Allosteric Modulation of Binding Specificity by Alternative Packing of Protein Cores.

Hydrophobic cores are often viewed as tightly packed and rigid, but they do show some plasticity and could thus be attractive targets for protein design. Here we explored the role of different functional pressures on the core packing and ligand recognition of the SH3 domain from human Fyn tyrosine kinase. We randomized the hydrophobic core and used phage display to select variants that bound to each of three distinct ligands. The three evolved groups showed remarkable differences in core composition, illustrating the effect of different selective pressures on the core. Changes in the core did not significantly alter protein stability, but were linked closely to changes in binding affinity and specificity. Structural analysis and molecular dynamics simulations revealed the structural basis for altered specificity. The evolved domains had significantly reduced core volumes, which in turn induced increased backbone flexibility. These motions were propagated from the core to the binding surface and induced significant conformational changes. These results show that alternative core packing and consequent allosteric modulation of binding interfaces could be used to engineer proteins with novel functions.

Data driven flexible backbone protein design.

Protein design remains an important problem in computational structural biology. Current computational protein design methods largely use physics-based methods, which make use of information from a single protein structure. This is despite the fact that multiple structures of many protein folds are now readily available in the PDB. While ensemble protein design methods can use multiple protein structures, they treat each structure independently. Here, we introduce a flexible backbone strategy, FlexiBaL-GP, which learns global protein backbone movements directly from multiple protein structures. FlexiBaL-GP uses the machine learning method of Gaussian Process Latent Variable Models to learn a lower dimensional representation of the protein coordinates that best represent backbone movements. These learned backbone movements are used to explore alternative protein backbones, while engineering a protein within a parallel tempered MCMC framework. Using the human ubiquitin-USP21 complex as a model we demonstrate that our design strategy outperforms current strategies for the interface design task of identifying tight binding ubiquitin variants for USP21.

Protein engineering by highly parallel screening of computationally designed variants.

Current combinatorial selection strategies for protein engineering have been successful at generating binders against a range of targets; however, the combinatorial nature of the libraries and their vast undersampling of sequence space inherently limit these methods due to the difficulty in finely controlling protein properties of the engineered region. Meanwhile, great advances in computational protein design that can address these issues have largely been underutilized. We describe an integrated approach that computationally designs thousands of individual protein binders for high-throughput synthesis and selection to engineer high-affinity binders. We show that a computationally designed library enriches for tight-binding variants by many orders of magnitude as compared to conventional randomization strategies. We thus demonstrate the feasibility of our approach in a proof-of-concept study and successfully obtain low-nanomolar binders using in vitro and in vivo selection systems.

Computational analysis of interactomes: current and future perspectives for bioinformatics approaches to model the host-pathogen interaction space.

Bacterial and viral pathogens affect their eukaryotic host partly by interacting with proteins of the host cell. Hence, to investigate infection from a systems' perspective we need to construct complete and accurate host-pathogen protein-protein interaction networks. Because of the paucity of available data and the cost associated with experimental approaches, any construction and analysis of such a network in the near future has to rely on computational predictions. Specifically, this challenge consists of a number of sub-problems: First, prediction of possible pathogen interactors (e.g. effector proteins) is necessary for bacteria and protozoa. Second, the prospective host binding partners have to be determined and finally, the impact on the host cell analyzed. This review gives an overview of current bioinformatics approaches to obtain and understand host-pathogen interactions. As an application example of the methods covered, we predict host-pathogen interactions of Salmonella and discuss the value of these predictions as a prospective for further research.

Network evolution: rewiring and signatures of conservation in signaling.

The analysis of network evolution has been hampered by limited availability of protein interaction data for different organisms. In this study, we investigate evolutionary mechanisms in Src Homology 3 (SH3) domain and kinase interaction networks using high-resolution specificity profiles. We constructed and examined networks for 23 fungal species ranging from Saccharomyces cerevisiae to Schizosaccharomyces pombe. We quantify rates of different rewiring mechanisms and show that interaction change through binding site evolution is faster than through gene gain or loss. We found that SH3 interactions evolve swiftly, at rates similar to those found in phosphoregulation evolution. Importantly, we show that interaction changes are sufficiently rapid to exhibit saturation phenomena at the observed timescales. Finally, focusing on the SH3 interaction network, we observe extensive clustering of binding sites on target proteins by SH3 domains and a strong correlation between the number of domains that bind a target protein (target in-degree) and interaction conservation. The relationship between in-degree and interaction conservation is driven by two different effects, namely the number of clusters that correspond to interaction interfaces and the number of domains that bind to each cluster leads to sequence specific conservation, which in turn results in interaction conservation. In summary, we uncover several network evolution mechanisms likely to generalize across peptide recognition modules.

