Catalysis of Creatine Kinase Refolding by Protein Disulfide Isomerase Involves Disulfide Cross-link and Dimer to Tetramer Switch

Abstract

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 M, 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 M, 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.In eukaryotes, all the nascent outer membrane and secreted proteins are synthesized and fold to their native structures in the lumen of endoplasmic reticulum. The specialized compartment provides a favorable environment for native disulfide bond formation, which is often the rate-limiting step in the folding of many proteins. Endoplasmic reticulum is abundant in molecular chaperones and folding catalysts, among which the most abundant and efficient catalyst of disulfide bond formation is protein disulfide isomerase (PDI, 1 EC 5.3.4.1) (1-3). PDI is a multidomain and multifunction homodimer. It comprises four thioredoxin-like domains, a, b, bЈ, and aЈ, plus a linker region between bЈ and aЈ and a C-terminal acid extension (2, 4, 5). Each of the two catalytic domains, a and aЈ, contains a WCGHCK motif, responsible for the isomerase activity (6). In vitro studies showed that the bЈ domain provides the principle peptide binding site, whereas the other domains should also be involved when the protein substrates have a substantial secondary structure (7, 8). The latest study (9) demonstrated the existence of a ligand binding site in the bЈ domain, which was a small hydrophobic pocket defined as Much effort has been devoted to investigating the catalytic mechanism of PDI since the first report published more than 40 years ago (10). As an enzyme, the specific binding to its substrate is a basic property of PDI, and the nature of the interaction between PDI and its substrates plays a crucial role in its activity of accelerating steps in protein fol...

Dissimilarity in the Folding of Human Cytosolic Creatine Kinase Isoenzymes

“…Consistent with previous results of RMCK [28], [30], [33], the folding of all five proteins were partially reversible as revealed by the inconsistency of the ANS fluorescence during folding and refolding. A comparison of the ANS fluorescence during CK folding and unfolding indicated that among the five proteins, the maximum ANS fluorescence of HMCK during refolding was significantly higher than that during unfolding.…”

Dissimilarity in the Folding of Human Cytosolic Creatine Kinase Isoenzymes

“…Consistent with previous results of RMCK [28], [30], [33], the folding of all five proteins were partially reversible as revealed by the inconsistency of the ANS fluorescence during folding and refolding. A comparison of the ANS fluorescence during CK folding and unfolding indicated that among the five proteins, the maximum ANS fluorescence of HMCK during refolding was significantly higher than that during unfolding.…”

eXL-MS: An Enhanced Cross-Linking Mass Spectrometry Workflow To Study Protein Complexes

“…An example of these intermolecular cross-linked peptides showing the interaction between the N-termini of two CK monomers is depicted in Figure S5. One hundred and thirty three other cross-linked peptides were found to be between 26 and 40 Å and could be explained by dynamic events such as breathing motions or large domain movements in the protein complex, as already observed in other studies. , Finally, we found 80 cross-linked peptides exceeding 40 Å. Looking in detail into these long cross-linked peptides, 46 were found to describe interactions occurring between a single patch constituted of 13 residues in close vicinity (K222, K241, K246, K297, S302, K303, K306, K318, K357, K364, K365, K368, S371) and the CK N-terminus (T5, K8, Y9, K10, K14, Y38, K39, and K40).…”

Designing an Antibody-Based Chaperoning System through Programming the Binding and Release of the Folding Intermediate

“…When DTT was added to the PDI− CK complexes, CK was released and the activity increased to nearly or slightly higher than that of the self-refolding CK. 38 We think the effect and mechanism of scFv A4 on folding of HCK in our present study are similar to those of PDI and a high concentration of Cyp18. They could prevent aggregation by forming a complex with HCK.…”

“…At concentrations above 1 μM, PDI acted as a protector against aggregation but as an inhibitor of reactivation by forming PDI–CK complexes through hydrophobic interaction and intermolecular disulfide bonds. When DTT was added to the PDI–CK complexes, CK was released and the activity increased to nearly or slightly higher than that of the self-refolding CK . We think the effect and mechanism of scFv A4 on folding of HCK in our present study are similar to those of PDI and a high concentration of Cyp18.…”

