A Model for the Proteolipid Ring and Bafilomycin/Concanamycin-binding Site in the Vacuolar ATPase ofNeurospora crassa

Barry J. Bowman, Mary E. McCall, Robert Baertsch, Emma Jean Bowman
2006 Journal of Biological Chemistry  
The vacuolar ATPase has been implicated in a variety of physiological processes in eukaryotic cells. Bafilomycin and concanamycin, highly potent and specific inhibitors of the vacuolar ATPase, have been widely used to investigate the enzyme. Derivatives have been developed as possible therapeutic drugs. We have used random mutagenesis and site-directed mutagenesis to identify 23 residues in the c subunit involved in binding these drugs. We generated a model for the structure of the ring of c
more » ... units in Neurospora crassa by using data from the crystal structure of the homologous subunits of the bacterium Enterococcus hirae (Murata, T., Yamato, I., Kakinuma, Y., Leslie, A. G., and Walker, J. E. (2005) Science 308, 654 -659). In the model 10 of the 11 mutation sites that confer the highest degree of resistance are closely clustered. They form a putative drug-binding pocket at the interface between helices 1 and 2 on one c subunit and helix 4 of the adjacent c subunit. The excellent fit of the N. crassa sequence to the E. hirae structure and the degree to which the structural model predicts the clustering of these residues suggest that the folding of the bacterial and eukaryotic polypeptides is very similar. The vacuolar ATPase (V-ATPase) 2 is a component of almost all the eukaryotic cell membranes derived from the endoplasmic reticulum, e.g. Golgi, vacuoles, and coated vesicles (1-4). It generates an electrochemical gradient for protons, providing a driving force for metabolite transport. It also plays a major role in adjusting the pH within organelles. In animals many diverse physiological processes depend on the function of the V-ATPase. For example, a single mutation of the enzyme in humans was shown to cause both loss of kidney function and deafness (5). The V-ATPase is critical for the growth of bone (6), and overexpression of the enzyme has been correlated with the metastasis of breast cancer cells (7). The common thread linking these physiological events is secretion of protons by specialized cells that have high amounts of V-ATPase in the plasma membrane. A high rate of proton transport by the V-ATPase appears to be a key element in the development of diseases such as osteoporosis and in the growth of certain types of tumors. These findings have led to an investigation of the use of V-ATPase inhibitors as therapeutic drugs (8, 9) . Several different classes of natural products, such as the benzolactone enamides and the chondropsins, have been shown to be potent and specific inhibitors of V-ATPases (10 -12). The most widely used inhibitors are two similar macrolide antibiotics, bafilomycin and concanamycin. These molecules inhibit the V-ATPase from all eukaryotic cells that have been tested, typically with half-maximal inhibition at 0.1-5 nM when assayed in vitro (13-15). At higher concentrations these antibiotics also inhibit in vivo, and derivatives of bafilomycin were effective in treating osteoporosis in a rat model system (16). We have been trying to identify the site on the V-ATPase at which bafilomycin and concanamycin bind and inhibit (17, 18) . A precise definition of this site could be useful for development of therapeutic drugs. In addition it would lead to a much better understanding of the structure and mechanism of the V-ATPase, a large and complex enzyme. The proton-translocating part of the enzyme, named Vo, is composed of six types of subunits embedded in the membrane, with a reported stoichiometry of a 1 c 4 -5 cЈ 1 cЉ 1 d 1 e 1 (1, 4). ATP is hydrolyzed within the V1 part of the enzyme, a roughly globular structure connected to Vo by at least two stalks (19, 20) . V1 contains eight types of subunits with a reported stoichiometry of A 3 B 3 CDEFG 2 H 1-2 . Like the F-type ATPase of mitochondria, chloroplasts, and eubacteria, the V-ATPase functions as a rotary motor. In current models, hydrolysis of ATP by the "motor" (primarily the A and B subunits) drives the rotation in the membrane of the "rotor" (composed of the D, F, c, cЈ, cЉ, and d subunits) (21-23). In previous work we developed methods to isolate mutant strains of Neurospora crassa that were resistant to inhibition by bafilomycin and concanamycin (17, 18) .
doi:10.1074/jbc.m605532200 pmid:16912037 fatcat:kkrr6xu53fgrnhypavlox3my7a