PDF(Pubmed)
Abstract：
Human peptidylarginine deiminase isoform VI (PAD6), which is predominantly limited to cytoplasmic lattices in the mammalian oocytes in ovarian tissue, is essential for female fertility. It belongs to the peptidylarginine deiminase (PAD) enzyme family that catalyzes the conversion of arginine residues to citrulline in proteins. In contrast to other members of the family, recombinant PAD6 was previously found to be catalytically inactive. We sought to provide structural insight into the human homologue to shed light on this observation. We report here the first crystal structure of PAD6, determined at 1.7 Å resolution. PAD6 follows the same domain organization as other structurally known PAD isoenzymes. Further structural analysis and size-exclusion chromatography show that PAD6 behaves as a homodimer similar to PAD4. Differential scanning fluorimetry suggests that PAD6 does not coordinate Ca2+ which agrees with acidic residues found to coordinate Ca2+ in other PAD homologs not being conserved in PAD6. The crystal structure of PAD6 shows similarities with the inactive state of apo PAD2, in which the active site conformation is unsuitable for catalytic citrullination. The putative active site of PAD6 adopts a non-productive conformation that would not allow protein-substrate binding due to steric hindrance with rigid secondary structure elements. This observation is further supported by the lack of activity on the histone H3 and cytokeratin 5 substrates. These findings suggest a different mechanism for enzymatic activation compared with other PADs; alternatively, PAD6 may exert a non-enzymatic function in the cytoplasmic lattice of oocytes and early embryos.
摘要：
人肽酰精氨酸脱亚胺酶亚型VI（PAD6），主要限于卵巢组织中哺乳动物卵母细胞的细胞质晶格，对女性生育至关重要。它属于肽基精氨酸脱亚胺酶（PAD）酶家族，可催化蛋白质中精氨酸残基向瓜氨酸的转化。与其他家庭成员相比，以前发现重组PAD6是无催化活性的。我们试图提供对人类同源物的结构见解，以阐明这一观察结果。我们在此报告PAD6的第一个晶体结构，以1.7µ分辨率确定。PAD6遵循与其他结构上已知的PAD同工酶相同的结构域组织。进一步的结构分析和尺寸排阻色谱显示PAD6表现为类似于PAD4的同源二聚体。差示扫描荧光法表明PAD6不与Ca2配位，这与在PAD6中不保守的其他PAD同系物中发现的与Ca2配位的酸性残基一致。PAD6的晶体结构与apoPAD2的非活性状态相似，其中活性位点构象不适合催化瓜氨酸化。PAD6的推定活性位点采用非生产性构象，由于具有刚性二级结构元件的空间位阻，因此不允许蛋白质-底物结合。对组蛋白H3和细胞角蛋白5底物缺乏活性进一步支持了这一观察结果。这些发现表明与其他PAD相比，酶促激活的机制不同；或者，PAD6可能在卵母细胞和早期胚胎的细胞质晶格中发挥非酶功能。
