Structure-function relationship of the Clostridium thermocellum calcium-binding dockerin domain.

摘要

Clostridium thermocellum produces an extracellular, multi-enzyme cellulase complex called the cellulosome. It consists of a scaffolding protein, CipA, containing nine cohesin domains and a cellulose binding domain, and at least fourteen different enzymatic subunits. Assembly of the cellulosome depends upon the strong interaction between the cohesin domains and a highly conserved domain, dockerin (DS), borne by each enzymatic subunit. DS consists of two 22-amino acid duplicated sequences, each bearing homology to the EF-hand calcium-binding motif. A similar DS (type II) is found at the C terminus of CipA.;The specificity of the cohesin-dockerin interaction was investigated by studying the binding of the DS of cellobiohydrolase CelS to four dissimilar cohesin domains. DS formed a stable complex on a nondenaturing polyacrylamide gel with each of the representative cohesin domains, indicating that binding of the catalytic subunits to the cohesin domains is nonselective. Conversely, the DS of CipA did not bind to any of its own cohesin domains.;To determine whether both conserved segments of DS are involved in cohesin binding, the binding of two subdomains (DS1 and DS2), each containing a putative calcium-binding motif, to R3 was analyzed by nondenaturing polyacrylamide electrophoresis. DS1 or DS2 alone were unable to form a stable complex with R3, indicating that both halves of DS are required for effective binding of DS to cohesin.;Further insights into the cohesin-dockerin interaction were obtained through NMR studies of DS. Changes in the 2D 1H-15N HSQC spectra of DS during Ca2+ titration revealed that Ca 2+ induces folding of DS into its tertiary structure. This Ca 2+-induced protein folding distinguishes DS from typical EF-hand-containing proteins. Lastly, the three-dimensional solution structure of DS was solved. The structure consists of two loop-helix motifs connected by a linker. The DS fold displays a dramatic departure from the conserved EF-hand fold due to the lack of E-helices. A putative cohesin-binding surface, comprised of conserved hydrophobic and basic residues, was indicated by structure analysis, chemical shift perturbation, and modeling of the complex. All of these results have furthered our understanding of the cohesin-dockerin interaction and have made additional contributions to the field of calcium-binding proteins.