Secondary Structure and Calcium-Induced Folding of the Clostridium thermocellum Dockerin Domain Determined by NMR Spectroscopy

Abstract

Assembly of the cellulosome, a large, extracellular cellulase complex, depends upon docking of a myriad of enzymatic subunits to homologous receptors, or cohesin domains, arranged in tandem along a noncatalytic scaffolding protein. Docking to the cohesin domains is mediated by 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 loop. To compare the DS structure with that of the EF-hand helix–loop–helix motif, we analyzed the solution secondary structure of the DS from the cellobiohydrolase CelS subunit of the Clostridium thermocellum cellulosome using multidimensional heteronuclear NMR spectroscopy. The effect of Ca2+-binding on the DS structure was first investigated by using 2D 15N–1H HSQC NMR spectroscopy. Changes in the spectra during Ca2+ titration revealed that Ca2+ induces folding of DS into its tertiary structure. This Ca2+-induced protein folding distinguishes DS from typical EF-hand-containing proteins. Sequential backbone assignments were determined for 63 of 69 residues. Analysis of the NOE connectivities and Hα chemical shifts revealed that each half of the dockerin contains just one α-helix, comparable to the F-helix of the EF-hand motif. Thus, the structure of the DS Ca2+-binding subdomain deviates from that of the canonical EF-hand motif.