PEDS Advance Access originally published online on December 13, 2004
Protein Engineering Design and Selection 2004 17(11):759-769; doi:10.1093/protein/gzh094
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Interactions between immunoglobulin-like and catalytic modules in Clostridium thermocellum cellulosomal cellobiohydrolase CbhA
1Department of Biochemistry and Molecular Biology, University of Georgia, Athens, GA 30602-7229, USA, 3Department of Chemistry and Biochemistry, University of California, Santa Cruz, CA 95064, USA, 4Institute for Biological Instrumentation, Russian Academy of Sciences, Pushchino, Moscow Region 142292, Russia and 5Macromolecular Crystallography Laboratory, National Cancer Institute at Frederick, Frederick, MD 21702, USA
2 To whom correspondence should be addressed. E-mail: kataeva{at}uga.edu
Cellobiohydrolase CbhA from Clostridium thermocellum cellulosome is a multi-modular protein composed starting from the N-terminus of a carbohydrate-binding module (CBM) of family 4, an immunoglobulin(Ig)-like module, a catalytic module of family 9 glycoside hydrolases (GH9), X11 and X12 modules, a CBM of family 3 and a dockerin module. Deletion of the Ig-like module from the Ig–GH9 construct results in complete inactivation of the GH9 module. The crystal structure of the Ig–GH9 module pair reveals the existence of an extensive module interface composed of over 40 amino acid residues of both modules and maintained through a large number of hydrophilic and hydrophobic interactions. To investigate the importance of these interactions between the two modules, we compared the secondary and tertiary structures and thermostabilities of the individual Ig-like and GH9 modules and the Ig–GH9 module pair using both circular dichroism (CD) spectroscopy and differential scanning calorimetry (DSC). Thr230, Asp262 and Asp264 of the Ig-like module are located in the module interface of the Ig–GH9 module pair and are suggested to be important in ‘communication’ between the modules. These residues were mutated to alanyl residues. The structure, stability and catalytic properties of the native Ig–GH9 and its D264A and T230A/D262A mutants were compared. The results indicate that despite being able to fold relatively independently, the Ig-like and GH9 modules interact and these interactions affect the final fold and stability of each module. Mutations of one or two amino acid residues lead to destabilization and change of the mechanism of thermal unfolding of the polypeptides. The enzymatic properties of native Ig–GH9, D264A and T230A/D262A mutants are similar. The results indicate that inactivation of the GH9 module occurs as a result of multiple structural disturbances finally affecting the topology of the catalytic center.
Received May 27, 2004; revised November 15, 2004; accepted November 30, 2004.
Edited by Daniel Raleigh
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