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REV7 is a HORMA (Hop1, Rev7, Mad2) family adaptor protein best known as an accessory subunit of the translesion synthesis (TLS) DNA polymerase ζ (Polζ). In this role, REV7 binds REV3, the catalytic subunit of Polζ, by locking REV7-binding motifs (RBMs) in REV3 underneath the REV7 safety-belt loop. The same mechanism is used by REV7 to interact with RBMs from other proteins in DNA damage response (DDR) and mitosis. Because of the importance of REV7 for TLS and other DDR pathways, targeting REV7:RBM protein-protein interactions (PPIs) with small molecules has emerged as a strategy to enhance cancer response to genotoxic chemotherapy. To identify druggable pockets at the REV7:RBM interface, we performed computational analyses of REV7 complexed with several RBM partners. The contributions of different interface regions to REV7:RBM stabilization were corroborated experimentally. These studies provide insights into key intermolecular interactions and establish targetable regions of REV7 for the design of REV7:RBM PPI inhibitors., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 Elsevier Inc. All rights reserved.)

The Rho GTPase (Ras homolog GTPases) system is a crucial signal transducer that regulates various cellular processes, including cell cycle and migration, genetic transcription, and apoptosis. In this study, we investigated the unfolded state of the first FF domain (FF1) of P190A RhoGAP, which features four tandem FF domains. For signal transduction, FF1 is phosphorylated at tyrosine 308 (Y308), which is buried in the hydrophobic core and is inaccessible to kinases in the folded domain. It was proposed, therefore, that the phosphorylation occurs in a transiently populated unfolded state of FF1. To probe the folding pathway of the RhoGAP FF1 domain, here we have performed a nearly complete backbone resonance assignments of a putative partially unfolded state of FF1 in 5 M urea and its fully unfolded state in 8 M urea., (© 2024. The Author(s), under exclusive licence to Springer Nature B.V.)

The 81 kDa E. coli β clamp is a ring-shaped head-to-tail homodimer that encircles DNA and plays a central role in bacterial DNA replication by serving as a processivity factor for DNA polymerases and a binding platform for other DNA replication and repair proteins. Here we report the backbone 1 H, 15 N, and 13 C NMR resonance assignments of the stabilized T45R/S107R β clamp variant obtained using standard TROSY-based triple-resonance experiments (BMRB 52548). The backbone assignments were aided by 13 C and 15 N edited NOESY experiments, allowing us to utilize our previously reported assignments of the β clamp ILV side-chain methyl groups (BMRB 51430, 51431). The backbone assignments of the T45R/S107R β clamp variant were transferred to the wild-type β clamp using a minimal set of TROSY-based 15 N edited NOESY, NHCO and NHCA experiments (BMRB 52549). The reported backbone and previous ILV side-chain resonance assignments will enable NMR studies of the β clamp interactions and dynamics using amide and methyl groups as probes., (© 2024. The Author(s), under exclusive licence to Springer Nature B.V.)

Translesion synthesis (TLS) is a mechanism of DNA damage tolerance utilized by eukaryotic cells to replicate DNA across lesions that impede the high-fidelity replication machinery. In TLS, a series of specialized DNA polymerases are employed, which recognize specific DNA lesions, insert nucleotides across the damage, and extend the distorted primer-template. This allows cells to preserve genetic integrity at the cost of mutations. In humans, TLS enzymes include the Y-family, inserter polymerases, Polη, Polι, Polκ, Rev1, and the B-family extender polymerase Polζ, while in S. cerevisiae only Polη, Rev1, and Polζ are present. To bypass DNA lesions, TLS polymerases cooperate, assembling into a complex on the eukaryotic sliding clamp, PCNA, termed the TLS mutasome. The mutasome assembly is contingent on protein-protein interactions (PPIs) between the modular domains and subunits of TLS enzymes, and their interactions with PCNA and DNA. While the structural mechanisms of DNA lesion bypass by the TLS polymerases and PPIs of their individual modules are well understood, the mechanisms by which they cooperate in the context of TLS complexes have remained elusive. This review focuses on structural studies of TLS polymerases and describes the case of TLS holoenzyme assemblies in action emerging from recent high-resolution Cryo-EM studies.

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 β-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., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024. Published by Elsevier Inc.)

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., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

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., Competing Interests: Declaration of interests The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

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., (© 2023. The Author(s), under exclusive licence to Springer Nature B.V.)

