPCRs are the largest fraction of the ''druggable proteome,'' and their ligand-binding and signaling properties remain of considerable interest to academia and industry (2). GPCRs catalyze activation of heterotrimeric G proteins comprising a guanine nucleotide-binding G␣ subunit and an obligate G␤␥ dimer (3). Receptor-promoted activation of G␣␤␥ causes exchange of GDP for GTP by G␣ and resultant dissociation of G␤␥. GTP-bound G␣ and freed G␤␥ then regulate intracellular effectors such as adenylyl cyclase, phospholipase C, ion channels, RhoGEFs, and phosphodiesterases (1, 4). This ''G protein cycle'' is reset by the intrinsic GTP hydrolysis activity of G␣, producing G␣⅐GDP that favors heterotrimer reformation and, consequently, signal termination. Thus, a major determinant of the duration and magnitude of GPCR signaling is the lifetime of G␣ in the GTP-bound state.Regulators of G protein signaling are GTPase-accelerating proteins (GAPs) for G␣ subunits and thus facilitate GPCR signal termination (5). GAP activity is conferred by an RGS domain present in one or more copies within members of this protein superfamily (5). The archetypal RGS domain is composed of nine ␣-helices (6) and binds most avidly to G␣ in the transi...

Protein kinase autophosphorylation of activation segment residues is a common regulatory mechanism in phosphorylation-dependent signalling cascades. However, the molecular mechanisms that guarantee specific and efficient phosphorylation of these sites have not been elucidated. Here, we report on three novel and diverse protein kinase structures that reveal an exchanged activation segment conformation. This dimeric arrangement results in an active kinase conformation in trans, with activation segment phosphorylation sites in close proximity to the active site of the interacting protomer. Analytical ultracentrifugation and chemical cross-linking confirmed the presence of dimers in solution. Consensus substrate sequences for each kinase showed that the identified activation segment autophosphorylation sites are non-consensus substrate sites. Based on the presented structural and functional data, a model for specific activation segment phosphorylation at non-consensus substrate sites is proposed that is likely to be common to other kinases from diverse subfamilies.