Woo S B, Neet K E
Department of Biological Chemistry, Finch University of Health Sciences/The Chicago Medical School, North Chicago, Illinois 60064, USA.
J Biol Chem. 1996 Oct 4;271(40):24433-41. doi: 10.1074/jbc.271.40.24433.
The role of the four histidine residues in receptor binding and activity of mouse nerve growth factor (NGF) was investigated using both site-directed mutagenesis and chemical modification with diethyl pyrocarbonate. Replacement of His-75 or His-84 with alanine resulted in decreased biological activity and decreased affinity for p140(trkA); however, with H75A only, a 5-fold increased affinity toward p75(LANR) was observed. The effect of simultaneous replacement of both His-75 and His-84 was neither additive nor synergistic. Slight perturbations in circular dichroism spectra and weakened self-association of the mutants indicated that His-75 and His-84 may be involved in stability, dimerization, and/or folding of NGF. Diethyl pyrocarbonate modification of His-4 and His-8 in the H75A/H84Q double mutant abolished neuritogenesis, binding to both receptors, and phosphorylation of p140(trkA) in PC12 cells. These chemical and mutational results confirm and clarify previous evidence for the involvement of His-75 and His-84 (Dunbar, J. C., Tregear, G. W., and Bradshaw, R. A. (1984) J. Protein Chem. 3, 349-356) or His-4 and His-8 (Shih, A., Laramee, G. R., Schmelzer, C. H., Burton, L. E., and Winslow, J. W. (1994) J. Biol. Chem. 269, 27679-27686) in receptor binding of NGF. At least three and possibly all four histidines, which are located in three spatially distinct regions, contribute to maintenance of functional sites that are essential for receptor binding and activity of NGF.
利用定点诱变和焦碳酸二乙酯化学修饰法，研究了4个组氨酸残基在小鼠神经生长因子（NGF）受体结合及活性中的作用。用丙氨酸取代His-75或His-84会导致生物活性降低以及对p140（trkA）的亲和力下降；然而，仅对于H75A而言，观察到对p75（LANR）的亲和力增加了5倍。同时取代His-75和His-84的效果既不是相加的也不是协同的。圆二色光谱的轻微扰动以及突变体自缔合减弱表明，His-75和His-84可能参与了NGF的稳定性、二聚化和/或折叠。在H75A/H84Q双突变体中，焦碳酸二乙酯对His-4和His-8的修饰消除了神经突生长、与两种受体的结合以及PC12细胞中p140（trkA）的磷酸化。这些化学和突变结果证实并阐明了先前关于His-75和His-84（邓巴，J.C.，特里吉尔，G.W.，和布拉德肖，R.A.（1984年）《蛋白质化学杂志》3，349 - 356）或His-4和His-8（施，A.，拉腊米，G.R.，施梅尔泽，C.H.，伯顿，L.E.，和温斯洛，J.W.（1994年）《生物化学杂志》269，27679 - 27686）参与NGF受体结合的证据。位于三个空间不同区域的至少三个且可能所有四个组氨酸，有助于维持对NGF受体结合和活性至关重要的功能位点。
