Michael R. Gryk

Solution structure of the single-strand break repair protein XRCC1 N-terminal domain.

XRCC1 functions in the repair of single-strand DNA breaks in mammalian cells and forms a repair complex with β-Pol, ligase III and PARP. Here we describe the NMR solution structure of the XRCC1 N-terminal domain (XRCC1 NTD). The structural core is a β-sandwich with β-strands connected by loops, three helices and two short two-stranded β-sheets at each connection side. We show, for the first time, that the XRCC1 NTD specifically binds single-strand break DNA (gapped and nicked). We also show that the XRCC1 NTD binds a gapped DNA–β-Pol complex. The DNA binding and β-Pol binding surfaces were mapped by NMR and found to be well suited for interaction with single-strand gap DNA containing a 90° bend, and for simultaneously making contacts with the palm-thumb of β-Pol in a ternary complex. The findings suggest a mechanism for preferential binding of the XRCC1 NTD to flexible single-strand break DNA.

Minimotif Miner: a tool for investigating protein function

In addition to large domains, many short motifs mediate functional post-translational modification of proteins as well as protein-protein interactions and protein trafficking functions. We have constructed a motif database comprising 312 unique motifs and a web-based tool for identifying motifs in proteins. Functional motifs predicted by MnM can be ranked by several approaches, and we validated these scores by analyzing thousands of confirmed examples and by confirming prediction of previously unidentified 14-3-3 motifs in EFF-1.

Minimotif miner 2nd release: a database and web system for motif search

Minimotif Miner (MnM) consists of a minimotif database and a web-based application that enables prediction of motif-based functions in user-supplied protein queries. We have revised MnM by expanding the database more than 10-fold to approximately 5000 motifs and standardized the motif function definitions. The web-application user interface has been redeveloped with new features including improved navigation, screencast-driven help, support for alias names and expanded SNP analysis. A sample analysis of prion shows how MnM 2 can be used. Weblink: http://mnm.engr.uconn.edu, weblink for version 1 is http://sms.engr.uconn.edu.

An automated tool for maximum entropy reconstruction of biomolecular NMR spectra

calculations are computationally costly (that is, efficiency is low). Modern large-scale ∆∆G prediction methods use heuristic algorithms with effective force fields and empirical parameters to estimate the stability changes caused by mutations in agreement with experimental data2–5. There are, however, two considerable drawbacks pertinent to the heuristic methods. First, most of these prediction methods rely on parameter training using available experimental ∆∆G data. Such training is usually biased toward mutations that feature large-to-small residue substitutions, such as alanine-scanning experiments (that is, poor transferability). Second, protein backbone flexibility, which is crucial for resolving atomic clashes and backbone strains in mutant proteins, is not considered in these methods, thereby reducing accuracy and limiting the application of heuristic methods (that is, limited applicability). To address the issues of efficiency, transferability and applicability, we developed the Eris method, which uses a physical force field with atomic modeling as well as fast side-chain packing and backbone relaxation algorithms. The free energy is expressed as a weighted sum of van der Waals forces, solvation, hydrogen bonding and backbone-dependent statistical energies6 (Supplementary Methods online). The weighting parameters are independently trained to recapitulate the native amino acid sequences for 34 proteins using high-resolution X-ray structures6. Additionally, an integral step of Eris is backbone relaxation when severe atom clashes or backbone strains are detected during calculation. We tested Eris on 595 mutants from five proteins, for which the ∆∆G values were documented (Fig. 1a). We found significant agreement between the predicted and measured ∆∆G values with a correlation coefficient of 0.75 (P = 2 × 10−108). The correlation between the predictions and experiments is comparable to that reported using other methods2–5. Unlike previous methods, Eris also has high predictive power for small-to-large3 sidechain-size mutations (Fig. 1b,c), owing to its ability to effectively relax backbone structures and resolve clashes introduced by mutations. As a direct comparison with other methods, we computed the stability changes of the small-to-large mutations using Eris and other web-based stability prediction servers. We found that Eris outperformed other available servers (Supplementary Discussion and Supplementary Tables 1 and 2 online). Additionally, Eris features a protein structure pre-relaxation option, which remarkably improves the prediction accuracy when a highresolution protein structure is not available (Supplementary Discussion and Supplementary Fig. 1 online). Our test validates the unbiased force field, side-chain packing and backbone relaxation algorithms in Eris. We anticipate Eris will be applicable to examining a much larger variety of mutations during protein engineering. We built a web-based Eris server for ∆∆G estimation. The server is freely accessible online (http:// eris.dokhlab.org).

Solution structure of a viral DNA repair polymerase.

DNA polymerase X (Pol X) from the African swine fever virus (ASFV) specifically binds intermediates in the single-nucleotide base-excision repair process, an activity indicative of repair function. In addition, Pol X catalyzes DNA polymerization with low nucleotide-insertion fidelity. The structural mechanisms by which DNA polymerases confer high or low fidelity in DNA polymerization remain to be elucidated. The three-dimensional structure of Pol X has been determined. Unlike other DNA polymerases, Pol X is formed from only a palm and a C-terminal subdomain. Pol X has a novel palm subdomain fold, containing a positively charged helix at the DNA binding surface. Purine deoxynucleoside triphosphate (dNTP) substrates bind between the palm and C-terminal subdomain, at a dNTP-binding helix, and induce a unique conformation in Pol X. The purine dNTP–bound conformation and high binding affinity for dGTP–Mg2+ of Pol X may contribute to its low fidelity.

