Ross I. Brinkworth

Structural basis and prediction of substrate specificity in protein serine/threonine kinases.

The large number of protein kinases makes it impractical to determine their specificities and substrates experimentally. Using the available crystal structures, molecular modeling, and sequence analyses of kinases and substrates, we developed a set of rules governing the binding of a heptapeptide substrate motif (surrounding the phosphorylation site) to the kinase and implemented these rules in a web-interfaced program for automated prediction of optimal substrate peptides, taking only the amino acid sequence of a protein kinase as input. We show the utility of the method by analyzing yeast cell cycle control and DNA damage checkpoint pathways. Our method is the only available predictive method generally applicable for identifying possible substrate proteins for protein serine/threonine kinases and helps in silico construction of signaling pathways. The accuracy of prediction is comparable to the accuracy of data from systematic large-scale experimental approaches.

Genetic and Histopathologic Evaluation of BRCA1 and BRCA2 DNA Sequence Variants of Unknown Clinical Significance

Classification of rare missense variants as neutral or disease causing is a challenge and has important implications for genetic counseling. A multifactorial likelihood model for classification of unclassified variants in BRCA1 and BRCA2 has previously been developed, which uses data on co-occurrence of the unclassified variant with pathogenic mutations in the same gene, cosegregation of the unclassified variant with affected status, and Grantham analysis of the fit between the missense substitution and the evolutionary range of variation observed at its position in the protein. We have further developed this model to take into account relevant features of BRCA1- and BRCA2-associated tumors, such as the characteristic histopathology and immunochemical profiles associated with pathogenic mutations in BRCA1, and the fact that approximately 80% of tumors from BRCA1 and BRCA2 carriers undergo inactivation of the wild-type allele by loss of heterozygosity. We examined 10 BRCA1 and 15 BRCA2 unclassified variants identified in Australian, multiple-case breast cancer families. By a combination of genetic, in silico, and histopathologic analyses, we were able to classify one BRCA1 variant as pathogenic and six BRCA1 and seven BRCA2 variants as neutral. Five of these neutral variants were also found in at least 1 of 180 healthy controls, suggesting that screening a large number of appropriate controls might be a useful adjunct to other methods for evaluation of unclassified variants.

Flavones are inhibitors of HIV-1 proteinase

Substituted gamma-chromones were found to weakly inhibit HIV-1 proteinase, an important enzyme in the replication and processing of the AIDS virus. Chromones bearing hydroxyl substituents and a phenolic group at the 2-position (flavones) were the most active compounds and structure-activity relationships for a limited series of flavone inhibitors are presented. Dixon plots are reported and a possible mechanism for flavone-induced inhibition is proposed. The results are also compared with those for some structurally related non-peptidic inhibitors of HIV-1 proteinase. Since some flavonoid compounds have already been shown to have antiviral activity against AIDS, the present observations of anti-HIV-1 proteinase activity may be particularly significant.

Multiple regions of human Fc gamma RII (CD32) contribute to the binding of IgG.

The low affinity receptor for IgG, FcγRII (CD32), has a wide distribution on hematopoietic cells where it is responsible for a diverse range of cellular responses crucial for immune regulation and resistance to infection. FcγRII is a member of the immunoglobulin superfamily, containing an extracellular region of two Ig-like domains. The IgG binding site of human FcγRII has been localized to an 8-amino acid segment of the second extracellular domain, Asn154-Ser161. In this study, evidence is presented to suggest that domain 1 and two additional regions of domain 2 also contribute to the binding of IgG by FcγRII. Chimeric receptors generated by exchanging the extracellular domains and segments of domain 2 between FcγRII and the structurally related FcεRI α chain were used to demonstrate that substitution of domain 1 in its entirety or the domain 2 regions encompassing residues Ser109-Val116 and Ser130-Thr135 resulted in a loss of the ability of these receptors to bind hIgG1 in dimeric form. Site-directed mutagenesis performed on individual residues within and flanking the Ser109-Val116 and Ser130-Thr135 domain 2 segments indicated that substitution of Lys113, Pro114, Leu115, Val116, Phe129, and His131 profoundly decreased the binding of hIgG1, whereas substitution of Asp133 and Pro134 increased binding. These findings suggest that not only is domain 1 contributing to the affinity of IgG binding by FcγRII but, importantly, that the domain 2 regions Ser109-Val116 and Phe129-Thr135 also play key roles in the binding of hIgG1. The location of these binding regions on a molecular model of the entire extracellular region of FcγRII indicates that they comprise loops that are juxtaposed in domain 2 at the interface with domain 1, with the putative crucial binding residues forming a hydrophobic pocket surrounded by a wall of predominantly aromatic and basic residues.

