<p>The structural plasticity conferred by conformational flexibility has increasingly been recognized as a likely determinant of function. For example, multiscale heterogeneity in the calmodulin central helix most likely helps it in binding over 100 protein targets, and a concerted motion seen in both nuclear magnetic resonance (NMR) and crystal structures of ubiquitin is proposed to underlie its functional plasticity of promiscuous binding to many different proteins with high affinity. However, flexibility is manifested in a variety of ways, depending both on the protein itself and on how it is observed. Conformational heterogeneity (the term we use for flexibility when studied by X-ray crystallography) is evident in electron density, either as fully separated peaks or as anisotropic density shapes showing fluctuation of atom groupings. Many phenomena contribute to conformational heterogeneity in crystal structures, from diverse crystal contacts to functionally relevant conformational fluctuations on a wide range of time and size scales.</p><p>In addition to ubiquitin and calmodulin, the Staphylococcus aureus virulence factor staphylococcal protein A (SpA) is an example of a highly heterogeneous protein. SpA is a major contributor to bacterial evasion of the host immune system, through high-affinity binding to host proteins such as antibodies, von Willebrand factor, and tumor necrosis factor receptor 1 (TNFR1). The protein includes five small three-helix-bundle domains (E-D-A-B-C) separated by conserved flexible linkers. Prior attempts to crystallize individual domains in the absence of a binding partner were apparently unsuccessful. There are also no previous structures of tandem domains. In this thesis, I report the high-resolution crystal structures of a single C domain (collected at both cryogenic and room temperatures), a single A domain, and two B domains connected by the conserved linker. All four apo structures exhibit extensive multiscale conformational heterogeneity, which required novel modeling protocols. Comparison of domain structures shows that helix1 orientation is especially heterogeneous, coordinated with changes in sidechain conformational networks and contacting protein interfaces.</p><p>The interaction between a SpA domain and the Fc fragment of IgG was partially elucidated previously in the crystal structure 1FC2. Although informative, the previous structure wasn't properly folded and left many substantial questions unanswered, such as a detailed description of the tertiary structure of SpA domains in complex with Fc and the structural changes that take place upon binding. In this thesis, I report the 2.3-A structure of a fully folded SpA domain in complex with Fc. My structure indicates that there are extensive structural rearrangements necessary for binding Fc, including concerted rotamer changes and coupled backbone rearrangements that lead to a difference in helix1 angle. The conformational heterogeneity of the helix1/2 interface is also eliminated in the complex, with previously poly-rotameric interfacial residues locking into single rotamer conformations. Such a loss of conformational heterogeneity upon formation of the protein-protein interface may occur in SpA and in its multiple binding partners and may be an important structural paradigm in other functionally plastic proteins.</p> / Dissertation

Mycobacterium tuberculosis, the causative agent of pulmonary tuberculosis, infects one-third of the world’s human population. Despite the multiplicity of antimicrobial mechanisms mounted by its host, M. tuberculosis shows a remarkable ability to survive either by evoking survival strategies or by interference with critical macrophage functions that are required to successfully respond to the infection. It has been postulated that the outcome of exposure to M. tuberculosis (in terms of disease symptoms) largely depends upon the selective gene expression of tuberculosis bacilli along with activation of specific signaling pathways in the infected host cells during different phases of infection. In this perspective, determination of the complete genome sequence of Mycobacterium tuberculosis has provided crucial information with respect to the physiology of this bacterium and the pathogenesis of tuberculosis. However, putative functional annotation to all hypothetical proteins coded by M. tuberculosis genome remains complex. One important outcome of the genome-sequencing project was the discovery of two new multigene families designated PE and PPE. About 10% of the M. tuberculosis coding capacity is devoted to the PE and PPE genes, named for the Pro-Glu (PE) and Pro-Pro-Glu (PPE) motifs near the N terminus of their gene products. In addition to these motifs, proteins of PE family share N-terminal domains of approximately 100 amino acids, whereas the PPE proteins possess an N-terminal domain of about 180 amino acids. Many PE and PPE proteins are composed only of these N-terminal homologous domains. However, other members possess an additional C-terminal segment of variable length, often composed of multiple copies of polymorphic GC rich sequences (PGRS). The uniqueness of the PE genes is further illustrated by the fact that these genes are restricted to mycobacteria. However, despite their abundance in mycobacteria, very little is known regarding the expression or the functions of PE family genes. Although the PE and PPE families of mycobacterial proteins are the focus of intense research, no precise function has so far been unraveled for any member of these families. In perspective of above-mentioned observations, we have chosen Rv0754 as a representative PE family gene. Rv0754 was shown to be upregulated in tubercle bacilli upon infection of bone marrow derived macrophages as well as in M. tuberculosis isolated from alveolar macrophages of infected mice. In the current investigation, we demonstrate that Rv0754 is hypoxia responsive gene based on promoter or transcript expression analysis. Further, extensive bioinformatics analysis predicated that Rv0754 posses possible Phosphoglycerate Mutase domain, an enzyme known for its significant role not only in the glycolytic pathway of the carbohydrate metabolism, but also for the crucial cell fate decision during conditions like oxidative stress as well as infection. Experimental data clearly suggests that hypoxic environment dependent expression of Rv0754 imparts resistance to macrophages from oxidative stress. These findings could be attributed to the presence of catalytically active Phosphoglycerate Mutase domain of Rv0754. More often, sophisticated regulation/modulation of key signaling events regulate the critical cell fate decisions during oxidative stress. In this context, TLR2 dependent triggering of PI3K-ERK1/2- NF-κB signaling axis by Rv0754 may be operative in imparting resistance to oxidative stress. Further, Rv0754 triggers COX-2 expression by activating PI3K-ERK1/2-NF-κB cascade in mouse macrophages. These observations are of relevance as Rv0754 is associated with cell wall and is exposed outside the surface of the bacterium suggesting the possible access to intracellular compartments of the infected macrophages. Additionally, Rv0754 elicited humoral antibody reactivities in a panel of human sera or in cerebrospinal fluid samples obtained from different clinical categories of tuberculosis patients. DNA immunizations experiments in mice clearly suggested that Rv0754 is an immunodominant antigen demonstrating significant T cell and humoral reactivity. These observations clearly advocate that Rv0754 protein is expressed in vivo during active infection with M. tuberculosis and that the Rv0754 is immunogenic. Taken together, our findings suggest that Rv0754 is a novel PE_PGRS protein with unique features which could generate conditions that favor survival of the mycobacteria.