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., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

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., (© 2022. The Author(s), under exclusive licence to Springer Nature B.V.)

The ring-shaped E. coli β-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 β-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 β-clamp interactions and dynamics., (© 2022. The Author(s), under exclusive licence to Springer Nature B.V.)

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.

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. Cu 2+ -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., (Copyright © 2022 Biophysical Society. Published by Elsevier Inc. All rights reserved.)

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.

The E. coli γ clamp loader is a pentameric complex of δ, δ' and three γ subunits that opens and loads β-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 β-clamp homodimer. Here we report the nearly complete backbone and side-chain 1 H, 13 C and 15 N 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., (© 2021. The Author(s), under exclusive licence to Springer Nature B.V.)

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., Competing Interests: There are no conflicts to declare., (This journal is © The Royal Society of Chemistry.)

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/Rev7 R124A /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., (© 2020 Wiley-VCH GmbH.)

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., (© 2019 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

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 β-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 β 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., (Copyright © 2019 Biophysical Society. Published by Elsevier Inc. All rights reserved.)

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., (© 2019 Elsevier Inc. All rights reserved.)

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 IC 50 values, validating this computational tool as a viable approach to predict the biological activity for inhibitors of the Rev1-CT/RIR PPI.

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-β (CSαβ) 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 15 N chemical shift changes point to a global conformational rearrangement such as a potential α- to β-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 CSαβ fold to expose and reorganize hydrophobic residues, which enables the creation of versatile binding sites.

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 p31 comet 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., Competing Interests: The authors declare no conflict of interest.

Translesion synthesis (TLS) is a DNA damage tolerance mechanism that allows replicative bypass of DNA lesions, including DNA adducts formed by cancer chemotherapeutics. Previous studies demonstrated that suppression of TLS can increase sensitivity of cancer cells to first-line chemotherapeutics and decrease mutagenesis linked to the onset of chemoresistance, marking the TLS pathway as an emerging therapeutic target. TLS is mediated by a heteroprotein complex consisting of specialized DNA polymerases, including the Y-family DNA polymerase Rev1. Previously, we developed a screening assay to identify the first small molecules that disrupt the protein-protein interaction between the C-terminal domain of Rev1 (Rev1-CT) and the Rev1-interacting region (RIR) present in multiple DNA polymerases involved in TLS. Herein we report additional hit scaffolds that inhibit this key TLS PPI. In addition, through a series of biochemical, computational, and cellular studies we have identified preliminary structure-activity relationships and determined initial pharmacokinetic parameters for our original hits., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

The β-clamp protein and the γ clamp loader complex are essential components of bacterial DNA replication machinery. The β-clamp is a ring-shaped homodimer that encircles DNA and increases the efficiency of replication by providing a binding platform for DNA polymerases and other replication-related proteins. The β-clamp is loaded onto DNA by the five-subunit γ clamp loader complex in a multi-step ATP-dependent process. The initial steps of this process involve the cooperative binding of the β-clamp by the five subunits of ATP-bound clamp loader, which induces or traps an open conformation of the clamp. Remarkably, the δ subunit of the E. coli clamp loader, or even its 140 residue N-terminal domain (called mini-δ), alone can shift conformational equilibrium of the β-clamp towards the open state. Here we report nearly complete backbone and side-chain 1 H, 13 C and 15 N NMR resonance assignments of mini-δ that will facilitate NMR studies of the mechanisms of β-clamp opening and its loading on DNA by the clamp loader.

Translesion synthesis (TLS) is an important mechanism through which proliferating cells tolerate DNA damage during replication. The mutagenic Rev1/Polζ-dependent branch of TLS helps cancer cells survive first-line genotoxic chemotherapy and introduces mutations that can contribute to the acquired resistance so often observed with standard anticancer regimens. As such, inhibition of Rev1/Polζ-dependent TLS has recently emerged as a strategy to enhance the efficacy of first-line chemotherapy and reduce the acquisition of chemoresistance by decreasing tumor mutation rate. The TLS DNA polymerase Rev1 serves as an integral scaffolding protein that mediates the assembly of the active multiprotein TLS complexes. Protein-protein interactions (PPIs) between the C-terminal domain of Rev1 (Rev1-CT) and the Rev1-interacting region (RIR) of other TLS DNA polymerases play an essential role in regulating TLS activity. To probe whether disrupting the Rev1-CT/RIR PPI is a valid approach for developing a new class of targeted anticancer agents, we designed a fluorescence polarization-based assay that was utilized in a pilot screen for small molecule inhibitors of this PPI. Two small molecule scaffolds that disrupt this interaction were identified, and secondary validation assays confirmed that compound 5 binds to Rev1-CT at the RIR interface. Finally, survival and mutagenesis assays in mouse embryonic fibroblasts and human fibrosarcoma HT1080 cells treated with cisplatin and ultraviolet light indicate that these compounds inhibit mutagenic Rev1/Polζ-dependent TLS in cells, validating the Rev1-CT/RIR PPI for future anticancer drug discovery and identifying the first small molecule inhibitors of TLS that target Rev1-CT.

