| 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 thesecond duplicated segment between the two polypeptides. These residues have been proposed to determinethe specificity of cohesin−dockerin interactions. The dockerin domain of CelD still bound to the seventhcohesin domain of CipA (CohCip7), provided that mutagenesis occurred in one segment only. Bindingwas no longer detected by nondenaturing gel electrophoresis when both segments were mutagenized. Thedockerin domain of CipA bound to the cohesin domain of SdbA as long as the second segment wasintact. 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 ahigh affinity [Ka = (2.6 ± 0.5) × 109 M-1] and a 1:1 stoichiometry. The reaction was weakly exothermic(ΔH° = −2.22 ± 0.2 kcal mol-1) and largely entropy driven (TΔS° = 10.70 ± 0.5 kcal mol-1). The heatcapacity change on complexation was negative (ΔCp = −305 ± 15 cal mol-1 K-1). These values showthat cohesin−dockerin binding is mainly hydrophobic. Mutations in the first or the second dockerin segmentreduced 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, thebinding affinity was strongly decreased when both segments were mutated, indicating strong negativecooperativity between the two mutated sites.
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