能量不利的蛋白质角度：磷酸丙糖异构酶保守二面角的探索,Biopolymers

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能量不利的蛋白质角度：磷酸丙糖异构酶保守二面角的探索
Biopolymers ( IF 3.2 ) Pub Date : 2022-09-15 , DOI: 10.1002/bip.23525
Patrick W Allen ₁ , Jordan A Cook ₁ , Anh N Colquhoun ₁ , Eric J Sorin ₁ , Enrico Tapavicza ₁ , Jason P Schwans ₁

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在过去 35 亿年的进化过程中，酶采用了无数的构象来适应地球上的生命。然而，蛋白质的扭转角已经进入 Ramachandran 图中观察到的能量有利二面角的有限区域。由于空间位阻，所述区域之外的区域被认为不允许使用除甘氨酸之外的所有氨基酸。磷酸丙糖异构酶 (TIM) 是一种催化速率接近扩散极限的同源二聚体，含有一个活性位点赖氨酸残基 (K13)，在第四象限内具有二面角 (Φ = +51/Ψ = -143)。无论配体如何，氨基酸和二面角在所有 TIM 和已知晶体结构中都是保守的。只有工程单体版本 (1MSS) 的晶体结构显示可接受的 β-折叠二面角值 Φ = -135/Ψ = +170，但实验显示活性损失 1000 倍。基于这些结果，我们假设对 K13 采用不利的扭转角有助于催化。使用计算和实验方法，将与 K13 相互作用的四个残基（N11、M14、E97 和 Q64）突变为丙氨酸。使用 2JK2 未结合的人 TIM 作为起始结构进行计算机分子动力学 (MD) 模拟。包含 K13 二面角值的 Ramachandran 图揭示了不允许的区域角的全部或部分损失。

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Energetically unfavorable protein angles: Exploration of a conserved dihedral angle in triosephosphate isomerase

Over the past 3.5 billion years of evolution, enzymes have adopted a myriad of conformations to suit life on earth. However, torsional angles of proteins have settled into limited zones of energetically favorable dihedrals observed in Ramachandran plots. Areas outside said zones are believed to be disallowed to all amino acids, except glycine, due to steric hindrance. Triosephosphate isomerase (TIM), a homodimer with a catalytic rate approaching the diffusion limit, contains an active site lysine residue (K13) with dihedrals within the fourth quadrant (Φ = +51/Ψ = −143). Both the amino acid and the dihedral angles are conserved across all species of TIM and known crystal structures regardless of ligand. Only crystal structures of the engineered monomeric version (1MSS) show accepted β-sheet dihedral values of Φ = -135/Ψ = +170 but experiments show a 1000-fold loss in activity. Based on these results, we hypothesized that adopting the unfavorable torsion angle for K13 contributes to catalysis. Using both, computational and experimental approaches, four residues that interact with K13 (N11, M14, E97, and Q64) were mutated to alanine. In silico molecular dynamics (MD) simulations were performed using 2JK2 unliganded human TIM as a starting structure. Ramachandran plots, containing K13 dihedral values reveal full or partial loss of disallowed zone angles. N11A showed no detectable catalytic activity and lost the unfavorable K13 dihedral angles across four separate force fields during simulation while all other mutants plus wild type retained activity and retained the conserved K13 dihedral angles.

更新日期：2022-09-15

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