Curriculum in Bioinformatics and Computational Biology, University of North Carolina, Chapel Hill, North Carolina 27599-7260, USA.
Proteins. 2013 May;81(5):884-95. doi: 10.1002/prot.24247. Epub 2013 Feb 27.
The ability to generate and design antibodies recognizing specific targets has revolutionized the pharmaceutical industry and medical imaging. Engineering antibody therapeutics in some cases requires modifying their constant domains to enable new and altered interactions. Engineering novel specificities into antibody constant domains has proved challenging due to the complexity of inter-domain interactions. Covarying networks of residues that tend to cluster on the protein surface and near binding sites have been identified in some proteins. However, the underlying role these networks play in the protein resulting in their conservation remains unclear in most cases. Resolving their role is crucial, because residues in these networks are not viable design targets if their role is to maintain the fold of the protein. Conversely, these networks of residues are ideal candidates for manipulating specificity if they are primarily involved in binding, such as the myriad interdomain interactions maintained within antibodies. Here, we identify networks of evolutionarily-related residues in C-class antibody domains by evaluating covariation, a measure of propensity with which residue pairs vary dependently during evolution. We computationally test whether mutation of residues in these networks affects stability of the folded antibody domain, determining their viability as design candidates. We find that members of covarying networks cluster at domain-domain interfaces, and that mutations to these residues are diverse and frequent during evolution, precluding their importance to domain stability. These results indicate that networks of covarying residues exist in antibody domains for functional reasons unrelated to thermodynamic stability, making them ideal targets for antibody design.
生成和设计识别特定靶标的抗体的能力彻底改变了制药行业和医学成像。在某些情况下，工程抗体治疗剂需要修饰其恒定结构域以实现新的和改变的相互作用。由于结构域间相互作用的复杂性，将新的特异性工程设计到抗体恒定结构域中一直具有挑战性。已经在一些蛋白质中鉴定到倾向于在蛋白质表面和结合位点附近聚集的残基共变网络。然而，在大多数情况下，这些网络在导致其保守性的蛋白质中所起的作用仍然不清楚。确定它们的作用至关重要，因为如果这些网络中的残基的作用是维持蛋白质的折叠，则它们不是可行的设计靶标。相反，如果这些网络中的残基主要参与结合，例如抗体中维持的无数结构域间相互作用，则它们是操纵特异性的理想候选者。在这里，我们通过评估协变来鉴定 C 类抗体结构域中进化相关残基的网络，协变是衡量残基对在进化过程中依赖性变化的倾向的度量。我们通过计算测试这些网络中的残基突变是否会影响折叠抗体结构域的稳定性，从而确定它们作为设计候选者的可行性。我们发现共变网络的成员在结构域-结构域界面处聚集，并且这些残基在进化过程中发生的突变多样化且频繁，排除了它们对结构域稳定性的重要性。这些结果表明，抗体结构域中存在共变残基网络是出于与热力学稳定性无关的功能原因，这使它们成为抗体设计的理想靶标。
