Department of Chemistry, University of Missouri, Columbia, Missouri.
Department of Pharmacological Sciences, Icahn School of Medicine at Mount Sinai, New York, New York.
Biophys J. 2018 Apr 10;114(7):1579-1589. doi: 10.1016/j.bpj.2018.01.043.
Intramembrane-cleaving proteases (I-CLiPs) activate pools of single-pass helical membrane protein signaling precursors that are key in the physiology of prokaryotic and eukaryotic cells. Proteases typically cleave peptide bonds within extended or flexible regions of their substrates, and thus the mechanism underlying the ability of I-CLiPs to hydrolyze the presumably α-helical transmembrane domain (TMD) of these membrane proteins is unclear. Using deep-ultraviolet resonance Raman spectroscopy in combination with isotopic labeling, we show that although predominantly in canonical α-helical conformation, the TMD of the established I-CLiP substrate Gurken displays 3-helical geometry. As measured by microscale thermophoresis, this substrate binds with high affinity to the I-CLiPs GlpG rhomboid and MCMJR1 presenilin homolog in detergent micelles. Binding results in deep-ultraviolet resonance Raman spectra, indicating conformational changes consistent with unwinding of the 3-helical region of the substrate's TMD. This 3-helical conformation is key for intramembrane proteolysis, as the substitution of a single proline residue in the TMD of Gurken by alanine suppresses 3-helical content in favor of α-helical geometry and abolishes cleavage without affecting binding to the I-CLiP. Complemented by molecular dynamics simulations of the TMD of Gurken, our vibrational spectroscopy data provide biophysical evidence in support of a model in which the transmembrane region of cleavable I-CLiP substrates displays local deviations in canonical α-helical conformation characterized by chain flexibility, and binding to the enzyme results in conformational changes that facilitate local unwinding of the transmembrane helix for cleavage.
跨膜切割蛋白酶（I-CLiPs）激活单通道螺旋膜蛋白信号前体池，这些前体在原核和真核细胞的生理学中起着关键作用。蛋白酶通常在其底物的伸展或柔性区域内切割肽键，因此 I-CLiPs 水解这些膜蛋白的假定α-螺旋跨膜结构域（TMD）的机制尚不清楚。本研究使用深紫外共振拉曼光谱结合同位素标记，表明尽管 Gurken 是既定的 I-CLiP 底物，但它的 TMD 主要处于典型的α-螺旋构象，但显示出 3-螺旋几何形状。通过微尺度热泳法测量，该底物与 GlpG 菱形和 MCMJR1 早老素同源物 I-CLiP 以高亲和力结合在去污剂胶束中。结合导致深紫外共振拉曼光谱的变化，表明与底物 TMD 的 3-螺旋区域解旋一致的构象变化。这种 3-螺旋构象是跨膜水解的关键，因为 Gurken 的 TMD 中单个脯氨酸残基被丙氨酸取代会降低 3-螺旋含量，有利于α-螺旋构象，并消除切割而不影响与 I-CLiP 的结合。本研究的振动光谱数据与 Gurken 的 TMD 的分子动力学模拟相辅相成，为支持这样一种模型提供了生物物理证据，即可切割的 I-CLiP 底物的跨膜区域显示出局部偏离典型α-螺旋构象的特征，表现为链的柔韧性，与酶的结合导致构象变化，从而促进跨膜螺旋的局部解旋以进行切割。
