Lead compound profiling for small molecule inhibitors of the REV1-CT/RIR Translesion synthesis Protein-Protein interaction.

Translesion synthesis (TLS) is a cellular mechanism through which actively replicating cells recruit specialized, low-fidelity DNA polymerases to damaged DNA to allow for replication past these lesions. REV1 is one of these TLS DNA polymerases that functions primarily as a scaffolding protein to organize the TLS heteroprotein complex and ensure replication occurs in the presence of DNA lesions. The C-Terminal domain of REV1 (REV1-CT) forms many protein-protein interactions (PPIs) with other TLS polymerases, making it essential for TLS function and a promising drug target for anti-cancer drug development. We utilized several lead identification strategies to identify various small molecules capable of disrupting the PPI between REV1-CT and the REV1 Interacting Regions (RIR) present in several other TLS polymerases. These lead compounds were profiled in several in vitro potency and PK assays to identify two scaffolds (1 and 6) as the most promising for further development. Both 1 and 6 synergized with cisplatin in a REV1-dependent fashion and demonstrated promising in vivo PK and toxicity profiles.

Effects of Xylanase A double mutation on substrate specificity and structural dynamics.

While protein activity is traditionally studied with a major focus on the active site, the activity of enzymes has been hypothesized to be linked to the flexibility of adjacent regions, warranting more exploration into how the dynamics in these regions affects catalytic turnover. One such enzyme is Xylanase A (XylA), which cleaves hemicellulose xylan polymers by hydrolysis at internal ß-1,4-xylosidic linkages. It contains a "thumb" region whose flexibility has been suggested to affect the activity. The double mutation D11F/R122D was previously found to affect activity and potentially bias the thumb region to a more open conformation. We find that the D11F/R122D double mutation shows substrate-dependent effects, increasing activity on the non-native substrate ONPX2 but decreasing activity on its native xylan substrate. To characterize how the double mutant causes these kinetics changes, nuclear magnetic resonance (NMR) and molecular dynamics (MD) simulations were used to probe structural and flexibility changes. NMR chemical shift perturbations revealed structural changes in the double mutant relative to the wild-type, specifically in the thumb and fingers regions. Increased slow-timescale dynamics in the fingers region was observed as intermediate-exchange line broadening. Lipari-Szabo order parameters show negligible changes in flexibility in the thumb region in the presence of the double mutation. To help understand if there is increased energetic accessibility to the open state upon mutation, alchemical free energy simulations were employed that indicated thumb opening is more favorable in the double mutant. These studies aid in further characterizing how flexibility in adjacent regions affects the function of XylA.

Conformational exchange at a C2H2 zinc-binding site facilitates redox sensing by the PML protein.

The promyelocytic leukemia protein, PML, plays a vital role in the cellular response to oxidative stress; however, the molecular mechanism of its action remains poorly understood. Here, we identify redox-sensitive sites of PML. A molecule of PML is cysteine-rich and contains three zinc-binding domains including RING, B-box1, and B-box2. Using in vitro assays, we have compared the sensitivity of the isolated RING and B-box1 domains and shown that B-box1 is more sensitive to oxidation. NMR studies of PML dynamics showed that one of the Zn-coordination sites within the B-box1 undergoes significant conformational exchange, revealing a hotspot for exposure of reactive cysteines. In agreement with the in vitro data, enhancement of the B-box1 Zn-coordination dynamics led to more efficient recruitment of PML into PML nuclear bodies in cells. Overall, our results suggest that the increased sensitivity of B-box1 to oxidative stress makes this domain an important redox-sensing component of PML.

Backbone and ILV side-chain methyl NMR resonance assignments of human Rev7/Rev3-RBM1 and Rev7/Rev3-RBM2 complexes.

