Structures of REV1 UBM2 Domain Complex with Ubiquitin and with a Small-Molecule that Inhibits the REV1 UBM2-Ubiquitin Interaction.

REV1 is a DNA damage tolerance protein and encodes two ubiquitin-binding motifs (UBM1 and UBM2) that are essential for REV1 functions in cell survival under DNA-damaging stress. Here we report the first solution and X-ray crystal structures of REV1 UBM2 and its complex with ubiquitin, respectively. Furthermore, we have identified the first small-molecule compound, MLAF50, that directly binds to REV1 UBM2. In the heteronuclear single quantum coherence NMR experiments, peaks of UBM2 but not of UBM1 are significantly shifted by the addition of ubiquitin, which agrees to the observation that REV1 UBM2 but not UBM1 is required for DNA damage tolerance. REV1 UBM2 interacts with hydrophobic residues of ubiquitin such as L8 and L73. NMR data suggest that MLAF50 binds to the same residues of REV1 UBM2 that interact with ubiquitin, indicating that MLAF50 can compete with the REV1 UBM2-ubiquitin interaction orthosterically. Indeed, MLAF50 inhibited the interaction of REV1 UBM2 with ubiquitin and prevented chromatin localization of REV1 induced by cisplatin in U2OS cells. Our results structurally validate REV1 UBM2 as a target of a small-molecule inhibitor and demonstrate a new avenue to targeting ubiquitination-mediated protein interactions with a chemical tool.

Small-molecules that bind to the ubiquitin-binding motif of REV1 inhibit REV1 interaction with K164-monoubiquitinated PCNA and suppress DNA damage tolerance.

REV1 protein is a mutagenic DNA damage tolerance (DDT) mediator and encodes two ubiquitin-binding motifs (i.e., UBM1 and UBM2) that are essential for the DDT function. REV1 interacts with K164-monoubiquitinated PCNA (UbPCNA) in cells upon DNA-damaging stress. By using AlphaScreen assays to detect inhibition of REV1 and UbPCNA protein interactions along with an NMR-based strategy, we identified small-molecule compounds that inhibit the REV1/UbPCNA interaction and that directly bind to REV1 UBM2. In cells, one of the compound prevented recruitment of REV1 to PCNA foci on chromatin upon cisplatin treatment, delayed removal of UV-induced cyclopyrimidine dimers from nuclei, prevented UV-induced mutation of HPRT gene, and diminished clonogenic survival of cells that were challenged by cyclophosphamide or cisplatin. This study demonstrates the potential utility of a small-molecule REV1 UBM2 inhibitor for preventing DDT.

Identification of the first small-molecule inhibitor of the REV7 DNA repair protein interaction.

DNA interstrand crosslink (ICL) repair (ICLR) has been implicated in the resistance of cancer cells to ICL-inducing chemotherapeutic agents. Despite the clinical significance of ICL-inducing chemotherapy, few studies have focused on developing small-molecule inhibitors for ICLR. The mammalian DNA polymerase ζ, which comprises the catalytic subunit REV3L and the non-catalytic subunit REV7, is essential for ICLR. To identify small-molecule compounds that are mechanistically capable of inhibiting ICLR by targeting REV7, high-throughput screening and structure-activity relationship (SAR) analysis were performed. Compound 1 was identified as an inhibitor of the interaction of REV7 with the REV7-binding sequence of REV3L. Compound 7 (an optimized analog of compound 1) bound directly to REV7 in nuclear magnetic resonance analyses, and inhibited the reactivation of a reporter plasmid containing an ICL in between the promoter and reporter regions. The normalized clonogenic survival of HeLa cells treated with cisplatin and compound 7 was lower than that for cells treated with cisplatin only. These findings indicate that a small-molecule inhibitor of the REV7/REV3L interaction can chemosensitize cells by inhibiting ICLR.

Species-Specific Structural and Functional Divergence of α-Crystallins: Zebrafish αBa- and Rodent αA(ins)-Crystallin Encode Activated Chaperones.

