Institute for Biological Interfaces 4, Karlsruhe Institute of Technology, P.O. Box 3640, 76021 Karlsruhe, Germany.
Physics Department T38, Technical University of Munich, James-Frank-Str. 1, 85748 Garching, Germany.
ACS Chem Neurosci. 2020 Dec 16;11(24):4426-4433. doi: 10.1021/acschemneuro.0c00640. Epub 2020 Nov 24.
Cleavage of substrates by γ-secretase is an inherently slow process where substrate-enzyme affinities cannot be broken down into specific sequence requirements in contrast to soluble proteases. Nevertheless, despite its apparent sequence tolerance single point mutations in amyloid precursor protein can severely affect cleavage efficiencies and change product line preferences. We have determined by NMR spectroscopy the structures of the transmembrane domain of amyloid precursor protein in TFE/water and compared it to that of four mutants: two FAD mutants, V44M and I45T, and the two diglycine hinge mutants, G38L and G38P. In accordance with previous publications, the transmembrane domain is composed of two helical segments connected by the diglycine hinge. Mutations alter kink angles and structural flexibility. Furthermore, to our surprise, we observe different, but specific mutual orientations of N- and C-terminal helical segments in the four mutants compared to the wildtype. We speculate that the observed orientations for G38L, G38P, V44M, and I45T lead to unfavorable interactions with γ-secretase exosites during substrate movement to the enzyme's active site in presenilin and/or for the accommodation into the substrate-binding cavity of presenilin.
γ-分泌酶对底物的裂解是一个固有缓慢的过程，与可溶性蛋白酶不同，底物-酶亲和力不能分解为特定的序列要求。尽管如此，尽管其明显的序列容忍性，淀粉样前体蛋白中的单点突变可以严重影响裂解效率并改变产物谱偏好。我们通过 NMR 光谱法测定了跨膜结构域在 TFE/水中的结构，并与四个突变体进行了比较：两个 FAD 突变体 V44M 和 I45T，以及两个二肽铰链突变体 G38L 和 G38P。与先前的出版物一致，跨膜结构域由通过二肽铰链连接的两个螺旋段组成。突变会改变扭曲角和结构灵活性。此外，令我们惊讶的是，与野生型相比，我们在四个突变体中观察到 N-和 C-末端螺旋段的不同但特定的相互取向。我们推测观察到的 G38L、G38P、V44M 和 I45T 的取向导致在底物向早老素的活性位点移动期间与 γ-分泌酶外显子发生不利相互作用，和/或在早老素的底物结合腔中容纳。
