ABSTRACT
Wild-type and variant forms of HpmA265 (truncated hemolysin A) from Proteus mirabilis reveal a right-handed, parallel ß-helix capped and flanked by segments of antiparallel ß-strands. The low-salt crystal structures form a dimeric structure via the implementation of on-edge main-chain hydrogen bonds donated by residues 243-263 of adjacent monomers. Surprisingly, in the high-salt structures of two variants, Y134A and Q125A-Y134A, a new dimeric interface is formed via main-chain hydrogen bonds donated by residues 203-215 of adjacent monomers, and a previously unobserved tetramer is formed. In addition, an eight-stranded antiparallel ß-sheet is formed from the flap regions of crystallographically related monomers in the high-salt structures. This new interface is possible owing to additional proteolysis of these variants after Tyr240. The interface formed in the high-salt crystal forms of hemolysin A variants may mimic the on-edge ß-strand positioning used in template-assisted hemolytic activity.
Subject(s)
Bacterial Proteins/chemistry , Hemolysin Proteins/chemistry , Proteus mirabilis/chemistry , Amino Acid Sequence , Bacterial Proteins/genetics , Bacterial Proteins/metabolism , Binding Sites , Cloning, Molecular , Crystallography, X-Ray , Escherichia coli/genetics , Escherichia coli/metabolism , Gene Expression , Hemolysin Proteins/genetics , Hemolysin Proteins/metabolism , Hydrogen Bonding , Models, Molecular , Mutation , Plasmids/chemistry , Plasmids/metabolism , Protein Binding , Protein Conformation, beta-Strand , Protein Interaction Domains and Motifs , Protein Multimerization , Protein Structure, Quaternary , Protein Structure, Tertiary , Proteolysis , Proteus mirabilis/metabolism , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/metabolismABSTRACT
Protein secretion is a major contributor to Gram-negative bacterial virulence. Type Vb or two-partner secretion (TPS) pathways utilize a membrane bound ß-barrel B component (TpsB) to translocate large and predominantly virulent exoproteins (TpsA) through a nucleotide independent mechanism. We focused our studies on a truncated TpsA member termed hemolysin A (HpmA265), a structurally and functionally characterized TPS domain from Proteus mirabilis. Contrary to the expectation that the TPS domain of HpmA265 would denature in a single cooperative transition, we found that the unfolding follows a sequential model with three distinct transitions linking four states. The solvent inaccessible core of HpmA265 can be divided into two different regions. The C-proximal region contains nonpolar residues and forms a prototypical hydrophobic core as found in globular proteins. The N-proximal region of the solvent inaccessible core, however, contains polar residues. To understand the contributions of the hydrophobic and polar interiors to overall TPS domain stability, we conducted unfolding studies on HpmA265 and site-specific mutants of HpmA265. By correlating the effect of individual site-specific mutations with the sequential unfolding results we were able to divide the HpmA265 TPS domain into polar core, nonpolar core, and C-terminal subdomains. Moreover, the unfolding studies provide quantitative evidence that the folding free energy for the polar core subdomain is more favorable than for the nonpolar core and C-terminal subdomains. This study implicates the hydrogen bonds shared among these conserved internal residues as a primary means for stabilizing the N-proximal polar core subdomain.
