Department of Pharmacology, Howard Hughes Medical Institute.
Department of Biophysics, University of Texas Southwestern Medical Center, 6001 Forest Park Road, Dallas, TX 75390, USA.
Cell Rep. 2019 Mar 19;26(12):3336-3346.e4. doi: 10.1016/j.celrep.2019.02.082.
The DNA-dependent metalloprotease Spartan (SPRTN) cleaves DNA-protein crosslinks (DPCs) and protects cells from DPC-induced genome instability. Germline mutations of SPRTN are linked to human Ruijs-Aalfs syndrome (RJALS) characterized by progeria and early-onset hepatocellular carcinoma. The mechanism of DNA-mediated activation of SPRTN is not understood. Here, we report the crystal structure of the human SPRTN SprT domain bound to single-stranded DNA (ssDNA). Our structure reveals a Zn-binding sub-domain (ZBD) in SprT that shields its active site located in the metalloprotease sub-domain (MPD). The narrow catalytic groove between MPD and ZBD only permits cleavage of flexible substrates. The ZBD contains an ssDNA-binding site, with a DNA-base-binding pocket formed by aromatic residues. Mutations of ssDNA-binding residues diminish the protease activity of SPRTN. We propose that the ZBD contributes to the ssDNA specificity of SPRTN, restricts the access of globular substrates, and positions DPCs, which may need to be partially unfolded, for optimal cleavage.
依赖于 DNA 的金属蛋白酶 Spartan（SPRTN）可切割 DNA-蛋白质交联物（DPC），从而保护细胞免受 DPC 诱导的基因组不稳定性。SPRTN 的种系突变与人类 Ruijs-Aalfs 综合征（RJALS）有关，其特征为早衰和早发性肝细胞癌。SPRTN 的 DNA 介导的激活机制尚不清楚。在此，我们报告了与人 SPRTN SprT 结构域结合的单链 DNA（ssDNA）的晶体结构。我们的结构揭示了 SprT 中的一个 Zn 结合亚结构域（ZBD），该亚结构域可屏蔽位于金属蛋白酶亚结构域（MPD）中的活性位点。MPD 和 ZBD 之间狭窄的催化槽仅允许柔性底物的切割。ZBD 包含一个 ssDNA 结合位点，由芳香族残基形成 DNA-碱基结合口袋。ssDNA 结合残基的突变会降低 SPRTN 的蛋白酶活性。我们提出，ZBD 有助于 SPRTN 的 ssDNA 特异性，限制球状底物的进入，并定位 DPC，DPC 可能需要部分展开，以进行最佳切割。
