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Identification of an alcohol binding site in the first cysteine-rich domain of protein kinase Cdelta.

ABSTRACT: 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.

SUBMITTER: Das J

PROVIDER: S-EPMC2242605 | biostudies-literature | 2006 Sep

REPOSITORIES: biostudies-literature

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Identification of an alcohol binding site in the first cysteine-rich domain of protein kinase Cdelta.

Das Joydip J Zhou Xiaojuan X Miller Keith W KW

Protein science : a publication of the Protein Society 20060901 9

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 ...[more]

PMID: 16943444

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Project description:Plasmodium falciparum RH5 is a secreted parasite ligand that is essential for erythrocyte invasion through direct interaction with the host erythrocyte receptor basigin. RH5 forms a tripartite complex with two other secreted parasite proteins, CyRPA and RIPR, and is tethered to the surface of the parasite through membrane-anchored P113. Antibodies against RH5, CyRPA, and RIPR can inhibit parasite invasion, suggesting that vaccines containing these three components have the potential to prevent blood-stage malaria. To further explore the role of the P113-RH5 interaction, we selected monoclonal antibodies against P113 that were either inhibitory or noninhibitory for RH5 binding. Using a Fab fragment as a crystallization chaperone, we determined the crystal structure of the RH5 binding region of P113 and showed that it is composed of two domains with structural similarities to rhamnose-binding lectins. We identified the RH5 binding site on P113 by using a combination of hydrogen-deuterium exchange mass spectrometry and site-directed mutagenesis. We found that a monoclonal antibody to P113 that bound to this interface and inhibited the RH5-P113 interaction did not inhibit parasite blood-stage growth. These findings provide further structural information on the protein interactions of RH5 and will be helpful in guiding the development of blood-stage malaria vaccines that target RH5.IMPORTANCE Malaria is a deadly infectious disease primarily caused by the parasite Plasmodium falciparum It remains a major global health problem, and there is no highly effective vaccine. A parasite protein called RH5 is centrally involved in the invasion of host red blood cells, making it-and the other parasite proteins it interacts with-promising vaccine targets. We recently identified a protein called P113 that binds RH5, suggesting that it anchors RH5 to the parasite surface. In this paper, we use structural biology to locate and characterize the RH5 binding region on P113. These findings will be important to guide the development of new antimalarial vaccines to ultimately prevent this disease, which affects some of the poorest people on the planet.

Dataset Information

Identification of an alcohol binding site in the first cysteine-rich domain of protein kinase Cdelta.

Publications

Identification of an alcohol binding site in the first cysteine-rich domain of protein kinase Cdelta.

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