钙网蛋白转酰基酶P结构域中的定点诱变确定赖氨酸-207为活性位点残基。

Site-directed mutagenesis in the P-domain of calreticulin transacylase identifies Lys-207 as the active site residue.

作者信息

Joshi Rini, Singh Prabhjot, Sharma Naresh K, Ponnan Prija, Saluja Daman, Gambhir Jasvinder K, Rawat Diwan S, Parmar Virinder S, Dwarakanath Bilkere S, Prasad Ashok K, Raj Hanumantharao G

机构信息

Department of Biochemistry, V.P. Chest Institute, Delhi, India.

Department of Chemistry, University of Delhi, Delhi, 110 007 India.

出版信息

3 Biotech. 2021 Mar;11(3):113. doi: 10.1007/s13205-021-02659-1. Epub 2021 Feb 3.

DOI:10.1007/s13205-021-02659-1

PMID:33585151

原文链接:https://pmc.ncbi.nlm.nih.gov/articles/PMC7859019/

Abstract

UNLABELLED

In silico-docking studies from previous work have suggested that Lys-206 and lys-207 of calreticulin (CR) play a pivotal key role in its well-established transacetylation activity. To experimentally validate this prediction, we introduced three mutations at lysine residues of P-domain of CR: K → A, (K -206, -209), (K -206, -207) and (K -207, -209) and analyzed their immunoreactivity and acetylation potential. The clones of wild-type P-domain ( ) and three mutated P-domain ( , and ) were expressed in pTrcHis C vector and the recombinant , , and proteins were purified by Ni-NTA affinity chromatography. Screening of the transacylase activity (TAase) by the Glutathione S Transferase (GST) assay revealed that the TAase activity was associated with the and while and did not show any activity. The immune-reactivity to anti-lysine antibody was also retained only by the in which the Lys-207 was intact. Retention of the TAase activity and immunoreactivity of with mutations introduced at Lys-206, Lys-209, while its loss with a mutation at Lys-207 residue indicated that lysine-207 of P-domain constitutes the active site residue controlling TAase activity.

SUPPLEMENTARY INFORMATION

The online version contains supplementary material available at 10.1007/s13205-021-02659-1.

摘要

未标注

先前工作中的计算机对接研究表明，钙网蛋白（CR）的赖氨酸-206和赖氨酸-207在其已确立的转乙酰化活性中起关键作用。为了通过实验验证这一预测，我们在CR的P结构域的赖氨酸残基处引入了三个突变：K→A，（K -206，-209），（K -206，-207）和（K -207，-209），并分析了它们的免疫反应性和乙酰化潜力。野生型P结构域（）和三个突变的P结构域（、和）的克隆在pTrcHis C载体中表达，重组的、、和蛋白通过镍-氮三乙酸亲和层析法纯化。通过谷胱甘肽S-转移酶（GST）测定筛选转酰基酶活性（TAase），结果显示TAase活性与和相关，而和未显示任何活性。仅赖氨酸-207完整的保留了对抗赖氨酸抗体的免疫反应性。在赖氨酸-206、赖氨酸-209处引入突变的保留了TAase活性和免疫反应性，而在赖氨酸-207残基处引入突变则使其丧失，这表明P结构域的赖氨酸-207构成了控制TAase活性的活性位点残基。