The Tale of Two Domains

Abstract

Two-partner secretion (TPS) systems, harboring both A and B-components, are present in nearly all gram-negative bacteria. TpsB components selectively secrete their cognate TpsA exoprotein in an energy independent and vectorial manner across the outer membrane. Upon secretion, TpsA proteins function as adhesins, cytolysins, and heme binding proteins. In order to more fully understand the structure and folding of the TpsA protein family, a truncated form of hemolysin (HpmA265) was investigated. HpmA265 harbors a right-handed, parallel β-helix structure that is topped and flanked via segments of anti-parallel β-sheet. Previous structural studies have suggested a variety means to stabilize the β-helix structure including inner core electrostatic interactions and an inner core disulfide bond shared between cysteine 144 and 147. Equilibrium unfolding studies were implemented to investigate the impact of replacing glutamine 125 and the two cysteine residues on the stability of the HpmA265 β-helix structure. Multi-transitional denaturation results reported within this study provide evidence that the β-helix is comprised of two distinct and thermodynamically independent domains, termed the amino cap and β-helix core. The ΔG°H2O for folding the amino-cap is much larger than the β-helix core, implicating the buried and insulated hydrogen bonds as a primary means to stabilize the domain. Functionally, we propose the free-energy contribution from the stability within the amino-cap provides substantial selectivity and stability during the energy independent and vectorial folding of two-partner secretion pathway family members. This work was supported, in whole or in part, by National Science Foundation Grants MCB1050435 (to TMW) and MCB1434473 (to MRW), and a University Wisconsin – La Crosse Faculty Research Grant (to TMW).