Integrated Analysis of Residue Coevolution and Protein Structure in ABC Transporters

Intraprotein side chain contacts can couple the evolutionary process of amino acid substitution at one position to that at another. This coupling, known as residue coevolution, may vary in strength. Conserved contacts thus not only define 3-dimensional protein structure, but also indicate which residue-residue interactions are crucial to a protein’s function. Therefore, prediction of strongly coevolving residue-pairs helps clarify molecular mechanisms underlying function. Previously, various coevolution detectors have been employed separately to predict these pairs purely from multiple sequence alignments, while disregarding available structural information. This study introduces an integrative framework that improves the accuracy of such predictions, relative to previous approaches, by combining multiple coevolution detectors and incorporating structural contact information. This framework is applied to the ABC-B and ABC-C transporter families, which include the drug exporter P-glycoprotein involved in multidrug resistance of cancer cells, as well as the CFTR chloride channel linked to cystic fibrosis disease. The predicted coevolving pairs are further analyzed based on conformational changes inferred from outward- and inward-facing transporter structures. The analysis suggests that some pairs coevolved to directly regulate conformational changes of the alternating-access transport mechanism, while others to stabilize rigid-body-like components of the protein structure. Moreover, some identified pairs correspond to residues previously implicated in cystic fibrosis

Systematic Experimental Determination of Functional Constraints on Proteins and Adaptive Potential of Mutations: A Dissertation

Sequence-function relationship is a fundamental question for many branches of modern biomedical research. It connects the primary sequence of proteins to the function of proteins and fitness of organisms, holding answers for critical questions such as functional consequences of mutations identified in whole genome sequencing and adaptive potential of fast evolving pathogenic viruses and microbes. Many different approaches have been developed to delineate the genotype-phenotype map for different proteins, but are generally limited by their throughput or precision. To systematically quantify the fitness of large numbers of mutations, I modified a novel high throughput mutational scanning approach (EMPIRIC) to investigate the fitness landscape of mutations in important regions of essential proteins from the yeast or RNA viruses. Using EMPIRIC, I analyzed the interplay of the expression level and sequence of Hsp90 on the yeast growth and revealed latent effect of mutations at reduced expression levels of Hsp90. I also examined the functional constraint on the receptor binding site of the Env of Human Immunodeficiency Virus (HIV) and uncovered enhanced receptor binding capacity as a common pathway for adaptation of HIV to laboratory conditions. Moreover, I explored the adaptive potential of neuraminidase (NA) of influenza A virus to a NA inhibitor, oseltamivir, and identified novel oseltamivir resistance mutations with distinct molecular mechanisms. In summary, I applied a high throughput functional genomics approach to map the sequence-function relationship in various systems and examined the evolutionary constraints and adaptive potential of essential proteins ranging from molecular chaperones to drug-targetable viral proteins

Rigidity and flexibility of biological networks

The network approach became a widely used tool to understand the behaviour of complex systems in the last decade. We start from a short description of structural rigidity theory. A detailed account on the combinatorial rigidity analysis of protein structures, as well as local flexibility measures of proteins and their applications in explaining allostery and thermostability is given. We also briefly discuss the network aspects of cytoskeletal tensegrity. Finally, we show the importance of the balance between functional flexibility and rigidity in protein-protein interaction, metabolic, gene regulatory and neuronal networks. Our summary raises the possibility that the concepts of flexibility and rigidity can be generalized to all networks.Comment: 21 pages, 4 figures, 1 tabl

A gaussian mixture-based approach to synthesizing nonlinear feature functions for automated object detection

Feature design is an important part to identify objects of interest into a known number of categories or classes in object detection. Based on the depth-first search for higher order feature functions, the technique of automated feature synthesis is generally considered to be a process of creating more effective features from raw feature data during the run of the algorithms. This dynamic synthesis of nonlinear feature functions is a challenging problem in object detection. This thesis presents a combinatorial approach of genetic programming and the expectation maximization algorithm (GP-EM) to synthesize nonlinear feature functions automatically in order to solve the given tasks of object detection. The EM algorithm investigates the use of Gaussian mixture which is able to model the behaviour of the training samples during an optimal GP search strategy. Based on the Gaussian probability assumption, the GP-EM method is capable of performing simultaneously dynamic feature synthesis and model-based generalization. The EM part of the approach leads to the application of the maximum likelihood (ML) operation that provides protection against inter-cluster data separation and thus exhibits improved convergence. Additionally, with the GP-EM method, an innovative technique, called the histogram region of interest by thresholds (HROIBT), is introduced for diagnosing protein conformation defects (PCD) from microscopic imagery. The experimental results show that the proposed approach improves the detection accuracy and efficiency of pattern object discovery, as compared to single GP-based feature synthesis methods and also a number of other object detection systems. The GP-EM method projects the hyperspace of the raw data onto lower-dimensional spaces efficiently, resulting in faster computational classification processes

An interdomain sector mediating allostery in Hsp70 molecular chaperones

The Hsp70 family of molecular chaperones provides a well defined and experimentally powerful model system for understanding allosteric coupling between different protein domains.New extensions to the statistical coupling analysis (SCA) method permit identification of a group of co-evolving amino-acid positions—a sector—in the Hsp70 that is associated with allosteric function.Literature-based and new experimental studies support the notion that the protein sector identified through SCA underlies the allosteric mechanism of Hsp70.This work extends the concept of protein sectors by showing that two non-homologous protein domains can share a single sector when the underlying biological function is defined by the coupled activity of the two domains

