Preliminary X-ray characterization and phasing of a type II cohesin domain from the cellulosome ofAcetivibrio cellulolyticus

Abstract

The N-terminal type II cohesin from the cellulosomal ScaB subunit of Acetivibrio cellulolyticus was crystallized in two different crystal systems: orthorhombic (space group P2(1)2(1)2(1)), with unit-cell parameters a = 37.455, b = 55.780, c = 87.912 A, and trigonal (space group P3(1)21), with unit-cell parameters a = 55.088, b = 55.088, c = 112.553 A. The two crystals diffracted to 1.2 and 1.9 A, respectively. A selenomethionine derivative was also crystallized and exhibited trigonal symmetry (space group P3(1)21), with unit-cell parameters a = 55.281, b = 55.281, c = 112.449 A and a diffraction limit of 1.97 A. Initial phasing of the trigonal crystals was successfully performed by the SIRAS method using Cu Kalpha radiation with the selenomethionine derivative as a heavy-atom derivative. The structure of the orthorhombic crystal form was solved by molecular replacement using the coordinates of the trigonal form.

Evidence for a dual binding mode of dockerin modules to cohesins

“…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.…”

Crystallization and preliminary X-ray analysis ofAcetivibrio cellulolyticuscellulosomal type II cohesin module: two versions having different linker lengths

“…The type II cohesins differ somewhat from those of type I, having additional segments within the polypeptide and clear diversity in the latter half of the sequence (Leibovitz & Bé guin, 1996). The type II cohesin discussed in this communication is the second cohesin (enumerated from the N-terminus) of the ScaB scaffoldin polypeptide chain and shares 50% sequence identity with the first cohesin in this scaffoldin, the crystal structure of which has been solved previously (PDB code 1ohz; Noach et al, 2003). The cohesins on the scaffoldin subunits are separated by distinctive linker sequences, most of which are rich in proline and threonine residues.…”

“…The DNA encoding the second cohesin module from the scaB scaffoldin gene (gi:31540574; Noach et al, 2003;Xu et al, 2003) was cloned, with either the short or full-length C-terminal linker segment, from genomic DNA of A. cellulolyticus into the NcoI and XhoI sites of the pET28a expression vector (Novagen). The constructs, which contained two additional restriction-site residues (Leu-Glu) followed by a hexa-His tag attached to the 3 0 end, were expressed in Escherichia coli strain BL21 cells grown at 310 K in 1 l Luria-Bertani medium supplemented with 50 mg ml À1 kanamycin.…”

“…Following this work, the structure of a type I cohesin-dockerin complex from C. thermocellum was determined, which revealed a dual-binding mode of interaction between the two complementary modules (Carvalho et al, 2003). In addition, two type II cohesin structures from A. cellulolyticus and Bacteroides cellulosolvens (Noach et al, 2003(Noach et al, , 2005) have been described, followed by that of another type II cohesin from C. thermocellum, first as a single module (Carvalho et al, 2005) and subsequently in a trimodular complex with its counterpart dockerin together with an adjacent X module (Adams et al, 2006).…”

Structural characterization of a novel autonomous cohesin fromRuminococcus flavefaciens

“…Krissinel & Henrick, 2004) revealed that despite the relatively low sequence identity (9-25%), RfCohG can be structurally aligned to cohesin modules from all of the known types (I-III). RfCohG is the most similar to the type IIIe cohesin from R. flavefaciens ScaE (PDB entry 2zf9; Alber et al, 2009;Salama-Alber et al, 2013) in the midst of -strands 4 and 8 (Noach et al, 2003(Noach et al, , 2005Carvalho et al, 2005).…”