Crystal structures of Melanocarpus albomyces cellobiohydrolase Cel7B in complex with cello-oligomers show high flexibility in the substrate binding

Abstract

Cellobiohydrolase from Melanocarpus albomyces (Cel7B) is a thermostable, single-module, cellulose-degrading enzyme. It has relatively low catalytic activity under normal temperatures, which allows structural studies of the binding of unmodified substrates to the native enzyme. In this study, we have determined the crystal structure of native Ma Cel7B free and in complex with three different cello-oligomers: cellobiose (Glc(2)), cellotriose (Glc(3)), and cellotetraose (Glc(4)), at high resolution (1.6-2.1 A). In each case, four molecules were found in the asymmetric unit, which provided 12 different complex structures. The overall fold of the enzyme is characteristic of a glycoside hydrolase family 7 cellobiohydrolase, where the loops extending from the core beta-sandwich structure form a long tunnel composed of multiple subsites for the binding of the glycosyl units of a cellulose chain. The catalytic residues at the reducing end of the tunnel are conserved, and the mechanism is expected to be retaining similarly to the other family 7 members. The oligosaccharides in different complex structures occupied different subsite sets, which partly overlapped and ranged from -5 to +2. In four cellotriose and one cellotetraose complex structures, the cello-oligosaccharide also spanned over the cleavage site (-1/+1). There were surprisingly large variations in the amino acid side chain conformations and in the positions of glycosyl units in the different cello-oligomer complexes, particularly at subsites near the catalytic site. However, in each complex structure, all glycosyl residues were in the chair (4C(1)) conformation. Implications in relation to the complex structures with respect to the reaction mechanism are discussed.

Figure 1.

(A) The overall structure of Ma Cel7B complexed with cellobiose. The cellobiose is bound at glycosyl subsites +1 and +2. (B) The superimposition of Cα backbone of all 16 Ma Cel7B structures. Native structures are in black, cellobiose complex structures are in orange, cellotriose complex structures are in red, and cellotetraose complex structures are in purple. (C) The final electron density (2|F _o| − |F _c|) maps for Glc₄, after the SHELX refinement (all model atoms were used for phase calculation, the level of contouring 1.0 σ), resolution 2.1 Å.

Figure 2.

(A–C) Active site superposition of the four molecules in the asymmetric unit in stereo. (A) Cellobiose, (B) cellotriose, and (C) cellotetraose soaked structures. Observed cello-oligomers are in green and red. The side chains of amino acid residues in the cellulose-binding site are in gray. (D) Superposition of all 12 active sites with cellulose chain modeled in Tr Cel7A structure (blue) in stereoview.

Figure 3.

Protein–carbohydrate-hydrogen-bonding interactions in the active site tunnel of Ma Cel7B. The interactions are collected from all 12 complex structures. Hydrogen bonds are indicated with broken lines.

Figure 4.

The discussed reaction mechanisms for Ma Cel7B (see text for further discussion).