ENHANCing the limb: from micro- to macro-evolution

Abstract Understanding the molecular basis of the diverse morphological forms found within and across species is a longstanding goal in evolutionary biology. One especially relevant class of cis-regulatory elements are enhancers. This is because mutations affecting enhancers tend to be tissue- or stage-specific, which allows adaptation to proceed with relatively less harmful side effects in other organs or tissues. In Chapter 2 I explore how enhancers help drive morphological selection response within-species. We scanned the genomes of the Longshanks mice, which are mice selectively bred over 20 generations, for a 13% increase in tibiae. Against a backdrop of polygenic response, we found the bone repressor Nkx3-2, and specifically its enhancers, to be among the strongest contributor towards increased tibia length. I used transgenics to compare the enhancer activity of the F0 and F17 alleles at 3 candidate enhancers (two near the Nkx3-2 gene; and one near the limb developmental regulator gene, Gli3). We found that both loss-of-function (Nkx3-2) and gain-of-function (Gli3) alleles contributed to the selection response. In Chapter 3, we explored an approach to study macro-evolutionary variations across species. One of the major barriers to such study is the inability to perform direct genetic crosses due to hybrid sterility. We tackle the species barrier problem by inducing mitotic recombination in vitro in hybrid embryonic stem cells (including cross-species hybrids between Mus musculus and Mus spretus). This was achieved via Blm inhibition by the small molecule ML216. We further show, that the resultant mitotic recombinant cells can be used for genetic mapping by connecting tioguanine drug resistance to variations at the Hprt locus. Furthermore, in vitro recombinant stem cells can be used for rederivation of animals through laser-assisted morula injection, thus allowing the acquisition of morphological data. Here, through a multidisciplinary approach, we show that enhancer modulation contributes to morphological diversity and selection response within-species and provide a new methodology for enhancer study across-species, thus enabling the study of evolutionary developmental variations in genetic backgrounds that would otherwise be challenging to obtain. Overall, these studies highlight the relevance of enhancers in morphological diversification and provide new tools for their study

Comprehensively Surveying Structure and Function of RING Domains from Drosophila melanogaster

Using a complete set of RING domains from Drosophila melanogaster, all the solved RING domains and cocrystal structures of RING-containing ubiquitin-ligases (RING-E3) and ubiquitin-conjugating enzyme (E2) pairs, we analyzed RING domains structures from their primary to quarternary structures. The results showed that: i) putative orthologs of RING domains between Drosophila melanogaster and the human largely occur (118/139, 84.9%); ii) of the 118 orthologous pairs from Drosophila melanogaster and the human, 117 pairs (117/118, 99.2%) were found to retain entirely uniform domain architectures, only Iap2/Diap2 experienced evolutionary expansion of domain architecture; iii) 4 evolutionary structurally conserved regions (SCRs) are responsible for homologous folding of RING domains at the superfamily level; iv) besides the conserved Cys/His chelating zinc ions, 6 equivalent residues (4 hydrophobic and 2 polar residues) in the SCRs possess good-consensus and conservation- these 4 SCRs function in the structural positioning of 6 equivalent residues as determinants for RING-E3 catalysis; v) members of these RING proteins located nucleus, multiple subcellular compartments, membrane protein and mitochondrion are respectively 42 (42/139, 30.2%), 71 (71/139, 51.1%), 22 (22/139, 15.8%) and 4 (4/139, 2.9%); vi) CG15104 (Topors) and CG1134 (Mul1) in C3HC4, and CG3929 (Deltex) in C3H2C3 seem to display broader E2s binding profiles than other RING-E3s; vii) analyzing intermolecular interfaces of E2/RING-E3 complexes indicate that residues directly interacting with E2s are all from the SCRs in RING domains. Of the 6 residues, 2 hydrophobic ones contribute to constructing the conserved hydrophobic core, while the 2 hydrophobic and 2 polar residues directly participate in E2/RING-E3 interactions. Based on sequence and structural data, SCRs, conserved equivalent residues and features of intermolecular interfaces were extracted, highlighting the presence of a nucleus for RING domain fold and formation of catalytic core in which related residues and regions exhibit preferential evolutionary conservation

Detecting Repetitions and Periodicities in Proteins by Tiling the Structural Space

The notion of energy landscapes provides conceptual tools for understanding the complexities of protein folding and function. Energy Landscape Theory indicates that it is much easier to find sequences that satisfy the "Principle of Minimal Frustration" when the folded structure is symmetric (Wolynes, P. G. Symmetry and the Energy Landscapes of Biomolecules. Proc. Natl. Acad. Sci. U.S.A. 1996, 93, 14249-14255). Similarly, repeats and structural mosaics may be fundamentally related to landscapes with multiple embedded funnels. Here we present analytical tools to detect and compare structural repetitions in protein molecules. By an exhaustive analysis of the distribution of structural repeats using a robust metric we define those portions of a protein molecule that best describe the overall structure as a tessellation of basic units. The patterns produced by such tessellations provide intuitive representations of the repeating regions and their association towards higher order arrangements. We find that some protein architectures can be described as nearly periodic, while in others clear separations between repetitions exist. Since the method is independent of amino acid sequence information we can identify structural units that can be encoded by a variety of distinct amino acid sequences