Sequential unfolding of the hemolysin two-partner secretion domain from Proteus mirabilis.
Protein secretion is a major contributor to Gram-negative bacterial virulence. Type Vb or two-partner secretion (TPS) pathways utilize a membrane bound β-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.
Department of Chemistry and Biochemistry, University Wisconsin - La Crosse, La Crosse, Wisconsin, 54601.
Megan R
MR
Wimmer
Department of Chemistry and Biochemistry, University Wisconsin - La Crosse, La Crosse, Wisconsin, 54601.
Christopher N
CN
Woods
Department of Chemistry and Biochemistry, University Wisconsin - La Crosse, La Crosse, Wisconsin, 54601.
Kyle J
KJ
Adamczak
Department of Chemistry and Biochemistry, University Wisconsin - La Crosse, La Crosse, Wisconsin, 54601.
Evan M
EM
Glasgow
Department of Chemistry, Wabash College, Crawfordsville, Indiana, 47933.
Walter R P
WR
Novak
Department of Chemistry and Biochemistry, University Wisconsin - La Crosse, La Crosse, Wisconsin, 54601.
Daniel P
DP
Grilley
Department of Chemistry and Biochemistry, University Wisconsin - La Crosse, La Crosse, Wisconsin, 54601.
Todd M
TM
Weaver
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry* / Q000378:metabolism*; D006460:Hemolysin Proteins / Q000737:chemistry* / Q000378:metabolism*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D017434:Protein Structure, Tertiary; D058767:Protein Unfolding; D011513:Proteus mirabilis / Q000737:chemistry*; D016415:Sequence Alignment
D001426:Bacterial Proteins; D006460:Hemolysin Proteins
beta-helix; domain; protein folding; sequential unfolding; subdomain; two-partner secretion