Metalloprotease SPRTN/DVC1 Orchestrates Replication-Coupled DNA-Protein Crosslink Repair.

The cytotoxicity of DNA-protein crosslinks (DPCs) is largely ascribed to their ability to block the progression of DNA replication. DPCs frequently occur in cells, either as a consequence of metabolism or exogenous agents, but the mechanism of DPC repair is not completely understood. Here, we characterize SPRTN as a specialized DNA-dependent and DNA replication-coupled metalloprotease for DPC repair. SPRTN cleaves various DNA binding substrates during S-phase progression and thus protects proliferative cells from DPC toxicity. Ruijs-Aalfs syndrome (RJALS) patient cells with monogenic and biallelic mutations in SPRTN are hypersensitive to DPC-inducing agents due to a defect in DNA replication fork progression and the inability to eliminate DPCs. We propose that SPRTN protease represents a specialized DNA replication-coupled DPC repair pathway essential for DNA replication progression and genome stability. Defective SPRTN-dependent clearance of DPCs is the molecular mechanism underlying RJALS, and DPCs are contributing to accelerated aging and cancer.

Activation of a primed RING E3-E2-ubiquitin complex by non-covalent ubiquitin.

RING ubiquitin ligases (E3) recruit ubiquitin-conjugate enzymes (E2) charged with ubiquitin (Ub) to catalyze ubiquitination. Non-covalent Ub binding to the backside of certain E2s promotes processive polyUb formation, but the mechanism remains elusive. Here, we show that backside bound Ub (Ub(B)) enhances both RING-independent and RING-dependent UbcH5B-catalyzed donor Ub (Ub(D)) transfer, but with a more prominent effect in RING-dependent transfer. Ub(B) enhances RING E3s' affinities for UbcH5B-Ub, and RING E3-UbcH5B-Ub complex improves Ub(B)'s affinity for UbcH5B. A comparison of the crystal structures of a RING E3, RNF38, bound to UbcH5B-Ub in the absence and presence of Ub(B), together with molecular dynamics simulation and biochemical analyses, suggests Ub(B) restricts the flexibility of UbcH5B's α1 and α1ß1 loop. Ub(B) supports E3 function by stabilizing the RING E3-UbcH5B-Ub complex, thereby improving the catalytic efficiency of Ub transfer. Thus, Ub(B) serves as an allosteric activator of RING E3-mediated Ub transfer.

The crystal structure of human forkhead box N1 in complex with DNA reveals the structural basis for forkhead box family specificity.

Forkhead box N1 (FOXN1) is a member of the forkhead box family of transcription factors and plays an important role in thymic epithelial cell differentiation and development. FOXN1 mutations in humans and mice give rise to the "nude" phenotype, which is marked by athymia. FOXN1 belongs to a subset of the FOX family that recognizes an alternative forkhead-like (FHL) consensus sequence (GACGC) that is different from the more widely recognized forkhead (FKH) sequence RYAAAYA (where R is purine, and Y is pyrimidine). Here, we present the FOXN1 structure in complex with DNA containing an FHL motif at 1.6 Å resolution, in which the DNA sequence is recognized by a mixture of direct and water-mediated contacts provided by residues in an α-helix inserted in the DNA major groove (the recognition helix). Comparisons with the structure of other FOX family members revealed that the FKH and FHL DNA sequences are bound in two distinct modes, with partially different registers for the protein DNA contacts. We identified a single alternative rotamer within the recognition helix itself as an important determinant of DNA specificity and found protein sequence features in the recognition helix that could be used to predict the specificity of other FOX family members. Finally, we demonstrate that the C-terminal region of FOXN1 is required for high-affinity DNA binding and that FOXN1 has a significantly reduced affinity for DNA that contains 5'-methylcytosine, which may have implications for the role of FOXN1 in thymic involution.

Insights into the RecQ helicase mechanism revealed by the structure of the helicase domain of human RECQL5.