The nucleation-condensation mechanism represents a major paradigm to understand the folding process of many small globular proteins. Although substantial evidence has been acquired for this mechanism, it has remained very challenging to characterize the initial events leading to the formation of a folding nucleus. To achieve this goal, we used a combination of relaxation dispersion NMR spectroscopy and molecular dynamics simulations to determine ensembles of conformations corresponding to the denatured, transition, and native states in the folding of the activation domain of human procarboxypeptidase A2 (ADA2h). We found that the residues making up the folding nucleus tend to interact in the denatured state in a transient manner and not simultaneously, thereby forming incomplete and distorted versions of the folding nucleus. Only when all the contacts between these key residues are eventually formed can the protein reach the transition state and continue folding. Overall, our results elucidate the mechanism of formation of the folding nucleus of a protein and provide insights into how its folding rate can be modified during evolution by mutations that modulate the strength of the interactions between the residues forming the folding nucleus.

Protein folding is a highly complex process proceeding through a number of disordered and partially folded nonnative states with various degrees of structural organization. These transiently and sparsely populated species on the protein folding energy landscape play crucial roles in driving folding toward the native conformation, yet some of these nonnative states may also serve as precursors for protein misfolding and aggregation associated with a range of devastating diseases, including neuro-degeneration, diabetes and cancer. Therefore, in vivo protein folding is often reshaped co- and post-translationally through interactions with the ribosome, molecular chaperones and/or other cellular components. Owing to developments in instrumentation and methodology, solution NMR spectroscopy has emerged as the central experimental approach for the detailed characterization of the complex protein folding processes in vitro and in vivo. NMR relaxation dispersion and saturation transfer methods provide the means for a detailed characterization of protein folding kinetics and thermodynamics under native-like conditions, as well as modeling high-resolution structures of weakly populated short-lived conformational states on the protein folding energy landscape. Continuing development of isotope labeling strategies and NMR methods to probe high molecular weight protein assemblies, along with advances of in-cell NMR, have recently allowed protein folding to be studied in the context of ribosome-nascent chain complexes and molecular chaperones, and even inside living cells. Here we review solution NMR approaches to investigate the protein folding energy landscape, and discuss selected applications of NMR methodology to studying protein folding in vitro and in vivo. Together, these examples highlight a vast potential of solution NMR in providing atomistic insights into molecular mechanisms of protein folding and homeostasis in health and disease., (Copyright © 2016 Elsevier B.V. All rights reserved.)

HLTF is a SWI2/SNF2-family ATP-dependent chromatin remodeling enzyme that acts in the error-free branch of DNA damage tolerance (DDT), a cellular mechanism that enables replication of damaged DNA while leaving damage repair for a later time. Human HLTF and a closely related protein SHPRH, as well as their yeast homologue Rad5, are multi-functional enzymes that share E3 ubiquitin-ligase activity required for activation of the error-free DDT. HLTF and Rad5 also function as ATP-dependent dsDNA translocases and possess replication fork reversal activities. Thus, they can convert Y-shaped replication forks into X-shaped Holliday junction structures that allow error-free replication over DNA lesions. The fork reversal activity of HLTF is dependent on 3'-ssDNA-end binding activity of its N-terminal HIRAN domain. Here we present the solution NMR structure of the human HLTF HIRAN domain, an OB-like fold module found in organisms from bacteria (as a stand-alone domain) to plants, fungi and metazoan (in combination with SWI2/SNF2 helicase-like domain). The obtained structure of free HLTF HIRAN is similar to recently reported structures of its DNA bound form, while the NMR analysis also reveals that the DNA binding site of the free domain exhibits conformational heterogeneity. Sequence comparison of N-terminal regions of HLTF, SHPRH and Rad5 aided by knowledge of the HLTF HIRAN structure suggests that the SHPRH N-terminus also includes an uncharacterized structured module, exhibiting weak sequence similarity with HIRAN regions of HLTF and Rad5, and potentially playing a similar functional role.

