Reconstitution of human Ero1-Lalpha/protein-disulfide isomerase oxidative folding pathway in vitro. Position-dependent differences in role between the a and a' domains of protein-disulfide isomerase.
Protein-disulfide isomerase (PDI), a critical enzyme responsible for oxidative protein folding in the eukaryotic endoplasmic reticulum, is composed of four thioredoxin domains a, b, b', a', and a linker x between b' and a'. Ero1-Lalpha, an oxidase for human PDI (hPDI), has been determined to have one molecular flavin adenine dinucleotide (FAD) as its prosthetic group. Oxygen consumption assays with purified recombinant Ero1-Lalpha revealed that it utilizes oxygen as a terminal electron acceptor producing one disulfide bond and one molecule of hydrogen peroxide per dioxygen molecule consumed. Exogenous FAD is not required for recombinant Ero1-Lalpha activity. By monitoring the reactivation of denatured and reduced RNase A, we reconstituted the Ero1-Lalpha/hPDI oxidative folding system in vitro and determined the enzymatic activities of hPDI in this system. Mutagenesis studies suggested that the a' domain of hPDI is much more active than the a domain in Ero1-Lalpha-mediated oxidative folding. A domain swapping study revealed that one catalytic thioredoxin domain to the C-terminal of bb'x, whether a or a', is essential in Ero1-Lalpha-mediated oxidative folding. These data, combined with a pull-down assay and isothermal titration calorimetry measurements, enabled the minimal element for binding with Ero1-Lalpha to be mapped to the b'xa' fragment of hPDI.
Wang
Lei
L
National Laboratory of Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, Beijing 100101, China, the Graduate School of the Chinese Academy of Sciences, Beijing 100049, China, the Department of Biological Sciences, Warwick University, Coventry CV4 7AL, United Kingdom, and the State Key Laboratory of Virology, College of Life Sciences, Wuhan University, Wuhan 430072, China; National Laboratory of Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, Beijing 100101, China, the Graduate School of the Chinese Academy of Sciences, Beijing 100049, China, the Department of Biological Sciences, Warwick University, Coventry CV4 7AL, United Kingdom, and the State Key Laboratory of Virology, College of Life Sciences, Wuhan University, Wuhan 430072, China.
Li
Sheng-Jian
SJ
National Laboratory of Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, Beijing 100101, China, the Graduate School of the Chinese Academy of Sciences, Beijing 100049, China, the Department of Biological Sciences, Warwick University, Coventry CV4 7AL, United Kingdom, and the State Key Laboratory of Virology, College of Life Sciences, Wuhan University, Wuhan 430072, China; National Laboratory of Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, Beijing 100101, China, the Graduate School of the Chinese Academy of Sciences, Beijing 100049, China, the Department of Biological Sciences, Warwick University, Coventry CV4 7AL, United Kingdom, and the State Key Laboratory of Virology, College of Life Sciences, Wuhan University, Wuhan 430072, China.
Sidhu
Ateesh
A
National Laboratory of Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, Beijing 100101, China, the Graduate School of the Chinese Academy of Sciences, Beijing 100049, China, the Department of Biological Sciences, Warwick University, Coventry CV4 7AL, United Kingdom, and the State Key Laboratory of Virology, College of Life Sciences, Wuhan University, Wuhan 430072, China.
Zhu
Li
L
National Laboratory of Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, Beijing 100101, China, the Graduate School of the Chinese Academy of Sciences, Beijing 100049, China, the Department of Biological Sciences, Warwick University, Coventry CV4 7AL, United Kingdom, and the State Key Laboratory of Virology, College of Life Sciences, Wuhan University, Wuhan 430072, China; National Laboratory of Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, Beijing 100101, China, the Graduate School of the Chinese Academy of Sciences, Beijing 100049, China, the Department of Biological Sciences, Warwick University, Coventry CV4 7AL, United Kingdom, and the State Key Laboratory of Virology, College of Life Sciences, Wuhan University, Wuhan 430072, China.
Liang
Yi
Y
National Laboratory of Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, Beijing 100101, China, the Graduate School of the Chinese Academy of Sciences, Beijing 100049, China, the Department of Biological Sciences, Warwick University, Coventry CV4 7AL, United Kingdom, and the State Key Laboratory of Virology, College of Life Sciences, Wuhan University, Wuhan 430072, China.
Freedman
Robert B
RB
National Laboratory of Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, Beijing 100101, China, the Graduate School of the Chinese Academy of Sciences, Beijing 100049, China, the Department of Biological Sciences, Warwick University, Coventry CV4 7AL, United Kingdom, and the State Key Laboratory of Virology, College of Life Sciences, Wuhan University, Wuhan 430072, China.
Wang
Chih-Chen
CC
National Laboratory of Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, Beijing 100101, China, the Graduate School of the Chinese Academy of Sciences, Beijing 100049, China, the Department of Biological Sciences, Warwick University, Coventry CV4 7AL, United Kingdom, and the State Key Laboratory of Virology, College of Life Sciences, Wuhan University, Wuhan 430072, China. Electronic address: chihwang@sun5.ibp.ac.cn.
D005182:Flavin-Adenine Dinucleotide / Q000737:chemistry* / Q000235:genetics; D006801:Humans; D006861:Hydrogen Peroxide / Q000737:chemistry*; D008562:Membrane Glycoproteins / Q000737:chemistry* / Q000235:genetics; D010084:Oxidation-Reduction; D010088:Oxidoreductases / Q000737:chemistry* / Q000235:genetics; D010100:Oxygen / Q000737:chemistry*; D019704:Protein Disulfide-Isomerases / Q000737:chemistry* / Q000235:genetics; D017510:Protein Folding; D017434:Protein Structure, Tertiary / Q000502:physiology; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics; D012259:Ribonuclease, Pancreatic / Q000737:chemistry / Q000235:genetics
D008562:Membrane Glycoproteins; D011994:Recombinant Proteins; D005182:Flavin-Adenine Dinucleotide; D006861:Hydrogen Peroxide; C405327:ERO1A protein, human; D010088:Oxidoreductases; D012259:Ribonuclease, Pancreatic; D019704:Protein Disulfide-Isomerases; D010100:Oxygen