Understanding intramolecular signal transduction in regulated proteases of the High Temperature Requirement A family
Protein homeostasis in all organisms is a complex process involving regulatory mechanisms that govern protein synthesis, post-translational events and degradation. The protein degradation mechanism serves multiple functions ranging from maturation of cellular proteins, protein recycling and intracellular signal transduction. This mechanism, therefore, has a major role in diverse cellular and developmental contexts. In prokaryotes, the protein degradation mechanism has been shown to influence the cellular response to environmental stimuli and thus pathogenesis and virulence. The prokaryotic protein degradation machinery involves both regulated and processive proteases. Of these, the regulated proteases are particularly important as controlled and specific proteolysis are often key events in a signal transduction cascade triggered by intracellular or extracellular stimuli. The studies described in this thesis were designed to examine a specific aspect of regulated proteases wherein proteolytic activity could be either triggered or inhibited by a regulatory domain. These studies involve three regulatory proteases belonging to the High temperature requirement A (HtrA) family in Mycobacterium tuberculosis. These M. tuberculosis HtrA paralogues share common structural features with the well-characterized regulated protease E. coli DegS. HtrA proteases, in general, have a serine protease domain that is flexibly tethered to one or more PDZ (Post synaptic density protein-95; Drosophila disc-large tumor suppressor; Zonula occludens-1) domains at the C-terminus. HtrA proteases are membrane tethered. The membrane localization has been suggested to be significant from a functional perspective. The common features noted from multiple HtrA homologous characterized thus far include changes in quaternary structure and typically two distinct conformational states of catalytic triad (referred interchangeably as the inactive or tense state and the active or relaxed state). Tightly regulated proteolytic activity is thus achieved by an enzyme that is primarily in an inactive conformation and adopts an active conformation upon a specific trigger. Characterized triggers for HtrA activity are environmental triggers (such as high temperature), peptides or other effector ligands or substrate proteins. The modular organization of HtrA enzymes relies on the interactions between the sensory (tethered PDZ) domain with an effector ligand. Information from the PDZ domain is conveyed by concerted conformational changes to activate the catalytic domain. The intramolecular signal transduction paths thus play a key role in dictating the regulatory mechanism in HtrA enzymes. This thesis describes the structural features of M. tuberculosis HtrA and characterization of its biochemical features and regulatory mechanism(s). The first chapter of this thesis provides a brief introduction to regulated proteolytic mechanisms. Similarities and broad differences between the eukaryotic and prokaryotic mechanisms are discussed to phrase the research problem in the context of reported studies. The extensive studies performed on this enzyme, especially E. coli DegS, are summarized to place the known features and observations in the context of M. tuberculosis homologues. The characterized features of the M. tuberculosis HtrA paralogues are summarized to describe the scope of the studies reported in this thesis. A brief introduction to the diverse techniques employed in the course of these studies is also provided to place the methodology in context. The detailed methodology is included in different chapters alongside experimental information. Chapter two describes the crystal structure of M. tuberculosis HtrA at 1.83 Å resolution. This membrane-associated protease is essential for the survival of M. tuberculosis. The crystal structure revealed that the catalytic triad in HtrA is in an inactive conformation. This finding is consistent with the proposed role of M. tuberculosis HtrA as a regulatory protease that is conditionally activated upon appropriate environmental triggers. This structure provided a basis for directed studies to evaluate the role of this essential protein and the intracellular signal transduction mechanism that governs the activity of this protease. The intramolecular signal transduction process that is essential for regulated enzyme activity in HtrA proteases is described in chapter three. The first part of the study involves computational analysis and molecular dynamics (MD) simulations. This work is based on several ‘seed structures’ including those of E. coli DegS in peptide-bound and free forms, M. tuberculosis HtrA2 (PepD) in peptide-bound and apo forms and M. tuberculosis HtrA (HtrA1). These simulations provided multiple insights. The first was that of conformational sampling wherein the switch between the inactive (tense) arrangement of catalytic triad and the active (relaxed) arrangement of catalytic triad could be visualized. This finding, in effect, suggested that concerted conformational changes between the site in the PDZ domain that binds the effector ligand (peptide or any substrate) and the active site were essential for regulated proteolysis. While this aspect has long been discussed, the paths for intramolecular signal transduction remain poorly understood. In this study, we adopted a methodology involving difference energy calculations between the peptide bound and free forms of HtrA homologous. The emphasis in this context was on electrostatic interactions. Difference energy calculations, in turn, provided sites on the enzyme that could serve as nodes in the intramolecular signal transduction pathways. This analysis suggested multiple paths between the PDZ and the protease domain that could potentially be adopted for signal transduction. Three representative intramolecular signal transduction paths were experimentally validated by mutational analysis. Together, the computational studies and experimental observations provided a framework to understand intramolecular signal transduction paths that serve to transmit regulatory information. Chaperone activity in HtrA enzymes was suggested to be associated with the ability of these enzymes to adopt large oligomeric assemblies. The oligomeric assemblies of M. tuberculosis HtrA paralogues were evaluated in the presence of generic substrates such as β-casein or denatured lysozyme. These studies are described in chapter four of this thesis. Size exclusion chromatography suggested that M. tuberculosis HtrA (also referred to as HtrA1) and PepD (also referred to as HtrA2) existed in hexameric, trimeric and monomeric states. PepA (HtrA3) was prominently a trimer in solution. An important experimental insight on the differences between these paralogues came from an assay to evaluate the ability of these enzymes to inhibit protein aggregation. These assays suggested that PepD (HtrA2) was perhaps the best suited amongst three paralogues to serve this function. Small angle X-ray scattering methods provided information on the oligomeric assembly. While these were not conclusive due to experimental limitations, these studies suggested that higher order oligomers (such as dodecamer or di-dodecamer in E. coli DegP and DegQ) were not likely in the case of the M. tuberculosis paralogues. We note that conformational heterogeneity is positively correlated with chaperone activity as both HtrA and PepD adopt multiple oligomeric states. PepA (HtrA3), on the other hand, is the most homogeneous (trimer) and also the least effective in inhibiting protein aggregation. A broad summary of the findings from studies on regulated proteases is described in chapter five. The observations on the allosteric pathways also provide an insight into the mechanism of intramolecular signal transduction. These findings are likely to be useful from an enzyme engineering perspective wherein catalytic activity can be specifically triggered by effector ligand binding to the PDZ domain. Future studies based on the work reported in this thesis include identification of cognate triggers for the three M. tuberculosis HtrA paralogues. Together, the finding that the human pathogen M. tuberculosis has three paralogues of HtrA with diverse functional roles thus exemplifies how a functional diversity is embedded within a conserved structural scaffold. This thesis has four annexures. Annexure-I summarizes the experimental details and strategies that could not be incorporated in the main text of this thesis. Annexure-II enlisted the pairs of residues having difference electrostatic energy. Annexure-III describes a collaborative study performed on the M. tuberculosis σ factor σJ. Annexure-IV describes a collaborative project on the E. coli Arginine transporter ArgO.