Archazolid A Binds to the Equatorial Region of the c-Ring of the Vacuolar H+-ATPase

Autor(en): Bockelmann, Svenja 
Menche, Dirk 
Rudolph, Sven
Bender, Tobias
Grond, Stephanie
von Zezschwitz, Paultheo
Muench, Stephen P. 
Wieczorek, Helmut 
Huss, Markus 
Stichwörter: Biochemistry & Molecular Biology; CONCANAMYCIN-A; ELUCIDATION; INHIBITORS; MEMBRANE; MYXOBACTERIA; P-TYPE; PURIFICATION; SITE; SUBUNIT-C; V-ATPASES
Erscheinungsdatum: 2010
Herausgeber: AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC
Journal: JOURNAL OF BIOLOGICAL CHEMISTRY
Volumen: 285
Ausgabe: 49
Startseite: 38304
Seitenende: 38314
Zusammenfassung: 
The macrolactone archazolid is a novel, highly specific V-ATPase inhibitor with an IC50 value in the low nanomolar range. The binding site of archazolid is presumed to overlap with the binding site of the established plecomacrolide V-ATPase inhibitors bafilomycin and concanamycin in subunit c of the membrane-integral V-O complex. Using a semi-synthetic derivative of archazolid for photoaffinity labeling of the V1VO holoenzyme we confirmed binding of archazolid to the V-O subunit c. For the plecomacrolide binding site a model has been published based on mutagenesis studies of the c subunit of Neurospora crassa, revealing 11 amino acids that are part of the binding pocket at the interface of two adjacent c subunits (Bowman, B. J., McCall, M. E., Baertsch, R., and Bowman, E. J. (2006) J. Biol. Chem. 281, 31885-31893). To investigate the contribution of these amino acids to the binding of archazolid, we established in Saccharomyces cerevisiae mutations that in N. crassa had changed the IC50 value for bafilomycin 10-fold or more and showed that out of the amino acids forming the plecomacrolide binding pocket only one amino acid (tyrosine 142) contributes to the binding of archazolid. Using a fluorescent derivative of N, N'-dicyclohexylcarbodiimide, we found that the binding site for archazolid comprises the essential glutamate within helix 4 of subunit c. In conclusion the archazolid binding site resides within the equatorial region of the V-O rotor subunit c. This hypothesis was supported by an additional subset of mutations within helix 4 that revealed that leucine 144 plays a role in archazolid binding.
DOI: 10.1074/jbc.M110.137539

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