Recognition specificity of the duplicated segments present in Clostridium thermocellum endoglucanase CelD and in the cellulosome-integrating protein CipA.
The binding specificity of the duplicated segments borne by Clostridium thermocellum endoglucanase CelD and by the cellulosome-integrating protein CipA was investigated. The fusion protein CelC-DSCelD, in which the duplicated segment of CelD was fused to the COOH terminus of endoglucanase CelC, bound with an affinity of 4.7 x 10(7) M-1 to the fusion protein MalE-RDCipA, in which the seventh receptor domain of CipA was grafted onto the COOH terminus of the Escherichia coli maltose-binding protein MalE. The affinity of CelC-DSCelD for the homologous chimeric protein MalE-RDORF3p, carrying the receptor of the surface protein ORF3p, was 6.9 x 10(6) M-1. The fusion protein CelC-DSCipA, in which the duplicated segment of CipA was grafted onto the COOH terminus of CelC, did not bind detectably to MalE-RDCipA or MalE-RDORF3p. However, Western blotting (immunoblotting) experiments indicated that the duplicated segment of CipA was able to bind to a set of C. thermocellum proteins which are different from those recognized by the duplicated segment of CelD. These results argue against the hypothesis that ORF3p interacts with the duplicated segment of CipA. More probably, ORF3p binds to individual cellulases and hemicellulases harboring duplicated segments.
Salamitou
S
S
Unité de Physiologie Cellulaire, Institut Pasteur, Paris, France.
Raynaud
O
O
Lemaire
M
M
Coughlan
M
M
Béguin
P
P
Aubert
J P
JP
D018528:ATP-Binding Cassette Transporters; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D001483:Base Sequence; D002352:Carrier Proteins / Q000235:genetics / Q000378:metabolism; D002458:Cell Fractionation; D002480:Cellulase / Q000235:genetics / Q000378:metabolism*; D002482:Cellulose / Q000378:metabolism*; D003013:Clostridium / Q000235:genetics / Q000378:metabolism*; D029968:Escherichia coli Proteins; D005818:Genetic Engineering; D058619:Maltose-Binding Proteins; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D009004:Monosaccharide Transport Proteins; D015388:Organelles / Q000378:metabolism*; D033902:Periplasmic Binding Proteins; D011485:Protein Binding; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D012091:Repetitive Sequences, Nucleic Acid; D001617:beta-Glucosidase / Q000235:genetics / Q000378:metabolism
D018528:ATP-Binding Cassette Transporters; D001426:Bacterial Proteins; D002352:Carrier Proteins; C080369:CipA protein, Clostridium; D029968:Escherichia coli Proteins; C461619:MalE protein, E coli; D058619:Maltose-Binding Proteins; D008565:Membrane Proteins; D009004:Monosaccharide Transport Proteins; D033902:Periplasmic Binding Proteins; D011993:Recombinant Fusion Proteins; C461599:maltose transport system, E coli; D002482:Cellulose; C083712:celC protein, bacteria; C087879:endoglucanase CelD; D001617:beta-Glucosidase; D002480:Cellulase