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J. Biol. Chem., Vol. 281, Issue 4, 2205-2214, January 27, 2006

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Assembly of the Bi-component Leukocidin Pore Examined by Truncation Mutagenesis^*

George Miles1, Lakmal Jayasinghe, and Hagan Bayley, Holder of a Royal Society-Wolfson Research Merit Award2

From the Department of Medical Biochemistry & Genetics, Texas A&M University System Health Science Center, College Station, Texas 77843-1114 and the Department of Chemistry, University of Oxford, Chemistry Research Laboratory, Mansfield Road, Oxford OX1 3TA, United Kingdom

Staphylococcal leukocidin (Luk) and -hemolysin (HL) are members of the same family of barrel pore-forming toxins (PFTs). Although the HL pore is a homoheptamer, the Luk pore is formed by the co-assembly of four copies each of the two distantly related polypeptides, LukF and LukS, to form an octamer. Here, we examine N- and C-terminal truncation mutants of LukF and LukS. LukF subunits missing up to nineteen N-terminal amino acids are capable of producing stable, functional hetero-oligomers with WT LukS. LukS subunits missing up to fourteen N-terminal amino acids perform similarly in combination with WT LukF. Further, the simultaneous truncation of both LukF and LukS is tolerated. Both Luk subunits are vulnerable to short deletions at the C terminus. Interestingly, the N terminus of the LukS polypeptide becomes resistant to proteolytic digestion in the fully assembled Luk pore while the N terminus of LukF remains in an exposed conformation. The results from this work and related experiments on HL suggest that, although the N termini of PFTs may undergo reorganization during assembly, they are dispensable for the formation of functional pores.

Received for publication, October 4, 2005 , and in revised form, October 28, 2005.

^* This work was supported by the Medical Research Council/Engineering and Physical Sciences Research Council and the Office of Naval Research. Work at Texas A&M was supported by Defense Advanced Research Project Agency, the DoD Tri-Service Technology Program, the U.S. Department of Energy, NASA, the National Institutes of Health, and the ONR. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ Present address: Laboratory of Pathology, National Institutes of Health, Bethesda, MD 20892.

² To whom correspondence should be addressed: Tel.: 44-1865-285-101; Fax: 44-1865-275-708; E-mail: hagan.bayley{at}chem.ox.ac.uk.

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				L. Jayasinghe, G. Miles, and H. Bayley Role of the Amino Latch of Staphylococcal {alpha}-Hemolysin in Pore Formation: A CO-OPERATIVE INTERACTION BETWEEN THE N TERMINUS AND POSITION 217 J. Biol. Chem., January 27, 2006; 281(4): 2195 - 2204. [Abstract] [Full Text] [PDF]