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

Abstract

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.

Targeting the Initiator Protease of the Classical Pathway of Complement Using Fragment-Based Drug Discovery

“…P00736). C1r-domain truncations were purified under denaturing conditions using previously published protocols with some modifications [ 39 , 56 ]. Plasmids were transformed into E. coli BL21 (DE3) and cells were grown to an optical density of 0.6–0.8 OD600 at 37 °C in Terrific Broth supplemented with kanamycin.…”

Complement evasion strategies of Borrelia burgdorferi sensu lato

“…The latter part contains three residues available for C1r binding and inhibition. The binding of BBK32 to C1r was found to be calcium-dependent [85]. Binding and inhibition of C1r are a novel type of anticomplement activity by bacteria.…”

Complement Evasion in Borrelia spirochetes: Mechanisms and Opportunities for Intervention

“…Although the structure of BBK32 has not yet been fully determined, the crystal structure of the C-terminal binding region for C1r was resolved and published during the writing of this paper, indicating a high interest in this protein [83]. A BLAST search for the B. burgdorferi BBK32 protein amino acid sequence revealed no matches outside the Borrelia genus.…”