On the Role of Aggregation Prone Regions in Protein Evolution, Stability, and Enzymatic Catalysis: Insights from Diverse Analyses

The various roles that aggregation prone regions (APRs) are capable of playing in proteins are investigated here via comprehensive analyses of multiple non-redundant datasets containing randomly generated amino acid sequences, monomeric proteins, intrinsically disordered proteins (IDPs) and catalytic residues. Results from this study indicate that the aggregation propensities of monomeric protein sequences have been minimized compared to random sequences with uniform and natural amino acid compositions, as observed by a lower average aggregation propensity and fewer APRs that are shorter in length and more often punctuated by gate-keeper residues. However, evidence for evolutionary selective pressure to disrupt these sequence regions among homologous proteins is inconsistent. APRs are less conserved than average sequence identity among closely related homologues (≥80% sequence identity with a parent) but APRs are more conserved than average sequence identity among homologues that have at least 50% sequence identity with a parent. Structural analyses of APRs indicate that APRs are three times more likely to contain ordered versus disordered residues and that APRs frequently contribute more towards stabilizing proteins than equal length segments from the same protein. Catalytic residues and APRs were also found to be in structural contact significantly more often than expected by random chance. Our findings suggest that proteins have evolved by optimizing their risk of aggregation for cellular environments by both minimizing aggregation prone regions and by conserving those that are important for folding and function. In many cases, these sequence optimizations are insufficient to develop recombinant proteins into commercial products. Rational design strategies aimed at improving protein solubility for biotechnological purposes should carefully evaluate the contributions made by candidate APRs, targeted for disruption, towards protein structure and activity.

本研究通过对多组非冗余数据集开展全面系统分析，探究了聚集倾向区域（aggregation prone regions, APRs）在蛋白质中可发挥的多种作用，所涉数据集包含随机生成的氨基酸序列、单体蛋白、内在无序蛋白（intrinsically disordered proteins, IDPs）以及催化残基。本研究结果显示，相较于氨基酸组成均匀且符合天然特征的随机序列，单体蛋白序列的聚集倾向已得到有效降低：具体体现为平均聚集倾向更低，聚集倾向区域的数量更少、长度更短，且更常被门控残基所分隔。然而，关于同源蛋白中存在进化选择压力以破坏这类序列区域的相关证据并不一致。在与亲本序列一致性≥80%的近缘同源蛋白中，APRs的保守性低于序列平均一致性；而在与亲本序列一致性至少为50%的同源蛋白中，APRs的保守性则高于序列平均一致性。对APRs的结构分析表明，APRs包含有序残基的概率是无序残基的三倍，且相较于同一蛋白质中长度相同的片段，APRs通常对蛋白质结构的稳定贡献更为显著。研究同时发现，催化残基与APRs在结构上发生接触的频率，显著高于随机概率下的预期值。本研究结果提示，蛋白质通过两种方式进化以适配细胞环境中的聚集风险：一是尽可能减少聚集倾向区域的存在，二是保留对蛋白质折叠与功能至关重要的聚集倾向区域。在诸多场景下，这类序列优化不足以将重组蛋白开发为商业化产品。旨在通过生物技术手段提升蛋白质溶解度的理性设计策略，应当仔细评估拟靶向破坏的候选APRs对蛋白质结构与活性的影响。