Determination of surface-exposed, functional domains of gonococcal XXXX -binding protein A.

@entity20117 @entity69
The gonococcal @entity69 receptor is composed of two distinct proteins, @entity20117 and TbpB. @entity20117 is a member of the TonB-dependent family of integral outer membrane transporters, while TbpB is lipid modified and thought to be peripherally surface exposed. We previously proposed a hypothetical topology model for gonococcal @entity20117 that was based upon computer predictions and similarity with other TonB-dependent transporters for which crystal structures have been determined. In the present study, the hemagglutinin epitope was inserted into @entity20117 to probe the surface topology of this protein and secondarily to test the functional impacts of site-specific mutagenesis. Twelve epitope insertion mutants were constructed, five of which allowed us to confirm the surface exposure of loops 2, 3, 5, 7, and 10. In contrast to the predictions set forth by the hypothetical model, insertion into the plug region resulted in an epitope that was surface accessible, while epitope insertions into two putative loops (9 and 11) were not surface accessible. Insertions into putative loop 3 and beta strand 9 abolished @entity69 binding and utilization, and the plug insertion mutant exhibited decreased @entity69 -binding affinity concomitant with an inability to utilize it. Insertion into putative beta strand 16 generated a mutant that was able to bind @entity69 normally but that was unable to mediate utilization. Mutants with insertions into putative loops 2, 9, and 11 maintained wild-type binding affinity but could utilize only @entity69 in the presence of TbpB. This is the first demonstration of the ability of TbpB to compensate for a mutation in @entity20117 .