Human cells possess tightly controlled mechanisms to rescue DNA replication following DNA damage caused by environmental and endogenous carcinogens using a set of low-fidelity translesion synthesis (TLS) DNA polymerases. These polymerases can copy over replication blocking DNA lesions while temporarily leaving them unrepaired, preventing cell death at the expense of increasing mutation rates and contributing to the onset and progression of cancer. In addition, TLS has been implicated as a major cellular mechanism promoting acquired resistance to genotoxic chemotherapy. Owing to its central role in mutagenesis and cell survival after DNA damage, inhibition of the TLS pathway has emerged as a potential target for the development of anticancer agents. This review will recap our current understanding of the structure and regulation of DNA polymerase complexes that mediate TLS and describe how this knowledge is beginning to translate into the development of small molecule TLS inhibitors.

Translesion synthesis (TLS) is a mutagenic branch of cellular DNA damage tolerance that enables bypass replication over DNA lesions carried out by specialized low-fidelity DNA polymerases. The replicative bypass of most types of DNA damage is performed in a two-step process of Rev1/Polζ-dependent TLS. In the first step, a Y-family TLS enzyme, typically Polη, Polι, or Polκ, inserts a nucleotide across a DNA lesion. In the second step, a four-subunit B-family DNA polymerase Polζ (Rev3/Rev7/PolD2/PolD3 complex) extends the distorted DNA primer-template. The coordinated action of error-prone TLS enzymes is regulated through their interactions with the two scaffold proteins, the sliding clamp PCNA and the TLS polymerase Rev1. Rev1 interactions with all other TLS enzymes are mediated by its C-terminal domain (Rev1-CT), which can simultaneously bind the Rev7 subunit of Polζ and Rev1-interacting regions (RIRs) from Polη, Polι, or Polκ. In this work, we identified a previously unknown RIR motif in the C-terminal part of PolD3 subunit of Polζ whose interaction with the Rev1-CT is among the tightest mediated by RIR motifs. Three-dimensional structure of the Rev1-CT/PolD3-RIR complex determined by NMR spectroscopy revealed a structural basis for the relatively high affinity of this interaction. The unexpected discovery of PolD3-RIR motif suggests a mechanism of "inserter" to "extender" DNA polymerase switch upon Rev1/Polζ-dependent TLS, in which the PolD3-RIR binding to the Rev1-CT (i) helps displace the "inserter" Polη, Polι, or Polκ from its complex with Rev1, and (ii) facilitates assembly of the four-subunit "extender" Polζ through simultaneous interaction of Rev1-CT with Rev7 and PolD3 subunits.

Human ubiquitin-specific protease 7 (USP7) is a deubiquitinating enzyme that prevents protein degradation by removing polyubiquitin chains from its substrates. It regulates the stability of a number of human transcription factors and tumor suppressors and plays a critical role in the development of several types of cancer, including prostate and small cell lung cancer. In addition, human USP7 is targeted by several viruses of the Herpesviridae family and is required for effective herpesvirus infection. The USP7 C-terminal region (C-USP7) contains five ubiquitin-like domains (UBL1-5) that interact with several USP7 substrates. Although structures of the USP7 C terminus bound to its substrates could provide vital information for understanding USP7 substrate specificity, no such data has been available to date. In this work we have demonstrated that the USP7 ubiquitin-like domains can be studied in isolation by solution NMR spectroscopy, and we have determined the structure of the UBL1 domain. Furthermore, we have employed NMR and viral plaque assays to probe the interaction between the C-USP7 and HSV-1 immediate-early protein ICP0 (infected cell protein 0), which is essential for efficient lytic infection and virus reactivation from latency. We have shown that depletion of the USP7 in HFF-1 cells negatively affects the efficiency of HSV-1 lytic infection. We have also found that USP7 directly binds ICP0 via its C-terminal UBL1-2 domains and mapped the USP7-binding site for ICP0. Therefore, this study represents a first step toward understanding the molecular mechanism of C-USP7 specificity toward its substrates and may provide the basis for future development of novel antiviral and anticancer therapies., (© 2015 by The American Society for Biochemistry and Molecular Biology, Inc.)

