Structural Basis of the Regulation of the CbpA Co-chaperone by its Specific Modulator CbpM |
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Authors: | Naghmeh S. Sarraf Jason Baardsnes Maureen O'Connor-McCourt Irena Ekiel |
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Affiliation: | 1 Health Sector, Biotechnology Research Institute, National Research Council of Canada, 6100 Royalmount Avenue, Montreal, Quebec, Canada H4P 2R2 2 Department of Chemistry and Biochemistry, Concordia University, 7141 Sherbrooke W., Montreal, Quebec, Canada H4B 1R6 3 Department of Biochemistry, McGill University, Montreal, Quebec, Canada H3G 1Y6 |
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Abstract: | CbpA, one of the Escherichia coli DnaJ homologues, acts as a co-chaperone in the DnaK chaperone system. Despite its extensive similarity in domain structure and function to DnaJ, CbpA has a unique and specific regulatory mechanism mediated through the small protein CbpM. Both CbpA and CbpM are highly conserved in bacteria. Earlier studies showed that CbpM interacts with the N-terminal J-domain of CbpA inhibiting its co-chaperone activity but the structural basis of this interaction is not known. Here, we have combined NMR spectroscopy, site-directed mutagenesis and surface plasmon resonance to characterize the CbpA/CbpM interaction at the molecular level. We have determined the solution structure of the CbpA J-domain and mapped the residues that are perturbed upon CbpM binding. The NMR data defined a broad region on helices α2 and α3 as involved in the interactions. Site-directed mutagenesis has been used to further delineate the CbpA J-domain/CbpM interface. We show that the binding sites of CbpM and DnaK on CbpA J-domain overlap, which suggests a competition between DnaK and CbpM for binding to CbpA as a mechanism for CbpA regulation. This study also provides the explanation for the specificity of CbpM for CbpA versus DnaJ, by identifying the key residues for differential binding. |
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Keywords: | JdomCbpA, J-domain of CbpA MBP, maltose-binding protein SEC, size-exclusion chromatography SPR, surface plasmon resonance MBP, maltose-binding protein SEC, size-exclusion chromatography SPR, surface plasmon resonance HSQC, heteronuclear single quantum coherence NOE, nuclear Overhauser effect NOESY, NOE spectroscopy |
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