The long range objectives of this research are to determine molecular mechanisms for the pathogenic action of cytolytic toxins and for the folding, assembly, pore-forming properties and regulation of oligomeric transmembrane pores in general. alpha-Hemolysin (alpha-He), one of the important virulence factors from Staphylococcus aureus, provides an excellent system for elucidating the molecular mechanisms for pore-forming toxins, protein insertion into a membrane, folding and assembly of a defined multimeric aggregate, and for formation of a transmembrane pore which is regulated by divalent cations. alpha-He is secreted from S. aureus as a water-soluble monomer (MW 33 kDa) and upon encountering an appropriate membrane environment, such as that provided by an erythrocyte, the monomer binds to the membrane, oligomerizes to form a hexamer and the hexamer then inserts through the bilayer to create a water-filled transmembrane pore. This initiates the leakage of ions and small molecules and finally, cell lysis occurs.
The specific aims proposed in this application are to determine the 3-D structures of the monomer and the hexamer, to map the surfaces of the monomer and the hexamer that interact with the membrane, to elucidate the conformational changes that accompany the monomer to hexamer oligomerization, to identify the amino acids that line the interior of the pore, to locate the binding sites for inhibitory divalent cations and to formulate detailed molecular mechanisms to describe the assembly, function and regulation of alpha-He. These goals will be accomplished by determining 3-D structures of alpha-He using x-ray diffraction. Specifically, the structures of the detergent- solubilized hexamer will be solved using the multiple isomorphous replacement (MIR) technique, the structure of the monomer will be determined by either MIR or by molecular replacement (MR), and the structures of important site-directed mutants, protein-ligand complexes and alternative crystal forms will be determined by MR. Over 12 different crystal forms of the detergent-solubilized hexamer have been obtained, at least one of which is suitable for a high resolution structure determination, and two crystal forms of the monomer have been found. Interpretation of the structures will be aided by molecular graphics and computational techniques. On the basis of the proposed studies, general models will be developed for the assembly and function of oligomeric membrane proteins which will be relevant to other membrane proteins such as the acetylcholine receptor, the gap junction and the complement attack complex. In addition, an understanding of the structural mechanism of action of alpha-He may reveal new approaches for the treatment of bacterial infections. Furthermore, these structural studies will provide a starting point for the rational and systematic design of immunotoxins that could be used to selectively target and kill cancer cells.
