PDBsum entry: 1oh3

Carbohydrate binding domain

PDB-id: 1oh3

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Description

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PROCHECK

Protein chains

141 a.a.

Ligands

GLC-BGC-BGC-BGC-BGC-BGC

Waters ×280

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Clefts Calculation

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PDB id:

1oh3

Name:

Carbohydrate binding domain

Title:

E78r mutant of a carbohydrate binding module family 29

Structure:

Non-catalytic protein 1. Chain: a, b. Fragment: carbohydrate binding module, residues 334-477. Synonym: ncp1. Engineered: yes. Mutation: yes

Source:

Piromyces equi. Organism_taxid: 99929. Expressed in: escherichia coli. Expression_system_taxid: 562

UniProt:

Chains A, B: Q9C171 (Q9C171_PIREQ)

Seq:

Struc:

Seq:

478 a.a.

Struc:

141 a.a.*

Key:	PfamA domain
Secondary structure	CATH domain

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

Resolution:

1.5Å

R-factor:

0.171

R-free:

0.187

Authors:

D.Nurizzo,H.J.Gilbert,G.J.Davies

Key ref:

J.Flint et al. (2004). Ligand-mediated dimerization of a carbohydrate-binding molecule reveals a novel mechanism for protein-carbohydrate recognition.. J Mol Biol, 337, 417-426. [PubMed id: 15003456] [DOI: 10.1016/j.jmb.2003.12.081]

Date:

21-May-03

Release date:

27-May-04

Related entries:

1gwk carbohydrate binding module family29
1gwl carbohydrate binding module family29 complexed with mannohexaose
1gwm carbohydrate binding module family29 complexed with glucohexaose

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Key reference

DOI no: 10.1016/j.jmb.2003.12.081

J Mol Biol 337:417-426 (2004)

PubMed id: 15003456

Ligand-mediated dimerization of a carbohydrate-binding molecule reveals a novel mechanism for protein-carbohydrate recognition.

J.Flint, D.Nurizzo, S.E.Harding, E.Longman, G.J.Davies, H.J.Gilbert, D.N.Bolam.

ABSTRACT

The structural and thermodynamic basis for carbohydrate-protein recognition is of considerable importance. NCP-1, which is a component of the Piromyces equi cellulase/hemicellulase complex, presents a provocative model for analyzing how structural and mutational changes can influence the ligand specificity of carbohydrate-binding proteins. NCP-1 contains two "family 29" carbohydrate-binding modules designated CBM29-1 and CBM29-2, respectively, that display unusually broad specificity; the proteins interact weakly with xylan, exhibit moderate affinity for cellulose and mannan, and bind tightly to the beta-1,4-linked glucose-mannose heteropolymer glucomannan. The crystal structure of CBM29-2 in complex with cellohexaose and mannohexaose identified key residues involved in ligand recognition. By exploiting this structural information and the broad specificity of CBM29-2, we have used this protein as a template to explore the evolutionary mechanisms that can lead to significant changes in ligand specificity. Here, we report the properties of the E78R mutant of CBM29-2, which displays ligand specificity that is different from that of wild-type CBM29-2; the protein retains significant affinity for cellulose but does not bind to mannan or glucomannan. Significantly, E78R exhibits a stoichiometry of 0.5 when binding to cellohexaose, and both calorimetry and ultracentrifugation show that the mutant protein displays ligand-mediated dimerization in solution. The three-dimensional structure of E78R in complex with cellohexaose reveals the intriguing molecular basis for this "dimeric" binding mode that involves the lamination of the oligosaccharide between two CBM molecules. The 2-fold screw axis of the ligand is mirrored in the orientation of the two protein domains with adjacent sugar rings stacking against the equivalent aromatic residues in the binding site of each protein molecule of the molecular sandwich. The sandwiching of an oligosaccharide chain between two protein modules, leading to ligand-induced formation of the binding site, represents a completely novel mechanism for protein-carbohydrate recognition that may mimic that displayed by naturally dimeric protein-carbohydrate interactions.

Selected figure(s)

Figure 2.
Figure 2. Analytical ultracentrifugation of E78R in the presence and in the absence of cellohexaose. A, The NONLIN fit of the equilibrium data plotted as protein concentration in absorbance units against (radial displacement squared)/2. From the fit of the data, the weight average molecular masses were obtained for E78R with ( open ) and without (+) 1 mM cellohexaose. B, the MSTAR extrapolation to estimate the weight average molecular mass (M*(z->1)) for E78R with ( open ) and without (+) 1 mM cellohexaose. c is a normalized radial displacement square parameter (r2 -a^2)/(b^2 -a^2), at a given position in the ultracentrifuge cell, where r is the radial displacement at a given position in the ultracentrifuge cell with a and b the corresponding radial displacements at the cell meniscus and base, respectively. The rotor speed was 17,000 rpm and the temperature was 25.0 ° C (see Materials and Methods).

Figure 3.
Figure 3. Crystal structure of E78R in complex with cellohexaose. (a) The crystal structure of the CBM29-2 mutant E78R in complex with cellohexaose. The molecules are color-ramped from N to C terminus, whilst the ligand is shown in ball-and-stick representation. (b) The observed electron density for the cellohexaose ligand together with the complementary aromatic interface provided by two tryptophan residues and a tyrosine residue from each protein monomer (purple and yellow licorice). This Figure was drawn with BOBSCRIPT.[41.]

The above figures are reprinted by permission from Elsevier: J Mol Biol (2004, 337, 417-426) copyright 2004.

Figures were selected by the author.