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Duplicated Dockerin Subdomains of Clostridium thermocellum Endoglucanase CelD Bind to a Cohesin Domain of the Scaffolding Protein CipA with Distinct Thermodynamic Parameters and a Negative Cooperativity

Abstract : Mutagenized dockerin domains of endoglucanase CelD (type I) and of the cellulosome-integrating protein CipA (type II) were constructed by swapping residues 10 and 11 of the first or the second duplicated segment between the two polypeptides. These residues have been proposed to determine the specificity of cohesin-dockerin interactions. The dockerin domain of CelD still bound to the seventh cohesin domain of CipA (CohCip7), provided that mutagenesis occurred in one segment only. Binding was no longer detected by nondenaturing gel electrophoresis when both segments were mutagenized. The dockerin domain of CipA bound to the cohesin domain of SdbA as long as the second segment was intact. None of the mutated dockerins displayed detectable binding to the noncognate cohesin domain. Isothermal titration calorimetry showed that binding of the CelD dockerin to CohCip7 occurred with a high affinity [K(a) = (2.6 +/- 0.5) x 10(9) M(-1)] and a 1:1 stoichiometry. The reaction was weakly exothermic (DeltaHdegrees = -2.22 +/- 0.2 kcal x mol(-1)) and largely entropy driven (TDeltaSdegrees = 10.70 +/- 0.5 kcal x mol(-1)). The heat capacity change on complexation was negative (DeltaC(p) = -305 +/- 15 cal x mol(-1) x K(-1)). These values show that cohesin-dockerin binding is mainly hydrophobic. Mutations in the first or the second dockerin segment reduced or enhanced, respectively, the hydrophobic character of the interaction. Due to partial enthalpy-entropy compensation, these mutations induced only small changes in binding affinity. However, the binding affinity was strongly decreased when both segments were mutated, indicating strong negative cooperativity between the two mutated sites.
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Contributor : Marie de Tarragon <>
Submitted on : Thursday, February 18, 2021 - 3:43:08 PM
Last modification on : Saturday, March 20, 2021 - 3:18:58 AM

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Francis Schaeffer, Markus Matuschek, Gérard Guglielmi, Isabelle Miras, Pedro Alzari, et al.. Duplicated Dockerin Subdomains of Clostridium thermocellum Endoglucanase CelD Bind to a Cohesin Domain of the Scaffolding Protein CipA with Distinct Thermodynamic Parameters and a Negative Cooperativity. Biochemistry, American Chemical Society, 2002, 41 (7), pp.2106-2114. ⟨10.1021/bi011853m⟩. ⟨pasteur-03145754⟩

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