Thursday, May 15, 2025
5/15/2025
Streptococcus pneumoniae HtrA and its target interactions - PROJECT SUMMARY High temperature requirement A (HtrA) enzymes represent a diverse family of serine proteases found across prokaryotes and eukaryotes that are critical for cellular homeostasis. In pathogenic bacteria, these enzymes are also shed from membranes after their N-terminal anchoring domain to target the host cell machinery for infection. However, the molecular mechanisms of the entire phylogenetic branch of Gram-positive bacteria that include S. pneumoniae HtrA remain largely unexplored. Considering that our biological studies have confirmed that S. pneumoniae HtrA is a virulence factor critical for infection, we propose to elucidate its molecular mechanism for the first time. The exploratory nature of this R21 is ideal for investigating the unique mechanistic features of the first family member from this phylogenetic branch of pathogenic Gram-positive bacteria. Our discovery that S. pneumoniae HtrA is a stable monomer provides a unique opportunity to address fundamental questions. For example, most HtrAs form trimers and some higher order oligomers. HtrAs have been crystallized in both an “open” form that is substrate-accessible and “closed” form where the PDZ blocks the PD active site, prompting questions as to how this occurs. Our preliminary findings of S. pneumoniae HtrA resolve such issues, as we can directly detect a dynamic process of “opened” and “closed” conformational sampling through NMR within the monomer and observe a stepwise oligomer formation used for substrate engagement through EM. Thus, we propose to fully elucidate this dynamic “opening”/“closing” of S. pneumoniae HtrA (Aim 1) and determine how this first family member within this largely unexplored Gram-positive phylogenetic branch oligomerizes for target engagement (Aim 2). We hypothesize that S. pneumoniae HtrA PDZ “opens” and “closes” over the PD active site while oligomerization and target formation involve unique conformational changes specific for this phylogenic branch. Our hypothesis will be addressed through the following specific aims: Aim 1: What are the structural rearrangements and associated dynamics of the S. pneumoniae HtrA monomer? NMR will be used to determine the conformational changes that occur between “opened” and “closed” conformations within the HtrA monomer along with relaxation experiments used to probe the underlying dynamics and cooperativity of such changes. Aim 2: What is the structural basis of S. pneumoniae HtrA oligomerization and substrate engagement? Cryo-EM will be used to elucidate the structural basis of HtrA oligomerization. Specifically, we will determine the structure of HtrA S234A mutant, which allosterically triggers trimer formation, and HtrA in complex with -casein, which induces hexamer formation. These studies will identify the molecular determinants responsible for the hierarchy of oligomerization.
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