Table_1_Investigation of the Importance of Protein 3D Structure for Assessing Conservation of Lysine Acetylation Sites in Protein Homologs.XLSX

Acetylation is a protein post-translational modification (PTM) that can affect a variety of cellular processes. In bacteria, two PTM Nε-acetylation mechanisms have been identified: non-enzymatic/chemical acetylation via acetyl phosphate or acetyl coenzyme A and enzymatic acetylation via protein acetyltransferases. Prior studies have shown that extensive acetylation of Nε-lysine residues of numerous proteins from a variety of bacteria occurs via non-enzymatic acetylation. In Escherichia coli, new Nε-lysine acetyltransferases (KATs) that enzymatically acetylate other proteins have been identified, thus expanding the repertoire of protein substrates that are potentially regulated by acetylation. Therefore, we designed a study to leverage the wealth of structural data in the Protein Data Bank (PDB) to determine: (1) the 3D location of lysine residues on substrate proteins that are acetylated by E. coli KATs, and (2) investigate whether these residues are conserved on 3D structures of their homologs. Five E. coli KAT substrate proteins that were previously identified as being acetylated by YiaC and had 3D structures in the PDB were selected for further analysis: adenylate kinase (Adk), isocitrate dehydrogenase (Icd), catalase HPII (KatE), methionyl-tRNA formyltransferase (Fmt), and a peroxide stress resistance protein (YaaA). We methodically compared over 350 protein structures of these E. coli enzymes and their homologs; to accurately determine lysine residue conservation requires a strategy that incorporates both flexible structural alignments and visual inspection. Moreover, our results revealed discrepancies in conclusions about lysine residue conservation in homologs when examining linear amino acid sequences compared to 3D structures.

乙酰化作为一种蛋白质翻译后修饰（PTM），能够影响多种细胞过程。在细菌中，已鉴定出两种PTM修饰机制：非酶促/化学乙酰化，通过乙酰磷酸或乙酰辅酶A实现；以及酶促乙酰化，通过蛋白质乙酰转移酶实现。先前的研究表明，多种细菌中大量蛋白质的Nε-赖氨酸残基的广泛乙酰化是通过非酶促乙酰化发生的。在大肠杆菌中，已鉴定出新的Nε-赖氨酸乙酰转移酶（KATs），这些酶可以酶促乙酰化其他蛋白质，从而扩展了可能通过乙酰化进行调控的蛋白质底物库。因此，我们设计了一项研究，旨在利用蛋白质数据银行（PDB）中的丰富结构数据来确定：（1）由大肠杆菌KATs乙酰化的底物蛋白质中赖氨酸残基的3D位置；（2）研究这些残基是否在它们的同源蛋白的3D结构中保持一致。我们选择了五个先前被鉴定为通过YiaC乙酰化且在PDB中具有3D结构的E. coli KAT底物蛋白质进行进一步分析：腺苷酸激酶（Adk）、异柠檬酸脱氢酶（Icd）、过氧化氢酶HPII（KatE）、蛋氨酸-tRNA甲酰转移酶（Fmt）以及一种过氧化物应激抵抗蛋白（YaaA）。我们系统地比较了这些E. coli酶及其同源蛋白的350多种蛋白质结构；要准确确定赖氨酸残基的一致性，需要一种结合灵活的结构对齐和视觉检查的策略。此外，我们的结果表明，当比较线性氨基酸序列与3D结构时，在关于同源蛋白中赖氨酸残基一致性的结论上存在差异。