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
| Autor: | Isabelle Miras, Pedro M. Alzari, Gerard Guglielmi, Francis Schaeffer, Pierre Béguin, Markus Matuschek |
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| Přispěvatelé: | Biochimie Structurale, Centre National de la Recherche Scientifique (CNRS)-Institut Pasteur [Paris], Microbiologie et Environnement, Institut Pasteur [Paris]-Centre National de la Recherche Scientifique (CNRS), Institut Pasteur [Paris] (IP)-Centre National de la Recherche Scientifique (CNRS) |
| Rok vydání: | 2002 |
| Předmět: |
Cohesin domain
Chromosomal Proteins Non-Histone Cell Cycle Proteins Dockerin MESH: Amino Acid Sequence MESH: Base Sequence MESH: Multienzyme Complexes Biochemistry Cellulosome assembly MESH: Protein Structure Tertiary MESH: Peptide Fragments MESH: Bacterial Proteins Gel electrophoresis 0303 health sciences [SDV.BBM.BS]Life Sciences [q-bio]/Biochemistry Molecular Biology/Structural Biology [q-bio.BM] 030302 biochemistry & molecular biology Nuclear Proteins MESH: Amino Acid Substitution MESH: Mutagenesis Site-Directed Thermodynamics Clostridium thermocellum MESH: Fungal Proteins MESH: Membrane Proteins MESH: Thermodynamics Protein Binding Repetitive Sequences Amino Acid [PHYS.PHYS.PHYS-BIO-PH]Physics [physics]/Physics [physics]/Biological Physics [physics.bio-ph] Molecular Sequence Data [SDV.BC]Life Sciences [q-bio]/Cellular Biology Biology Fungal Proteins 03 medical and health sciences MESH: Cell Cycle Proteins Bacterial Proteins Cellulase Multienzyme Complexes MESH: Chromosomal Proteins Non-Histone [CHIM.CRIS]Chemical Sciences/Cristallography MESH: Protein Binding [SDV.BBM]Life Sciences [q-bio]/Biochemistry Molecular Biology Amino Acid Sequence Cysteine 030304 developmental biology Clostridium Binding Sites MESH: Molecular Sequence Data Base Sequence MESH: Repetitive Sequences Amino Acid MESH: Clostridium Mutagenesis Membrane Proteins MESH: Cellulase Cooperative binding Isothermal titration calorimetry MESH: Cysteine biology.organism_classification Peptide Fragments Protein Structure Tertiary Amino Acid Substitution MESH: Binding Sites Mutagenesis Site-Directed [INFO.INFO-BI]Computer Science [cs]/Bioinformatics [q-bio.QM] MESH: Nuclear Proteins |
| Zdroj: | Biochemistry Biochemistry, American Chemical Society, 2002, 41 (7), pp.2106-2114. ⟨10.1021/bi011853m⟩ Biochemistry, 2002, 41 (7), pp.2106-2114. ⟨10.1021/bi011853m⟩ |
| ISSN: | 1520-4995 0006-2960 |
| Popis: | International audience; 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. |
| Databáze: | OpenAIRE |
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