Department of Biological Sciences, Trinity Washington University, Washington, District of Columbia, USA.
Department of Chemistry, Howard University, Washington, District of Columbia, USA.
Luminescence. 2022 Nov;37(11):1853-1863. doi: 10.1002/bio.4362. Epub 2022 Aug 31.
Calreticulin (CRT) is a protein found mainly in the endoplasmic reticulum (ER) that maintains calcium levels and controls protein folding, but has recently been found at the cell surface, cytoplasm, and in the extracellular matrix. CRT participates in multiple physiological processes such as gene expression, the immune response, and cancer. Calreticulin has been shown to autoacetylate with the binding of preferred ligand 7,8-diacetoxy-4-methylcoumarin (DAMC). This project aims to develop a chemical biology approach to investigate importance of CRT acylating abilities on its nonendoplasmic reticulum functions by targeting the downstream substrates of CRT acetylation. Our goal was to use CRT to transfer a pentynoyl tag (using a novel ligand, DPeMC) to its substrates, which can then be used as a handle for protein identification. Molecular modelling using available data in the literature was used to approximate the binding interface between CRT and the acylation ligands. Molecular Operating Environment (MOE) software was used to perform sequence alignment, simulated annealing, positional refinement, and blind docking of acylated coumarins with the CRT model. Docking studies pointed to the P domain as the most thermodynamically and sterically favourable region for acylated coumarin binding with tryptophan residue 200 within the active site on CRT. Absorption and fluorescence spectra of all coumarin compounds in ethanol:PBS (1:9 v/v) solution were investigated. Stern-Volmer quenching constant (K ), binding constant (K), and number of binding sites (n) of each coumarin compound with CRT was determined. Our studies demonstrated that acyl coumarin compounds bind to CRT using a dynamic quenching mechanism, bind to a single binding site on the P domain, and that the protein-ligand interaction is spontaneous and exothermic.
钙网蛋白（CRT）主要存在于内质网（ER）中，维持钙离子水平并控制蛋白质折叠，但最近在细胞表面、细胞质和细胞外基质中也有发现。CRT 参与多种生理过程，如基因表达、免疫反应和癌症。钙网蛋白已被证明与优选配体 7,8-二乙酰氧基-4-甲基香豆素（DAMC）结合时会自动乙酰化。本项目旨在开发一种化学生物学方法，通过靶向 CRT 乙酰化的下游底物来研究 CRT 酰化能力对其非内质网功能的重要性。我们的目标是使用 CRT 将戊炔酰基标签（使用新型配体 DPeMC）转移到其底物上，然后可以将其用作蛋白质鉴定的处理。使用文献中可用的数据进行分子建模，以近似 CRT 与酰化配体之间的结合界面。使用分子操作环境（MOE）软件对酰化香豆素与 CRT 模型进行序列比对、模拟退火、位置精修和盲目对接。对接研究指出 P 结构域是与 CRT 上的色氨酸残基 200 结合的最热力学和立体有利区域。研究了所有香豆素化合物在乙醇：PBS（1:9 v/v）溶液中的吸收和荧光光谱。确定了每个香豆素化合物与 CRT 的 Stern-Volmer 猝灭常数（K）、结合常数（K）和结合位点数（n）。我们的研究表明，酰基香豆素化合物通过动态猝灭机制与 CRT 结合，与 P 结构域上的单个结合位点结合，并且蛋白质-配体相互作用是自发和放热的。
