Ion-translocating ATPases are key cellular enzymes that utilize the free energy of ATP hydrolysis to generate transmembrane electrochemical ion gradients that in turn fuel a variety of vital biological processes including absorption, secretion, transmembrane signalling, nerve impulse transmission, excitation/contraction coupling, and growth and differentiation. A major family of ion-translocating ATPase with about 80 members from all parts of the evolutionary tree is the P-type ATPase family, so named for the participation of a high energy aspartyl- phosphoryl-enzyme intermediate in their catalytic cycle. An elucidation Of the molecular mechanism by which the P-type ATPases transduce the chemical energy of ATP hydrolysis into transmembrane ion gradients is a primary goal remaining in the transport field. A great deal of experimental effort has been invested to this end, but progress has been hindered by a lack of structural information about these enzymes due to the notorious difficulty of obtaining crystals of membrane proteins useful for X-ray and electron microscopic structure analysis. In this laboratory, both three-dimensional (3D) and two-dimensional (2D) crystals of the P- type proton-translocating ATPase from the plasma membrane of neurospora have been obtained, and the structure of this transporter has been determined by electron cryomicroscopy of the 2D crystals at a resolution of 8 Angstroms in the membrane plane. The experiments described in this proposal are designed to extend the resolution of the current H+-ATPase structure and elucidate the precise nature of the conformational changes that are known to occur during the H+-ATPase transport cycle, so that a feasible molecular mechanism can be formulated. Specifically, we hope l) to obtain a 6 Angstrom structure of the H+-ATPase by X-ray crystallography of the 3D crystals, 2) to extend the resolution of the H+ATPase structure and determine the path of the polypeptide chain and the localization of the active site, and 3) to determine the structures of the H+-ATPase in two additional conformations it assumes during its transport cycle. Another very important transport ATPase family, particularly relevant to human disease states, is the ATP binding cassette (ABC) transporter family. Prominent members of this family include the human multidrug resistance protein, or P-glycoprotein (Pgp), and the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR). In the past several years, we have developed a very effective yeast expression system for producing the Pgp and the CFTR in quantities adequate for biochemical studies, and have worked out procedures for their detergent solubilization and purification. The fourth specific aim of this proposal is to develop procedures for crystallizing the Pgp and the CFT'R, utilizing the methods we have developed with the H+-ATPase, and other methods as well. We shall then proceed with structure determinations of these two important human membrane proteins.
