THE STRUCTURE OF THE [[alpha]]-HEMOLYSIN TRANSMEMBRANE PORE IN NATIVE AND DIVALENT CATION INHIBITED FORMS. Michael R. Hobaugh¹, Langzhou Song¹, Christopher Shustak², Steven Cheley², Hagan Bayley², and J.Eric Gouaux¹. Department of Biochemistry and Molecular Biology, University of Chicago, Chicago, IL 60637¹, Worcester Foundation for Biomedical Research,Shrewsbury, MA 015452.

[[alpha]]-Hemolysin ([[alpha]]HL), a primary virulence factor in Staphylococcus aureus infection, forms a heptameric transmembrane pore ([[alpha]]₇HL) on susceptible mammalian cells leading to cell lysis. This activity is inhibited by mM concentrations of divalent cations (M²⁺)¹. In addition, assembly of [[alpha]]₇HL from the water-soluble monomer is inhibited by M²⁺ and mutants have been engineered which contain a M²⁺ actuated switch whose inhibition may be relieved by addition of EDTA².

We have solved the structure of the native [[alpha]]₇HL crystallized from ß-OG, ammonium sulfate, cacodylate, and PEG by MIR³. [[alpha]]₇HL is predominantly ß-sheet and forms a mushroom-like structure where the hydrophobic stem of the mushroom penetrates the host cell membrane. Examination of the interior of the pore reveals that the diameter ranges from a maximum of ~65 Å to a constriction of ~12 Å in which seven glutamic acids (Glu 111) protrude into the channel at the base of the stem where it joins the head of the mushroom.

Because the divalent polycation uranyl binds at this position in the structure of our uranyl derivative we believe that this position may be the site of binding for M²⁺. Crystals utilizing a variety of M²⁺, buffers, and PEGs have been characterized. Their diffraction limit is ~2.8 Å using conventional sources and all occur in the same space group (P1) with the same unit cell dimensions (a=173.5 Å, b=172.5 Å, c=101.1 Å, a=90.5deg., ß=89.5deg., [[gamma]]=95deg. 11'). Data collection on these crystal is ongoing and the structure will be solved by molecular replacement.

¹S.Harshman, N.Sugg, Infec. and Immun. 47: 37-40 (1985).

²B.Walker, J.Kasianowicz, M.Krishnasastry, H.Bayley,Prot. Engin. 7:655-662 (1994).

³Manuscript in preparation.