PDBsum entry: 1w8u

Carbohydrate-binding domain

PDB id: 1w8u

Contents

Protein chain

141 a.a.

Ligands

BMA-BMA-BMA-BMA-BMA

Waters ×166

PDB id:

1w8u

Name:

Carbohydrate-binding domain

Title:

Cbm29-2 mutant d83a complexed with mannohexaose: probing the mechanism of ligand recognition by family 29 carbohydrate binding modules

Structure:

Non catalytic protein 1. Chain: a. Fragment: carbohydrate binding module 2, residues 334-478. Engineered: yes. Mutation: yes

Source:

Piromyces equi. Organism_taxid: 99929. Expressed in: escherichia coli. Expression_system_taxid: 469008.

Resolution:

1.3Å R-factor: 0.162 R-free: 0.175

Authors:

J.Flint,D.N.Bolam,D.Nurizzo,E.J.Taylor,M.P.Williamson, C.Walters,G.J.Davies,H.J.Gilbert

Key ref:

J.Flint et al. (2005). Probing the mechanism of ligand recognition in family 29 carbohydrate-binding modules. J Biol Chem, 280, 23718-23726. PubMed id: 15784618 DOI: 10.1074/jbc.M501551200

Date:

28-Sep-04 Release date: 22-Mar-05

Protein chain

Q9C171  (Q9C171_PIREQ) -  Non-catalytic protein 1

Seq: 478 a.a.

Struc: 141 a.a.*

* PDB and UniProt seqs differ at 1 residue position (black cross)

DOI no: 10.1074/jbc.M501551200

J Biol Chem 280:23718-23726 (2005)

PubMed id: 15784618

Probing the mechanism of ligand recognition in family 29 carbohydrate-binding modules.

J.Flint, D.N.Bolam, D.Nurizzo, E.J.Taylor, M.P.Williamson, C.Walters, G.J.Davies, H.J.Gilbert.

ABSTRACT

The recycling of photosynthetically fixed carbon, by the action of microbial plant cell wall hydrolases, is integral to one of the major geochemical cycles and is of considerable industrial importance. Non-catalytic carbohydrate-binding modules (CBMs) play a key role in this degradative process by targeting hydrolytic enzymes to their cognate substrate within the complex milieu of polysaccharides that comprise the plant cell wall. Family 29 CBMs have, thus far, only been found in an extracellular multienzyme plant cell wall-degrading complex from the anaerobic fungus Piromyces equi, where they exist as a CBM29-1:CBM29-2 tandem. Here we present both the structure of the CBM29-1 partner, at 1.5 A resolution, and examine the importance of hydrophobic stacking interactions as well as direct and solvent-mediated hydrogen bonds in the binding of CBM29-2 to different polysaccharides. CBM29 domains display unusual binding properties, exhibiting specificity for both beta-manno- and beta-gluco-configured ligands such as mannan, cellulose, and glucomannan. Mutagenesis reveals that "stacking" of tryptophan residues in the n and n+2 subsites plays a critical role in ligand binding, whereas the loss of tyrosine-mediated stacking in the n+4 subsite reduces, but does not abrogate, polysaccharide recognition. Direct hydrogen bonds to ligand, such as those provided by Arg-112 and Glu-78, play a pivotal role in the interaction with both mannan and cellulose, whereas removal of water-mediated interactions has comparatively little effect on carbohydrate binding. The interactions of CBM29-2 with the O2 of glucose or mannose contribute little to binding affinity, explaining why this CBM displays dual gluco/manno specificity.

Selected figure(s)

Figure 2.
FIG. 2. Schematic representation of interactions of CBM29-2 with cellohexaose. Solvent-mediated interactions are shown in blue. The numbers at the top of the figure refer to the sugar binding subsites.

Figure 5.
FIG. 5. AGE of wild type and mutants of CBM29-2. Affinities of wild type and mutants of CBM29-2 for the polysaccharides hvKGM, high viscosity CGM, and HEC were assessed by AGE. Nondenaturing gels containing 0%, 0.1%, 0.05%, 0.025%, and 0.0125% of the polysaccharides were run with 20 µg of CBM29-2 proteins with BSA as the control nonbinding protein. All gels show BSA (1), CBM29-2 wild type (2), W24A (3), W26A (4), Y46A (5), E78A (6), R112A (7), Q116A (8), K74A (9), D83A (10), K85A (11), and D114A (12).

The above figures are reprinted by permission from the ASBMB: J Biol Chem (2005, 280, 23718-23726) copyright 2005.

Figures were selected by an automated process.