The ligand-binding b' domain of human protein disulphide-isomerase mediates homodimerization.
Purified preparations of the recombinant b'x domain fragment of human protein-disulphide isomerase (PDI), which are homogeneous by mass spectrometry and sodium dodecyl sulfate polyacrylamide gel electrophoresis, comprise more than one species when analyzed by ion-exchange chromatography and nondenaturing polyacrylamide gel electrophoresis. These species were resolved and shown to be monomer and dimer by analytical ultracentrifugation and analytical size-exclusion chromatography. Spectroscopic properties indicate that the monomeric species corresponds to the "capped" conformation observed in the x-ray structure of the I272A mutant of b'x (Nguyen, Wallis, Howard, Haapalainen, Salo, Saaranen, Sidhu, Wierenga, Freedman, Ruddock, and Williamson, J Mol Biol 2008;383:1144-1155) in which the x region binds to a hydrophobic patch on the surface of the b' domain; conversely, the dimeric species has an "open" or "uncapped" conformation in which the x region does not bind to this surface. The larger bb'x fragment of human PDI shows very similar behavior to b'x and can be resolved into a capped monomeric species and an uncapped dimer. Preparations of recombinant b' domain of human PDI and of the bb' domain pair are found exclusively as dimers. Full-length PDI is known to comprise a mixture of monomeric and dimeric species, whereas the isolated a, b, and a' domains of PDI are found exclusively as monomers. These results show that the b' domain of human PDI tends to form homodimers--both in isolation and in other contexts--and that this tendency is moderated by the adjacent x region, which can bind to a surface patch on the b' domain.
Department of Biological Sciences, Warwick University, Coventry, CV4 7AL, United Kingdom.
Anne Katrine
AK
Wallis
Ateesh
A
Sidhu
Lee J
LJ
Byrne
Mark J
MJ
Howard
Lloyd W
LW
Ruddock
Richard A
RA
Williamson
Robert B
RB
Freedman
D006801:Humans; D008024:Ligands; D019704:Protein Disulfide-Isomerases / Q000737:chemistry*; D055503:Protein Multimerization; D017434:Protein Structure, Tertiary
D008024:Ligands; D019704:Protein Disulfide-Isomerases