The Huck Institutes of the Life Sciences

Structural basis for entropy-driven cellulose binding by a type-A cellulose-binding module (CBM) and bacterial expansin

Georgelis N, Yennawar NH, and Cosgrove DJ (2012) PNAS 109(37): 14830-14835


Components of modular cellulases, type-A cellulose-binding modules (CBMs) bind to crystalline cellulose and enhance enzyme effectiveness, but structural details of the interaction are uncertain. We analyzed cellulose binding by EXLX1, a bacterial expansin with ability to loosen plant cell walls and whose domain D2 has type-A CBM characteristics. EXLX1 strongly binds to crystalline cellulose via D2, whereas its affinity for soluble cellooligosaccharides is weak. Calorimetry indicated cellulose binding was largely entropically driven. We solved the crystal structures of EXLX1 complexed with cellulose-like oligosaccharides to find that EXLX1 binds the ligands through hydrophobic interactions of three linearly arranged aromatic residues in D2. The crystal structures revealed a unique form of ligand-mediated dimerization, with the oligosaccharide sandwiched between two D2 domains in opposite polarity. This report clarifies the molecular target of expansin and the specific molecular interactions of a type-A CBM with cellulose.

Structure of EXLX1 with three different ligands

Georgelis et al. 2012 Figure S6
Sandwich structures consisting of two molecules of EXLX1 (A and B) and one molecule of MLG, hemithiocellodextrin (HTC), or cellotetraose. Glucose numbering starts from the reducing end of each cellooligosaccharide.

Structure of EXLX1 with cellohexaose

Georgelis et al. 2012 Figure 3
(A) Sandwich structure consisting of two molecules of EXLX1 (A and B) and one molecule of cellohexaose (green). (B) Direct hydrogen bonds and CH-π interactions between each EXLX1 molecule and cellohexaose. Glucose numbering starts from the reducing end of cellohexaose.

Materials and Methods

Protein Crystallization, Data Collection, Structure Determination, and Refinement

EXLX1 was concentrated to 30 mg/mL in the presence of cellohexaose or hemithiocellodextrin or MLG or cellotetraose, crystallized by hanging drops, and data were collected at an MM007 rotating anode X-ray generator with CuKα radiation, operating at 1.2 kW of power and Saturn944+ CCD detector (Rigaku Americas).

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