Rev7 is a versatile HORMA (Hop1, Rev7, Mad2) family adaptor protein with multiple roles in mitotic regulation and DNA damage response, and an essential accessory subunit of the translesion synthesis (TLS) DNA polymerase Polζ employed in replication of damaged DNA. Within Polζ, the two copies of Rev7 interact with the two Rev7-bonding motifs (RBM1 and RBM2) of the catalytic subunit Rev3 by a mechanism characteristic of HORMA proteins whereby the "safety-belt" loop of Rev7 closes on the top of the ligand. Here we report the nearly complete backbone and Ile, Val, Leu side-chain methyl NMR resonance assignments of the 27 kDa human Rev7/Rev3-RBM1 and Rev7/Rev3-RBM2 complexes (BMRB deposition numbers 51651 and 51652) that will facilitate future NMR studies of Rev7 dynamics and interactions.

Activation Dynamics of Ubiquitin Specific Protease 7.

Ubiquitin-specific protease 7 (USP7) is a deubiquitinating enzyme responsible for the regulation of key human oncoproteins and tumor suppressors including Mdm2 and p53, respectively. Unlike other members of the USP family of proteases, the isolated catalytic domain of USP7 adopts an enzymatically inactive conformation that has been well characterized using X-ray crystallography. The catalytic domain also samples an active conformation, which has only been captured upon USP7 substrate-binding. Here, we utilized CPMG NMR relaxation dispersion studies to observe the dynamic motions of USP7 in solution. Our results reveal that the catalytic domain of USP7 exchanges between two distinct conformations, the inactive conformation populated at 95% and the active conformation at 5%. The largest structural changes are localized within functionally important regions of the enzyme including the active site, the ubiquitin-binding fingers, and the allosteric helix of the enzyme, suggesting that USP7 can adopt its active conformation in the absence of a substrate. Furthermore, we show that the allosteric L299A activating mutation disturbs this equilibrium, slows down the exchange, and increases the residence time of USP7 in its active conformation, thus, explaining the elevated activity of the mutant. Overall, this work shows that the isolated USP7 catalytic domain pre-samples its "invisible" active conformation in solution, which may contribute to its activation mechanism.

Evolution of Rev7 interactions in eukaryotic TLS DNA polymerase Polζ.

Translesion synthesis (TLS) DNA polymerase Polζ is crucial for the bypass replication over sites of DNA damage. The Rev7 subunit of Polζ is a HORMA (Hop1, Rev7, Mad2) protein that facilitates recruitment of Polζ to the replication fork via interactions with the catalytic subunit Rev3 and the translesion synthesis scaffold protein Rev1. Human Rev7 (hRev7) interacts with two Rev7-binding motifs (RBMs) of hRev3 by a mechanism conserved among HORMA proteins whereby the safety-belt loop of hRev7 closes on the top of the ligand. The two copies of hRev7 tethered by the two hRev3-RBMs form a symmetric head-to-head dimer through the canonical HORMA dimerization interface. Recent cryo-EM structures reveal that Saccharomyces cerevisiae Polζ (scPolζ) also includes two copies of scRev7 bound to distinct regions of scRev3. Surprisingly, the HORMA dimerization interface is not conserved in scRev7, with the two scRev7 protomers forming an asymmetric head-to-tail dimer with a much smaller interface than the hRev7 dimer. Here, we validated the two adjacent RBM motifs in scRev3, which bind scRev7 with affinities that differ by two orders of magnitude and confirmed the 2:1 stoichiometry of the scRev7:Rev3 complex in solution. However, our biophysical studies reveal that scRev7 does not form dimers in solution either on its own accord or when tethered by the two RBMs in scRev3. These findings imply that the scRev7 dimer observed in the cryo-EM structures is induced by scRev7 interactions with other Polζ subunits and that Rev7 homodimerization via the HORMA interface is a mechanism that emerged later in evolution.

ILV methyl NMR resonance assignments of the 81 kDa E. coli ß-clamp.

The ring-shaped E. coli ß-clamp protein is an 81 kDa head-to-tail homodimer, which serves as a processivity factor anchoring the replicative polymerase to DNA, thereby increasing replication processivity and speed. In addition, it facilitates numerous protein transactions that take place on DNA during replication, repair, and damage response. We used a structure-based approach to obtain nearly complete Ile, Leu and Val side-chain methyl NMR resonance assignments of the wild-type ß-clamp and its stabilized T45R/S107R variant based on site directed mutagenesis and the analysis of methyl-methyl NOESY data. The obtained assignments will facilitate future studies of the ß-clamp interactions and dynamics.

