Department of Chemistry and Biochemistry, University of Delaware, Newark, Delaware 19716, United States.
Department of Biological Sciences, University of Delaware, Newark, Delaware 19716, United States.
Biochemistry. 2022 Dec 20;61(24):2856-2860. doi: 10.1021/acs.biochem.2c00305. Epub 2022 Jul 11.
The evolutionarily conserved leucine rich repeat (LRR) protein domain is a unique structural motif found in many viral, bacterial, archaeal, and eukaryotic proteins. The LRR domain serves many roles, including being a signaling domain and a pathogen recognition receptor. In the human innate immune system, it serves an essential role by recognizing fragments of bacterial cell walls. Interestingly, the human fungal pathogen also uses an LRR domain-containing protein, Cyrp1, to sense bacterial cell wall fragments. However, the dynamics of signaling and detection of bacterial peptidoglycan fragments by the LRR of Cyr1p remains poorly characterized. Here we develop optimal recombinant expression workflows and provide characterization of the entire region of the LRR domain of Cyr1p as a peripheral membrane protein. Using a newly designed peptidoglycan enrichment bead assay, we demonstrate that this domain can bind bacterial peptidoglycan fragments under native conditions. The new membrane-associated Cyr1p-LRR construct sets the stage for the development of antifungal agents via high-throughput campaigns to inhibit cell wall-Cyr1p interactions.
进化保守的亮氨酸丰富重复 (LRR) 蛋白结构域是一种存在于许多病毒、细菌、古菌和真核生物蛋白中的独特结构基序。LRR 结构域具有多种功能，包括作为信号结构域和病原体识别受体。在人类先天免疫系统中，它通过识别细菌细胞壁片段发挥重要作用。有趣的是，人类真菌病原体 也使用含有 LRR 结构域的蛋白 Cyrp1 来感知细菌细胞壁片段。然而，Cyr1p 的 LRR 对细菌肽聚糖片段的信号转导和检测的动力学仍未得到充分描述。在这里，我们开发了最佳的重组表达工作流程，并对 Cyr1p 的 LRR 结构域的整个区域作为外周膜蛋白进行了表征。使用新设计的肽聚糖富集珠测定法，我们证明该结构域可以在天然条件下结合细菌肽聚糖片段。新的膜相关 Cyr1p-LRR 构建体为通过高通量抑制细胞壁-Cyr1p 相互作用的方法开发抗真菌药物奠定了基础。