RecQ helicases are important maintainers of genome integrity with distinct roles in almost every cellular process requiring access to DNA. RECQL5 is one of five human RecQ proteins and is particularly versatile in this regard, forming protein complexes with a diverse set of cellular partners in order to coordinate its helicase activity to various processes including replication, recombination and DNA repair. In this study, we have determined crystal structures of the core helicase domain of RECQL5 both with and without the nucleotide ADP in two distinctly different ('Open' and 'Closed') conformations. Small angle X-ray scattering studies show that the 'Open' form of the protein predominates in solution and we discuss implications of this with regards to the RECQL5 mechanism and conformational changes. We have measured the ATPase, helicase and DNA binding properties of various RECQL5 constructs and variants and discuss the role of these regions and residues in the various RECQL5 activities. Finally, we have performed a systematic comparison of the RECQL5 structures with other RecQ family structures and based on these comparisons we have constructed a model for the mechano-chemical cycle of the common catalytic core of these helicases.

Structures of the Ets Protein DNA-binding Domains of Transcription Factors Etv1, Etv4, Etv5, and Fev: DETERMINANTS OF DNA BINDING AND REDOX REGULATION BY DISULFIDE BOND FORMATION.

Ets transcription factors, which share the conserved Ets DNA-binding domain, number nearly 30 members in humans and are particularly involved in developmental processes. Their deregulation following changes in expression, transcriptional activity, or by chromosomal translocation plays a critical role in carcinogenesis. Ets DNA binding, selectivity, and regulation have been extensively studied; however, questions still arise regarding binding specificity outside the core GGA recognition sequence and the mode of action of Ets post-translational modifications. Here, we report the crystal structures of Etv1, Etv4, Etv5, and Fev, alone and in complex with DNA. We identify previously unrecognized features of the protein-DNA interface. Interactions with the DNA backbone account for most of the binding affinity. We describe a highly coordinated network of water molecules acting in base selection upstream of the GGAA core and the structural features that may account for discrimination against methylated cytidine residues. Unexpectedly, all proteins crystallized as disulfide-linked dimers, exhibiting a novel interface (distant to the DNA recognition helix). Homodimers of Etv1, Etv4, and Etv5 could be reduced to monomers, leading to a 40-200-fold increase in DNA binding affinity. Hence, we present the first indication of a redox-dependent regulatory mechanism that may control the activity of this subset of oncogenic Ets transcription factors.

Structural insights into the autoregulation and cooperativity of the human transcription factor Ets-2.

Ets-2, like its closely related homologue Ets-1, is a member of the Ets family of DNA binding transcription factors. Both proteins are subject to multiple levels of regulation of their DNA binding and transactivation properties. One such regulatory mechanism is the presence of an autoinhibitory module, which in Ets-1 allosterically inhibits the DNA binding activity. This inhibition can be relieved by interaction with protein partners or cooperative binding to closely separated Ets binding sites in a palindromic arrangement. In this study we describe the 2.5 Å resolution crystal structure of a DNA complex of the Ets-2 Ets domain. The Ets domain crystallized with two distinct species in the asymmetric unit, which closely resemble the autoinhibited and DNA bound forms of Ets-1. This discovery prompted us to re-evaluate the current model for the autoinhibitory mechanism and the structural basis for cooperative DNA binding. In contrast to Ets-1, in which the autoinhibition is caused by a combination of allosteric and steric mechanisms, we were unable to find clear evidence for the allosteric mechanism in Ets-2. We also demonstrated two possibly distinct types of cooperative binding to substrates with Ets binding motifs separated by four and six base pairs and suggest possible molecular mechanisms for this behavior.

Structural comparison of typical and atypical E2 pestivirus glycoproteins.

Pestiviruses, within the family Flaviviridae, are economically important viruses of livestock. In recent years, new pestiviruses have been reported in domestic animals and non-cloven-hoofed animals. Among them, atypical porcine pestivirus (APPV) and Norway rat pestivirus (NRPV) have relatively little sequence conservation in their surface glycoprotein E2. Despite E2 being the main target for neutralizing antibodies and necessary for cell attachment and viral fusion, the mechanism of viral entry remains elusive. To gain further insights into the pestivirus E2 mechanism of action and to assess its diversity within the genus, we report X-ray structures of the pestivirus E2 proteins from APPV and NRPV. Despite the highly divergent structures, both are able to dimerize through their C-terminal domain and contain a solvent-exposed ß-hairpin reported to be involved in host receptor binding. Functional analysis of this ß-hairpin in the context of BVDV revealed its ability to rescue viral infectivity.

Structure of Bovine CD46 Ectodomain.

