The Ras gene is frequently mutated in cancer, and mutant Ras drives tumorigenesis. Although Ras is a central oncogene, small molecules that bind to Ras in a well-defined manner and exert inhibitory effects have not been uncovered to date. Through an NMR-based fragment screen, we identified a group of small molecules that all bind to a common site on Ras. High-resolution cocrystal structures delineated a unique ligand-binding pocket on the Ras protein that is adjacent to the switch I/II regions and can be expanded upon compound binding. Structure analysis predicts that compound-binding interferes with the Ras/SOS interactions. Indeed, selected compounds inhibit SOS-mediated nucleotide exchange and prevent Ras activation by blocking the formation of intermediates of the exchange reaction. The discovery of a small-molecule binding pocket on Ras with functional significance provides a new direction in the search of therapeutically effective inhibitors of the Ras oncoprotein.small G protein | guanine nucleotide exchange | nuclear magnetic resonance | crystal structure | small-molecule inhibitors R as is a small GTP-binding protein that functions as a nucleotide-dependent switch for central growth signaling pathways (1, 2). In response to extracellular signals, Ras is converted from a GDP-bound (Ras GDP ) to a GTP-bound (Ras GTP ) state, as catalyzed by guanine nucleotide exchange factors (GEFs), notably the SOS1 protein. Active Ras GTP mediates its diverse growth-stimulating functions through its direct interactions with effectors including Raf, PI3K, and Ral guanine nucleotide dissociation stimulator. The intrinsic GTPase activity of Ras then hydrolyzes GTP to GDP to terminate Ras signaling. The Ras GTPase activity can be further accelerated by its interactions with GTPase-activating proteins (GAPs), including the neurofibromin 1 tumor suppressor (2).Ras, a human oncogene identified and characterized over 30 y ago, is mutated in more than 20% of human cancers. Among the three Ras isoforms (K, N, and H), KRas is most frequently mutated (2). Mutant Ras has a reduced GTPase activity, which prolongs its activated conformation, thereby promoting Rasdependent signaling and cancer cell survival or growth (1, 2).Mutations of Ras in cancer are associated with poor prognosis (2). Inactivation of oncogenic Ras in mice results in tumor shrinkage. Thus, Ras is widely considered an oncology target of exceptional importance. However, development of small-molecule inhibitors against Ras has thus far proven unsuccessful. Given the picomolar affinity between guanine nucleotides and Ras and the high cytosolic concentration of guanine nucleotides, it is very challenging to develop a conventional inhibitor competitive against nucleotide binding (1, 2). Outside of the nucleotide-binding pocket, the Ras protein does not contain obvious cavities for small-molecule binding. A number of small molecules have been reported to bind to Ras (3-7), but their mechanisms of action and the structural basis to achieve Ras inhibition remain elusive.Fra...

The ubiquitin system regulates essential cellular processes in eukaryotes. Ubiquitin is ligated to substrate proteins as monomers or chains and the topology of ubiquitin modifications regulates substrate interactions with specific proteins. Thus ubiquitination directs a variety of substrate fates including proteasomal degradation. Deubiquitinase enzymes cleave ubiquitin from substrates and are implicated in disease; for example, ubiquitin-specific protease-7 (USP7) regulates stability of the p53 tumour suppressor and other proteins critical for tumour cell survival. However, developing selective deubiquitinase inhibitors has been challenging and no co-crystal structures have been solved with small-molecule inhibitors. Here, using nuclear magnetic resonance-based screening and structure-based design, we describe the development of selective USP7 inhibitors GNE-6640 and GNE-6776. These compounds induce tumour cell death and enhance cytotoxicity with chemotherapeutic agents and targeted compounds, including PIM kinase inhibitors. Structural studies reveal that GNE-6640 and GNE-6776 non-covalently target USP7 12 Å distant from the catalytic cysteine. The compounds attenuate ubiquitin binding and thus inhibit USP7 deubiquitinase activity. GNE-6640 and GNE-6776 interact with acidic residues that mediate hydrogen-bond interactions with the ubiquitin Lys48 side chain, suggesting that USP7 preferentially interacts with and cleaves ubiquitin moieties that have free Lys48 side chains. We investigated this idea by engineering di-ubiquitin chains containing differential proximal and distal isotopic labels and measuring USP7 binding by nuclear magnetic resonance. This preferential binding protracted the depolymerization kinetics of Lys48-linked ubiquitin chains relative to Lys63-linked chains. In summary, engineering compounds that inhibit USP7 activity by attenuating ubiquitin binding suggests opportunities for developing other deubiquitinase inhibitors and may be a strategy more broadly applicable to inhibiting proteins that require ubiquitin binding for full functional activity.

Protein-protein interactions are often studied by chemical shift mapping using solution NMR spectroscopy. When heteronuclear data are available the interaction interface is usually predicted by combining the chemical shift changes of different nuclei to a single quantity, the combined chemical shift perturbation Deltadelta comb In this paper different procedures (published and non-published) to calculate Deltadelta comb are examined that include a variety of different functional forms and weighting factors for each nucleus. The predictive power of all shift mapping methods depends on the magnitude of the overlap of the chemical shift distributions of interacting and non-interacting residues and the cut-off criterion used. In general, the quality of the prediction on the basis of chemical shift changes alone is rather unsatisfactory but the combination of chemical shift changes on the basis of the Hamming or the Euclidian distance can improve the result. The corrected standard deviation to zero of the combined chemical shift changes can provide a reasonable cut-off criterion. As we show combined chemical shifts can also be applied for a more reliable quantitative evaluation of titration data.

