ATP-site inhibitors induce unique conformations of the acute myeloid leukemia-associated Src-family kinase, Fgr.

The Src-family kinase Fgr is expressed primarily in myeloid hematopoietic cells and contributes to myeloid leukemia. Here, we present X-ray crystal structures of Fgr bound to the ATP-site inhibitors A-419259 and TL02-59, which show promise as anti-leukemic agents. A-419259 induces a closed Fgr conformation, with the SH3 and SH2 domains engaging the SH2-kinase linker and C-terminal tail, respectively. In the Fgr:A-419259 complex, the activation loop of one monomer inserts into the active site of the other, providing a snapshot of trans-autophosphorylation. By contrast, TL02-59 binding induced SH2 domain displacement from the C-terminal tail and SH3 domain release from the linker. Solution studies using HDX MS were consistent with the crystal structures, with A-419259 reducing and TL02-59 enhancing solvent exposure of the SH3 domain. These structures demonstrate that allosteric connections between the kinase and regulatory domains of Src-family kinases are regulated by the ligand bound to the active site.

Structural basis for defective membrane targeting of mutant enzyme in human VLCAD deficiency.

Very long-chain acyl-CoA dehydrogenase (VLCAD) is an inner mitochondrial membrane enzyme that catalyzes the first and rate-limiting step of long-chain fatty acid oxidation. Point mutations in human VLCAD can produce an inborn error of metabolism called VLCAD deficiency that can lead to severe pathophysiologic consequences, including cardiomyopathy, hypoglycemia, and rhabdomyolysis. Discrete mutations in a structurally-uncharacterized C-terminal domain region of VLCAD cause enzymatic deficiency by an incompletely defined mechanism. Here, we conducted a structure-function study, incorporating X-ray crystallography, hydrogen-deuterium exchange mass spectrometry, computational modeling, and biochemical analyses, to characterize a specific membrane interaction defect of full-length, human VLCAD bearing the clinically-observed mutations, A450P or L462P. By disrupting a predicted α-helical hairpin, these mutations either partially or completely impair direct interaction with the membrane itself. Thus, our data support a structural basis for VLCAD deficiency in patients with discrete mutations in an α-helical membrane-binding motif, resulting in pathologic enzyme mislocalization.

Allosteric inhibition of PPM1D serine/threonine phosphatase via an altered conformational state.

PPM1D encodes a serine/threonine phosphatase that regulates numerous pathways including the DNA damage response and p53. Activating mutations and amplification of PPM1D are found across numerous cancer types. GSK2830371 is a potent and selective allosteric inhibitor of PPM1D, but its mechanism of binding and inhibition of catalytic activity are unknown. Here we use computational, biochemical and functional genetic studies to elucidate the molecular basis of GSK2830371 activity. These data confirm that GSK2830371 binds an allosteric site of PPM1D with high affinity. By further incorporating data from hydrogen deuterium exchange mass spectrometry and sedimentation velocity analytical ultracentrifugation, we demonstrate that PPM1D exists in an equilibrium between two conformations that are defined by the movement of the flap domain, which is required for substrate recognition. A hinge region was identified that is critical for switching between the two conformations and was directly implicated in the high-affinity binding of GSK2830371 to PPM1D. We propose that the two conformations represent active and inactive forms of the protein reflected by the position of the flap, and that binding of GSK2830371 shifts the equilibrium to the inactive form. Finally, we found that C-terminal truncating mutations proximal to residue 400 result in destabilization of the protein via loss of a stabilizing N- and C-terminal interaction, consistent with the observation from human genetic data that nearly all PPM1D mutations in cancer are truncating and occur distal to residue 400. Taken together, our findings elucidate the mechanism by which binding of a small molecule to an allosteric site of PPM1D inhibits its activity and provides insights into the biology of PPM1D.

Allosteric control of Ubp6 and the proteasome via a bidirectional switch.

