Residue proximity information and protein model discrimination using saturation-suppressor mutagenesis

Identification of residue-residue contacts from primary sequence can be used to guide protein structure prediction. Using Escherichia coli CcdB as the test case, we describe an experimental method termed saturation-suppressor mutagenesis to acquire residue contact information. In this methodology, for each of five inactive CcdB mutants, exhaustive screens for suppressors were performed. Proximal suppressors were accurately discriminated from distal suppressors based on their phenotypes when present as single mutants. Experimentally identified putative proximal pairs formed spatial constraints to recover >98% of native-like models of CcdB from a decoy dataset. Suppressor methodology was also applied to the integral membrane protein, diacylglycerol kinase A where the structures determined by X-ray crystallography and NMR were significantly different. Suppressor as well as sequence co-variation data clearly point to the X-ray structure being the functional one adopted in vivo. The methodology is applicable to any macromolecular system for which a convenient phenotypic assay exists.

基于一级序列的残基间接触（residue-residue contacts）鉴定，可用于指导蛋白质结构预测。以大肠杆菌（Escherichia coli）的CcdB蛋白为测试案例，本文介绍了一种名为饱和抑制子诱变（saturation-suppressor mutagenesis）的实验方法，用于获取残基间接触信息。在该方法中，针对5种失活的CcdB突变体，均开展了抑制子的穷尽筛选。根据单个突变体的表型，可准确区分近端抑制子与远端抑制子。实验鉴定得到的推定近端接触对，可形成空间约束，从诱饵数据集中恢复出98%以上的CcdB类天然构象模型。该抑制子方法还被应用于整合膜蛋白二酰甘油激酶A（diacylglycerol kinase A），该蛋白经X射线晶体学（X-ray crystallography）与核磁共振（NMR）解析得到的结构存在显著差异。抑制子数据与序列共变异（sequence co-variation）数据均明确表明，X射线晶体学解析的结构是该蛋白在活体内发挥功能时采用的构象。该方法适用于所有可便捷开展表型检测的大分子体系。