Cellulosome assembly revealed by the crystal structure of the cohesin–dockerin complex

Abstract

The utilization of organized supramolecular assemblies to exploit the synergistic interactions afforded by close proximity, both for enzymatic synthesis and for the degradation of recalcitrant substrates, is an emerging theme in cellular biology. Anaerobic bacteria harness a multiprotein complex, termed the ''cellulosome,'' for efficient degradation of the plant cell wall. This megadalton catalytic machine organizes an enzymatic consortium on a multifaceted molecular scaffold whose ''cohesin'' domains interact with corresponding ''dockerin'' domains of the enzymes. Here we report the structure of the cohesin-dockerin complex from Clostridium thermocellum at 2.2-Å resolution. The data show that the ␤-sheet cohesin domain interacts predominantly with one of the helices of the dockerin. Whereas the structure of the cohesin remains essentially unchanged, the loop-helix-helix-loop-helix motif of the dockerin undergoes conformational change and ordering compared with its solution structure, although the classical 12-residue EF-hand coordination to two calcium ions is maintained. Significantly, internal sequence duplication within the dockerin is manifested in near-perfect internal twofold symmetry, suggesting that both ''halves'' of the dockerin may interact with cohesins in a similar manner, thus providing a higher level of structure to the cellulosome and possibly explaining the presence of ''polycellulosomes.'' The structure provides an explanation for the lack of cross-species recognition between cohesin-dockerin pairs and thus provides a blueprint for the rational design, construction, and exploitation of these catalytic assemblies.

Evidence for a dual binding mode of dockerin modules to cohesins

“…Coh-DocS45A-T46A contains three ␣-helices (1-3) and two calcium ions coordinated by residues at the C-and N-terminal ends of the dockerin that display typical EF-hand Ca 2ϩ -binding motifs. Calcium coordination in CohDocS45A-T46A is essentially identical to the Coh-DocWT complex reported previously (18), and details of this coordination are provided in supplementary information (SI Table 3). …”

“…The first ␤-sheet comprises ␤-strands 5, 6, 3 and 8 and defines the interacting surface with the dockerin, whereas ␤-strands 4, 7, 2, 1 and 9 define the second ␤-sheet. Comparison of both type I or type II cohesin structures of C. thermocellum, solved either as discrete entities (10,(20)(21)(22)(23)(24)(25), or in complex with the corresponding dockerin modules (18,19), show that the conformation of both cohesins do not change upon binding its protein ligand. Coh-DocS45A-T46A contains three ␣-helices (1-3) and two calcium ions coordinated by residues at the C-and N-terminal ends of the dockerin that display typical EF-hand Ca 2ϩ -binding motifs.…”

“…The crystal structures of C. thermocellum type I (18) and type II (19) cohesin-dockerin complexes have been solved providing insight into the mechanism of cellulosome assembly and cell surface attachment, respectively. In both complexes, cohesin-dockerin recognition is dominated by hydrophobic interactions, augmented through an extensive hydrogen-bonding network.…”

Understanding the structural and functional properties of carbohydrate esterases with a special focus on hemicellulose deacetylating acetyl xylan esterases

“…Mature SlCE4 CE-4 enzyme consists of 233 amino acid residues forming a bidomain enzyme with C-terminus of the enzyme encodes for CBM2 module (Carbohydrate binding module) with first 41 residues coding for a cleaved signal peptide. The CtCE4 was reported to be derived from the xylanase (Xyn11A) of C. thermocellum , where the C-terminus of the protein contains CE class-4 domain and N-terminus of the protein consists of endoxylanase catalytic domain, CBM6 (Carbohydrate binding module) and a dockerin domain, which is involved in direction of the enzyme to the cellulosome machinery (Carvalho et al 2003). Both CtCE4 and SlCE4 display (β/α) 8 distorted structures of barrel folds.…”

Structural and biochemical properties of lichenase from Clostridium thermocellum

“…The over-expressed enzyme was soluble and upon sonication comes out in cell-free extract as soluble fraction. The protein was purifi ed by immobilized metal ion affi nity chromatography (IMAC) as described previously [7]. For crystallization, CtLic26A was further purifi ed by size exclusion chromatography [2].…”