FROM HOMOHEPTAMERS TO HETEROHEPTAMERS: AN APPROACH TO THE STRUCTURE DETERMINATION OF HETEROMERIC TRANSMEMBRANE CHANNELS. L. Song*, M.R. Hobaugh*, O. Braha#, B.J. Walker#, H. Bayley# and J.E. Gouaux*, *Dept. of Biochem. & Mol. Biol., University of Chicago, 920 E. 58th St., Chicago IL 60637, #Worcester Found. for Biomedical Research, 222 Maple Avenue, Shrewsbury MA 01545.

Staphylococcus aureus [[alpha]]-hemolysin ([[alpha]]HL) is a lytic toxin that forms transmembrane channels by assembling 7 identical, water-soluble subunits on the surface of erythrocytes or lipid bilayers. [[alpha]]HL is an ideal system for engineering channels and for understanding mechanisms of selectivity, gating and inhibition of ion channels. The mushroom-shaped heptamer structure has been determined in the Gouaux lab to a resolution of 1.9 Å. Defining the transmembrane channel is a 14-strand antiparallel ß-barrel that is approximately 20 Å in diameter and 50 Å long, measured from C_[[alpha]] to C_[[alpha]].

We aim to determine structures of heteroheptamers in which only one or a few amino acids on a single subunit have been changed. To do this, we must break the 7-fold axis of noncrystallographic symmetry. By examination of interheptamer contacts in the wild-type C2 crystal form¹, we found that S69 is located within a short section of interheptamer antiparallel ß-sheet close to the 2-fold crystallographic axis.

To test whether this site is suitable for introduction of a disulfide bond, coordinates for cysteine residues comprising a disulfide bond between 2 antiparallel strands were obtained from the 1.7 Å resolution crystal structure of restrictocin². Perturbations to this interface and estimation of disulfide-bond strain were evaluated by energy minimization of wild-type and disulfide bonded [[alpha]]HL subunits. The resulting structures were essentially identical and the disulfide bond adopts favorable stereochemistry. We will present data on the formation, crystallization and crystallographic analysis of heteroheptameric transmembrane channels.

¹Gouaux, J.E., Braha, O., Hobaugh, M.R., Song, L., Cheley, S., Shustak, C. and Bayley, H. (1994) Proc. Natl. Acad. Sci. USA 91 12828-31.

²Yang, X.-J. (1995) Ph.D. Thesis, University of Chicago, Chicago IL 60637.