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Pi‐system Stabilization of a 220 Å 3 Buried Solvent Network
Author(s) -
Woods Christopher,
Novak Walter,
Grilley Daniel,
Weaver Todd
Publication year - 2015
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.29.1_supplement.882.2
Subject(s) - hemolysin , chemistry , hydrogen bond , crystallography , solvent , molecule , crystal structure , stereochemistry , mutant , biochemistry , organic chemistry , virulence , gene
Two‐partner secretion (TPS) systems are ubiquitous among gram‐negative bacteria and are composed of an A‐component (TpsA) and a B‐component (TpsB). The subject of this study is hemolysin A, a TpsA component from P. mirabilis , which is secreted and concomitantly activated by its TpsB partner. Once activated, hemolysin A functions to lyse red blood cells. Previous work in our lab has revealed the structure of truncated hemolysin A (HpmA265), which forms a right‐handed parallel β‐helix. Peculiarly, a buried solvent, which forms hydrogen bonds with Q125 and Y134, and a 38 Å 3 anhydrous void were found near the first two complete beta‐circuits of HpmA265. In this work, site‐directed mutants, which replace Q125, Y134 or both Q125 and Y134 with serine residues, have been built into the HpmA265 framework to disrupt both the hydrogen bond network and expand the internal void. The thermodynamic unfolding results for each mutant support the preservation of native stability and the two‐domain architecture. Of most importance, the double serine replacement crystal structure fills the unoccupied space left by the removal of the Q125 carboxamide and Y134 aryl functional groups with six completely solvent inaccessible waters. The unprecedented organizational arrangement of these six water molecules (1) occupies a completely solvent inaccessible volume of 220 Å 3 and (2) involves pi‐bonding to two buried phenylalanine residues, F80 and F149. The adaptations in this region have led to the preservation of the inherent β‐helix stability and structural water, S1, as observed in the wild type HpmA265 crystal structure. The research was supported by NSF‐RUI award 1050435 (TW), ASBMB UAN Summer Research Award (CW), and a UW‐La Crosse URC grant (CW).