A model for the proteolipid ring and bafilomycin/concanamycin-binding site in the vacuolar ATPase of Neurospora crassa.
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 subunits 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.
Bowman
Barry J
BJ
Department of Molecular, Cell, and Developmental Biology, University of California, Santa Cruz, California 95064, USA. bowman@biology.ucsc.edu
McCall
Mary E
ME
Baertsch
Robert
R
Bowman
Emma Jean
EJ
D000255:Adenosine Triphosphate / Q000737:chemistry; D000595:Amino Acid Sequence; D001665:Binding Sites; D016983:Enterococcus / Q000378:metabolism; D004791:Enzyme Inhibitors / Q000494:pharmacology; D018942:Macrolides / Q000737:chemistry*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009492:Neurospora crassa / Q000201:enzymology* / Q000378:metabolism; D017510:Protein Folding; D017386:Sequence Homology, Amino Acid; D025262:Vacuolar Proton-Translocating ATPases / Q000737:chemistry*
D004791:Enzyme Inhibitors; D018942:Macrolides; C057620:bafilomycin A; C036978:concanamycin A; D000255:Adenosine Triphosphate; D025262:Vacuolar Proton-Translocating ATPases