Mapping by Site-Directed Mutagenesis of the Region Responsible for Cohesin−Dockerin Interaction on the Surface of the Seventh Cohesin Domain of Clostridium thermocellum CipA - Institut Pasteur Access content directly
Journal Articles Biochemistry Year : 2002

Mapping by Site-Directed Mutagenesis of the Region Responsible for Cohesin−Dockerin Interaction on the Surface of the Seventh Cohesin Domain of Clostridium thermocellum CipA

Abstract

To locate the region involved in binding dockerin domains, 15 mutations were introduced across the surface of the seventh cohesin domain of the scaffolding protein CipA, which holds together the cellulosome of Clostridium thermocellum. Mutated residues were located on both faces of the nine-stranded beta-sandwich forming the cohesin domain and on the loops connecting beta-strands 4 and 5, 6 and 7, and 8 and 9. The loop region was previously proposed, on the basis of sequence comparisons, to form a contiguous "recognition strip". Individual mutants of four residues, D39, Y74, E86, and G89, formed no complexes detectable by nondenaturing gel electrophoresis after incubation with CelD664, a shortened form of endoglucanase CelD lacking the residues linking the catalytic domain with the dockerin domain. The four sensitive residues encompass a hydrophobic region on the 5-6-3-8 face of the molecule, which overlaps partially with the recognition strip and with a hydrophobic zone involved in the formation of cohesin-cohesin dimers. Isothermal titration calorimetry showed that single cohesin mutations affecting the binding of CelD664 had significant effects on the enthalpy or entropy of binding of wild-type CelD but much lesser effects on the association constant, owing to enthalpy-entropy compensation. However, the affinity for wild-type CelD of the triple mutant affecting D39, Y74, and E86 was reduced by 2 orders of magnitude, due to negative cooperativity between mutations affecting D39 + Y74 on one hand and E86 on the other hand.

Dates and versions

pasteur-03144723 , version 1 (17-02-2021)

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Isabelle Miras, Francis Schaeffer, Pierre Béguin, Pedro Alzari. Mapping by Site-Directed Mutagenesis of the Region Responsible for Cohesin−Dockerin Interaction on the Surface of the Seventh Cohesin Domain of Clostridium thermocellum CipA. Biochemistry, 2002, 41 (7), pp.2115-2119. ⟨10.1021/bi011854e⟩. ⟨pasteur-03144723⟩

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