Differential Contribution of the Repeats to Heparin Binding of HBHA, a Major Adhesin of Mycobacterium tuberculosis

BackgroundTuberculosis remains one of the most important causes of global mortality and morbidity, and the molecular mechanisms of the pathogenesis are still incompletely understood. Only few virulence factors of the causative agent Mycobacterium tuberculosis are known. One of them is the heparin-binding haemagglutinin (HBHA), an important adhesin for epithelial cells and an extrapulmonary dissemination factor. HBHA mediates mycobacterial adherence to epithelial cells via the interactions of its C-terminal, lysine rich repeat domain with sulfated glycoconjugates on the surface of epithelial cells. Methodology/Principal FindingsUsing defined heparin sulfate (HS) analogs, we determined the minimal heparin fragment length for HBHA binding and structural adaptations of the HBHA heparin-binding domain (HBD) upon binding to heparin. The NMR studies show significant shifts of all residues in the HBD upon interaction with heparin, with stronger shifts in the last repeats compared to the upstream repeats, and indicated that the HS fragments with 14 sugar units cover the entire C-terminal lysine-rich domain of HBHA. The differential implication of the repeats is determined by the relative position of prolines and lysines within each repeat, and may contribute to binding specificity. GAG binding induces a non-homogeneous structural rearrangement in the HBD, with stabilization of a nascent α-helix only in the last penta-repeats. Conclusion/SignificanceMycobacterial HBHA undergoes structural adaptation upon interaction with GAGs, which is likely involved in binding specificities of the adhesin, and mycobacterial pathogens may use HBD polymorphisms for host or organ specificity. Further studies will aim at decoding the complementarity between HBD repeats and HS sequence.

背景：结核病仍是全球致死与致残的首要病因之一，其发病的分子机制仍未完全阐明。目前已知的致病菌结核分枝杆菌（Mycobacterium tuberculosis）毒力因子寥寥无几，肝素结合血凝素（heparin-binding haemagglutinin, HBHA）便是其中之一，它是一类重要的上皮细胞黏附素，同时也是肺外播散因子。HBHA通过其羧基末端富赖氨酸重复结构域，与上皮细胞表面的硫酸化糖缀合物相互作用，介导分枝杆菌黏附至上皮细胞。
方法与主要结果：本研究使用结构明确的肝素硫酸盐（heparin sulfate, HS）类似物，明确了HBHA结合所需的最小肝素片段长度，以及HBHA肝素结合结构域（heparin-binding domain, HBD）在结合肝素时的结构适配变化。核磁共振（NMR）研究显示，HBHA-HBD内所有残基在与肝素结合后均出现显著化学位移，且末端重复序列的位移幅度较上游重复序列更为显著；结果同时证实，包含14个糖单元的HS片段可覆盖HBHA完整的羧基末端富赖氨酸结构域。各重复序列的差异化作用由每个重复序列内脯氨酸与赖氨酸的相对位置决定，这可能有助于提升结合特异性。糖胺聚糖（glycosaminoglycan, GAG）的结合会诱导HBD发生非均一的结构重排，仅在末端五联重复序列中稳定新生的α螺旋结构。
结论与意义：分枝杆菌来源的HBHA在与糖胺聚糖相互作用时会发生结构适配，这可能与该黏附素的结合特异性密切相关；分枝杆菌病原菌或可通过HBD多态性实现宿主或器官特异性靶向。后续研究将致力于解析HBD重复序列与HS序列之间的互补关系。