In addition to contributing to lens optical properties, the α-crystallins are small heat shock proteins that possess chaperone activity and are predicted to bind and sequester destabilized proteins to delay cataract formation. The current model of α-crystallin chaperone mechanism envisions a transition from the native oligomer to an activated form that has higher affinity to non-native states of the substrate. Previous studies have suggested that this oligomeric plasticity is encoded in the primary sequence and controls access to high affinity binding sites within the N-terminal domain. Here, we further examined the role of sequence variation in the context of species-specific α-crystallins from rat and zebrafish. Alternative splicing of the αA gene in rodents produces αA(ins), which is distinguished by a longer N-terminal domain. The zebrafish genome includes duplicate αB-crystallin genes, αBa and αBb, which display divergent primary sequence and tissue expression patterns. Equilibrium binding experiments were employed to quantitatively define chaperone interactions with a destabilized model substrate, T4 lysozyme. In combination with multiangle light scattering, we show that rat αA(ins) and zebrafish α-crystallins display distinct global structural properties and chaperone activities. Notably, we find that αA(ins) and αBa demonstrate substantially enhanced chaperone function relative to other α-crystallins, binding the same substrate more than 2 orders of magnitude higher affinity and mimicking the activity of fully activated mammalian small heat shock proteins. These results emphasize the role of sequence divergence as an evolutionary strategy to tune chaperone function to the requirements of the tissues and organisms in which they are expressed.

Cryoelectron microscopy analysis of small heat shock protein 16.5 (Hsp16.5) complexes with T4 lysozyme reveals the structural basis of multimode binding.

Small heat shock proteins (sHSPs) are ubiquitous chaperones that bind and sequester non-native proteins preventing their aggregation. Despite extensive studies of sHSPs chaperone activity, the location of the bound substrate within the sHSP oligomer has not been determined. In this paper, we used cryoelectron microscopy (cryoEM) to visualize destabilized mutants of T4 lysozyme (T4L) bound to engineered variants of the small heat shock protein Hsp16.5. In contrast to wild type Hsp16.5, binding of T4L to these variants does not induce oligomer heterogeneity enabling cryoEM analysis of the complexes. CryoEM image reconstruction reveals the sequestration of T4L in the interior of the Hsp16.5 oligomer primarily interacting with the buried N-terminal domain but also tethered by contacts with the α-crystallin domain shell. Analysis of Hsp16.5-WT/T4L complexes uncovers oligomer expansion as a requirement for high affinity binding. In contrast, a low affinity mode of binding is found to involve T4L binding on the outer surface of the oligomer bridging the formation of large complexes of Hsp16.5. These mechanistic principles were validated by cryoEM analysis of an expanded variant of Hsp16.5 in complex with T4L and Hsp16.5-R107G, which is equivalent to a mutant of human αB-crystallin linked to cardiomyopathy. In both cases, high affinity binding is found to involve conformational changes in the N-terminal region consistent with a central role of this region in substrate recognition.

Sequence, structure, and dynamic determinants of Hsp27 (HspB1) equilibrium dissociation are encoded by the N-terminal domain.

Human small heat shock protein 27 (Hsp27) undergoes concentration-dependent equilibrium dissociation from an ensemble of large oligomers to a dimer. This phenomenon plays a critical role in Hsp27 chaperone activity in vitro enabling high affinity binding to destabilized proteins. In vivo dissociation, which is regulated by phosphorylation, controls Hsp27 role in signaling pathways. In this study, we explore the sequence determinants of Hsp27 dissociation and define the structural basis underlying the increased affinity of Hsp27 dimers to client proteins. A systematic cysteine mutagenesis is carried out to identify residues in the N-terminal domain important for the equilibrium between Hsp27 oligomers and dimers. In addition, spin-labels were attached to the cysteine mutants to enable electron paramagnetic resonance (EPR) analysis of residue environment and solvent accessibility in the context of the large oligomers, upon dissociation to the dimer, and following complex formation with the model substrate T4 Lysozyme (T4L). The mutagenic analysis identifies residues that modulate the equilibrium dissociation in favor of the dimer. EPR analysis reveals that oligomer dissociation disrupts subunit contacts leading to the exposure of Hsp27 N-terminal domain to the aqueous solvent. Moreover, regions of this domain are highly dynamic with no evidence of a packed core. Interaction between T4L and sequences in this domain is inferred from transition of spin-labels to a buried environment in the substrate/Hsp27 complex. Together, the data provide the first structural analysis of sHSP dissociation and support a model of chaperone activity wherein unstructured and highly flexible regions in the N-terminal domain are critical for substrate binding.