Backbone and ILV side-chain NMR resonance assignments of the catalytic domain of human deubiquitinating enzyme USP7.

Ubiquitin specific protease 7 (USP7) is a deubiquitinating enzyme, which removes ubiquitin tag from numerous protein substrates involved in diverse cellular processes such as cell cycle regulation, apoptosis and DNA damage response. USP7 affects stability, interaction network and cellular localization of its cellular and viral substrates by controlling their ubiquitination status. The large 41 kDa catalytic domain of USP7 harbors the active site of the enzyme. Here we present a nearly complete (93%) NMR resonance assignment of isoleucine, leucine and valine (ILV) side-chains of the USP7 catalytic domain along with a refined nearly complete (93%) assignment of its backbone resonances. The reported ILV methyl group assignment will facilitate further NMR investigations of structure, interactions and conformational dynamics of the USP7 enzyme.

DNA Sequence Specificity Reveals a Role of the HLTF HIRAN Domain in the Recognition of Trinucleotide Repeats.

DNA damage tolerance (DDT) pathways enable cells to cope with a variety of replication blocks that threaten their ability to complete DNA replication. Helicase-like transcription factor (HLTF) plays a central role in the error-free DDT pathway, template switching (TS), by serving as a ubiquitin ligase to polyubiquitinate the DNA sliding clamp PCNA, which promotes TS initiation. HLTF also serves as an ATP-dependent DNA translocase facilitating replication fork remodeling. The HIP116, Rad5p N-terminal (HIRAN) domain of HLTF specifically recognizes the unmodified 3'-end of single-stranded DNA (ssDNA) at stalled replication forks to promote fork regression. Several crystal structures of the HIRAN domain in complex with ssDNA have been reported; however, optimal ssDNA sequences for high-affinity binding with the domain have not been described. Here we elucidated DNA sequence preferences of HLTF HIRAN through systematic studies of its binding to ssDNA substrates using fluorescence polarization assays and a computational analysis of the ssDNA:HIRAN interaction. These studies reveal that the HLTF HIRAN domain preferentially recognizes a (T/C)TG sequence at the 3'-hydroxyl ssDNA end, which occurs in the CTG trinucleotide repeat (TNR) regions that are susceptible to expansion and deletion mutations identified in neuromuscular and neurodegenerative disorders. These findings support a role for HLTF in maintaining the stability of difficult to replicate TNR microsatellite regions.

Architecture of the two metal-binding sites in prolactin.

Metal binding by members of the growth hormone (GH) family of hematopoietic cytokines has been a subject of considerable interest. However, beyond appreciation of its role in reversible packing of GH proteins in secretory granules, the molecular mechanisms of metal binding and granule formation remain poorly understood. Here, we investigate metal binding by a GH family member prolactin (PRL) using paramagnetic metal titration and chelation experiments. Cu2+-mediated paramagnetic relaxation enhancement measurements identified two partial metal-binding sites on the opposite faces of PRL composed of residues H30/H180 and E93/H97, respectively. Coordination of metal ions by these two sites causes formation of inter-molecular bridges between the PRL protomers and enables formation of reversible higher aggregates. These findings in vitro suggest a model for reversible packaging of PRL in secretory granules. The proposed mechanism of metal-promoted PRL aggregation lends insight and support to the previously suggested role of metal coordination in secretory granule formation by GH proteins.

REV1 Inhibition Enhances Radioresistance and Autophagy.

Cancer therapy resistance is a persistent clinical challenge. Recently, inhibition of the mutagenic translesion synthesis (TLS) protein REV1 was shown to enhance tumor cell response to chemotherapy by triggering senescence hallmarks. These observations suggest REV1's important role in determining cancer cell response to chemotherapy. Whether REV1 inhibition would similarly sensitize cancer cells to radiation treatment is unknown. This study reports a lack of radiosensitization in response to REV1 inhibition by small molecule inhibitors in ionizing radiation-exposed cancer cells. Instead, REV1 inhibition unexpectedly triggers autophagy, which is a known biomarker of radioresistance. We report a possible role of the REV1 TLS protein in determining cancer treatment outcomes depending upon the type of DNA damage inflicted. Furthermore, we discover that REV1 inhibition directly triggers autophagy, an uncharacterized REV1 phenotype, with a significant bearing on cancer treatment regimens.

