Department of Biochemistry and Genetics, La Trobe Institute for Molecular Science, La Trobe University, Melbourne, Victoria 3086, Australia.
Bio21 Molecular Science and Biotechnology Institute, The University of Melbourne, Parkville, Australia.
Biochem J. 2020 Dec 11;477(23):4527-4541. doi: 10.1042/BCJ20200776.
Premature apoptosis of cells is a strategy utilized by multicellular organisms to counter microbial threats. Orf virus (ORFV) is a large double-stranded DNA virus belonging to the poxviridae. ORFV encodes for an apoptosis inhibitory protein ORFV125 homologous to B-cell lymphoma 2 or Bcl-2 family proteins, which has been shown to inhibit host cell encoded pro-apoptotic Bcl-2 proteins. However, the structural basis of apoptosis inhibition by ORFV125 remains to be clarified. We show that ORFV125 is able to bind to a range of peptides spanning the BH3 motif of human pro-apoptotic Bcl-2 proteins including Bax, Bak, Puma and Hrk with modest to weak affinity. We then determined the crystal structures of ORFV125 alone as well as bound to the highest affinity ligand Bax BH3 motif. ORFV125 adopts a globular Bcl-2 fold comprising 7 α-helices, and utilizes the canonical Bcl-2 binding groove to engage pro-apoptotic host cell Bcl-2 proteins. In contrast with a previously predicted structure, ORFV125 adopts a domain-swapped dimeric topology, where the α1 helix from one protomer is swapped into a neighbouring unit. Furthermore, ORFV125 differs from the conserved architecture of the Bcl-2 binding groove and instead of α3 helix forming one of the binding groove walls, ORFV125 utilizes an extended α2 helix that comprises the equivalent region of helix α3. This results in a subtle variation of previously observed dimeric Bcl-2 architectures in other poxvirus and human encoded Bcl-2 proteins. Overall, our results provide a structural and mechanistic basis for orf virus-mediated inhibition of host cell apoptosis.
细胞的过早凋亡是多细胞生物对抗微生物威胁的一种策略。口疮病毒（ORFV）是一种大型双链 DNA 病毒，属于痘病毒科。ORFV 编码一种凋亡抑制蛋白 ORFV125，与 B 细胞淋巴瘤 2 或 Bcl-2 家族蛋白同源，已被证明可抑制宿主细胞编码的促凋亡 Bcl-2 蛋白。然而，ORFV125 抑制细胞凋亡的结构基础仍有待阐明。我们表明，ORFV125 能够结合一系列跨越人类促凋亡 Bcl-2 蛋白 Bax、Bak、Puma 和 Hrk 的 BH3 基序的肽段，亲和力适中至较弱。然后，我们确定了 ORFV125 单独以及与最高亲和力配体 Bax BH3 基序结合的晶体结构。ORFV125 采用球形 Bcl-2 折叠结构，由 7 个α-螺旋组成，并利用典型的 Bcl-2 结合槽与促凋亡的宿主细胞 Bcl-2 蛋白结合。与先前预测的结构不同，ORFV125 采用了一种交换的二聚体拓扑结构，其中一个原体的α1 螺旋被交换到相邻的单位中。此外，ORFV125 与 Bcl-2 结合槽的保守结构不同，α3 螺旋不形成结合槽壁之一，而是 ORFV125 利用扩展的α2 螺旋来包含相当于α3 螺旋的区域。这导致了先前在其他痘病毒和人类编码的 Bcl-2 蛋白中观察到的二聚体 Bcl-2 结构的细微变化。总体而言，我们的研究结果为 ORFV 介导的宿主细胞凋亡抑制提供了结构和机制基础。