Evolution of biological interaction networks: from models to real data.

We are beginning to uncover common mechanisms leading to the evolution of biological networks. The driving force behind these advances is the increasing availability of comparative data in several species.

deFuse: an algorithm for gene fusion discovery in tumor RNA-Seq data.

Gene fusions created by somatic genomic rearrangements are known to play an important role in the onset and development of some cancers, such as lymphomas and sarcomas. RNA-Seq (whole transcriptome shotgun sequencing) is proving to be a useful tool for the discovery of novel gene fusions in cancer transcriptomes. However, algorithmic methods for the discovery of gene fusions using RNA-Seq data remain underdeveloped. We have developed deFuse, a novel computational method for fusion discovery in tumor RNA-Seq data. Unlike existing methods that use only unique best-hit alignments and consider only fusion boundaries at the ends of known exons, deFuse considers all alignments and all possible locations for fusion boundaries. As a result, deFuse is able to identify fusion sequences with demonstrably better sensitivity than previous approaches. To increase the specificity of our approach, we curated a list of 60 true positive and 61 true negative fusion sequences (as confirmed by RT-PCR), and have trained an adaboost classifier on 11 novel features of the sequence data. The resulting classifier has an estimated value of 0.91 for the area under the ROC curve. We have used deFuse to discover gene fusions in 40 ovarian tumor samples, one ovarian cancer cell line, and three sarcoma samples. We report herein the first gene fusions discovered in ovarian cancer. We conclude that gene fusions are not infrequent events in ovarian cancer and that these events have the potential to substantially alter the expression patterns of the genes involved; gene fusions should therefore be considered in efforts to comprehensively characterize the mutational profiles of ovarian cancer transcriptomes.

Diverse protein kinase interactions identified by protein microarrays reveal novel connections between cellular processes.

Protein kinases are key regulators of cellular processes. In spite of considerable effort, a full understanding of the pathways they participate in remains elusive. We globally investigated the proteins that interact with the majority of yeast protein kinases using protein microarrays. Eighty-five kinases were purified and used to probe yeast proteome microarrays. One-thousand-twenty-three interactions were identified, and the vast majority were novel. Coimmunoprecipitation experiments indicate that many of these interactions occurred in vivo. Many novel links of kinases to previously distinct cellular pathways were discovered. For example, the well-studied Kss1 filamentous pathway was found to bind components of diverse cellular pathways, such as those of the stress response pathway and the Ccr4-Not transcriptional/translational regulatory complex; genetic tests revealed that these different components operate in the filamentation pathway in vivo. Overall, our results indicate that kinases operate in a highly interconnected network that coordinates many activities of the proteome. Our results further demonstrate that protein microarrays uncover a diverse set of interactions not observed previously.

SNVMix: predicting single nucleotide variants from next-generation sequencing of tumors.

MOTIVATION: Next-generation sequencing (NGS) has enabled whole genome and transcriptome single nucleotide variant (SNV) discovery in cancer. NGS produces millions of short sequence reads that, once aligned to a reference genome sequence, can be interpreted for the presence of SNVs. Although tools exist for SNV discovery from NGS data, none are specifically suited to work with data from tumors, where altered ploidy and tumor cellularity impact the statistical expectations of SNV discovery. RESULTS: We developed three implementations of a probabilistic Binomial mixture model, called SNVMix, designed to infer SNVs from NGS data from tumors to address this problem. The first models allelic counts as observations and infers SNVs and model parameters using an expectation maximization (EM) algorithm and is therefore capable of adjusting to deviation of allelic frequencies inherent in genomically unstable tumor genomes. The second models nucleotide and mapping qualities of the reads by probabilistically weighting the contribution of a read/nucleotide to the inference of a SNV based on the confidence we have in the base call and the read alignment. The third combines filtering out low-quality data in addition to probabilistic weighting of the qualities. We quantitatively evaluated these approaches on 16 ovarian cancer RNASeq datasets with matched genotyping arrays and a human breast cancer genome sequenced to >40x (haploid) coverage with ground truth data and show systematically that the SNVMix models outperform competing approaches. AVAILABILITY: Software and data are available at http://compbio.bccrc.ca CONTACT: sshah@bccrc.ca SUPPLEMANTARY INFORMATION: Supplementary data are available at Bioinformatics online.