Characterization of guanine nucleotide exchange activity of DH domain of human FGD2

Junlan Chuan; Shiyu He; Tian Xie; Ganggang Wang; Zhenglin Yang

doi:10.1016/j.pep.2020.105693

Characterization of guanine nucleotide exchange activity of DH domain of human FGD2

Protein Expr Purif. 2020 Dec:176:105693. doi: 10.1016/j.pep.2020.105693. Epub 2020 Jul 16.

Authors

Junlan Chuan¹, Shiyu He², Tian Xie³, Ganggang Wang³, Zhenglin Yang⁴

Affiliations

¹ Sichuan Provincial Key Laboratory for Human Disease Gene Study and Institute of Laboratory Medicine, Sichuan Provincial People's Hospital, University of Electronic Science and Technology of China, Chengdu, 610072, China; Personalized Drug Therapy Key Laboratory of Sichuan Province, Department of Pharmacy, Sichuan Provincial People's Hospital, University of Electronic Science and Technology of China, Chengdu, 610072, China.
² School of Clinical Medicine, Southwest Medical University, Luzhou, Sichuan, 646000, PR China.
³ Key Laboratory of Environmental and Applied Microbiology of Chinese Academy of Sciences, Chengdu Institute of Biology, Chinese Academy of Sciences, Chengdu, 610041, China; Key Laboratory of Environmental Microbiology of Sichuan Province, Chengdu Institute of Biology, Chinese Academy of Sciences, Chengdu, 610041, China.
⁴ Sichuan Provincial Key Laboratory for Human Disease Gene Study and Institute of Laboratory Medicine, Sichuan Provincial People's Hospital, University of Electronic Science and Technology of China, Chengdu, 610072, China; Research Unit for Blindness Prevention of Chinese Academy of Medical Sciences (2019RU026), Sichuan Academy of Medical Sciences & Sichuan Provincial People's Hospital, Chengdu, Sichuan, China; Chengdu Institute of Biology, Sichuan Translational Medicine Hospital, Chinese Academy of Science, Chengdu, 610072, China. Electronic address: zliny@yahoo.com.

PMID: 32681954
DOI: 10.1016/j.pep.2020.105693

Abstract

FGD2, a member of FGD family, contains a Dbl homology domain (DH) and two pleckstrin homology domains segregated by a FYVE domain. The DH domain has been deduced to be responsible for guanine nucleotide exchange of CDC42 to activate downstream factors. Our aim was to build a prokaryotic expression system for the DH domain and to examine its guanine nucleotide exchange activity toward CDC42 in vitro. A recombinant vector, which was successfully constructed based on pGEX-6P-1, was employed to express the DH domain of human FGD2 (FGD2-DH) in E. coli BL21 (DE3). Purified FGD2-DH behaved as a homogeneous monomer with an estimated molecular weight that corresponded to the theoretical molecular weight and was predicted to be an α-helix protein by circular dichroism spectroscopy. FGD2-DH displayed weak guanine nucleotide exchange activity in vitro and very weak interactions with CDC42 following glutaraldehyde cross-linking.

Keywords: CDC42; Circular dichroism spectroscopy; Dbl homology domain; FGD2; Glutaraldehyde cross-linking; Guanine nucleotide exchange activity.

Publication types

Research Support, Non-U.S. Gov't

MeSH terms

Escherichia coli / genetics
Escherichia coli / metabolism
Guanine Nucleotide Exchange Factors / biosynthesis
Guanine Nucleotide Exchange Factors / chemistry*
Guanine Nucleotide Exchange Factors / genetics
Guanine Nucleotide Exchange Factors / isolation & purification
Guanine Nucleotides / chemistry*
Guanine Nucleotides / metabolism
Humans
Protein Domains
Recombinant Proteins / biosynthesis
Recombinant Proteins / chemistry
Recombinant Proteins / genetics
Recombinant Proteins / isolation & purification
cdc42 GTP-Binding Protein / chemistry*
cdc42 GTP-Binding Protein / genetics
cdc42 GTP-Binding Protein / metabolism

Substances

FGD2 protein, human
Guanine Nucleotide Exchange Factors
Guanine Nucleotides
Recombinant Proteins
CDC42 protein, human
cdc42 GTP-Binding Protein