Recognition and coacervation of G-quadruplexes by a multifunctional disordered region in RECQ4 helicase

Biomolecular polyelectrolyte complexes can be formed between oppositely charged intrinsically disordered regions (IDRs) of proteins or between IDRs and nucleic acids. Highly charged IDRs are abundant in the nucleus, yet few have been functionally characterized. Here, we show that a positively charged IDR within the human ATP-dependent DNA helicase Q4 (RECQ4) forms coacervates with G-quadruplexes (G4s). We describe a three-step model of charge-driven coacervation by integrating equilibrium and kinetic binding data in a global numerical model. The oppositely charged IDR and G4 molecules form a complex in the solution that follows a rapid nucleation-growth mechanism leading to a dynamic equilibrium between dilute and condensed phases. We also discover a physical interaction with Replication Protein A (RPA) and demonstrate that the IDR can switch between the two extremes of the structural continuum of complexes. The structural, kinetic, and thermodynamic profile of its interactions revealed a dynamic disordered complex with nucleic acids and a static ordered complex with RPA protein. The two mutually exclusive binding modes suggest a regulatory role for the IDR in RECQ4 function by enabling molecular handoffs. Our study extends the functional repertoire of IDRs and demonstrates a role of polyelectrolyte complexes involved in G4 binding

Crystal Structure of TDRD3 and Methyl-Arginine Binding Characterization of TDRD3, SMN and SPF30

SMN (Survival motor neuron protein) was characterized as a dimethyl-arginine binding protein over ten years ago. TDRD3 (Tudor domain-containing protein 3) and SPF30 (Splicing factor 30 kDa) were found to bind to various methyl-arginine proteins including Sm proteins as well later on. Recently, TDRD3 was shown to be a transcriptional coactivator, and its transcriptional activity is dependent on its ability to bind arginine-methylated histone marks. In this study, we systematically characterized the binding specificity and affinity of the Tudor domains of these three proteins quantitatively. Our results show that TDRD3 preferentially recognizes asymmetrical dimethylated arginine mark, and SMN is a very promiscuous effector molecule, which recognizes different arginine containing sequence motifs and preferentially binds symmetrical dimethylated arginine. SPF30 is the weakest methyl-arginine binder, which only binds the GAR motif sequences in our library. In addition, we also reported high-resolution crystal structures of the Tudor domain of TDRD3 in complex with two small molecules, which occupy the aromatic cage of TDRD3

Association between XPF Polymorphisms and Cancer Risk: A Meta-Analysis

Background: Xeroderma pigmentosum complementation group F (XPF or ERCC4) plays a key role in DNA repair that protects against genetic instability and carcinogenesis. A series of epidemiological studies have examined associations between XPF polymorphisms and cancer risk, but the findings remain inconclusive. Methodology/Principal Findings: In this meta-analysis of 47,639 cancer cases and 51,915 controls, by searching three electronic databases (i.e., MEDLINE, EMBASE and CNKI), we summarized 43 case-control studies from 29 publications on four commonly studied polymorphisms of XPF (i.e., rs1800067, rs1799801, rs2020955 and rs744154), and we did not find statistical evidence of any significant association with overall cancer risk. However, in stratification analyses, we found a significant association of XPF-rs1799801 with a reduced cancer risk in Caucasian populations (4,845 cases and 5,556 controls; recessive model: OR = 0.87, 95% CI = 0.76–1.00, P = 0.049, P = 0.723 for heterogeneity test, I2 = 0). Further genotype-phenotype correlation analysis showed that the homozygous variant CC genotype carriers had higher XPF expression levels than that of the TT genotype carriers (Student’s t test for a recessive model: P = 0.046). No publication bias was found by using the funnel plot and Egger’s test. Conclusion: This meta-analysis suggests a lack of statistical evidence for the association between the four XPF SNPs and overall risk of cancers. However, XPF-rs1799801 may be associated with cancer risk in Caucasian populations, which needs to be further validated in single large, well-designed prospective studies

