PDF(Pubmed)
Abstract：
The complement system serves as the first line of defense against invading pathogens by promoting opsonophagocytosis and bacteriolysis. Antibody-dependent activation of complement occurs through the classical pathway and relies on the activity of initiating complement proteases of the C1 complex, C1r and C1s. The causative agent of Lyme disease, Borrelia burgdorferi, expresses two paralogous outer surface lipoproteins of the OspEF-related protein family, ElpB and ElpQ, that act as specific inhibitors of classical pathway activation. We have previously shown that ElpB and ElpQ bind directly to C1r and C1s with high affinity and specifically inhibit C2 and C4 cleavage by C1s. To further understand how these novel protease inhibitors function, we carried out a series of hydrogen-deuterium exchange mass spectrometry (HDX-MS) experiments using ElpQ and full-length activated C1s as a model of Elp-protease interaction. Comparison of HDX-MS profiles between unbound ElpQ and the ElpQ/C1s complex revealed a putative C1s-binding site on ElpQ. HDX-MS-guided, site-directed ElpQ mutants were generated and tested for direct binding to C1r and C1s using surface plasmon resonance. Several residues within the C-terminal region of ElpQ were identified as important for protease binding, including a single conserved tyrosine residue that was required for ElpQ- and ElpB-mediated complement inhibition. Collectively, our study identifies key molecular determinants for classical pathway protease recognition by Elp proteins. This investigation improves our understanding of the unique complement inhibitory mechanism employed by Elp proteins which serve as part of a sophisticated complement evasion system present in Lyme disease spirochetes.
摘要：
补体系统通过促进调理吞噬作用和细菌溶解而充当抵抗入侵病原体的第一道防线。补体的抗体依赖性激活通过经典途径发生，并且依赖于C1复合物的补体蛋白酶的启动活性，C1r和C1s。莱姆病的病原体，伯氏疏螺旋体,表达OspEF相关蛋白家族的两个旁系外表面脂蛋白，ElpB和ElpQ,作为经典途径激活的特异性抑制剂。我们先前已经证明ElpB和ElpQ以高亲和力直接结合C1r和C1s,并特异性抑制C1s对C2和C4的切割。为了进一步了解这些新型蛋白酶抑制剂的功能，我们使用ElpQ和全长激活的C1s作为Elp/蛋白酶相互作用的模型进行了一系列氢-氘交换质谱（HDX-MS）实验。未结合的ElpQ和ElpQ/C1s复合物之间的HDX-MS图谱的比较揭示了ElpQ上推定的C1s结合位点。HDX-MS引导,产生定点ElpQ突变体,并使用表面等离子体共振测试与C1r和C1s的直接结合。ElpQ的C端区域内的几个残基被鉴定为对蛋白酶结合重要,包括ElpQ-和ElpB-介导的补体抑制所需的单个保守酪氨酸残基。总的来说，我们的研究确定了Elp蛋白识别经典途径蛋白酶的关键分子决定因素。这项研究提高了我们对Elp蛋白所采用的独特补体抑制机制的理解，Elp蛋白是莱姆病螺旋体中复杂的补体逃避系统的一部分。
