To what degree are individual structural elements within proteins modular such that similar structures from unrelated proteins can be interchanged? We study subdomain modularity by creating 20 chimeras of an enzyme, Escherichia coli dihydrofolate reductase (DHFR), in which a catalytically important, 10-residue α-helical sequence is replaced by α-helical sequences from a diverse set of proteins. The chimeras stably fold but have a range of diminished thermal stabilities and catalytic activities. Evolutionary coupling analysis indicates that the residues of this α-helix are under selection pressure to maintain catalytic activity in DHFR. Reversion to phenylalanine at key position 31 was found to partially restore catalytic activity, which could be explained by evolutionary coupling values. We performed molecular dynamics simulations using replica exchange with solute tempering. Chimeras with low catalytic activity exhibit nonhelical conformations that block the binding site and disrupt the positioning of the catalytically essential residue D27. Simulation observables and in vitro measurements of thermal stability and substrate-binding affinity are strongly correlated. Several E. coli strains with chromosomally integrated chimeric DHFRs can grow, with growth rates that follow predictions from a kinetic flux model that depends on the intracellular abundance and catalytic activity of DHFR. Our findings show that although α-helices are not universally substitutable, the molecular and fitness effects of modular segments can be predicted by the biophysical compatibility of the replacement segment.

蛋白质内的各个结构元件的模块化程度如何，使得不相关蛋白质的相似结构可以互换？我们通过创建大肠杆菌二氢叶酸还原酶 (DHFR) 酶的 20 个嵌合体来研究子域模块化，其中催化重要的 10 个残基α螺旋序列被来自不同蛋白质组的α螺旋序列取代。嵌合体稳定折叠，但热稳定性和催化活性一系列降低。进化耦合分析表明，该α螺旋的残基处于选择压力下，以维持 DHFR 的催化活性。发现关键位置 31 处恢复为苯丙氨酸可部分恢复催化活性，这可以通过进化耦合值来解释。我们使用复制品交换和溶质回火进行分子动力学模拟。具有低催化活性的嵌合体表现出非螺旋构象，可阻断结合位点并破坏催化必需残基 D27 的定位。热稳定性和底物结合亲和力的模拟观察值和体外测量值密切相关。一些带有染色体整合嵌合 DHFR 的大肠杆菌菌株可以生长，其生长速率遵循动态通量模型的预测，该模型取决于 DHFR 的细胞内丰度和催化活性。我们的研究结果表明，尽管α-螺旋不是普遍可替代的，但模块化片段的分子和适应度效应可以通过替代片段的生物物理相容性来预测。