Targeting protein-protein interactions in the DNA damage response pathways for cancer chemotherapy.

Cellular DNA damage response (DDR) is an extensive signaling network that orchestrates DNA damage recognition, repair and avoidance, cell cycle progression and cell death. DDR alteration is a hallmark of cancer, with the deficiency in one DDR capability often compensated by a dependency on alternative pathways endowing cancer cells with survival and growth advantage. Targeting these DDR pathways has provided multiple opportunities for the development of cancer therapies. Traditional drug discovery has mainly focused on catalytic inhibitors that block enzyme active sites, which limits the number of potential drug targets within the DDR pathways. This review article describes the emerging approach to the development of cancer therapeutics targeting essential protein-protein interactions (PPIs) in the DDR network. The overall strategy for the structure-based design of small molecule PPI inhibitors is discussed, followed by an overview of the major DNA damage sensing, DNA repair, and DNA damage tolerance pathways with a specific focus on PPI targets for anti-cancer drug design. The existing small molecule inhibitors of DDR PPIs are summarized that selectively kill cancer cells and/or sensitize cancers to front-line genotoxic therapies, and a range of new PPI targets are proposed that may lead to the development of novel chemotherapeutics.

NMR resonance assignments for the nucleotide binding domains of the E. coli clamp loader complex γ subunit.

The E. coli γ clamp loader is a pentameric complex of δ, δ' and three γ subunits that opens and loads ß-clamp proteins onto DNA in an ATP-dependent process essential for efficient DNA replication. ATP binding to the γ subunits promotes conformational changes that enable the clamp loader to bind and open the ring-shaped ß-clamp homodimer. Here we report the nearly complete backbone and side-chain 1H, 13C and 15N NMR resonance assignments of the 242-residue truncated γ subunit of the clamp loader complex, which includes the N-terminal mini (domain I) and lid (domain II) domains. This construct represents the nucleotide binding module in the clamp loader complex and provides a model system for studies of conformational rearrangements of the clamp loader induced by nucleotide binding.

Structure-Based Drug Design of Phenazopyridine Derivatives as Inhibitors of Rev1 Interactions in Translesion Synthesis.

Rev1 is a protein scaffold of the translesion synthesis (TLS) pathway, which employs low-fidelity DNA polymerases for replication of damaged DNA. The TLS pathway helps cancers tolerate DNA damage induced by genotoxic chemotherapy, and increases mutagenesis in tumors, thus accelerating the onset of chemoresistance. TLS inhibitors have emerged as potential adjuvant drugs to enhance the efficacy of first-line chemotherapy, with the majority of reported inhibitors targeting protein-protein interactions (PPIs) of the Rev1 C-terminal domain (Rev1-CT). We previously identified phenazopyridine (PAP) as a scaffold to disrupt Rev1-CT PPIs with Rev1-interacting regions (RIRs) of TLS polymerases. To explore the structure-activity relationships for this scaffold, we developed a protocol for co-crystallization of compounds that target the RIR binding site on Rev1-CT with a triple Rev1-CT/Rev7R124A /Rev3-RBM1 complex, and solved an X-ray crystal structure of Rev1-CT bound to the most potent PAP analogue. The structure revealed an unexpected binding pose of the compound and informed changes to the scaffold to improve its affinity for Rev1-CT. We synthesized eight additional PAP derivatives, with modifications to the scaffold driven by the structure, and evaluated their binding to Rev1-CT by microscale thermophoresis (MST). Several second-generation PAP derivatives showed an affinity for Rev1-CT that was improved by over an order of magnitude, thereby validating the structure-based assumptions that went into the compound design.

The Rev1-Polζ translesion synthesis mutasome: Structure, interactions and inhibition.

