Involvement of both dockerin subdomains in assembly of the Clostridium thermocellum cellulosome.
Clostridium thermocellum produces an extracellular cellulase complex termed the cellulosome. It consists of a scaffolding protein, CipA, containing nine cohesin domains and a cellulose-binding domain, and at least 14 different enzymatic subunits, each containing a conserved duplicated sequence, or dockerin domain. The cohesin-dockerin interaction is responsible for the assembly of the catalytic subunits into the cellulosome structure. Each duplicated sequence of the dockerin domain contains a region bearing homology to the EF-hand calcium-binding motif. Two subdomains, each containing a putative calcium-binding motif, were constructed from the dockerin domain of CelS, a major cellulosomal catalytic subunit. These subdomains, called DS1 and DS2, were cloned by PCR and expressed in Escherichia coli. The binding of DS1 and DS2 to R3, the third cohesin domain of CipA, was analyzed by nondenaturing gel electrophoresis. A stable complex was formed only when R3 was combined with both DS1 and DS2, indicating that the two halves of the dockerin domain interact with each other and such interaction is required for effective binding of the dockerin domain to the cohesin domain.
Lytle
B
B
Department of Chemical Engineering, University of Rochester Rochester, New York 14627-0166, USA.
Wu
J H
JH
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000378:metabolism*; D001665:Binding Sites; D002480:Cellulase / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D003001:Cloning, Molecular; D003013:Clostridium / Q000201:enzymology / Q000235:genetics / Q000378:metabolism*; D015870:Gene Expression; D008565:Membrane Proteins / Q000378:metabolism*; D008969:Molecular Sequence Data; D017186:Titrimetry
D001426:Bacterial Proteins; C080369:CipA protein, Clostridium; D008565:Membrane Proteins; C071223:endoglucanase Ss; D002480:Cellulase