The movement of core-lipopolysaccharide across the inner membrane of Gram-negative bacteria is catalysed by an essential ATP-binding cassette transporter, MsbA. Recent structures of MsbA and related transporters have provided insights into the molecular basis of active lipid transport; however, structural information about their pharmacological modulation remains limited. Here we report the 2.9 Å resolution structure of MsbA in complex with G907, a selective small-molecule antagonist with bactericidal activity, revealing an unprecedented mechanism of ABC transporter inhibition. G907 traps MsbA in an inward-facing, lipopolysaccharide-bound conformation by wedging into an architecturally conserved transmembrane pocket. A second allosteric mechanism of antagonism occurs through structural and functional uncoupling of the nucleotide-binding domains. This study establishes a framework for the selective modulation of ABC transporters and provides rational avenues for the design of new antibiotics and other therapeutics targeting this protein family.

Addition and removal of ubiquitin or ubiquitin chains to and from proteins is a tightly regulated process that contributes to cellular signaling and protein stability. Here we show that phosphorylation of the human deubiquitinase DUBA (OTUD5) at a single residue, Ser177, is both necessary and sufficient to activate the enzyme. The crystal structure of the ubiquitin aldehyde adduct of active DUBA reveals a marked cooperation between phosphorylation and substrate binding. An intricate web of interactions involving the phosphate and the C-terminal tail of ubiquitin cause DUBA to fold around its substrate, revealing why phosphorylation is essential for deubiquitinase activity. Phosphoactivation of DUBA represents an unprecedented mode of protease regulation and a clear link between two major cellular signal transduction systems: phosphorylation and ubiquitin modification.

The deubiquitinating enzyme USP7 has a pivotal role in regulating the stability of proteins involved in fundamental cellular processes of normal biology and disease. Despite the importance of USP7, the mechanisms underlying substrate recognition and catalytic activation are poorly understood. Here we present structural, biochemical, and biophysical analyses elucidating the molecular mechanism by which the C-terminal 19 amino acids of USP7 (residues 1084-1102) enhance the ubiquitin cleavage activity of the deubiquitinase (DUB) domain. Our data demonstrate that the C-terminal peptide binds the activation cleft in the catalytic domain and stabilizes the catalytically competent conformation of USP7. Additional structures of longer fragments of USP7, as well as solution studies, provide insight into full-length USP7, the role of the UBL domains, and demonstrate that both substrate recognition and deubiquitinase activity are highly regulated by the catalytic and noncatalytic domains of USP7, a feature that could be essential for the proper function of multi-domain DUBs.

Accumulation of amyloid-β (Aβ) peptides and amyloid plaque deposition in brain is postulated as a cause of Alzheimer’s disease (AD). The precise pathological species of Aβ remains elusive although evidence suggests soluble oligomers may be primarily responsible for neurotoxicity. Crenezumab is a humanized anti-Aβ monoclonal IgG4 that binds multiple forms of Aβ, with higher affinity for aggregated forms, and that blocks Aβ aggregation, and promotes disaggregation. To understand the structural basis for this binding profile and activity, we determined the crystal structure of crenezumab in complex with Aβ. The structure reveals a sequential epitope and conformational requirements for epitope recognition, which include a subtle but critical element that is likely the basis for crenezumab’s versatile binding profile. We find interactions consistent with high affinity for multiple forms of Aβ, particularly oligomers. Of note, crenezumab also sequesters the hydrophobic core of Aβ and breaks an essential salt-bridge characteristic of the β-hairpin conformation, eliminating features characteristic of the basic organization in Aβ oligomers and fibrils, and explains crenezumab’s inhibition of aggregation and promotion of disaggregation. These insights highlight crenezumab’s unique mechanism of action, particularly regarding Aβ oligomers, and provide a strong rationale for the evaluation of crenezumab as a potential AD therapy.

Activity-based probes (ABPs) are widely used to monitor the activity of enzyme families in biological systems. Inferring enzyme activity from probe reactivity requires that the probe reacts with the enzyme at its active site; however, probe-labeling sites are rarely verified. Here we present an enhanced chemoproteomic approach to evaluate the activity and probe reactivity of deubiquitinase enzymes, using bioorthogonally tagged ABPs and a sequential on-bead digestion protocol to enhance the identification of probe-labeling sites. We confirm probe labeling of deubiquitinase catalytic Cys residues and reveal unexpected labeling of deubiquitinases on non-catalytic Cys residues and of non-deubiquitinase proteins. In doing so, we identify ZUFSP (ZUP1) as a previously unannotated deubiquitinase with high selectivity toward cleaving K63-linked chains. ZUFSP interacts with and modulates ubiquitination of the replication protein A (RPA) complex. Our reactive-site-centric chemoproteomics method is broadly applicable for identifying the reaction sites of covalent molecules, which may expand our understanding of enzymatic mechanisms.