Crystallographic evidence for substrate ring distortion and protein conformational changes during catalysis in cellobiohydrolase Cel6A from Trichoderma reesei

Background: Cel6A is one of the two cellobiohydrolases produced by Trichode rma reesei, The catalytic core has a structure that is a variation of the c lassic TIM barrel. The active site is located inside a tunnel, the roof of which is formed mainly by a pair of loops. Results: We describe three new ligand complexes. One is the structure of th e wild-type enzyme in complex with a nonhydrolysable cello-oligosaccharide, methyl 4-S-beta-cellobiosyl-4-thio-beta-cellobioside (Glc)(2)-S-(Glc)(2), which differs from a cellotetraose in the nature of the central glycosidic linkage where a sulphur atom replaces an oxygen atom. The second structure is a mutant, Y169F, in complex with the same ligand, and the third is the w ild-type enzyme in complex with m-iodobenzyl beta-D-glucopyranosyl-beta(1,4 )-D-xylopyranoside (IBXG). Conclusions: The (Glc)(2)-S-(Glc)(2) ligand binds in the -2 to +2 sites in both the wild-type and mutant enzymes. The glucosyl unit in the -1 site is distorted from the usual chair conformation in both structures. The IBXG li gand binds in the -2 to +1 sites, with the xylosyl unit in the -1 site wher e it adopts the energetically favourable chair conformation, The -1 site gl ucosyl of the (Glc)(2)-S-(Glc)(2) ligand is unable to take on this conforma tion because of steric clashes with the protein. The crystallographic resul ts show that one of the tunnel-forming loops in Cel6A is sensitive to modif ications at the active site, and is able to take on a number of different c onformations. One of the conformational changes disrupts a set of interacti ons at the active site that we propose is an integral part of the reaction mechanism.