The structural characterization of low populated states of proteins with accuracy comparable to that achievable for native states is important for understanding the mechanisms of protein folding and function, as well as misfolding and aggregation. Because of the transient nature of these low populated states, they are seldom detected directly under conditions that favor folding. The activation domain of human procarboxypeptidase A2 (ADA2h) is an α/β-protein that forms amyloid fibrils at low pH, presumably initiated from a denatured state with a considerable amount of residual structure. Here we used Carr-Parcell-Meiboom-Gill relaxation dispersion (CPMG RD) nuclear magnetic resonance (NMR) spectroscopy to characterize the structure of the denatured state of the ADA2h I71V mutant under conditions that favor folding. Under these conditions, the lifetime of the denatured state of I71V ADA2h is on the order of milliseconds and its population is approximately several percent, which makes this mutant amenable to studies by CPMG RD methods. The nearly complete set of CPMG RD-derived backbone (15)N, (13)C, and (1)H NMR chemical shifts in the I71V ADA2h denatured state reveals that it retains a significant fraction (up to 50-60%) of nativelike α-helical structure, while the regions encompassing native β-strands are structured to a much lesser extent. The nativelike α-helical structure of the denatured state can bring together hydrophobic residues on the same sides of α-helices, making them available for intra- or intermolecular interactions. CPMG RD data analysis thus allowed a detailed structural characterization of the ADA2h denatured state under folding conditions not previously achieved for this protein.

Stalled replication forks are a critical problem for the cell because they can lead to complex genome rearrangements that underlie cell death and disease. Processes such as DNA damage tolerance and replication fork reversal protect stalled forks from these events. A central mediator of these DNA damage responses in humans is the Rad5-related DNA translocase, HLTF. Here, we present biochemical and structural evidence that the HIRAN domain, an ancient and conserved domain found in HLTF and other DNA processing proteins, is a modified oligonucleotide/oligosaccharide (OB) fold that binds to 3' ssDNA ends. We demonstrate that the HIRAN domain promotes HLTF-dependent fork reversal in vitro through its interaction with 3' ssDNA ends found at forks. Finally, we show that HLTF restrains replication fork progression in cells in a HIRAN-dependent manner. These findings establish a mechanism of HLTF-mediated fork reversal and provide insight into the requirement for distinct fork remodeling activities in the cell., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Ubiquitin-mediated interactions are critical for the cellular DNA damage response (DDR). Therefore, many DDR-related proteins contain ubiquitin-binding domains, including ubiquitin-binding zinc fingers (UBZs). The majority of these UBZ domains belong to the C2H2 (type 3 Polη-like) or C2HC (type 4 Rad18-like) family. We have used nuclear magnetic resonance (NMR) spectroscopy to characterize the binding to ubiquitin and determine the structure of the type 4 UBZ domain (UBZ4) from human Rad18, which is a key ubiquitin ligase in the DNA damage tolerance pathway responsible for monoubiquitination of the DNA sliding clamp PCNA. The Rad18-UBZ domain binds ubiquitin with micromolar affinity and adopts a β1-β2-α fold similar to the previously characterized type 3 UBZ domain (UBZ3) from the translesion synthesis DNA polymerase Polη. However, despite nearly identical structures, a disparity in the location of binding-induced NMR chemical shift perturbations shows that the Rad18-UBZ4 and Polη-UBZ3 domains bind ubiquitin in distinctly different modes. The Rad18-UBZ4 domain interacts with ubiquitin with the α-helix and strand β1 as shown by the structure of the Rad18-UBZ domain-ubiquitin complex determined in this work, while the Polη-UBZ3 domain exclusively utilizes the α-helix. Our findings suggest the existence of two classes of UBZ domains in DDR-related proteins with similar structures but unique ubiquitin binding properties and provide context for further study to establish the differential roles of these domains in the complex cellular response to DNA damage.

