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J. Biol. Chem., Vol. 281, Issue 4, 2195-2204, January 27, 2006

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Role of the Amino Latch of Staphylococcal -Hemolysin in Pore Formation

A CO-OPERATIVE INTERACTION BETWEEN THE N TERMINUS AND POSITION 217^*

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

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

Staphylococcal -hemolysin (HL) is a barrel pore-forming toxin that is secreted by the bacterium as a water-soluble monomeric protein. Upon binding to susceptible cells, HL assembles via an inactive prepore to form a water-filled homoheptameric transmembrane pore. The N terminus of HL, which in the crystal structure of the fully assembled pore forms a latch between adjacent subunits, has been thought to play a vital role in the prepore to pore conversion. For example, the deletion of two N-terminal residues produced a completely inactive protein that was arrested in assembly at the prepore stage. In the present study, we have re-examined assembly with a comprehensive set of truncation mutants. Surprisingly, we found that after truncation of up to 17 amino acids, the ability of HL to form functional pores was diminished, but still substantial. We then discovered that the mutation Ser²¹⁷ Asn, which was present in our original set of truncations but not in the new ones, promotes complete inactivation upon truncation of the N terminus. Therefore, the N terminus of HL cannot be critical for the prepore to pore transformation as previously thought. Residue 217 is involved in the assembly process and must interact indirectly with the distant N terminus during the last step in pore formation. In addition, we provide evidence that an intact N terminus prevents the premature oligomerization of HL monomers in solution.

Received for publication, October 4, 2005

^* This work was supported by the MRC/EPSRC and the ONR. Work at Texas A&M was supported by DARPA, the U.S. Department of Defense 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, NCI, National Institutes of Health, Bldg. 10, Rm. 2N206, 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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