DNA contains information that must be safeguarded, but also accessed for transcription and replication. To perform replication, eukaryotic cells use the B-family DNA polymerase enzymes Polδ and Polɛ, which are optimized for accuracy, speed, and processivity. The molecular basis of these high-performance characteristics causes these replicative polymerases to fail at sites of DNA damage (lesions), which would lead to genomic instability and cell death. To avoid this, cells possess additional DNA polymerases such as the Y-family of polymerases and the B-family member Polζ that can replicate over sites of DNA damage in a process called translesion synthesis (TLS). While able to replicate over DNA lesions, the TLS polymerases exhibit low-fidelity on undamaged DNA and, consequently, must be prevented from replicating DNA under normal circumstances and recruited only when necessary. The replicative bypass of most types of DNA lesions requires the consecutive action of these specialized TLS polymerases assembled into a dynamic multiprotein complex called the Rev1/Polζ mutasome. To this end, posttranslational modifications and a network of protein-protein interactions mediated by accessory domains/subunits of the TLS polymerases control the assembly and rearrangements of the Rev1/Polζ mutasome and recruitment of TLS proteins to sites of DNA damage. This chapter focuses on the structures and interactions that control these processes underlying the function of the Rev1/Polζ mutasome, as well as the development of small molecule inhibitors of the Rev1/Polζ-dependent TLS holding promise as a potential anticancer therapy.

Dynamics of the E. coli ß-Clamp Dimer Interface and Its Influence on DNA Loading.

The ring-shaped sliding clamp proteins have crucial roles in the regulation of DNA replication, recombination, and repair in all organisms. We previously showed that the Escherichia coli ß-clamp is dynamic in solution, transiently visiting conformational states in which Domain 1 at the dimer interface is more flexible and prone to unfolding. This work aims to understand how the stability of the dimer interface influences clamp-opening dynamics and clamp loading by designing and characterizing stabilizing and destabilizing mutations in the clamp. The variants with stabilizing mutations conferred similar or increased thermostability and had similar quaternary structure as compared to the wild type. These variants stimulated the ATPase function of the clamp loader, complemented cell growth of a temperature-sensitive strain, and were successfully loaded onto a DNA substrate. The L82D and L82E I272A variants with purported destabilizing mutations had decreased thermostability, did not complement the growth of a temperature-sensitive strain, and had weakened dimerization as determined by native trapped ion mobility spectrometry-mass spectrometry. The ß L82E variant had a reduced melting temperature but dimerized and complemented growth of a temperature-sensitive strain. All three clamps with destabilizing mutations had perturbed loading on DNA. Molecular dynamics simulations indicate altered hydrogen-bonding patterns at the dimer interface, and cross-correlation analysis showed the largest perturbations in the destabilized variants, consistent with the observed change in the conformations and functions of these clamps.

Virtual Pharmacophore Screening Identifies Small-Molecule Inhibitors of the Rev1-CT/RIR Protein-Protein Interaction.

Translesion synthesis (TLS) has emerged as a mechanism through which several forms of cancer develop acquired resistance to first-line genotoxic chemotherapies by allowing replication to continue in the presence of damaged DNA. Small molecules that inhibit TLS hold promise as a novel class of anticancer agents that can serve to enhance the efficacy of these front-line therapies. We previously used a structure-based rational design approach to identify the phenazopyridine scaffold as an inhibitor of TLS that functions by disrupting the protein-protein interaction (PPI) between the C-terminal domain of the TLS DNA polymerase Rev1 (Rev1-CT) and the Rev1 interacting regions (RIR) of other TLS DNA polymerases. To continue the identification of small molecules that disrupt the Rev1-CT/RIR PPI, we generated a pharmacophore model based on the phenazopyridine scaffold and used it in a structure-based virtual screen. In vitro analysis of promising hits identified several new chemotypes with the ability to disrupt this key TLS PPI. In addition, several of these compounds were found to enhance the efficacy of cisplatin in cultured cells, highlighting their anti-TLS potential.

Structural Approach To Identify a Lead Scaffold That Targets the Translesion Synthesis Polymerase Rev1.