Rev1 is a Y-family translesion synthesis (TLS) DNA polymerase involved in bypass replication across sites of DNA damage and postreplicational gap filling. In the process of TLS, high-fidelity replicative DNA polymerases stalled by DNA damage are replaced by error-prone TLS enzymes responsible for the majority of mutagenesis in eukaryotic cells. The polymerase exchange that gains low-fidelity TLS polymerases access to DNA is mediated by their interactions with proliferating cell nuclear antigen (PCNA). Rev1 stands alone from other Y-family TLS enzymes since it lacks the consensus PCNA-interacting protein box (PIP-box) motif, instead utilizing other modular domains for PCNA binding. Here we report solution NMR structure of an 11-kDa BRCA1 C-terminus (BRCT) domain from Saccharomyces cerevisiae Rev1 and demonstrate with the use of transverse relaxation optimized spectroscopy (TROSY) NMR methods that Rev1-BRCT domain directly interacts with an 87-kDa PCNA in solution. The domain adopts α/β fold (β1-α1-β2-β3-α2-β4-α3-α4) typical for BRCT domain superfamily. PCNA-binding interface of the Rev1-BRCT domain comprises conserved residues of the outer surface of the α1-helix and the α1-β1, β2-β3 and β3-α2 loops. On the other hand, Rev1-BRCT binds to the inter-domain region of PCNA that overlaps with the binding site for the PIP-box motif. Furthermore, Rev1-BRCT domain bound to PCNA can be displaced by increasing amounts of the PIP-box peptide from TLS DNA polymerase polη, suggesting that Rev1-BRCT and polη PIP-box interactions with the same PCNA monomer are mutually exclusive. These results provide structural insights into PCNA recognition by TLS DNA polymerases that help better understand TLS regulation in eukaryotes., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Studies of protein folding and the intermediates that are formed along the folding pathway provide valuable insights into the process by which an unfolded ensemble forms a functional native conformation. However, because intermediates on folding pathways can serve as initiation points of aggregation (implicated in a number of diseases), their characterization assumes an even greater importance. Establishing the role of such intermediates in folding, misfolding, and aggregation remains a major challenge due to their often low populations and short lifetimes. We recently used NMR relaxation dispersion methods and computational techniques to determine an atomic resolution structure of the folding intermediate of a small protein module--the FF domain--with an equilibrium population of 2-3% and a millisecond lifetime, 25 °C. Based on this structure a variant FF domain has been designed in which the native state is selectively destabilized by removing the carboxyl-terminal helix in the native structure to produce a highly populated structural mimic of the intermediate state. Here, we show via solution NMR studies of the designed mimic that the mimic forms distinct conformers corresponding to monomeric and dimeric (K(d) = 0.2 mM) forms of the protein. The conformers exchange on the seconds timescale with a monomer association rate of 1.1 · 10(4) M(-1) s(-1) and with a region responsible for dimerization localized to the amino-terminal residues of the FF domain. This study establishes the FF domain intermediate as a central player in both folding and misfolding pathways and illustrates how incomplete folding can lead to the formation of higher-order structures.

Human Rev1 is a translesion synthesis (TLS) DNA polymerase involved in bypass replication across sites of DNA damage and postreplicational gap-filling. Rev1 plays an essential structural role in TLS by providing a binding platform for other TLS polymerases that insert nucleotides across DNA lesions (polη, polι, polκ) and extend the distorted primer-terminus (polς). We use NMR spectroscopy to demonstrate that the Rev1 C-terminal domain utilizes independent interaction interfaces to simultaneously bind a fragment of the 'inserter' polη and Rev7 subunit of the 'extender' polς, thereby serving as a cassette that may accommodate several polymerases making them instantaneously available for TLS., (Copyright © 2012 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.)

Rev1 is a translesion synthesis (TLS) DNA polymerase essential for DNA damage tolerance in eukaryotes. In the process of TLS stalled high-fidelity replicative DNA polymerases are temporarily replaced by specialized TLS enzymes that can bypass sites of DNA damage (lesions), thus allowing replication to continue or postreplicational gaps to be filled. Despite its limited catalytic activity, human Rev1 plays a key role in TLS by serving as a scaffold that provides an access of Y-family TLS polymerases polη, ι, and κ to their cognate DNA lesions and facilitates their subsequent exchange to polζ that extends the distorted DNA primer-template. Rev1 interaction with the other major human TLS polymerases, polη, ι, κ, and the regulatory subunit Rev7 of polζ, is mediated by Rev1 C-terminal domain (Rev1-CT). We used NMR spectroscopy to determine the spatial structure of the Rev1-CT domain (residues 1157-1251) and its complex with Rev1 interacting region (RIR) from polη (residues 524-539). The domain forms a four-helix bundle with a well-structured N-terminal β-hairpin docking against helices 1 and 2, creating a binding pocket for the two conserved Phe residues of the RIR motif that upon binding folds into an α-helix. NMR spin-relaxation and NMR relaxation dispersion measurements suggest that free Rev1-CT and Rev1-CT/polη-RIR complex exhibit μs-ms conformational dynamics encompassing the RIR binding site, which might facilitate selection of the molecular configuration optimal for binding. These results offer new insights into the control of TLS in human cells by providing a structural basis for understanding the recognition of the Rev1-CT by Y-family DNA polymerases.

