Structural and Functional Characterization of Ubiquitin Variant Inhibitors of USP15.

The multi-domain deubiquitinase USP15 regulates diverse eukaryotic processes and has been implicated in numerous diseases. We developed ubiquitin variants (UbVs) that targeted either the catalytic domain or each of three adaptor domains in USP15, including the N-terminal DUSP domain. We also designed a linear dimer (diUbV), which targeted the DUSP and catalytic domains, and exhibited enhanced specificity and more potent inhibition of catalytic activity than either UbV alone. In cells, the UbVs inhibited the deubiquitination of two USP15 substrates, SMURF2 and TRIM25, and the diUbV inhibited the effects of USP15 on the transforming growth factor ß pathway. Structural analyses revealed that three distinct UbVs bound to the catalytic domain and locked the active site in a closed, inactive conformation, and one UbV formed an unusual strand-swapped dimer and bound two DUSP domains simultaneously. These inhibitors will enable the study of USP15 function in oncology, neurology, immunology, and inflammation.

Real-time tracking of complex ubiquitination cascades using a fluorescent confocal on-bead assay.

BACKGROUND: The ubiquitin-proteasome system (UPS) controls the stability, localization and/or activity of the proteome. However, the identification and characterization of complex individual ubiquitination cascades and their modulators remains a challenge. Here, we report a broadly applicable, multiplexed, miniaturized on-bead technique for real-time monitoring of various ubiquitination-related enzymatic activities. The assay, termed UPS-confocal fluorescence nanoscanning (UPS-CONA), employs a substrate of interest immobilized on a micro-bead and a fluorescently labeled ubiquitin which, upon enzymatic conjugation to the substrate, is quantitatively detected on the bead periphery by confocal imaging. RESULTS: UPS-CONA is suitable for studying individual enzymatic activities, including various E1, E2, and HECT-type E3 enzymes, and for monitoring multi-step reactions within ubiquitination cascades in a single experimental compartment. We demonstrate the power of the UPS-CONA technique by simultaneously following ubiquitin transfer from Ube1 through Ube2L3 to E6AP. We applied this multi-step setup to investigate the selectivity of five ubiquitination inhibitors reportedly targeting different classes of ubiquitination enzymes. Using UPS-CONA, we have identified a new activity of a small molecule E2 inhibitor, BAY 11-7082, and of a HECT E3 inhibitor, heclin, towards the Ube1 enzyme. CONCLUSIONS: As a sensitive, quantitative, flexible, and reagent-efficient method with a straightforward protocol, UPS-CONA constitutes a powerful tool for interrogation of ubiquitination-related enzymatic pathways and their chemical modulators, and is readily scalable for large experiments.

Structural basis for the autoinhibition of focal adhesion kinase.

Appropriate tyrosine kinase signaling depends on coordinated sequential coupling of protein-protein interactions with catalytic activation. Focal adhesion kinase (FAK) integrates signals from integrin and growth factor receptors to regulate cellular responses including cell adhesion, migration, and survival. Here, we describe crystal structures representing both autoinhibited and active states of FAK. The inactive structure reveals a mechanism of inhibition in which the N-terminal FERM domain directly binds the kinase domain, blocking access to the catalytic cleft and protecting the FAK activation loop from Src phosphorylation. Additionally, the FERM domain sequesters the Tyr397 autophosphorylation and Src recruitment site, which lies in the linker connecting the FERM and kinase domains. The active phosphorylated FAK kinase adopts a conformation that is immune to FERM inhibition. Our biochemical and structural analysis shows how the architecture of autoinhibited FAK orchestrates an activation sequence of FERM domain displacement, linker autophosphorylation, Src recruitment, and full catalytic activation.

Non-canonical interaction of phosphoinositides with pleckstrin homology domains of Tiam1 and ArhGAP9.

