Evidence for a dual binding mode of dockerin modules to cohesins

Abstract

The assembly of proteins that display complementary activities into macromolecular complexes is critical to cellular function. One such enzyme complex, of environmental significance, is the plant cell wall degrading apparatus of anaerobic bacteria, termed the cellulosome. The complex assembles through the interaction of enzyme-derived "type I dockerin" modules with the multiple "cohesin" modules of the scaffolding protein. Clostridium thermocellum type I dockerin modules contain a duplicated 22-residue sequence that comprises helix-1 and helix-3, respectively. The crystal structure of a C. thermocellum type I cohesin-dockerin complex showed that cohesin recognition was predominantly through helix-3 of the dockerin. The sequence duplication is reflected in near-perfect 2-fold structural symmetry, suggesting that both repeats could interact with cohesins by a common mechanism in wild-type (WT) proteins. Here, a helix-3 disrupted mutant dockerin is used to visualize the reverse binding in which the dockerin mutant is indeed rotated 180 degrees relative to the WT dockerin such that helix-1 now dominates recognition of its protein partner. The dual binding mode is predicted to impart significant plasticity into the orientation of the catalytic subunits within this supramolecular assembly, which reflects the challenges presented by the degradation of a heterogeneous, recalcitrant, insoluble substrate by a tethered macromolecular complex.

Fig. 1.

The cellulosome. (a) Schematic of the cellulosome. The type I dockerins, appended to the catalytic subunits, interact with the cohesin modules on the scaffoldin (CipA) leading to the formation of the supramolecular cellulosome complex. The type II dockerin on CipA, by binding to a type II cohesin on the bacterial membrane, tethers the cellulosome to the surface of C. thermocellum. (b) Internal symmetry of the WT dockerin in complex with cohesin. Not only do residues 1–22 overlap with 35–56, but the reverse is also true, because the dockerin shows internal 2-fold symmetry (panel b adapted from ref. 18).

Fig. 2.

The dual binding mode of the Xyn10B dockerin. (a) Ribbon representation of the superposition of the type I Coh-DocWT complex (in orange) with its S45A-T46A mutant complex (in blue). In the mutant complex, helix-1 (containing Ser-11 and Thr-12) dominates binding whereas, in the WT complex, helix-3 (containing Ser-45 and Thr-46) plays a key role in ligand recognition. Ser-11, Thr-12, Ser-45, and Thr-46, which interact with the cohesin module, are depicted as stick models and colored accordingly. The second molecule of the mutant complex, generated by the 2-fold NCS, is represented in light-gray ribbon. The Ca²⁺ ions are depicted as spheres and colored orange, in the case of the WT complex, and light blue, in the case of the mutant. The N- and C-terminal ends are labeled and colored accordingly. (b) The structure-based sequence alignment of the WT (in red) and S45A-T46A mutant (in blue) type I dockerins. Mutated residues, Ala-45 and Ala-46, are shown in green. Because of internal 2-fold symmetry of each dockerin module, the two structures overlap almost perfectly in their α1/α3 regions. The N- and C-terminal ends of each module are indicated, as well as the α-helix regions. Numbering is indicated for every 10th residue.

Fig. 3.

The Coh-Doc interface of the native (in orange) and S45A-T46A mutant (in blue) type I complexes. (a) Stick representation of the hydrophobic residues on the surface of the cohesin modules (in ribbon representation). The dockerin modules are represented by their molecular surfaces. (b) Stick representation of the hydrophobic residues on the surface of the dockerin modules (in ribbon representation). The cohesin modules are represented by their molecular surfaces. (c) Stick representation of the hydrogen-bond network in the interface of the Coh-DocS45A-T46A complex (in ribbon representation). Carbon atoms are shown in yellow, oxygens are shown in red, and nitrogens are shown in blue. All pictures were produced with the CCP4 mg program (42).

Fig. 4.

The type I and type II dockerin modules. (a) The S45A-T46A mutant type I dockerin module. (b) The WT type I dockerin from the type I Coh-DocWT complex (PDB code 1ohz). (c) The type II dockerin from the type II Coh-Doc complex (PDB code 2b59). The α-helices in each module are numbered. Residues in positions 11, 12, 45, and 46 are shown as ball-and-stick models (carbon atoms in green and oxygen atoms in red) and labeled in green. The calcium ions in each module are colored according to structure and labeled as Ca1 and Ca2. Residues coordinating each calcium ion are depicted as stick models (carbon atoms are shown in white, oxygens are shown in red, and nitrogens are shown in blue). The N- and C-terminal ends are labeled and colored accordingly.