β‐helix structural stability and malleability affords both a buried water network and an unoccupied 200 Å3 void
Authors: Weaver Todd , Bhattacharyya Basudeb , Novak Walter 
Published: November 2023

β‐helix structures are a unique structural domain typified by the helical arrangement of parallel β‐strands. The uniqueness of the β‐helix structure can be further deconstructed into individual β‐circuits, where each β‐circuit describes a single revolution of three contiguous parallel β‐strands. Inherently, the cooperative formation of consecutive β‐circuits is driven by on‐edge main chain hydrogen bonds between adjacent parallel β‐strands. This cooperatively folded domain affords both structural (1) stability and (2) malleability. Therefore, the β‐helix structure has been recruited as a robust structural domain for numerous bacterial virulence factors, including adhesins, hemolysins, and heme‐binding proteins.A truncated version of hemolysin A (HpmA265) from Proteus mirabilis has been implemented as a model to probe the stability and malleability of the β‐helix structure. Hemolysin A belongs to the two‐partner secretion pathway, which is the most widely distributed protein secretion system within gram‐negative bacteria. Hemolysin A is secreted and concomitantly activated via hemolysin B, its cognate TPS outer membrane β‐barrel component. Structurally, HpmA265 harbors the putative TPS domain, but lacks the functional pore‐forming domain. Recent equilibrium unfolding studies have dissected the larger TPS domain into three sequentially folding subdomains, termed the polar core, non‐polar core, and carboxy‐terminal subdomains. Moreover, polar core subdomain was proposed to serve as a template to couple the vectorial alignment of parallel β‐strands with nucleotide independent protein secretion across the outer membrane.In order to probe key interactions as associated with β‐helix stability and malleability, a series of HpmA265 variants were prepared and analyzed functionally and structurally. The first set of variants was constructed to investigate the importance of a previously observed buried hydrogen bond between Gln125 and Tyr134 within the polar core subdomain. Additionally, the importance of the on‐edge hydrogen bonds was investigated through the limited proteolysis of HpmA265. Replacement of Gln125 and Tyr134 was conducted singly and in tandem with alanine, serine or phenylalanine. Each of the polar core variants was analyzed structurally via equilibrium unfolding and x‐ray crystallographic studies. Interestingly, the double alanine replacement at Gln125 and Tyr134 establishes a 200 Å3 cavity buried within the HpmA265 β‐helix structure. Moreover, the double serine replacement establishes an unprecedented inner core hydrogen bond network involving seven well‐resolved water molecules. The high resolution crystallographic results allowed visualization and placement of each water molecule buried within the HpmA265 β‐helix structure, while the equilibrium unfolding studies supported destabilization within the polar core subdomain. The limited proteolysis study has established a previously unobserved HpmA265 dimerization interface facilitated via newly exposed on‐edge main chain hydrogen bond partners. This newly observed dimerization interface extends cooperative β‐circuit formation via the implementation of exposed parallel β‐strands donated by adjacent monomers. Collectively, the results support the β‐helix as a stable and malleable structural domain.Support or Funding InformationThis research used resources of the Advanced Photon Source, a U.S. Department of Energy (DOE) Office of Science User Facility operated for the DOE Office of Science by Argonne National Laboratory under Contract No. DE‐AC02‐06CH11357. Use of the LS‐CAT Sector 21 was supported by the Michigan Economic Development Corporation and the Michigan Technology Tri‐Corridor (Grant 085P1000817). GM/CA@APS has been funded in whole or in part with Federal funds from the National Cancer Institute (ACB‐12002) and the National Institute of General Medical Sciences (AGM‐12006). The research was supported in part by National Science Foundation Grant: MCB1050435 (TW) and a University Wisconsin – La Crosse Faculty Research Grant (TW).