Department of Biochemistry and Molecular Biology, The University of Kansas Medical Center, Kansas City, Kansas, USA.
Department of Molecular Biosciences, The University of Kansas, Lawrence, Kansas, USA.
Protein Sci. 2022 Jul;31(7):e4336. doi: 10.1002/pro.4336.
Various protein properties are often illuminated using sequence comparisons of protein homologs. For example, in analyses of the pyruvate kinase multiple sequence alignment, the set of positions that changed during speciation ("phylogenetic" positions) were enriched for "rheostat" positions in human liver pyruvate kinase (hLPYK). (Rheostat positions are those which, when substituted with various amino acids, yield a range of functional outcomes). However, the correlation was moderate, which could result from multiple biophysical constraints acting on the same position during evolution and/or various sources of noise. To further examine this correlation, we here tested Zymomonas mobilis PYK (ZmPYK), which has <65% sequence identity to any other PYK sequence. Twenty-six ZmPYK positions were selected based on their phylogenetic scores, substituted with multiple amino acids, and assessed for changes in K . Although we expected to identify multiple, strong rheostat positions, only one moderate rheostat position was detected. Instead, nearly half of the 271 ZmPYK variants were inactive and most others showed near wild-type function. Indeed, for the active ZmPYK variants, the total range of K values ("tunability") was 40-fold less than that observed for hLPYK variants. The combined functional studies and sequence comparisons suggest that ZmPYK has evolved functional and/or structural attributes that differ from the rest of the family. We hypothesize that including such "orphan" sequences in MSA analyses obscures the correlations used to predict rheostat positions. Finally, results raise the intriguing biophysical question as to how the same protein fold can support rheostat positions in one homolog but not another.
各种蛋白质特性通常通过蛋白质同源物的序列比较来揭示。例如，在丙酮酸激酶多序列比对的分析中，在物种形成过程中发生变化的位置（“变阻器”位置）在人肝丙酮酸激酶（hLPYK）中富集了“变阻器”位置。（变阻器位置是指用各种氨基酸取代时会产生一系列功能结果的位置）。然而，相关性适中，这可能是由于进化过程中同一位置受到多种生物物理限制的影响，以及各种来源的噪声。为了进一步研究这种相关性，我们在这里测试了运动发酵单胞菌 PYK（ZmPYK），它与任何其他 PYK 序列的序列同一性<65%。根据它们的系统发育得分，选择了 26 个 ZmPYK 位置，用多种氨基酸取代，并评估了 K 值的变化。虽然我们预计会识别出多个强变阻器位置，但只检测到一个中等变阻器位置。相反，271 个 ZmPYK 变体中有近一半没有活性，大多数其他变体的功能接近野生型。事实上，对于活性 ZmPYK 变体，K 值的总变化范围（“可调谐性”）比 hLPYK 变体观察到的小 40 倍。综合功能研究和序列比较表明，ZmPYK 已经进化出与家族其他成员不同的功能和/或结构属性。我们假设在 MSA 分析中包括这种“孤儿”序列会掩盖用于预测变阻器位置的相关性。最后，结果提出了一个有趣的生物物理问题，即同一蛋白质折叠如何在一个同源物中支持变阻器位置，而在另一个同源物中不支持。