We have recently reported the atomic resolution structure of a low populated and transiently formed on-pathway folding intermediate of the FF domain from human HYPA/FBP11 [Korzhnev, D. M.; Religa, T. L.; Banachewicz, W.; Fersht, A. R.; Kay, L.E. Science 2011, 329, 1312-1316]. The structure was determined on the basis of backbone chemical shift and bond vector orientation restraints of the invisible intermediate state measured using relaxation dispersion nuclear magnetic resonance (NMR) spectroscopy that were subsequently input into the database structure determination program, CS-Rosetta. As a cross-validation of the structure so produced, we present here the solution structure of a mimic of the folding intermediate that is highly populated in solution, obtained from the wild-type domain by mutagenesis that destabilizes the native state. The relaxation dispersion/CS-Rosetta structures of the intermediate are within 2 Å of those of the mimic, with the nonnative interactions in the intermediate also observed in the mimic. This strongly confirms the structure of the FF domain folding intermediate, in particular, and validates the use of relaxation dispersion derived restraints in structural studies of invisible excited states, in general.

Several all-helical single-domain proteins have been shown to fold rapidly (microsecond time scale) to a compact intermediate state and subsequently rearrange more slowly to the native conformation. An understanding of this process has been hindered by difficulties in experimental studies of intermediates in cases where they are both low-populated and only transiently formed. One such example is provided by the on-pathway folding intermediate of the small four-helix bundle FF domain from HYPA/FBP11 that is populated at several percent with a millisecond lifetime at room temperature. Here we have studied the L24A mutant that has been shown previously to form nonnative interactions in the folding transition state. A suite of Carr-Purcell-Meiboom-Gill relaxation dispersion NMR experiments have been used to measure backbone chemical shifts and amide bond vector orientations of the invisible folding intermediate that form the input restraints in calculations of atomic resolution models of its structure. Despite the fact that the intermediate structure has many features that are similar to that of the native state, a set of nonnative contacts is observed that is even more extensive than noted previously for the wild-type (WT) folding intermediate. Such nonnative interactions, which must be broken prior to adoption of the native conformation, explain why the transition from the intermediate state to the native conformer (millisecond time scale) is significantly slower than from the unfolded ensemble to the intermediate and why the L24A mutant folds more slowly than the WT.

The Wilson disease protein (ATP7B) is a copper-transporting member of the P-type ATPase superfamily, which plays a central role in copper homeostasis and interacts with the copper chaperone Atox1. The N-terminus of ATP7B is comprised of six copper-binding domains (WCBDs), each capable of binding one copper atom in the +1 oxidation state. To better understand the regulatory effect of copper binding to these domains, we have performed NMR characterization of WCBD4-6 (domains 4-6 of ATP7B). (15)N relaxation measurements on the apo and Cu(I)-bound WCBD4-6 show that there is no dramatic change in the dynamic properties of this three-domain construct; the linker between domains 4 and 5 remains flexible, domains 5 and 6 do not form a completely rigid dimer but rather have some flexibility with respect to each other, and there is minimal change in the relative orientation of the domains in the two states. We also show that, contrary to previous reports, the protein-protein interaction between Atox1 and the copper-binding domains takes place even in the absence of copper. Comparison of apo and Cu(I)-bound spectra of WCBD1-6 shows that binding of Cu(I) does not induce the formation of a unit that tumbles as a single entity, consistent with our results for WCBD4-6. We propose that copper transfer to and between the N-terminal domains of the Wilson Cu-ATPase occurs via protein interactions that are facilitated by the flexibility of the linkers and the motional freedom of the domains with respect to each other.

