Department of Microbial Pathogenesis and Immunology, College of Medicine, Texas A&M Health Sciences Center, Bryan, Texas, United States of America.
Department of Microbiology and Immunology, Brody School of Medicine, East Carolina University, Greenville, North Carolina, United States of America.
PLoS Pathog. 2019 Mar 21;15(3):e1007659. doi: 10.1371/journal.ppat.1007659. eCollection 2019 Mar.
The carboxy-terminal domain of the BBK32 protein from Borrelia burgdorferi sensu stricto, termed BBK32-C, binds and inhibits the initiating serine protease of the human classical complement pathway, C1r. In this study we investigated the function of BBK32 orthologues of the Lyme-associated Borrelia burgdorferi sensu lato complex, designated BAD16 from B. afzelii strain PGau and BGD19 from B. garinii strain IP90. Our data show that B. afzelii BAD16-C exhibits BBK32-C-like activities in all assays tested, including high-affinity binding to purified C1r protease and C1 complex, and potent inhibition of the classical complement pathway. Recombinant B. garinii BGD19-C also bound C1 and C1r with high-affinity yet exhibited significantly reduced in vitro complement inhibitory activities relative to BBK32-C or BAD16-C. Interestingly, natively produced BGD19 weakly recognized C1r relative to BBK32 and BAD16 and, unlike these proteins, BGD19 did not confer significant protection from serum killing. Site-directed mutagenesis was performed to convert BBK32-C to resemble BGD19-C at three residue positions that are identical between BBK32 and BAD16 but different in BGD19. The resulting chimeric protein was designated BXK32-C and this BBK32-C variant mimicked the properties observed for BGD19-C. To query the disparate complement inhibitory activities of BBK32 orthologues, the crystal structure of BBK32-C was solved to 1.7Å limiting resolution. BBK32-C adopts an anti-parallel four-helix bundle fold with a fifth alpha-helix protruding from the helical core. The structure revealed that the three residues targeted in the BXK32-C chimera are surface-exposed, further supporting their potential relevance in C1r binding and inhibition. Additional binding assays showed that BBK32-C only recognized C1r fragments containing the serine protease domain. The structure-function studies reported here improve our understanding of how BBK32 recognizes and inhibits C1r and provide new insight into complement evasion mechanisms of Lyme-associated spirochetes of the B. burgdorferi sensu lato complex.
伯氏疏螺旋体严格意义上的 BBK32 蛋白的羧基末端结构域，称为 BBK32-C，与人类经典补体途径的起始丝氨酸蛋白酶 C1r 结合并抑制其活性。在这项研究中，我们研究了莱姆病相关伯氏疏螺旋体亚种复合体中的 BBK32 同源物，即阿菲型伯氏疏螺旋体菌株 PGau 的 BAD16 和伽氏伯氏疏螺旋体菌株 IP90 的 BGD19 的功能。我们的数据表明，阿菲型伯氏疏螺旋体 BAD16-C 在所有测试的实验中都表现出 BBK32-C 样的活性，包括与纯化的 C1r 蛋白酶和 C1 复合物的高亲和力结合，以及对经典补体途径的强烈抑制作用。重组伯氏疏螺旋体 BGD19-C 也与 C1 和 C1r 高亲和力结合，但与 BBK32-C 或 BAD16-C 相比，体外补体抑制活性显著降低。有趣的是，天然产生的 BGD19 与 BBK32 和 BAD16 相比，对 C1r 的识别能力较弱，与这些蛋白不同的是，BGD19 并不能显著防止血清杀伤。我们进行了定点突变，将 BBK32-C 转化为与 BGD19-C 中三个残基相同的结构，而这三个残基在 BBK32 和 BAD16 中是相同的，但在 BGD19 中不同。由此产生的嵌合蛋白被命名为 BXK32-C，这种 BBK32-C 变体模拟了 BGD19-C 的特性。为了探究 BBK32 同源物不同的补体抑制活性，我们将 BBK32-C 的晶体结构解析至 1.7Å 的限制分辨率。BBK32-C 采用反平行四螺旋束折叠结构，第五个α-螺旋从螺旋核心中伸出。该结构表明，在 BXK32-C 嵌合体中靶向的三个残基是表面暴露的，进一步支持了它们在 C1r 结合和抑制中的潜在作用。其他结合实验表明，BBK32-C 仅识别含有丝氨酸蛋白酶结构域的 C1r 片段。本研究的结构功能研究提高了我们对 BBK32 如何识别和抑制 C1r 的理解，并为莱姆病相关螺旋体逃避补体的机制提供了新的见解。