The proteasome recognizes ubiquitinated proteins and can also edit ubiquitin marks, allowing substrates to be rejected based on ubiquitin chain topology. In yeast, editing is mediated by deubiquitinating enzyme Ubp6. The proteasome activates Ubp6, whereas Ubp6 inhibits the proteasome through deubiquitination and a noncatalytic effect. Here, we report cryo-EM structures of the proteasome bound to Ubp6, based on which we identify mutants in Ubp6 and proteasome subunit Rpt1 that abrogate Ubp6 activation. The Ubp6 mutations define a conserved region that we term the ILR element. The ILR is found within the BL1 loop, which obstructs the catalytic groove in free Ubp6. Rpt1-ILR interaction opens the groove by rearranging not only BL1 but also a previously undescribed network of three interconnected active-site-blocking loops. Ubp6 activation and noncatalytic proteasome inhibition are linked in that they are eliminated by the same mutations. Ubp6 and ubiquitin together drive proteasomes into a unique conformation associated with proteasome inhibition. Thus, a multicomponent allosteric switch exerts simultaneous control over both Ubp6 and the proteasome.

Discovery of a First-in-Class Inhibitor of the PRMT5-Substrate Adaptor Interaction.

PRMT5 and its substrate adaptor proteins (SAPs), pICln and Riok1, are synthetic lethal dependencies in MTAP-deleted cancer cells. SAPs share a conserved PRMT5 binding motif (PBM) which mediates binding to a surface of PRMT5 distal to the catalytic site. This interaction is required for methylation of several PRMT5 substrates, including histone and spliceosome complexes. We screened for small molecule inhibitors of the PRMT5-PBM interaction and validated a compound series which binds to the PRMT5-PBM interface and directly inhibits binding of SAPs. Mode of action studies revealed the formation of a covalent bond between a halogenated pyridazinone group and cysteine 278 of PRMT5. Optimization of the starting hit produced a lead compound, BRD0639, which engages the target in cells, disrupts PRMT5-RIOK1 complexes, and reduces substrate methylation. BRD0639 is a first-in-class PBM-competitive inhibitor that can support studies of PBM-dependent PRMT5 activities and the development of novel PRMT5 inhibitors that selectively target these functions.

The Src family kinase Fgr is a transforming oncoprotein that functions independently of SH3-SH2 domain regulation.

Fgr is a member of the Src family of nonreceptor tyrosine kinases, which are overexpressed and constitutively active in many human cancers. Fgr expression is restricted to myeloid hematopoietic cells and is markedly increased in a subset of bone marrow samples from patients with acute myeloid leukemia (AML). Here, we investigated the oncogenic potential of Fgr using Rat-2 fibroblasts that do not express the kinase. Expression of either wild-type or regulatory tail-mutant constructs of Fgr promoted cellular transformation (inferred from colony formation in soft agar), which was accompanied by phosphorylation of the Fgr activation loop, suggesting that the kinase domain of Fgr functions independently of regulation by its noncatalytic SH3-SH2 region. Unlike other family members, recombinant Fgr was not activated by SH3-SH2 domain ligands. However, hydrogen-deuterium exchange mass spectrometry data suggested that the regulatory SH3 and SH2 domains packed against the back of the kinase domain in a Src-like manner. Sequence alignment showed that the activation loop of Fgr was distinct from that of all other Src family members, with proline rather than alanine at the +2 position relative to the activation loop tyrosine. Substitution of the activation loop of Fgr with the sequence from Src partially inhibited kinase activity and suppressed colony formation. Last, Fgr expression enhanced the sensitivity of human myeloid progenitor cells to the cytokine GM-CSF. Because its kinase domain is not sensitive to SH3-SH2-mediated control, simple overexpression of Fgr without mutation may contribute to oncogenic transformation in AML and other blood cancers.

A Discovery Strategy for Selective Inhibitors of c-Src in Complex with the Focal Adhesion Kinase SH3/SH2-binding Region.

The c-Src tyrosine kinase co-operates with the focal adhesion kinase to regulate cell adhesion and motility. Focal adhesion kinase engages the regulatory SH3 and SH2 domains of c-Src, resulting in localized kinase activation that contributes to tumor cell metastasis. Using assay conditions where c-Src kinase activity required binding to a tyrosine phosphopeptide based on the focal adhesion kinase SH3-SH2 docking sequence, we screened a kinase-biased library for selective inhibitors of the Src/focal adhesion kinase peptide complex versus c-Src alone. This approach identified an aminopyrimidinyl carbamate compound, WH-4-124-2, with nanomolar inhibitory potency and fivefold selectivity for c-Src when bound to the phospho-focal adhesion kinase peptide. Molecular docking studies indicate that WH-4-124-2 may preferentially inhibit the 'DFG-out' conformation of the kinase active site. These findings suggest that interaction of c-Src with focal adhesion kinase induces a unique kinase domain conformation amenable to selective inhibition.

