Structural and functional studies of truncated hemolysin A from Proteus mirabilis

Todd M. Weaver, Jason M. Hocking, Lucas J. Bailey, Grayson T. Wawrzyn, David R. Howard, Laura A. Sikkink, Marina Ramirez-Alvarado, James R. Thompson

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Abstract

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.

Original languageEnglish (US)
Pages (from-to)22297-22309
Number of pages13
JournalJournal of Biological Chemistry
Volume284
Issue number33
DOIs
StatePublished - Aug 14 2009

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Proteus mirabilis
Hemolysin Proteins
Secretory Pathway
Hemolysis
Molecules
Confocal microscopy
Ladders
Gram-Negative Bacteria
Confocal Microscopy
Disulfides
Bacteria
Monomers
Crystal structure
X-Rays
Scanning
X rays
Lasers

ASJC Scopus subject areas

  • Biochemistry
  • Cell Biology
  • Molecular Biology

Cite this

Weaver, T. M., Hocking, J. M., Bailey, L. J., Wawrzyn, G. T., Howard, D. R., Sikkink, L. A., ... Thompson, J. R. (2009). Structural and functional studies of truncated hemolysin A from Proteus mirabilis. Journal of Biological Chemistry, 284(33), 22297-22309. https://doi.org/10.1074/jbc.M109.014431

Structural and functional studies of truncated hemolysin A from Proteus mirabilis. / Weaver, Todd M.; Hocking, Jason M.; Bailey, Lucas J.; Wawrzyn, Grayson T.; Howard, David R.; Sikkink, Laura A.; Ramirez-Alvarado, Marina; Thompson, James R.

In: Journal of Biological Chemistry, Vol. 284, No. 33, 14.08.2009, p. 22297-22309.

Research output: Contribution to journalArticle

Weaver, TM, Hocking, JM, Bailey, LJ, Wawrzyn, GT, Howard, DR, Sikkink, LA, Ramirez-Alvarado, M & Thompson, JR 2009, 'Structural and functional studies of truncated hemolysin A from Proteus mirabilis', Journal of Biological Chemistry, vol. 284, no. 33, pp. 22297-22309. https://doi.org/10.1074/jbc.M109.014431
Weaver TM, Hocking JM, Bailey LJ, Wawrzyn GT, Howard DR, Sikkink LA et al. Structural and functional studies of truncated hemolysin A from Proteus mirabilis. Journal of Biological Chemistry. 2009 Aug 14;284(33):22297-22309. https://doi.org/10.1074/jbc.M109.014431
Weaver, Todd M. ; Hocking, Jason M. ; Bailey, Lucas J. ; Wawrzyn, Grayson T. ; Howard, David R. ; Sikkink, Laura A. ; Ramirez-Alvarado, Marina ; Thompson, James R. / Structural and functional studies of truncated hemolysin A from Proteus mirabilis. In: Journal of Biological Chemistry. 2009 ; Vol. 284, No. 33. pp. 22297-22309.
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