Department of Chemistry, Virginia Tech, Blacksburg, Virginia, USA.
University Libraries, Virginia Tech, Blacksburg, Virginia, USA.
Protein Sci. 2021 Apr;30(4):804-817. doi: 10.1002/pro.4031. Epub 2021 Feb 15.
Designed protein receptors hold diagnostic and therapeutic promise. We now report the design of five consensus leucine-rich repeat proteins (CLRR4-8) based on the LRR domain of nucleotide-binding oligomerization domain (NOD)-like receptors involved in the innate immune system. The CLRRs bind muramyl dipeptide (MDP), a bacterial cell wall component, with micromolar affinity. The overall K values ranged from 1.0 to 57 μM as measured by fluorescence quenching experiments. Biphasic fluorescence quenching curves were observed in all CLRRs, with higher affinity K values ranging from 0.04 to 4.5 μM, and lower affinity K values ranging from 3.1 to 227 μM. These biphasic binding curves, along with the docking studies of MDP binding to CLRR4, suggest that at least two MDPs bind to each protein. Previously, only single MDP binding was reported. This high-capacity binding of MDP promises small, soluble, stable CLRR scaffolds as candidates for the future design of pathogen biosensors.
设计的蛋白受体具有诊断和治疗的潜力。我们现在报告了基于参与先天免疫系统的核苷酸结合寡聚化结构域（NOD）样受体的 LRR 结构域设计的五个共识亮氨酸丰富重复蛋白（CLRR4-8）。CLRRs 以微摩尔亲和力结合细菌细胞壁成分 muramyl dipeptide (MDP)。通过荧光猝灭实验测量，总体 K 值范围为 1.0 至 57μM。所有 CLRR 中均观察到双相荧光猝灭曲线，高亲和力 K 值范围为 0.04 至 4.5μM，低亲和力 K 值范围为 3.1 至 227μM。这些双相结合曲线以及 MDP 与 CLRR4 结合的对接研究表明，至少有两个 MDP 结合到每个蛋白上。此前，仅报道了单个 MDP 结合。MDP 的这种高容量结合有望成为小分子、可溶性、稳定的 CLRR 支架，作为未来病原体生物传感器设计的候选物。
