Structural determination of the complement inhibitory domain of Borrelia burgdorferi BBK32 provides insight into classical pathway complement evasion by Lyme disease spirochetes

• Published in: PLoS pathogens 2019-03, Vol.15 (3), p.e1007659
• Main Authors: Xie, Jialei, Zhi, Hui, Garrigues, Ryan J, Keightley, Andrew, Garcia, Brandon L, Skare, Jon T
• Format: Article
• Language: English
• Subjects: Affinity; Antigens; Automation; Bacterial Proteins - genetics; Bacterial Proteins - immunology; Bacterial Proteins - ultrastructure; Binding; Biology and Life Sciences; Borrelia burgdorferi; Borrelia burgdorferi - genetics; Borrelia burgdorferi - immunology; Borrelia burgdorferi Group; Care and treatment; Classical pathway; Complement; Complement C1r - metabolism; Complement component C1r; Complement Pathway, Classical - genetics; Complement Pathway, Classical - physiology; Complement System Proteins - metabolism; Crystal structure; Gene expression; Health sciences; Humans; Immunology; Inhibition; Lectins; Lyme disease; Lyme Disease - physiopathology; Medicine; Medicine and Health Sciences; Mutagenesis; Pathogenesis; Pattern recognition; Physical Sciences; Protease; Proteases; Protein Domains - physiology; Protein structure; Proteins; Recombinant Proteins; Risk factors; Sequence Analysis, Protein; Serine; Serine proteinase; Site-directed mutagenesis; Spirochetes; Structure-function relationships; Thrombin; Vector-borne diseases
• ISSN: 1553-7374; 1553-7366; 1553-7374
• DOI: 10.1371/journal.ppat.1007659

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.