1N3W

Engineered High-Affinity Maltose-Binding Protein

Summary for 1N3W

• Entry DOI: 10.2210/pdb1n3w/pdb
• Related: 1N3X 1NL5 1PEB
• Related PRD ID: PRD_900001
• Descriptor: Maltose-binding periplasmic protein, alpha-D-glucopyranose-(1-4)-alpha-D-glucopyranose (2 entities in total)
• Functional Keywords: mbp, maltose-binding protein, high-affinity mutant, engineered, mbpdel-liganded, mbpdel, sugar binding protein
• Biological source: Escherichia coli
• Total number of polymer chains: 1
• Total formula weight: 40559.89

Authors

Telmer, P.G.,Shilton, B.H. (deposition date: 2002-10-29, release date: 2003-08-12, Last modification date: 2024-02-14)

Primary citation

Telmer, P.G.,Shilton, B.H.
Insights into the Conformational Equilibria of Maltose-binding Protein by Analysis of High Affinity Mutants.
J.Biol.Chem., 278:34555-34567, 2003

PubMed 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.

PubMed: 12794084
DOI: 10.1074/jbc.M301004200

Experimental method

X-RAY DIFFRACTION (2.6 Å)