Proteins can sample conformational states that are critical for function but are seldom detected directly because of their low occupancies and short lifetimes. In this work, we used chemical shifts and bond-vector orientation constraints obtained from nuclear magnetic resonance relaxation dispersion spectroscopy, in concert with a chemical shift-based method for structure elucidation, to determine an atomic-resolution structure of an "invisible" folding intermediate of a small protein module: the FF domain. The structure reveals non-native elements preventing formation of the native conformation in the carboxyl-terminal part of the protein. This is consistent with the kinetics of folding in which a well-structured intermediate forms rapidly and then rearranges slowly to the native state. The approach introduces a general strategy for structure determination of low-populated and transiently formed protein states.

NMR relaxation dispersion spectroscopy is a powerful method for studying protein conformational dynamics whereby visible, ground and invisible, excited conformers interconvert on the millisecond time-scale. In addition to providing kinetics and thermodynamics parameters of the exchange process, the CPMG dispersion experiment also allows extraction of the absolute values of the chemical shift differences between interconverting states, /Delta(omega)/, opening the way for structure determination of excited state conformers. Central to the goal of structural analysis is the availability of the chemical shifts of the excited state that can only be obtained once the signs of Delta(omega) are known. Herein we describe a very simple method for determining the signs of (1)H(N) Delta(omega) values based on a comparison of peak positions in the directly detected dimensions of a pair of (1)H(N)-(15)N correlation maps recorded at different static magnetic fields. The utility of the approach is demonstrated for three proteins that undergo millisecond time-scale conformational rearrangements. Although the method provides fewer signs than previously published techniques it does have a number of strengths: (1) Data sets needed for analysis are typically available from other experiments, such as those required for measuring signs of (15)N Delta(omega) values, thus requiring no additional experimental time, (2) acquisition times in the critical detection dimension can be as long as necessary and (3) the signs obtained can be used to cross-validate those from other approaches.

Carr-Purcell-Meiboom-Gill (CPMG) relaxation dispersion NMR spectroscopy has emerged as a powerful tool for quantifying the kinetics and thermodynamics of millisecond exchange processes between a major, populated ground state and one or more minor, low populated and often invisible 'excited' conformers. Analysis of CPMG data-sets also provides the magnitudes of the chemical shift difference(s) between exchanging states (|Deltavarpi|), that inform on the structural properties of the excited state(s). The sign of Deltavarpi is, however, not available from CPMG data. Here we present one-dimensional NMR experiments for measuring the signs of (1)H(N) and (13)C(alpha) Deltavarpi values using weak off-resonance R (1rho ) relaxation measurements, extending the spin-lock approach beyond previous applications focusing on the signs of (15)N and (1)H(alpha) shift differences. The accuracy of the method is established by using an exchanging system where the invisible, excited state can be converted to the visible, ground state by altering conditions so that the signs of Deltavarpi values obtained from the spin-lock approach can be validated with those measured directly. Further, the spin-lock experiments are compared with the established H(S/M)QC approach for measuring the signs of chemical shift differences. For the Abp1p and Fyn SH3 domains considered here it is found that while H(S/M)QC measurements provide signs for more residues than the spin-lock data, the two different methodologies are complementary, so that combining both approaches frequently produces signs for more residues than when the H(S/M)QC method is used alone.

Marginally and transiently populated conformational states of biomolecules can play important functional roles in biochemical processes. It is of significant interest, therefore, to develop tools for characterizing the structural and dynamical properties of these excited states. One recent development has been the emergence of spin-state-selective relaxation dispersion methods for quantifying dipolar vector orientations in invisible excited-state conformers through measurement of residual dipolar couplings (RDCs). Particularly powerful are 1HN-(15)N RDCs that can be measured with high sensitivity on fractionally aligned, deuterated, uniformly 15N-labeled protein samples. Fractional alignment also produces nonzero 1HN-(1)HN dipolar couplings. These can be problematic for the extraction of robust 1HN-(15)N RDC values, and hence amide bond vector orientations, in cases where the amide proton of interest and a proximal amide proton have small chemical shift differences and a significant 1HN-(1)HN dipolar coupling. Here, we show that while this strong coupling effect leads to aberrant relaxation dispersion profiles, extracted excited-state 1HN-(15)N RDCs are for the most part only marginally affected. Experimental examples of such aberrant profiles are provided, as well as a theoretical consideration of the influence of this strong coupling effect and numerical simulations that assess its impact on extracted parameters.