The transamidase complex is a molecular machine in the endoplasmic reticulum of eukaryotes that attaches a glycosylphosphatidylinositol (GPI) lipid anchor to substrate proteins after cleaving a C-terminal propeptide with a defined sequence signal. Its five subunits are very hydrophobic; thus, solubility, heterologous expression and complex reconstruction are difficult. Therefore, theoretical approaches are currently the main source of insight into details of 3D structure and of the catalytic process. In this work, we generated model 3D structures of the lumenal domain of human GPAA1, the M28-type metallo-peptide-synthetase subunit of the transamidase, including zinc ion and model substrate positions. In comparative molecular dynamics (MD) simulations of M28-type structures and our GPAA1 models, we estimated the metal ion binding energies with evolutionary conserved amino acid residues in the catalytic cleft. We find that canonical zinc binding sites 2 and 3 are strongest binders for Zn1 and, where a second zinc is available, sites 2 and 4 for Zn2. Zinc interaction of site 5 with Zn1 enhances upon substrate binding in structures with only one zinc. Whereas a previously studied glutaminyl cyclase structure, the best known homologue to GPAA1, binds only one zinc ion at the catalytic site, GPAA1 can sterically accommodate two. The M28-type metallopeptidases segregate into two independent branches with regard to one/two zinc ion binding modality in a phylogenetic tree where the GPAA1 family is closer to the joint origin of both groups. For GPAA1 models, MD studies revealed two large loops (flaps) surrounding the active site being involved in an anti-correlated, breathing-like dynamics. In the light of combined sequence-analytic and phylogenetic arguments as well as 3D structural modelling results, GPAA1 is most likely a single zinc ion metallopeptidase. Two large flaps environ the catalytic site restricting access to large substrates. This article was reviewed by Thomas Dandekar (MD) and Michael Gromiha.

中文翻译：

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人 GPAA1 腔结构域的结构建模，转酰胺酶复合物的金属肽合成酶亚基，揭示锌结合模式和活性位点周围的两个皮瓣

转酰胺酶复合物是真核生物内质网中的一种分子机器，它在切割具有确定序列信号的 C 端前肽后将糖基磷脂酰肌醇 (GPI) 脂质锚连接到底物蛋白上。它的五个亚基非常疏水；因此，溶解性、异源表达和复杂的重建是困难的。因此，理论方法目前是深入了解 3D 结构和催化过程细节的主要来源。在这项工作中，我们生成了人类 GPAA1 腔结构域的模型 3D 结构，转酰胺酶的 M28 型金属肽合成酶亚基，包括锌离子和模型底物位置。在 M28 型结构和我们的 GPAA1 模型的比较分子动力学 (MD) 模拟中，我们用催化裂隙中进化保守的氨基酸残基估计了金属离子结合能。我们发现，规范的锌结合位点 2 和 3 是 Zn1 的最强结合剂，并且在第二个锌可用的情况下，位点 2 和 4 是 Zn2 的结合剂。位点 5 与 Zn1 的锌相互作用在只有一种锌的结构中与底物结合时增强。先前研究的谷氨酰胺酰环化酶结构（GPAA1 最著名的同系物）仅在催化位点结合一个锌离子，而 GPAA1 可以在空间上容纳两个。M28 型金属肽酶在系统发育树中就一个/两个锌离子结合方式分为两个独立的分支，其中 GPAA1 家族更接近两个群体的联合起源。对于 GPAA1 型号，MD 研究表明，围绕活动部位的两个大环（瓣）参与了一种反相关的、类似呼吸的动力学。根据组合的序列分析和系统发育论点以及 3D 结构建模结果，GPAA1 很可能是单一的锌离子金属肽酶。两个大襟翼环绕催化位点，限制接触大底物。本文由 Thomas Dandekar (MD) 和 Michael Gromiha 审阅。