Structural and functional studies of the human ERCC1-XPF DNA repair complex

During eukaryotic Nucleotide Excision Repair, DNA cleavage by XPF requires heterodimer formation with ERCC1. The aim of the current thesis was to address the structure-function relationship of the obligate ERCC1/XPF heterodimer. In this line, we have determined the structure of ERCC1/XPF C-terminal interacting domains by NMR. ERCC1 and XPF partners share the same fold, the double helix-hairpin-helix motif (HhH2), and complex in a symmetry related manner. The same dimer interface is observed in the archaeal XPF, which forms homodimers. The archaeal HhH2 domains bind equally to DNA minor grooves, in agreement with the substrate specificity of the archaeal XPF nuclease. In contrast, human ERCC1/XPF cleaves relevant DNA substrates, which consist of one minor groove. We have shown that exclusively the HhH2 domain of ERCC1 mediates minor groove binding. The functional asymmetry in humans is related to the altered second hairpin of XPF, revealed by the structure. Though non-functional this domain acts as scaffold for the folding of the ERCC1 corresponding domain. We concluded that ERCC1 possesses the DNA binding activity that serves to localize the XPF nuclease activity to the NER pre-incision complex and allow for damage excision. Next, we determined the structure of the ERCC1 central domain and investigate its contribution to the heterodimeric function. Although this domain shows structural homology with the catalytically active XPF nuclease domain, it has a distinct function by performing interactions with XPA, another NER protein. This function is crucial as it anchors the XPF nuclease in the pre-incision NER complex and subsequently allows cleavage to occur. Remarkably, the XPA binding by ERCC1 and the catalytic function of XPF are dependent on structurally homologous regions. Furthermore the ERCC1 central domain can bind ss-DNA. XPA and DNA interactions can happen simultaneously through distinct surfaces and indicate the significant dual role of the ERCC1 central domain in targeting the XPF nuclease to the sites of DNA damage. Overall, the structural data from human and archaeal species show that the human ERCC1 and XPF proteins share the architectural subunits of the archaeal XPF homodimer. The structural similarities provide evidence for the common origin of the human proteins. Based on the functional data we proposed that ERCC1 and XPF genes evolved by subfunctionalization, which resulted in the partitioning of the original functions after duplication of the ancestral XPF gene. This evolutionary scenario is able to explain the preservation of the ERCC1 gene and the concomitant transition from homo- to obligate hetero-association in eukaryotes. Structural and biochemical work on the first reported ERCC1 deficiency in humans is on going to understand the mutant ERCC1/XPF dysfunction

Repeat recognition.

CUG-binding protein 1 (CUGBP1) is a ubiquitous RNA-binding protein implicated in altered RNA metabolism linked to myotonic dystrophy type 1. Crystal structures of the RRM domains in complex with cognate RNAs (Teplova et al., 2010) reveal molecular details for the selectivity of CUGBP1 toward GU-rich mRNA elements

Robust and highly accurate automatic NOESY assignment and structure determination with Rosetta.

We have developed a novel and robust approach for automatic and unsupervised simultaneous nuclear Overhauser effect (NOE) assignment and structure determination within the CS-Rosetta framework. Starting from unassigned peak lists and chemical shift assignments, autoNOE-Rosetta determines NOE cross-peak assignments and generates structural models. The approach tolerates incomplete and raw NOE peak lists as well as incomplete or partially incorrect chemical shift assignments, and its performance has been tested on 50 protein targets ranging from 50 to 200 residues in size. We find a significantly improved performance compared to established programs, particularly for larger proteins and for NOE data obtained on perdeuterated protein samples. X-ray crystallographic structures allowed comparison of Rosetta and conventional, PDB-deposited, NMR models in 20 of 50 test cases. The unsupervised autoNOE-Rosetta models were often of significantly higher accuracy than the corresponding expert-supervised NMR models deposited in the PDB. We also tested the method with unrefined peak lists and found that performance was nearly as good as for refined peak lists. Finally, demonstrating our method's remarkable robustness against problematic input data, we provided correct models for an incorrect PDB-deposited NMR solution structure

Studying weak and dynamic interactions of posttranslationally modified proteins using expressed protein ligation.

Many cellular processes are regulated by posttranslational modifications that are recognized by specific domains in protein binding partners. These interactions are often weak, thus allowing a highly dynamic and combinatorial regulatory network of protein-protein interactions. We report an efficient strategy that overcomes challenges in structural analysis of such a weak transient interaction between the Tudor domain of the Survival of Motor Neuron (SMN) protein and symmetrically dimethylated arginine (sDMA). The posttranslational modification is chemically introduced and covalently linked to the effector module by a one-pot expressed protein ligation (EPL) procedure also enabling segmental incorporation of NMR-active isotopes for structural analysis. Covalent coupling of the two interacting moieties shifts the equilibrium to the bound state, and stoichiometric interactions are formed even for low affinity interactions. Our approach should enable the structural analysis of weak interactions by NMR or X-ray crystallography to better understand the role of posttranslational modifications in dynamic biological processes

A novel RNA binding surface of the TAM domain of TIP5/BAZ2A mediates epigenetic regulation of rRNA genes.

The chromatin remodeling complex NoRC, comprising the subunits SNF2h and TIP5/BAZ2A, mediates heterochromatin formation at major clusters of repetitive elements, including rRNA genes, centromeres and telomeres. Association with chromatin requires the interaction of the TAM (TIP5/ARBP/MBD) domain of TIP5 with noncoding RNA, which targets NoRC to specific genomic loci. Here, we show that the NMR structure of the TAM domain of TIP5 resembles the fold of the MBD domain, found in methyl-CpG binding proteins. However, the TAM domain exhibits an extended MBD fold with unique C-terminal extensions that constitute a novel surface for RNA binding. Mutation of critical amino acids within this surface abolishes RNA binding in vitro and in vivo. Our results explain the distinct binding specificities of TAM and MBD domains to RNA and methylated DNA, respectively, and reveal structural features for the interaction of NoRC with non-coding RNA