Identification of an alcohol binding site in the first cysteine-rich domain of protein kinase Cdelta.
Protein kinase C (PKC) is an important signal transduction protein whose cysteine-rich regulatory domain C1 has been proposed to interact with general anesthetics in both of its diacylglycerol/phorbol ester-binding subdomains, the tandem repeats C1A and C1B. Previously, we identified an allosteric binding site on one of the two cysteine-rich domains, PKCdelta C1B. To test the hypothesis that there is an additional anesthetic site on the other cysteine-rich subdomain, C1A, we subcloned, expressed in Escherichia coli, purified, and characterized mouse PKCdelta C1A. Octanol and butanol both quenched the intrinsic fluorescence of PKCdelta C1A in a saturable manner, suggesting the presence of a binding site. To locate this site, PKCdelta C1A was photolabeled with three diazirine-containing alkanols, 3-azioctanol, 7-azioctanol, and 3-azibutanol. Mass spectrometry revealed that at low concentrations all three photoincorporated into PKCdelta C1A with a stoichiometry of 1:1 in the labeled fraction, but higher stoichiometries occurred at higher concentrations, particularly with azibutanol. Photocomplexes of PKCdelta C1A with azioctanols were separated from the unlabeled protein by HPLC, reduced, alkylated, digested with trypsin, and sequenced by mass spectrometry. All the azioctanols photolabeled PKCdelta C1A at residue Tyr-29, corresponding to Tyr-187 of the full-length PKCdelta, and at a neighboring residue, Lys-40, suggesting there is an alcohol site in this vicinity. In addition, Glu-2 was photolabeled more efficiently by 3-azibutanol than by the azioctanols, suggesting the existence of a second, smaller site.
Das
Joydip
J
Department of Anesthesia and Critical Care, Massachusetts General Hospital, Boston, MA 02114, USA. joydip_das@hms.harvard.edu
Zhou
Xiaojuan
X
Miller
Keith W
KW
D000438:Alcohols / Q000737:chemistry*; D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D002942:Circular Dichroism; D003978:Diazomethane / Q000737:chemistry; D004075:Diglycerides / Q000494:pharmacology; D013058:Mass Spectrometry; D051379:Mice; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010703:Phorbol Esters / Q000737:chemistry; D011485:Protein Binding; D051745:Protein Kinase C-delta / Q000737:chemistry*; D017434:Protein Structure, Tertiary; D017386:Sequence Homology, Amino Acid; D013194:Staining and Labeling
D000438:Alcohols; D004075:Diglycerides; D010703:Phorbol Esters; C044295:1,2-dioctanoylglycerol; D003978:Diazomethane; D051745:Protein Kinase C-delta