Journal of Biological Chemistry
Volume 284, Issue 33, 14 August 2009, Pages 22297-22309
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Protein Structure and Folding
Structural and Functional Studies of Truncated Hemolysin A from Proteus mirabilis*

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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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The atomic coordinates and structure factors (code3FY3) have been deposited in the Protein Data Bank, Research Collaboratory for Structural Bioinformatics, Rutgers University, New Brunswick, NJ (http://www.rcsb.org/).

*

This work was supported, in whole or in part, by National Institutes of Health Grant AI057437 (to T. W.). This work was also supported by National Science Foundation Grant MCB0744754 (to T. W.), by the University of Wisconsin (UW)-La Crosse Faculty Research Program (to T. W.), and by a UW-La Crosse, College of Science and Health Dean舗s Summer Fellowship (to G. W.) supported the work. A National Science Foundation Grant DBI619289 (to Jennifer Miskowski, UW-La Crosse, Dept. of Biology) supported the laser scanning confocal microscopy studies.