Translesion synthesis (TLS) is a mechanism of replication past damaged DNA through which multiple forms of human cancer survive and acquire resistance to first-line genotoxic chemotherapies. As such, TLS is emerging as a promising target for the development of a new class of anticancer agents. The C-terminal domain of the DNA polymerase Rev1 (Rev1-CT) mediates assembly of the functional TLS complex through protein-protein interactions (PPIs) with Rev1 interacting regions (RIRs) of several other TLS DNA polymerases. Utilizing structural knowledge of the Rev1-CT/RIR interface, we have identified the phenazopyridine scaffold as an inhibitor of this essential TLS PPI. We demonstrate direct binding of this scaffold to Rev1-CT, and the synthesis and evaluation of a small series of analogues have provided important structure-activity relationships for further development of this scaffold. Furthermore, we utilized the umbrella sampling method to predict the free energy of binding to Rev1-CT for each of our analogues. Binding energies calculated through umbrella sampling correlated well with experimentally determined IC50 values, validating this computational tool as a viable approach to predict the biological activity for inhibitors of the Rev1-CT/RIR PPI.

Conformational Dynamics of a Cysteine-Stabilized Plant Defensin Reveals an Evolutionary Mechanism to Expose Hydrophobic Residues.

Sugar cane defensin 5 (Sd5) is a small antifungal protein, whose structure is held together by four conserved disulfide bridges. Sd5 and other proteins sharing a cysteine-stabilized α-ß (CSαß) fold lack a regular hydrophobic core. Instead, they are stabilized by tertiary contacts formed by surface-exposed hydrophilic and hydrophobic residues. Despite excessive cross-links, Sd5 exhibits complex millisecond conformational dynamics involving all secondary structure elements. We used Carr-Purcell-Meiboom-Gill (CPMG) NMR relaxation dispersion (RD) measurements performed at different temperatures and denaturant concentrations to probe brief excursions of Sd5 to a sparsely populated "excited" state. Temperature-dependent CPMG RD experiments reveal that the excited state is enthalpically unfavorable, suggesting a rearrangement of stabilizing contacts formed by surface-exposed side chains and/or secondary structure, while the experiments performed at different denaturant concentrations suggest a decrease in accessible surface area of Sd5 in the excited state. The measured backbone 15N chemical shift changes point to a global conformational rearrangement such as a potential α- to ß-transition of the Sd5 α-helix or other major secondary structure reorganization and concomitant conformational changes in other parts of the protein. Overall, the emerging picture of Sd5 dynamics suggests this protein can populate two alternative well-ordered conformational states, with the excited conformer being more compact than the native state and having a distinct secondary structure and side-chain arrangements. The observation of an energetically unfavorable yet more compact excited state reveals a remarkable evolution of the CSαß fold to expose and reorganize hydrophobic residues, which enables the creation of versatile binding sites.

Rev7 dimerization is important for assembly and function of the Rev1/Polζ translesion synthesis complex.

The translesion synthesis (TLS) polymerases Polζ and Rev1 form a complex that enables replication of damaged DNA. The Rev7 subunit of Polζ, which is a multifaceted HORMA (Hop1, Rev7, Mad2) protein with roles in TLS, DNA repair, and cell-cycle control, facilitates assembly of this complex by binding Rev1 and the catalytic subunit of Polζ, Rev3. Rev7 interacts with Rev3 by a mechanism conserved among HORMA proteins, whereby an open-to-closed transition locks the ligand underneath the "safety belt" loop. Dimerization of HORMA proteins promotes binding and release of this ligand, as exemplified by the Rev7 homolog, Mad2. Here, we investigate the dimerization of Rev7 when bound to the two Rev7-binding motifs (RBMs) in Rev3 by combining in vitro analyses of Rev7 structure and interactions with a functional assay in a Rev7-/- cell line. We demonstrate that Rev7 uses the conventional HORMA dimerization interface both to form a homodimer when tethered by the two RBMs in Rev3 and to heterodimerize with other HORMA domains, Mad2 and p31comet Structurally, the Rev7 dimer can bind only one copy of Rev1, revealing an unexpected Rev1/Polζ architecture. In cells, mutation of the Rev7 dimer interface increases sensitivity to DNA damage. These results provide insights into the structure of the Rev1/Polζ TLS assembly and highlight the function of Rev7 homo- and heterodimerization.