Arutyunova Elena, Panwar Pankaj, Skiba Pauline M, Gale Nicola, Mak Michelle W, Lemieux M Joanne
Department of Biochemistry, Faculty of Medicine & Dentistry, Membrane Protein Disease Research Group, University of Alberta, Edmonton, AB, Canada.
Department of Biochemistry, Faculty of Medicine & Dentistry, Membrane Protein Disease Research Group, University of Alberta, Edmonton, AB, Canada
EMBO J. 2014 Sep 1;33(17):1869-81. doi: 10.15252/embj.201488149. Epub 2014 Jul 9.
Proteolysis within the lipid bilayer is poorly understood, in particular the regulation of substrate cleavage. Rhomboids are a family of ubiquitous intramembrane serine proteases that harbour a buried active site and are known to cleave transmembrane substrates with broad specificity. In vitro gel and Förster resonance energy transfer (FRET)-based kinetic assays were developed to analyse cleavage of the transmembrane substrate psTatA (TatA from Providencia stuartii). We demonstrate significant differences in catalytic efficiency (kcat/K0.5) values for transmembrane substrate psTatA (TatA from Providencia stuartii) cleavage for three rhomboids: AarA from P. stuartii, ecGlpG from Escherichia coli and hiGlpG from Haemophilus influenzae demonstrating that rhomboids specifically recognize this substrate. Furthermore, binding of psTatA occurs with positive cooperativity. Competitive binding studies reveal an exosite-mediated mode of substrate binding, indicating allostery plays a role in substrate catalysis. We reveal that exosite formation is dependent on the oligomeric state of rhomboids, and when dimers are dissociated, allosteric substrate activation is not observed. We present a novel mechanism for specific substrate cleavage involving several dynamic processes including positive cooperativity and homotropic allostery for this interesting class of intramembrane proteases.
脂质双分子层中的蛋白水解作用目前还知之甚少，尤其是底物切割的调控机制。菱形蛋白酶是一类广泛存在的膜内丝氨酸蛋白酶，其活性位点深埋于膜内，已知能以广泛的特异性切割跨膜底物。我们开发了基于体外凝胶和福斯特共振能量转移（FRET）的动力学分析方法，以分析跨膜底物psTatA（来自斯氏普罗威登斯菌的TatA）的切割情况。我们证明，对于三种菱形蛋白酶，即来自斯氏普罗威登斯菌的AarA、来自大肠杆菌的ecGlpG和来自流感嗜血杆菌的hiGlpG，跨膜底物psTatA（来自斯氏普罗威登斯菌的TatA）切割的催化效率（kcat/K0.5）值存在显著差异，这表明菱形蛋白酶能特异性识别该底物。此外，psTatA的结合具有正协同性。竞争性结合研究揭示了一种由别构位点介导的底物结合模式，表明别构作用在底物催化中发挥作用。我们发现别构位点的形成取决于菱形蛋白酶的寡聚状态，当二聚体解离时，未观察到别构底物激活现象。我们提出了一种新的特异性底物切割机制，涉及多个动态过程，包括这种有趣的膜内蛋白酶的正协同性和同促别构作用。