Minimotif Miner 3.0: database expansion and significantly improved reduction of false-positive predictions from consensus sequences.

Minimotif Miner (MnM available at http://minimotifminer.org or http://mnm.engr.uconn.edu) is an online database for identifying new minimotifs in protein queries. Minimotifs are short contiguous peptide sequences that have a known function in at least one protein. Here we report the third release of the MnM database which has now grown 60-fold to approximately 300 000 minimotifs. Since short minimotifs are by their nature not very complex we also summarize a new set of false-positive filters and linear regression scoring that vastly enhance minimotif prediction accuracy on a test data set. This online database can be used to predict new functions in proteins and causes of disease.

Mapping of the Interaction Interface of DNA Polymerase β with XRCC1

Abstract Residues of DNA polymerase β (β-Pol) that interact with the DNA repair protein XRCC1 have been determined by NMR chemical shift mapping (CSM) and mutagenesis. 15 N/ 13 C/ 2 H/ 1 H, 13 C-methyl Leu,Ile,Val -labeled β-Pol palm-thumb domain was used for assignments of the 1 H, 15 N, and 13 C resonances used for CSM of the palm-thumb on forming the 40 kDa complex with the XRCC1 N-terminal domain (NTD). Large chemical shift changes were observed in the thumb on complexation. 15 N relaxation data indicate reduction in high-frequency motion for a thumb loop and three palm turn/loops, which showed concomitant chemical shift changes on complexation. A ΔV303–V306 deletion and an L301R/V303R/V306R triple mutation abolished complex formation due to loss in hydrophobicity. In an updated model, the thumb-loop of β-Pol contacts an edge/face region of the β sheet of the XRCC1 NTD, while the β-Pol palm weakly contacts the α2 helix.

VENN, a tool for titrating sequence conservation onto protein structures

Residue conservation is an important, established method for inferring protein function, modularity and specificity. It is important to recognize that it is the 3D spatial orientation of residues that drives sequence conservation. Considering this, we have built a new computational tool, VENN that allows researchers to interactively and graphically titrate sequence homology onto surface representations of protein structures. Our proposed titration strategies reveal critical details that are not readily identified using other existing tools. Analyses of a bZIP transcription factor and receptor recognition of Fibroblast Growth Factor using VENN revealed key specificity determinants. Weblink: http://sbtools.uchc.edu/venn/.

High-performance exact algorithms for motif search

Objective. The human genome project has resulted in the generation of voluminous biological data. Novel computational techniques are called for to extract useful information from this data. One such technique is that of finding patterns that are repeated over many sequences (and possibly over many species). In this paper we study the problem of identifying meaningful patterns (i.e., motifs) from biological data, the motif search problem. Methods. The general version of the motif search problem is NP-hard. Numerous algorithms have been proposed in the literature to solve this problem. Many of these algorithms fall under the category of heuristics. We concentrate on exact algorithms in this paper. In particular, we concentrate on two different versions of the motif search problem and offer exact algorithms for them. Results. In this paper we present algorithms for two versions of the motif search problem. All of our algorithms are elegant and use only such simple data structures as arrays. For the first version of the problem described as Problem 1 in the paper, we present a simple sorting based algorithm, SMS (Simple Motif Search). This algorithm has been coded and experimental results have been obtained. For the second version of the problem (described in the paper as Problem 2), we present two different algorithms – a deterministic algorithm (called DMS) and a randomized algorithm (Monte Carlo algorithm). We also show how these algorithms can be parallelized.Conclusions. All the algorithms proposed in this paper are improvements over existing algorithms for these versions of motif search in biological sequence data. The algorithms presented have the potential of performing well in practice.

A proposed syntax for Minimotif Semantics, version 1.

BackgroundOne of the most important developments in bioinformatics over the past few decades has been the observation that short linear peptide sequences (minimotifs) mediate many classes of cellular functions such as protein-protein interactions, molecular trafficking and post-translational modifications. As both the creators and curators of a database which catalogues minimotifs, Minimotif Miner, the authors have a unique perspective on the commonalities of the many functional roles of minimotifs. There is an obvious usefulness in standardizing functional annotations both in allowing for the facile exchange of data between various bioinformatics resources, as well as the internal clustering of sets of related data elements. With these two purposes in mind, the authors provide a proposed syntax for minimotif semantics primarily useful for functional annotation.ResultsHerein, we present a structured syntax of minimotifs and their functional annotation. A syntax-based model of minimotif function with established minimotif sequence definitions was implemented using a relational database management system (RDBMS). To assess the usefulness of our standardized semantics, a series of database queries and stored procedures were used to classify SH3 domain binding minimotifs into 10 groups spanning 700 unique binding sequences.ConclusionOur derived minimotif syntax is currently being used to normalize minimotif covalent chemistry and functional definitions within the MnM database. Analysis of SH3 binding minimotif data spanning many different studies within our database reveals unique attributes and frequencies which can be used to classify different types of binding minimotifs. Implementation of the syntax in the relational database enables the application of many different analysis protocols of minimotif data and is an important tool that will help to better understand specificity of minimotif-driven molecular interactions with proteins.