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.
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.
Miras
Isabelle
I
Unité Microbiologie et Environnement, URA 2172, CNRS, Institut Pasteur, 28, rue du Dr. Roux, 75724 Paris Cedex 15, France.
Schaeffer
Francis
F
Béguin
Pierre
P
Alzari
Pedro M
PM
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution / Q000235:genetics; D001426:Bacterial Proteins / Q000235:genetics* / Q000378:metabolism*; D001665:Binding Sites / Q000235:genetics; D018797:Cell Cycle Proteins; D002480:Cellulase / Q000378:metabolism; D002868:Chromosomal Proteins, Non-Histone; D003013:Clostridium / Q000201:enzymology* / Q000235:genetics*; D005656:Fungal Proteins; D008565:Membrane Proteins / Q000235:genetics* / Q000378:metabolism*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009687:Nuclear Proteins / Q000235:genetics* / Q000378:metabolism*; D010446:Peptide Fragments / Q000235:genetics / Q000378:metabolism; D010449:Peptide Mapping / Q000379:methods; D011485:Protein Binding / Q000235:genetics; D017434:Protein Structure, Tertiary / Q000235:genetics; D000097722:Cohesins
D001426:Bacterial Proteins; D018797:Cell Cycle Proteins; D002480:Cellulase; D002868:Chromosomal Proteins, Non-Histone; D005656:Fungal Proteins; D008565:Membrane Proteins; D009687:Nuclear Proteins; D010446:Peptide Fragments; D000097722:Cohesins; C080369:CipA protein, Clostridium; C087879:endoglucanase CelD