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

被引:62
作者
Schaeffer, F
Matuschek, M
Guglielmi, G
Miras, I
Alzari, PM
Béguin, P
机构
[1] Inst Pasteur, URA 2172 CNRS, Dept Biotechnol, Unite Microbiol & Environm, F-75724 Paris 15, France
[2] Inst Pasteur, URA 2185, Dept Immunol, Unite Biochim Struct, F-75724 Paris 15, France
关键词
D O I
10.1021/bi011853m
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
Mutagenized dockerin domains of endoglucanase CelD (type I) and of the cellulosomeintegrating 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 docket-in 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 mol(-1)) and largely entropy driven (TDeltaSdegrees = 10.70 +/- 0.5 kcal mol(-1)). The heat capacity change on complexation was negative (DeltaC(p) = -305 +/- 15 cal mol(-1) 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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页码:2106 / 2114
页数:9
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