Homology model of the dengue 2 virus NS3 protease: putative interactions with both substrate and NS2B cofactor.

The crystal structure coordinates of the hepatitis C virus NS3 protease (HCVpro) were used to develop an homology model of the dengue 2 virus NS3 protease (DEN2pro). The amino acid sequence of DEN2pro accommodates the same alpha-helices, beta-sheets and protein-binding domains as its HCVpro counterpart, but the model predicts a number of significant differences for DEN2pro and its interactions with substrates and cofactor. Whereas HCVpro contains a Zn2+-binding site, there is no equivalent metal-binding motif in DEN2pro. It is possible that the structural role played by the zinc ion may be provided by a salt bridge between Glu93 and Lys145. The two-component viral protease comprises NS3 and a virus-encoded cofactor, NS4A for HCV and NS2B for DEN2. Previous studies have identified a central 40 amino acid cofactor domain of the dengue virus NS2B that is required for protease activity. Modelling of the putative interactions between DEN2pro and its cofactor suggests that a 12 amino acid hydrophobic region within this sequence (70GSSPILSITISE81) may associate directly with NS3. Modelling also suggests that the substrate binds in an extended conformation to the solvent-exposed surface of the protease, with a P1-binding site that is significantly different from its HCV counterpart. The model described in this study not only reveals unique features of the flavivirus protease but also provides a structural basis for both cofactor and substrate binding that should prove useful in the early design and development of inhibitors.

Clinical Classification of BRCA1 and BRCA2 DNA Sequence Variants: The Value of Cytokeratin Profiles and Evolutionary Analysis—A Report From the kConFab Investigators

PURPOSE Rare missense substitutions and in-frame deletions of BRCA1 and BRCA2 genes present a challenge for genetic counseling of individuals carrying such unclassified variants. We assessed the value of tumor immunohistochemical markers in conjunction with genetic and evolutionary approaches for investigating the clinical significance of unclassified variants. PATIENTS AND METHODS We studied 10 BRCA1 and 12 BRCA2 variants identified in Australian families with breast cancer. Analyses assumed a prior probability based on revised cross-species sequence alignment methods assessing amino acid evolutionary conservation and position, combined with likelihoods from data on co-occurrence with pathogenic mutations in the same gene, segregation analysis, and immunohistochemistry. We specifically explored the value of estrogen receptor, cytokeratin 5/6, and cytokeratin 14 as tumor markers of BRCA1 mutation status. RESULTS Posterior probabilities classified 72% of variants. BRCA1 variants IVS18+1 G>T (del exon 18) and 5632 T >A (V1838E) were classified as pathogenic, with >99% posterior probability of being deleterious, and tumor histopathology was particularly important for their classification. BRCA2 variant classification was improved over previous studies, largely by incorporating the prior probability of pathogenicity based on amino acid cross-species sequence alignments. CONCLUSION Variant classification was considerably improved by analysis of estrogen receptor, cytokeratin 5/6, and cytokeratin 14 tumor expression, and use of updated methods estimating the clinical relevance of amino acid evolutionary conservation and position. These methodologies may assist genetic counseling of individuals with unclassified sequence variants.

The Role of Receptor Dimerization Domain Residues in Growth Hormone Signaling

While there is a considerable amount of evidence that signal transduction by the growth hormone (GH) receptor requires receptor homodimerization, there has been no systematic study of the role of receptor dimerization domain residues in this process. In conjunction with the distances derived from the crystal structure of the hGH-hGH receptor (extracellular domain) complex, we have used a luciferase-based c-fos promoter reporter assay in transiently transfected Chinese hamster ovary (CHO) cells, and stable receptor expressing CHO cell populations to define the dimerization domain residues needed for effective signaling. In addition to alanine substitution, we have used both aspartate and lysine substitutions to allow us to provide evidence for proximity relations through charge complementation. Introduced cysteine substitutions were also used, but unlike the erythropoietin receptor, these were unable to generate constitutively active receptor. We conclude that serine 145, histidine 150, aspartate 152, tyrosine 200, and serine 201, but not leucine 146 or threonine 147 are required for effective signal transduction through the dimerization domain. This information may be valuable in designing small molecule antagonists of GH and other cytokines that block dimerization by binding to the dimerization domain.

