1N3W

Engineered High-Affinity Maltose-Binding Protein

1N3W の概要

• エントリーDOI: 10.2210/pdb1n3w/pdb
• 関連するPDBエントリー: 1N3X 1NL5 1PEB
• 関連するBIRD辞書のPRD_ID: PRD_900001
• 分子名称: Maltose-binding periplasmic protein, alpha-D-glucopyranose-(1-4)-alpha-D-glucopyranose (2 entities in total)
• 機能のキーワード: mbp, maltose-binding protein, high-affinity mutant, engineered, mbpdel-liganded, mbpdel, sugar binding protein
• 由来する生物種: Escherichia coli
• タンパク質・核酸の鎖数: 1
• 化学式量合計: 40559.89

構造登録者

Telmer, P.G.,Shilton, B.H. (登録日: 2002-10-29, 公開日: 2003-08-12, 最終更新日: 2024-02-14)

主引用文献

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

実験手法

X-RAY DIFFRACTION (2.6 Å)