Structure-function analysis of the endoplasmic reticulum oxidoreductase TMX3 reveals interdomain stabilization of the N-terminal redox-active domain.
Disulfide bond formation in the endoplasmic reticulum is catalyzed by enzymes of the protein disulfide-isomerase family that harbor one or more thioredoxin-like domains. We recently discovered the transmembrane protein TMX3, a thiol-disulfide oxidoreductase of the protein disulfide-isomerase family. Here, we show that the endoplasmic reticulum-luminal region of TMX3 contains three thioredoxin-like domains, an N-terminal redox-active domain (named a) followed by two enzymatically inactive domains (b and b'). Using the recombinantly expressed TMX3 domain constructs a, ab, and abb', we compared structural stability and enzymatic properties. By structural and biophysical methods, we demonstrate that the reduced a domain has features typical of a globular folded domain that is, however, greatly destabilized upon oxidization. Importantly, interdomain stabilization by the b domain renders the a domain more resistant toward chemical denaturation and proteolysis in both the oxidized and reduced form. In combination with molecular modeling studies of TMX3 abb', the experimental results provide a new understanding of the relationship between the multidomain structure of TMX3 and its function as a redox enzyme. Overall, the data indicate that in addition to their role as substrate and co-factor binding domains, redox-inactive thioredoxin-like domains also function in stabilizing neighboring redox-active domains.
Haugstetter
Johannes
J
Institute of Biochemistry, ETH Zurich, 8093 Zurich, Switzerland.
Maurer
Michael Andreas
MA
Institute of Biochemistry, ETH Zurich, 8093 Zurich, Switzerland.
Blicher
Thomas
T
BioCentrum-DTU, Technical University of Denmark, 2800 Lyngby, Denmark.
Pagac
Martin
M
Institute of Biochemistry, ETH Zurich, 8093 Zurich, Switzerland.
Wider
Gerhard
G
Institute of Molecular Biology and Biophysics, ETH Zurich, 8093 Zurich, Switzerland.
Ellgaard
Lars
L
Institute of Biochemistry, ETH Zurich, 8093 Zurich, Switzerland. Electronic address: lellgaard@aki.ku.dk.
D000595:Amino Acid Sequence; D001665:Binding Sites; D004721:Endoplasmic Reticulum / Q000378:metabolism*; D006801:Humans; D009682:Magnetic Resonance Spectroscopy; D008954:Models, Biological; D008968:Molecular Conformation; D008969:Molecular Sequence Data; D010084:Oxidation-Reduction; D019704:Protein Disulfide-Isomerases / Q000378:metabolism*; D017510:Protein Folding; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry; D017386:Sequence Homology, Amino Acid; D013329:Structure-Activity Relationship
D011994:Recombinant Proteins; D019704:Protein Disulfide-Isomerases; C498879:TMX3 protein, human