From the Max F. Perutz Laboratories, Medical University of Vienna, Dr. Bohr-Gasse 9/3, A-1030 Vienna, Austria and.
J Biol Chem. 2014 Feb 7;289(6):3749-62. doi: 10.1074/jbc.M113.512756. Epub 2013 Dec 19.
Successful vaccinia virus (VACV) replication in the host requires expression of viral proteins that interfere with host immunity, such as antagonists of the activation of the proinflammatory transcription factor NF-κB. Two such VACV proteins are A46 and A52. A46 interacts with the Toll-like receptor/interleukin-1R (TIR) domain of Toll-like receptors and intracellular adaptors such as MAL (MyD88 adapter-like), TRAM (TIR domain-containing adapter-inducing interferon-β (TRIF)-related adaptor molecule), TRIF, and MyD88, whereas A52 binds to the downstream signaling components TRAF6 and IRAK2. Here, we characterize A46 biochemically, determine by microscale thermophoresis binding constants for the interaction of A46 with the TIR domains of MyD88 and MAL, and present the 2.0 Å resolution crystal structure of A46 residues 87-229. Full-length A46 behaves as a tetramer; variants lacking the N-terminal 80 residues are dimeric. Nevertheless, both bind to the Toll-like receptor domains of MAL and MyD88 with KD values in the low μm range. Like A52, A46 also shows a Bcl-2-like fold but with biologically relevant differences from that of A52. Thus, A46 uses helices α4 and α6 to dimerize, compared with the α1-α6 face used by A52 and other Bcl-2 like VACV proteins. Furthermore, the loop between A46 helices α4-α5 is flexible and shorter than in A52; there is also evidence for an intramolecular disulfide bridge between consecutive cysteine residues. We used molecular docking to propose how A46 interacts with the BB loop of the TRAM TIR domain. Comparisons of A46 and A52 exemplify how subtle changes in viral proteins with the same fold lead to crucial differences in biological activity.
痘苗病毒（VACV）在宿主中的成功复制需要表达干扰宿主免疫的病毒蛋白，例如促炎转录因子 NF-κB 激活的拮抗剂。VACV 中有两种这样的蛋白，即 A46 和 A52。A46 与 Toll 样受体/白细胞介素-1R（TIR）域的 Toll 样受体和细胞内衔接子相互作用，如 MAL（MyD88 衔接样）、TRAM（TIR 域包含衔接诱导干扰素-β（TRIF）相关衔接分子）、TRIF 和 MyD88，而 A52 与下游信号传导成分 TRAF6 和 IRAK2 结合。在这里，我们对 A46 进行了生化特性表征，通过微量热泳动测定了 A46 与 MyD88 和 MAL 的 TIR 域相互作用的结合常数，并呈现了 A46 残基 87-229 的 2.0 Å 分辨率晶体结构。全长 A46 表现为四聚体；缺乏 N 端 80 个残基的变体为二聚体。尽管如此，两者都以 KD 值在低 μm 范围内与 MAL 和 MyD88 的 Toll 样受体域结合。与 A52 一样，A46 也表现出 Bcl-2 样折叠，但与 A52 和其他 Bcl-2 样 VACV 蛋白有生物学相关的差异。因此，与 A52 和其他 Bcl-2 样 VACV 蛋白使用 α1-α6 面不同，A46 使用 α4 和 α6 螺旋来二聚化。此外，A46 中 α4-α5 螺旋之间的环是灵活的，比 A52 短；也有证据表明连续半胱氨酸残基之间存在分子内二硫键。我们使用分子对接来提出 A46 如何与 TRAM TIR 域的 BB 环相互作用。A46 和 A52 的比较说明了具有相同折叠的病毒蛋白的微小变化如何导致生物学活性的关键差异。
