Structural and Functional Effects of Altering the Nonpolar Core of Hemolysin A

Abstract

The protein structure and function paradigm is a foundational tenet of biomolecular science that underlies many infectious diseases. Hemolysin A (HpmA), a hemolytic protein produced by Proteus mirabilis, was used as a model to investigate the protein structure‐function paradigm. HpmA is a member of the two‐partner secretion (TPS) pathway, which is used by gram‐negative bacteria to export predominantly virulent proteins outside of the cell. Through this mechanism, the A‐component (HpmA) is translocated, folded, and activated by its cognate B‐component in the absence of high‐energy bond and electrochemical gradient dependency. All TpsA components are relatively large and can be further divided into two domains, the two‐partner secretion domain and the functional domain. Universally, all known TPS domains harbor a right‐handed, parallel b‐helix architecture, which has been shown critical for cognate TpsB‐dependent recognition and secretion. Conversely, functional domains provide TpsA diversification including, cytolysis, host cell adhesion, contact‐growth inhibition, and iron sequestration. A truncated version of hemolysin A (HpmA265) was implemented to define the contributions of the TPS domain toward TpsA structure and function. Recently, our group has further dissected HpmA265 into three sequentially folded structural units termed the polar core, non‐polar core, and carboxy‐terminal subdomains. This research project aims to expand upon our recent results and structurally map the role of the nonpolar core during TPS domain dependent secretion, folding, and function. Specifically, residues within the non‐polar core subdomain have been selectively targeted and modified. The structural and functional effects of these site‐selective modifications have been evaluated via chemical denaturation, protease sensitivity, and template‐assisted hemolytic activity. Site‐selective alterations within the non‐polar core subdomain shift four‐state b‐helix unfolding to a three‐state model, increased protease sensitivity, and decrease template‐assisted hemolysis. In contrast, site‐selective alterations on the exterior of the non‐polar core subdomain maintain four‐state unfolding and protease sensitivity, and increase template‐assisted hemolysis. Collectively, these results point toward TPS b‐helix stability as a requisite for robust template‐assisted hemolysis. Conservation of the TPS domain across gram‐negative bacteria makes development of antibiotics targeting b‐helix structure and function attractive.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.