Telmer Patrick G, Shilton Brian H
Department of Biochemistry, University of Western Ontario, London, Ontario N6A 5C1, Canada.
J Biol Chem. 2003 Sep 5;278(36):34555-67. doi: 10.1074/jbc.M301004200. Epub 2003 Jun 6.
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.
通过去除与配体结合裂隙相对的界面中的基团，麦芽糖结合蛋白（MBP）对麦芽糖及相关碳水化合物的亲和力大幅提高。野生型蛋白对麦芽糖的解离常数（KD）为1200 nM；将残基Met-321和Gln-325都突变为丙氨酸后，对麦芽糖的KD为70 nM；缺失4个残基Glu-172、Asn-173、Lys-175和Tyr-176（它们是一个无序环的一部分），对麦芽糖的KD为110 nM。将这些突变组合起来，对麦芽糊精的亲和力增加，对麦芽三糖的KD为6 nM。使用表面等离子体共振光谱对突变体的配体结合进行比较，结果表明解离速率的降低是亲和力增加的原因。小角X射线散射用于证明，无论有无麦芽糖，这些突变都不会显著影响MBP的溶液构象。所选突变体的晶体结构表明，这些突变在MBP的闭合或开放构象中均不会引起显著的结构变化。这些研究表明，在与配体结合裂隙相对的界面（我们称之为“平衡界面”）中的相互作用负责调节MBP对其配体的亲和力。我们的结果与一个模型一致，即配体结合的蛋白在闭合和开放构象之间交替，平衡界面中相互作用的去除降低了开放构象的稳定性，而不影响闭合构象。