CD46, or membrane cofactor protein, is a type-one transmembrane protein from the complement regulatory protein family. Alongside its role in complement activation, CD46 is involved in many other processes, from T-cell activation to reproduction. It is also referred to as a pathogen magnet, because it is used as a receptor by multiple bacteria and viruses. Bovine CD46 (bovCD46) in particular is involved in bovine viral diarrhoea virus entry, an economically important disease in cattle industries. This study presents the X-ray crystallographic structure of the extracellular region of bovCD46, revealing a four-short-consensus-repeat (SCR) structure similar to that in human CD46. SCR1-3 are arranged linearly, while SCR 4 has a reduced interface angle, resulting in a hockey stick-like appearance. The structure also reveals the bovine viral diarrhoea virus interaction site in SCR1, which is likely to confer pestivirus specificity for their target host, CD46. Insights gained from the structural information on pestivirus receptors, such as CD46, could offer valuable guidance for future control strategies.

Atypical Porcine Pestiviruses: Relationships and Conserved Structural Features.

For two decades, the genus pestivirus has been expanding and the host range now extends to rodents, bats and marine mammals. In this review, we focus on one of the most diverse pestiviruses, atypical porcine pestivirus or pestivirus K, comparing its special traits to what is already known at the structural and functional level from other pestiviruses.

A Ubiquitin-Binding Domain that Binds a Structural Fold Distinct from that of Ubiquitin.

Ubiquitylation, the posttranslational linkage of ubiquitin moieties to lysines in target proteins, helps regulate a myriad of biological processes. Ubiquitin, and sometimes ubiquitin-homology domains, are recognized by ubiquitin-binding domains, including CUE domains. CUE domains are thus generally thought to function by mediating interactions with ubiquitylated proteins. The chromatin remodeler, SMARCAD1, interacts with KAP1, a transcriptional corepressor. The SMARCAD1-KAP1 interaction is direct and involves the first SMARCAD1 CUE domain (CUE1) and the RBCC domain of KAP1. Here, we present a structural model of the KAP1 RBCC-SMARCAD1 CUE1 complex based on X-ray crystallography. Remarkably, CUE1, a canonical CUE domain, recognizes a cluster of exposed hydrophobic and surrounding charged/amphipathic residues on KAP1, which are presented in the context of a coiled-coil domain, not in a structure resembling ubiquitin. Together, these data suggest that CUE domains may have a wider function than simply recognizing ubiquitin and the ubiquitin-fold.

Structures of Two Melanoma-Associated Antigens Suggest Allosteric Regulation of Effector Binding.

The MAGE (melanoma associated antigen) protein family are tumour-associated proteins normally present only in reproductive tissues such as germ cells of the testis. The human genome encodes over 60 MAGE genes of which one class (containing MAGE-A3 and MAGE-A4) are exclusively expressed in tumours, making them an attractive target for the development of targeted and immunotherapeutic cancer treatments. Some MAGE proteins are thought to play an active role in driving cancer, modulating the activity of E3 ubiquitin ligases on targets related to apoptosis. Here we determined the crystal structures of MAGE-A3 and MAGE-A4. Both proteins crystallized with a terminal peptide bound in a deep cleft between two tandem-arranged winged helix domains. MAGE-A3 (but not MAGE-A4), is predominantly dimeric in solution. Comparison of MAGE-A3 and MAGE-A3 with a structure of an effector-bound MAGE-G1 suggests that a major conformational rearrangement is required for binding, and that this conformational plasticity may be targeted by allosteric binders.

Structure of the Helicase Domain of DNA Polymerase Theta Reveals a Possible Role in the Microhomology-Mediated End-Joining Pathway.

DNA polymerase theta (Polθ) has been identified as a crucial alternative non-homologous end-joining factor in mammalian cells. Polθ is upregulated in a range of cancer cell types defective in homologous recombination, and knockdown has been shown to inhibit cell survival in a subset of these, making it an attractive target for cancer treatment. We present crystal structures of the helicase domain of human Polθ in the presence and absence of bound nucleotides, and a characterization of its DNA-binding and DNA-stimulated ATPase activities. Comparisons with related helicases from the Hel308 family identify several unique features. Polθ exists as a tetramer both in the crystals and in solution. We propose a model for DNA binding to the Polθ helicase domain in the context of the Polθ tetramer, which suggests a role for the helicase domain in strand annealing of DNA templates for subsequent processing by the polymerase domain.