PDBsum entry 1peb

Sugar binding protein

PDB id: 1peb

Contents

Protein chain

366 a.a. *

Waters ×91

* Residue conservation analysis

PDB id:

1peb

Name:

Sugar binding protein

Title:

Ligand-free high-affinity maltose-binding protein

Structure:

Maltose-binding periplasmic protein. Chain: a. Synonym: maltodextrin-binding protein, mmbp. Engineered: yes. Mutation: yes

Source:

Escherichia coli. Organism_taxid: 562. Gene: male or b4034 or z5632 or ecs5017. Expressed in: escherichia coli. Expression_system_taxid: 562.

Resolution:

2.60Å R-factor: 0.181 R-free: 0.242

Authors:

P.G.Telmer,B.H.Shilton

Key ref:

P.G.Telmer and B.H.Shilton (2003). Insights into the conformational equilibria of maltose-binding protein by analysis of high affinity mutants. J Biol Chem, 278, 34555-34567. PubMed id: 12794084 DOI: 10.1074/jbc.M301004200

Date:

21-May-03 Release date: 12-Aug-03

Protein chain

P0AEX9  (MALE_ECOLI) -  Maltose/maltodextrin-binding periplasmic protein from Escherichia coli (strain K12)

Seq: 396 a.a.

Struc: 366 a.a.*

* PDB and UniProt seqs differ at 2 residue positions (black crosses)

Enzyme reactions

• Enzyme class: E.C.?

DOI no: 10.1074/jbc.M301004200

J Biol Chem 278:34555-34567 (2003)

PubMed id: 12794084

Insights into the conformational equilibria of maltose-binding protein by analysis of high affinity mutants.

P.G.Telmer, B.H.Shilton.

ABSTRACT

The affinity of maltose-binding protein (MBP) for maltose and related carbohydrates was greatly increased by removal of groups in the interface opposite the ligand binding cleft. The wild-type protein has a KD of 1200 nM for maltose; mutation of residues Met-321 and Gln-325, both to alanine, resulted in a KD for maltose of 70 nM; deletion of 4 residues, Glu-172, Asn-173, Lys-175, and Tyr-176, which are part of a poorly ordered loop, results in a KD for maltose of 110 nM. Combining the mutations yields an increased affinity for maltodextrins and a KD of 6 nM for maltotriose. Comparison of ligand binding by the mutants, using surface plasmon resonance spectroscopy, indicates that decreases in the off-rate are responsible for the increased affinity. Small-angle x-ray scattering was used to demonstrate that the mutations do not significantly affect the solution conformation of MBP in either the presence or absence of maltose. The crystal structures of selected mutants showed that the mutations do not cause significant structural changes in either the closed or open conformation of MBP. These studies show that interactions in the interface opposite the ligand binding cleft, which we term the "balancing interface," are responsible for modulating the affinity of MBP for its ligand. Our results are consistent with a model in which the ligand-bound protein alternates between the closed and open conformations, and removal of interactions in the balancing interface decreases the stability of the open conformation, without affecting the closed conformation.

Selected figure(s)

Figure 9.
FIG. 9. Structural analysis of the open and closed conformations. For panels A and B, open, unliganded structures of MBP are illustrated on the left, whereas liganded and closed structures of MBP are illustrated on the right. Panel A shows the backbone structures of MBP-WT (9, 10), with residues 171 to 178 highlighted in black. Panel B shows the backbone structures of MBP-Del, in exactly the same orientation as the wild-type protein. Note that for MBP-Del, the truncated loop cannot make contact with the N-terminal domain in either the open or closed conformations. Panel C illustrates the cavity formed in the balancing interface of the open conformation when Met-321 and Gln-325 are mutated to alanine. Here, the molecular surface of open, unliganded MBP-DM is shown, with the side chains of Met-321 and Gln-325 from the structure of open, unliganded MBP-Del. The figures in panels A and B were made with SwissPDBViewer (39), whereas the figure in panel C was made with SPOCK (40) and Raster3D (41).

Figure 10.
FIG. 10. Mechanism of maltose binding by MBP. MBP is illustrated in the two conformations, closed and open, that have been crystallized (9, 10). The circular "bite" taken out of the balancing interface represents the removal of interactions in MBP-DM. A comprehensive equilibrium that includes an open, liganded conformation as well as a closed unliganded conformation provides a mechanism for the increased affinity and decreased k[OFF] observed for the balancing interface mutants (see "Discussion"). Unliganded MBP exists in equilibrium between the closed and open conformations, and the equilibrium is shifted toward the closed, liganded conformation in the presence of maltose. In the closed conformation, the ligand binding site is not accessible, and therefore maltose can only exchange with the open conformation. On this basis, the bimolecular rate constant, k[ON], comprises at least two steps: the binding of maltose, and the conformational change from open to closed. Similarly, the rate constant for dissociation of the complex, k[OFF], includes rates for opening of MBP (k[-][3]) and ligand dissociation (k[-][2]).

The above figures are reprinted by permission from the ASBMB: J Biol Chem (2003, 278, 34555-34567) copyright 2003.

Figures were selected by the author.

Author's comment

In this protein engineering experiment, the affinity of maltose binding protein for its ligands was increased approximately 100-fold by disrupting interactions in the "balancing interface", which altered the conformational equiilbria of the protein.
Brian Shilton