Structural and functional studies of truncated hemolysin A from Proteus mirabilis.
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 beta-helix with three adjoining segments of anti-parallel beta-sheet. A CXXC disulfide bond, four buried solvent molecules, and a carboxyamide ladder were all located at the third complete beta-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 beta-sheet interactions between neighboring beta-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, beta-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.
Weaver
Todd M
TM
Departments of Chemistry, La Crosse, Wisconsin 54601.
Hocking
Jason M
JM
Departments of Chemistry, La Crosse, Wisconsin 54601.
Bailey
Lucas J
LJ
Departments of Chemistry, La Crosse, Wisconsin 54601.
Wawrzyn
Grayson T
GT
Departments of Chemistry, La Crosse, Wisconsin 54601.
Howard
David R
DR
Biology, University Wisconsin-La Crosse, La Crosse, Wisconsin 54601.
Sikkink
Laura A
LA
the Departments of Biochemistry and Molecular Biology, Rochester, Minnesota 55905.
Ramirez-Alvarado
Marina
M
the Departments of Biochemistry and Molecular Biology, Rochester, Minnesota 55905.
Thompson
James R
JR
Physiology and Biomedical Imaging, College of Medicine, Mayo Clinic, Rochester, Minnesota 55905.
D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D002942:Circular Dichroism; D004220:Disulfides; D015964:Gene Expression Regulation, Bacterial; D006460:Hemolysin Proteins / Q000737:chemistry*; D006461:Hemolysis; D018613:Microscopy, Confocal / Q000379:methods; D008954:Models, Biological; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011487:Protein Conformation; D017510:Protein Folding; D017433:Protein Structure, Secondary; D011513:Proteus mirabilis / Q000378:metabolism*
D004220:Disulfides; D006460:Hemolysin Proteins