Systematic Disruption of the Nonpolar β‐Helix Core of Hemolysin A and its Site‐Specific Effect on Protein Structure, Function, and Secretion

Abstract

Gram‐negative bacteria utilize a two‐partner secretion (TPS) pathway to export disease causing virulence factors. This pathway is a member of the Type Vb secretion systems which are thought to utilize the energy of protein folding to accomplish secretion across the outer membrane. In the TPS pathway, the secreted protein (TpsA) is produced along with a specific outer membrane protein (TpsB), which uses POTRA domains to recognize nonpolar segments on the TpsA. In our current model, this recognition leads to transport and folding of the TpsA protein via a Brownian ratchet mechanism. Utilizing this TPS pathway, Hemolysin A (HpmA), a hemolytic protein from Proteus mirabilis, is able to cross the bacterial outer membrane. A version of this protein, comprising a conserved TPS domain and truncated at position 265 (HpmA265), serves as a model for secretion and folding. HpmA265 adopts a β‐helical fold that encompasses three subdomains. Folded HpmA265 can function in trans as a template to fold and activate unfolded full‐length HpmA into its hemolytically active form (Template Assisted Hemolytic Activity – TAHA). This templating is thought to mimic the Brownian ratchet mechanism of secretion‐coupled folding of the full‐length protein. Previous work suggests that destabilization of the lowest stability subdomain enhances templating while destabilization of the second subdomain reduces templating ability. To identify the positional dependence of this effect, we have systematically replaced nonpolar amino acids with polar amino acids in the nonpolar core of the protein. Using CD spectroscopy to quantify the destabilization caused by each of the introduced variants, we have correlated the folding free energies of each variant with measured values of templating ability determined via TAHA assays. These measurements have allowed us to refine our model for how HpmA265 templates the folding of full‐length HpmA. In vivo, the nonpolar amino acids that were targeted may also play a role in the POTRA domain recognition. Previous data has shown that introduction of polar residues can increase secretion, suggesting that any destabilization of the folded form is offset by changes to POTRA domain interactions. To more fully map this effect, we have quantitatively determined the levels of secretion for each variant protein. Together, these results have allowed us to refine our model of the driving mechanisms in the TPS pathway, down to an amino acid level.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.