Department of Cell and Systems Biology, University of Toronto, Toronto, Ontario, Canada.
Department of Chemical and Physical Sciences, University of Toronto Mississauga, Mississauga, Ontario, Canada.
J Bacteriol. 2020 Mar 26;202(8). doi: 10.1128/JB.00644-19.
The last gene in the genome of the bacteriophage HK97 encodes gp74, an HNH endonuclease. HNH motifs contain two conserved His residues and an invariant Asn residue, and they adopt a ββα structure. gp74 is essential for phage head morphogenesis, likely because gp74 enhances the specific endonuclease activity of the HK97 terminase complex. Notably, the ability of gp74 to enhance the terminase-mediated cleavage of the phage site requires an intact HNH motif in gp74. Mutation of H82, the conserved metal-binding His residue in the HNH motif, to Ala abrogates gp74-mediated stimulation of terminase activity. Here, we present nuclear magnetic resonance (NMR) studies demonstrating that gp74 contains an α-helical insertion in the Ω-loop, which connects the two β-strands of the ββα fold, and a disordered C-terminal tail. NMR data indicate that the Ω-loop insert makes contacts to the ββα fold and influences the ability of gp74 to bind divalent metal ions. Further, the Ω-loop insert and C-terminal tail contribute to gp74-mediated DNA digestion and to gp74 activity in phage morphogenesis. The data presented here enrich our molecular-level understanding of how HNH endonucleases enhance terminase-mediated digestion of the site and contribute to the phage replication cycle. This study demonstrates that residues outside the canonical ββα fold, namely, the Ω-loop α-helical insert and a disordered C-terminal tail, regulate the activity of the HNH endonuclease gp74. The increased divalent metal ion binding when the Ω-loop insert is removed compared to reduced site digestion and phage formation indicates that the Ω-loop insert plays multiple regulatory roles. The data presented here provide insights into the molecular basis of the involvement of HNH proteins in phage DNA packing.
噬菌体 HK97 基因组的最后一个基因编码 gp74，这是一种 HNH 内切核酸酶。HNH 基序含有两个保守的 His 残基和一个不变的 Asn 残基，它们采用 ββα 结构。gp74 对噬菌体头部形态发生至关重要，可能是因为 gp74 增强了 HK97 末端酶复合物的特异性内切核酸酶活性。值得注意的是，gp74 增强末端酶介导的噬菌体 位点切割的能力需要 gp74 中完整的 HNH 基序。HNH 基序中保守的金属结合 His 残基 H82 突变为 Ala 会破坏 gp74 介导的末端酶活性的刺激作用。在这里，我们提供了核磁共振（NMR）研究，证明 gp74 在 Ω 环中包含一个α-螺旋插入，该插入连接了ββα 折叠的两个β-链，以及一个无序的 C 端尾巴。NMR 数据表明，Ω 环插入与ββα 折叠相互作用，并影响 gp74 结合二价金属离子的能力。此外，Ω 环插入和 C 端尾巴有助于 gp74 介导的 DNA 消化以及 gp74 在噬菌体形态发生中的活性。这里呈现的数据丰富了我们对 HNH 内切核酸酶如何增强末端酶介导的 位点消化以及如何促进噬菌体复制周期的分子水平理解。这项研究表明，在经典的ββα 折叠之外的残基，即 Ω 环α-螺旋插入和无序的 C 端尾巴，调节 HNH 内切核酸酶 gp74 的活性。与减少的 位点消化和噬菌体形成相比，当 Ω 环插入被去除时增加的二价金属离子结合表明 Ω 环插入发挥了多种调节作用。这里呈现的数据为 HNH 蛋白参与噬菌体 DNA 包装的分子基础提供了见解。