Predikin and PredikinDB: a computational framework for the prediction of protein kinase peptide specificity and an associated database of phosphorylation sites

BackgroundWe have previously described an approach to predicting the substrate specificity of serine-threonine protein kinases. The method, named Predikin, identifies key conserved substrate-determining residues in the kinase catalytic domain that contact the substrate in the region of the phosphorylation site and so determine the sequence surrounding the phosphorylation site. Predikin was implemented originally as a web application written in Javascript.ResultsHere, we describe a new version of Predikin, completely revised and rewritten as a modular framework that provides multiple enhancements compared with the original. Predikin now consists of two components: (i) PredikinDB, a database of phosphorylation sites that links substrates to kinase sequences and (ii) a Perl module, which provides methods to classify protein kinases, reliably identify substrate-determining residues, generate scoring matrices and score putative phosphorylation sites in query sequences. The performance of Predikin as measured using receiver operator characteristic (ROC) graph analysis equals or surpasses that of existing comparable methods. The Predikin website has been redesigned to incorporate the new features.ConclusionNew features in Predikin include the use of SQL queries to PredikinDB to generate predictions, scoring of predictions, more reliable identification of substrate-determining residues and putative phosphorylation sites, extended options to handle protein kinase and substrate data and an improved web interface. The new features significantly enhance the ability of Predikin to analyse protein kinases and their substrates. Predikin is available at http://predikin.biosci.uq.edu.au.

Fine structure analysis of interaction of FcepsilonRI with IgE.

The high affinity receptor for IgE (FcεRI) plays an integral role in triggering IgE-mediated hypersensitivity reactions. The IgE-interactive site of human FcεRI has previously been broadly mapped to several large regions in the second extracellular domain (D2) of the α-subunit (FcεRIα). In this study, the IgE binding site of human FcεRIα has been further localized to subregions of D2, and key residues putatively involved in the interaction with IgE have been identified. Chimeric receptors generated between FcεRIα and the functionally distinct but structurally homologous low affinity receptor for IgG (FcγRIIa) have been used to localize two IgE binding regions of FcεRIα to amino acid segments Tyr129–His134 and Lys154–Glu161. Both regions were capable of independently binding IgE upon placement into FcγRIIa. Molecular modeling of the three-dimensional structure of FcεRIα-D2 has suggested that these binding regions correspond to the “exposed” C′-E and F-G loop regions at the membrane distal portion of the domain. A systematic site-directed mutagenesis strategy, whereby each residue in the Tyr129–His134 and Lys154–Glu161 regions of FcεRIα was replaced with alanine, has identified key residues putatively involved in the interaction with IgE. Substitution of Tyr131, Glu132, Val155, and Asp159decreased the binding of IgE, whereas substitution of Trp130, Trp156, Tyr160, and Glu161 increased binding. In addition, mutagenesis of residues Trp113, Val115, and Tyr116in the B-C loop region, which lies adjacent to the C′-E and F-G loops, has suggested Trp113 also contributes to IgE binding, since the substitution of this residue with alanine dramatically reduces binding. This information should prove valuable in the design of strategies to intervene in the FcεRIα-IgE interaction for the possible treatment of IgE-mediated allergic disease.

Protein kinases associated with the yeast phosphoproteome

BackgroundProtein phosphorylation is an extremely important mechanism of cellular regulation. A large-scale study of phosphoproteins in a whole-cell lysate of Saccharomyces cerevisiae has previously identified 383 phosphorylation sites in 216 peptide sequences. However, the protein kinases responsible for the phosphorylation of the identified proteins have not previously been assigned.ResultsWe used Predikin in combination with other bioinformatic tools, to predict which of 116 unique protein kinases in yeast phosphorylates each experimentally determined site in the phosphoproteome. The prediction was based on the match between the phosphorylated 7-residue sequence and the predicted substrate specificity of each kinase, with the highest weight applied to the residues or positions that contribute most to the substrate specificity. We estimated the reliability of the predictions by performing a parallel prediction on phosphopeptides for which the kinase has been experimentally determined.ConclusionThe results reveal that the functions of the protein kinases and their predicted phosphoprotein substrates are often correlated, for example in endocytosis, cytokinesis, transcription, replication, carbohydrate metabolism and stress response. The predictions link phosphoproteins of unknown function with protein kinases with known functions and vice versa, suggesting functions for the uncharacterized proteins. The study indicates that the phosphoproteins and the associated protein kinases represented in our dataset have housekeeping cellular roles; certain kinases are not represented because they may only be activated during specific cellular responses. Our results demonstrate the utility of our previously reported protein kinase substrate prediction approach (Predikin) as a tool for establishing links between kinases and phosphoproteins that can subsequently be tested experimentally.