Catalysis of creatine kinase refolding by protein disulfide isomerase involves disulfide cross-link and dimer to tetramer switch.
Protein disulfide isomerase (PDI) functions as an isomerase to catalyze thiol:disulfide exchange, as a chaperone to assist protein folding, and as a subunit of prolyl-4-hydroxylase and microsomal triglyceride transfer protein. At a lower concentration of 0.2 microm, PDI facilitated the aggregation of unfolded rabbit muscle creatine kinase (CK) and exhibited anti-chaperone activity, which was shown to be mainly due to the hydrophobic interactions between PDI and CK and was independent of the cross-linking of disulfide bonds. At concentrations above 1 microm, PDI acted as a protector against aggregation but an inhibitor of reactivation during CK refolding. The inhibition effect of PDI on CK reactivation was further characterized as due to the formation of PDI-CK complexes through intermolecular disulfide bonds, a process involving Cys-36 and Cys-295 of PDI. Two disulfide-linked complexes containing both PDI and CK were obtained, and the large, soluble aggregates around 400 kDa were composed of 1 molecule of tetrameric PDI and 2 molecules of inactive intermediate dimeric CK, whereas the smaller one, around 200 kDa, was formed by 1 dimeric PDI and 1 dimeric CK. To our knowledge this is the first study revealing that PDI could switch its conformation from dimer to tetramer in its functions as a foldase. According to the observations in this research and our previous study of the folding pathways of CK, a working model was proposed for the molecular mechanism of CK refolding catalyzed by PDI.
Zhao
Tong-Jin
TJ
Department of Biological Sciences and Biotechnology, Tsinghua University, Beijing, China.
Ou
Wen-Bin
WB
Xie
Qiang
Q
Liu
Yang
Y
Yan
Yong-Bin
YB
Zhou
Hai-Meng
HM
D000818:Animals; D003402:Creatine Kinase / Q000737:chemistry* / Q000378:metabolism*; D004229:Dithiothreitol / Q000737:chemistry; D019791:Guanidine / Q000737:chemistry; D006801:Humans; D007527:Isoenzymes / Q000737:chemistry / Q000378:metabolism; D008962:Models, Theoretical; D018832:Molecular Chaperones / Q000378:metabolism; D011489:Protein Denaturation; D019704:Protein Disulfide-Isomerases / Q000378:metabolism*; D017510:Protein Folding; D020673:Protein Renaturation; D020836:Protein Structure, Quaternary; D011817:Rabbits
D007527:Isoenzymes; D018832:Molecular Chaperones; D003402:Creatine Kinase; D019704:Protein Disulfide-Isomerases; D019791:Guanidine; D004229:Dithiothreitol