Pleckstrin homology (PH) domains are phosphoinositide (PI)-binding modules that target proteins to membrane surfaces. Here we define a family of PH domain proteins, including Tiam1 and ArhGAP9, that demonstrates specificity for PI(4,5)P(2), as well as for PI(3,4,5)P(3) and PI(3,4)P(2), the products of PI 3-kinase. These PH domain family members utilize a non-canonical phosphoinositide binding pocket related to that employed by beta-spectrin. Crystal structures of the PH domain of ArhGAP9 in complex with the headgroups of Ins(1,3,4)P(3), Ins(1,4,5)P(3), and Ins(1,3,5)P(3) reveal how two adjacent phosphate positions in PI(3,4)P(2), PI(4,5)P(2), and PI(3,4,5)P(3) are accommodated through flipped conformations of the bound phospholipid. We validate the non-canonical site of phosphoinositide interaction by showing that binding pocket mutations, which disrupt phosphoinositide binding in vitro, also disrupt membrane localization of Tiam1 in cells. We posit that the diversity in PI interaction modes displayed by PH domains contributes to their versatility of use in biological systems.

Structural and functional analysis of Saccharomyces cerevisiae Mob1.

The Mob proteins function as activator subunits for the Dbf2/Dbf20 family of protein kinases. Human and Xenopus Mob1 protein structures corresponding to the most conserved C-terminal core, but lacking the variable N-terminal region, have been reported and provide a framework for understanding the mechanism of Dbf2/Dbf20 regulation. Here, we report the 2.0 A X-ray crystal structure of Saccharomyces cerevisiae Mob1 containing both the conserved C-terminal core and the variable N-terminal region. Within the N-terminal region, three novel structural elements are observed; namely, an alpha-helix denoted H0, a strand-like element denoted S0 and a short beta strand denoted S-1. Helix H0 associates in an intermolecular manner with a second Mob1 molecule to form a Mob1 homodimer. Strand S0 binds to the core domain in an intramolecular manner across a putative Dbf2 binding site mapped by Mob1 temperature-sensitive alleles and NMR binding experiments. In vivo functional analysis demonstrates that Mob1 mutants that target helix H0 or its reciprocal binding site are biologically compromised. The N-terminal region of Mob1 thus contains structural elements that are functionally important.

Crystal structure of the FERM domain of focal adhesion kinase.

Focal adhesion kinase (FAK) is a non-receptor tyrosine kinase that localizes to focal adhesions in adherent cells. Through phosphorylation of proteins assembled at the cytoplasmic tails of integrins, FAK promotes signaling events that modulate cellular growth, survival, and migration. The amino-terminal region of FAK contains a region of sequence homology with band 4.1 and ezrin/radixin/moesin (ERM) proteins termed a FERM domain. FERM domains are found in a variety of signaling and cytoskeletal proteins and are thought to mediate intermolecular interactions with partner proteins and phospholipids at the plasma membrane and intramolecular regulatory interactions. Here we report two crystal structures of an NH2-terminal fragment of avian FAK containing the FERM domain and a portion of the regulatory linker that connects the FERM and kinase domains. The tertiary folds of the three subdomains (F1, F2, and F3) are similar to those of known FERM structures despite low sequence conservation. Differences in the sequence and relative orientation of the F3 subdomain alters the nature of the interdomain interface, and the phosphoinositide binding site found in ERM family FERM domains is not present in FAK. A putative protein interaction site on the F3 lobe is masked by the proximal region of the linker. Additionally, in one structure the adjacent Src SH3 and SH2 binding sites in the linker associate with the surfaces of the F3 and F1 lobes, respectively. These structural features suggest the possibility that protein interactions of the FAK FERM domain can be regulated by binding of Src kinases to the linker segment.

FERM domain interaction promotes FAK signaling.

From the results of deletion analyses, the FERM domain of FAK has been proposed to inhibit enzymatic activity and repress FAK signaling. We have identified a sequence in the FERM domain that is important for FAK signaling in vivo. Point mutations in this sequence had little effect upon catalytic activity in vitro. However, the mutant exhibits reduced tyrosine phosphorylation and dramatically reduced Src family kinase binding. Further, the abilities of the mutant to transduce biochemical signals and to promote cell migration were severely impaired. The results implicate a FERM domain interaction in cell adhesion-dependent activation of FAK and downstream signaling. We also show that the purified FERM domain of FAK interacts with full-length FAK in vitro, and mutation of this sequence disrupts the interaction. These findings are discussed in the context of models of FAK regulation by its FERM domain.