Dissimilarity in the Folding of Human Cytosolic Creatine Kinase Isoenzymes

“…Consistent with previous results of RMCK [28], [30], [33], the folding of all five proteins were partially reversible as revealed by the inconsistency of the ANS fluorescence during folding and refolding. A comparison of the ANS fluorescence during CK folding and unfolding indicated that among the five proteins, the maximum ANS fluorescence of HMCK during refolding was significantly higher than that during unfolding.…”

eXL-MS: An Enhanced Cross-Linking Mass Spectrometry Workflow To Study Protein Complexes

“…An example of these intermolecular cross-linked peptides showing the interaction between the N-termini of two CK monomers is depicted in Figure S5. One hundred and thirty three other cross-linked peptides were found to be between 26 and 40 Å and could be explained by dynamic events such as breathing motions or large domain movements in the protein complex, as already observed in other studies. , Finally, we found 80 cross-linked peptides exceeding 40 Å. Looking in detail into these long cross-linked peptides, 46 were found to describe interactions occurring between a single patch constituted of 13 residues in close vicinity (K222, K241, K246, K297, S302, K303, K306, K318, K357, K364, K365, K368, S371) and the CK N-terminus (T5, K8, Y9, K10, K14, Y38, K39, and K40).…”

Designing an Antibody-Based Chaperoning System through Programming the Binding and Release of the Folding Intermediate

“…When DTT was added to the PDI− CK complexes, CK was released and the activity increased to nearly or slightly higher than that of the self-refolding CK. 38 We think the effect and mechanism of scFv A4 on folding of HCK in our present study are similar to those of PDI and a high concentration of Cyp18. They could prevent aggregation by forming a complex with HCK.…”

“…At concentrations above 1 μM, PDI acted as a protector against aggregation but as an inhibitor of reactivation by forming PDI–CK complexes through hydrophobic interaction and intermolecular disulfide bonds. When DTT was added to the PDI–CK complexes, CK was released and the activity increased to nearly or slightly higher than that of the self-refolding CK . We think the effect and mechanism of scFv A4 on folding of HCK in our present study are similar to those of PDI and a high concentration of Cyp18.…”

Dissimilarity in the Folding of Human Cytosolic Creatine Kinase Isoenzymes

“…Consistent with previous results of RMCK [28], [30], [33], the folding of all five proteins were partially reversible as revealed by the inconsistency of the ANS fluorescence during folding and refolding. A comparison of the ANS fluorescence during CK folding and unfolding indicated that among the five proteins, the maximum ANS fluorescence of HMCK during refolding was significantly higher than that during unfolding.…”

eXL-MS: An Enhanced Cross-Linking Mass Spectrometry Workflow To Study Protein Complexes

“…An example of these intermolecular cross-linked peptides showing the interaction between the N-termini of two CK monomers is depicted in Figure S5. One hundred and thirty three other cross-linked peptides were found to be between 26 and 40 Å and could be explained by dynamic events such as breathing motions or large domain movements in the protein complex, as already observed in other studies. , Finally, we found 80 cross-linked peptides exceeding 40 Å. Looking in detail into these long cross-linked peptides, 46 were found to describe interactions occurring between a single patch constituted of 13 residues in close vicinity (K222, K241, K246, K297, S302, K303, K306, K318, K357, K364, K365, K368, S371) and the CK N-terminus (T5, K8, Y9, K10, K14, Y38, K39, and K40).…”

Designing an Antibody-Based Chaperoning System through Programming the Binding and Release of the Folding Intermediate

“…When DTT was added to the PDI− CK complexes, CK was released and the activity increased to nearly or slightly higher than that of the self-refolding CK. 38 We think the effect and mechanism of scFv A4 on folding of HCK in our present study are similar to those of PDI and a high concentration of Cyp18. They could prevent aggregation by forming a complex with HCK.…”

“…At concentrations above 1 μM, PDI acted as a protector against aggregation but as an inhibitor of reactivation by forming PDI–CK complexes through hydrophobic interaction and intermolecular disulfide bonds. When DTT was added to the PDI–CK complexes, CK was released and the activity increased to nearly or slightly higher than that of the self-refolding CK . We think the effect and mechanism of scFv A4 on folding of HCK in our present study are similar to those of PDI and a high concentration of Cyp18.…”