TY - JOUR
T1 - Structural and functional studies of truncated hemolysin A from Proteus mirabilis
AU - Weaver, Todd M.
AU - Hocking, Jason M.
AU - Bailey, Lucas J.
AU - Wawrzyn, Grayson T.
AU - Howard, David R.
AU - Sikkink, Laura A.
AU - Ramirez-Alvarado, Marina
AU - Thompson, James R.
PY - 2009/8/14
Y1 - 2009/8/14
N2 - In this study we analyzed the structure and function of a truncated form of hemolysin A (HpmA265) from Proteus mirabilis using a series of functional and structural studies. Hemolysin A belongs to the two-partner secretion pathway. The two-partner secretion pathway has been identified as the most common protein secretion pathway among Gram-negative bacteria. Currently, the mechanism of action for the two-partner hemolysin members is not fully understood. In this study, hemolysis experiments revealed a unidirectional, cooperative, biphasic activity profile after full-length, inactive hemolysin A was seeded with truncated hemolysin A. We also solved the first x-ray structure of a TpsA hemolysin. The truncated hemolysin A formed a right-handed parallel β-helix with three adjoining segments of anti-parallel β-sheet. A CXXC disulfide bond, four buried solvent molecules, and a carboxyamide ladder were all located at the third complete β-helix coil. Replacement of the CXXC motif led to decreased activity and stability according to hemolysis and CD studies. Furthermore, the crystal structure revealed a sterically compatible, dry dimeric interface formed via anti-parallel β-sheet interactions between neighboring β-helix monomers. Laser scanning confocal microscopy further supported the unidirectional interconversion of full-length hemolysin A. From these results, a model has been proposed, where cooperative, β-strand interactions between HpmA265 and neighboring full-length hemolysin A molecules, facilitated in part by the highly conserved CXXC pattern, account for the template-assisted hemolysis.
AB - In this study we analyzed the structure and function of a truncated form of hemolysin A (HpmA265) from Proteus mirabilis using a series of functional and structural studies. Hemolysin A belongs to the two-partner secretion pathway. The two-partner secretion pathway has been identified as the most common protein secretion pathway among Gram-negative bacteria. Currently, the mechanism of action for the two-partner hemolysin members is not fully understood. In this study, hemolysis experiments revealed a unidirectional, cooperative, biphasic activity profile after full-length, inactive hemolysin A was seeded with truncated hemolysin A. We also solved the first x-ray structure of a TpsA hemolysin. The truncated hemolysin A formed a right-handed parallel β-helix with three adjoining segments of anti-parallel β-sheet. A CXXC disulfide bond, four buried solvent molecules, and a carboxyamide ladder were all located at the third complete β-helix coil. Replacement of the CXXC motif led to decreased activity and stability according to hemolysis and CD studies. Furthermore, the crystal structure revealed a sterically compatible, dry dimeric interface formed via anti-parallel β-sheet interactions between neighboring β-helix monomers. Laser scanning confocal microscopy further supported the unidirectional interconversion of full-length hemolysin A. From these results, a model has been proposed, where cooperative, β-strand interactions between HpmA265 and neighboring full-length hemolysin A molecules, facilitated in part by the highly conserved CXXC pattern, account for the template-assisted hemolysis.
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U2 - 10.1074/jbc.M109.014431
DO - 10.1074/jbc.M109.014431
M3 - Article
C2 - 19494116
AN - SCOPUS:69249201386
SN - 0021-9258
VL - 284
SP - 22297
EP - 22309
JO - Journal of Biological Chemistry
JF - Journal of Biological Chemistry
IS - 33
ER -