Allosteric regulation of rhomboid intramembrane proteolysis
Allosteric regulation of rhomboid intramembrane proteolysis
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DOI：
10.15252/embj.201488149
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发表时间：
2014-09-01
期刊：
影响因子：
11.4
通讯作者：
Lemieux, M. Joanne
中科院分区：
文献类型：
--
作者：
Arutyunova, Elena;Panwar, Pankaj;Lemieux, M. Joanne
脂双层内的蛋白水解知之甚少，特别是底物裂解的调节。菱形蛋白酶是一个普遍存在的膜内丝氨酸蛋白酶家族，其具有埋藏的活性位点，并且已知以广泛的特异性切割跨膜底物。开发了基于体外凝胶和福斯特共振能量转移（FRET）的动力学测定以分析跨膜底物psTatA（来自斯氏普罗威登斯菌的TatA）的裂解。我们证明了三种菱形蛋白对跨膜底物psTatA（来自斯氏普罗威登斯菌的TatA）裂解的催化效率（k（cat）/K-0.5）值的显著差异：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，表明菱形蛋白特异性识别该底物。此外，psTatA的结合以正协同性发生。竞争性结合研究揭示了一种外来位点介导的底物结合模式，表明变构在底物催化中起作用。我们发现，exosite的形成是依赖于低聚状态的菱形，当二聚体解离，变构底物激活没有观察到。我们提出了一种新的机制，具体的底物切割涉及几个动态过程，包括积极的协同性和同型变构这类有趣的膜内蛋白酶。
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 Forster 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 (k(cat)/K-0.5) 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.