Differential sensitivity of Src-family kinases to activation by SH3 domain displacement.

Src-family kinases (SFKs) are non-receptor protein-tyrosine kinases involved in a variety of signaling pathways in virtually every cell type. The SFKs share a common negative regulatory mechanism that involves intramolecular interactions of the SH3 domain with the PPII helix formed by the SH2-kinase linker as well as the SH2 domain with a conserved phosphotyrosine residue in the C-terminal tail. Growing evidence suggests that individual SFKs may exhibit distinct activation mechanisms dictated by the relative strengths of these intramolecular interactions. To elucidate the role of the SH3:linker interaction in the regulation of individual SFKs, we used a synthetic SH3 domain-binding peptide (VSL12) to probe the sensitivity of downregulated c-Src, Hck, Lyn and Fyn to SH3-based activation in a kinetic kinase assay. All four SFKs responded to VSL12 binding with enhanced kinase activity, demonstrating a conserved role for SH3:linker interaction in the control of catalytic function. However, the sensitivity and extent of SH3-based activation varied over a wide range. In addition, autophosphorylation of the activation loops of c-Src and Hck did not override regulatory control by SH3:linker displacement, demonstrating that these modes of activation are independent. Our results show that despite the similarity of their downregulated conformations, individual Src-family members show diverse responses to activation by domain displacement which may reflect their adaptation to specific signaling environments in vivo.

Phosphotyrosine-dependent coupling of Tim-3 to T-cell receptor signaling pathways.

The transmembrane protein Tim-3 has been shown to negatively regulate T-cell-dependent immune responses and was recently demonstrated to be associated with the phenomenon of immune exhaustion, which can occur as a consequence of chronic viral infection. Unlike other negative regulators of T-cell function (e.g., PD-1), Tim-3 does not contain any obvious inhibitory signaling motifs. We have found that ectopic expression of Tim-3 in T cells leads to enhancement of T-cell receptor (TCR)-dependent signaling pathways, which was observed at the level of transcriptional reporters and endogenous cytokine production. We have exploited this observation to dissect what elements within the cytoplasmic tail of Tim-3 are required for coupling to downstream signaling pathways. Here we have demonstrated that two of the more membrane-proximal cytoplasmic tail tyrosines are required for Tim-3 signaling to T-cell activation pathways in a redundant fashion. Furthermore, we show that Tim-3 can directly bind to the Src family tyrosine kinase Fyn and the p85 phosphatidylinositol 3-kinase (PI3K) adaptor. Thus, at least under conditions of short-term stimulation, Tim-3 can augment T-cell activation, although this effect can be blocked by the inclusion of an agonistic antibody to Tim-3. These findings should help further the study of Tim-3 function in other physiological settings, such as those that lead to immune exhaustion.

Crystal structure of the Src family kinase Hck SH3-SH2 linker regulatory region supports an SH3-dominant activation mechanism.

Most mammalian cell types depend on multiple Src family kinases (SFKs) to regulate diverse signaling pathways. Strict control of SFK activity is essential for normal cellular function, and loss of kinase regulation contributes to several forms of cancer and other diseases. Previous x-ray crystal structures of the SFKs c-Src and Hck revealed that intramolecular association of their Src homology (SH) 3 domains and SH2 kinase linker regions has a key role in down-regulation of kinase activity. However, the amino acid sequence of the Hck linker represents a suboptimal ligand for the isolated SH3 domain, suggesting that it may form the polyproline type II helical conformation required for SH3 docking only in the context of the intact structure. To test this hypothesis directly, we determined the crystal structure of a truncated Hck protein consisting of the SH2 and SH3 domains plus the linker. Despite the absence of the kinase domain, the structures and relative orientations of the SH2 and SH3 domains in this shorter protein were very similar to those observed in near full-length, down-regulated Hck. However, the SH2 kinase linker adopted a modified topology and failed to engage the SH3 domain. This new structure supports the idea that these noncatalytic regions work together as a "conformational switch" that modulates kinase activity in a manner unique to the SH3 domain and linker topologies present in the intact Hck protein. Our results also provide fresh structural insight into the facile induction of Hck activity by HIV-1 Nef and other Hck SH3 domain binding proteins and implicate the existence of innate conformational states unique to individual Src family members that "fine-tune" their sensitivities to activation by SH3-based ligands.