A single-domain cyclophilin from Leishmania donovani reactivates soluble aggregates of adenosine kinase by isomerase-independent chaperone function.
Disaggregation and reactivation of aggregated proteins by chaperones is well established. However, little is known regarding such kind of function of single-domain small cyclophilins (CyPs). Here we demonstrate that, with increasing concentrations, fully active adenosine kinase (AdK) of Leishmania donovani tends to form soluble aggregates, resulting in inactivation. Using this inactive enzyme as the substrate, it is shown that a CyP from L. donovani (LdCyP) alone can cause complete disaggregation, leading to reactivation of the enzyme. The reactivating ability of LdCyP remains unaffected even in the presence of cyclosporin A and macromolecular crowding agents. The reactivation occurs noncatalytically and is reversible. A truncated LdCyP, devoid of 88 amino acids from the N terminus, is found to be required in near stoichiometric proportion to reactivate AdK, suggesting essentiality of the C-terminal region. Gel filtration and light-scattering experiments together with protein cross-linking studies revealed that both full-length LdCyP and the truncated form directly interact with AdK and convert oligomeric forms of the enzyme to monomeric state. Homology modeling studies suggest that the exposed hydrophobic residues of LdCyP, by interacting with solvent-accessible hydrophobic surface of AdK, pull apart its aggregated inactive oligomers to functional monomers. Clearly, the results are consistent with the interpretation that the higher efficiency of the truncated LdCyP is most likely due to increased exposure of the hydrophobic residues on its surface. These observations, besides establishing L. donovani AdK as one of the model enzymes to study aggregation-disaggregation of proteins, raise the possibility that single-domain small CyPs, under physiological conditions, may regulate the activity of aggregation-prone proteins by ensuring their disaggregation.
Chakraborty
Anutosh
A
Division of Infectious Diseases, Leishmania Group, Indian Institute of Chemical Biology, Kolkata 700 032, India.
Das
Ishita
I
Datta
Rupak
R
Sen
Banibrata
B
Bhattacharyya
Debasish
D
Mandal
Chhabinath
C
Datta
Alok K
AK
D000248:Adenosine Kinase / Q000378:metabolism*; D000818:Animals; D002850:Chromatography, Gel; D003432:Cross-Linking Reagents / Q000494:pharmacology; D021983:Cyclophilins / Q000378:metabolism*; D016572:Cyclosporine / Q000378:metabolism / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D020104:Immunophilins / Q000737:chemistry; D007700:Kinetics; D007893:Leishmania donovani / Q000378:metabolism*; D008027:Light; D008954:Models, Biological; D008958:Models, Molecular; D010455:Peptides / Q000737:chemistry; D010766:Phosphorylation; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D012542:Scattering, Radiation; D013379:Substrate Specificity; D013997:Time Factors
D003432:Cross-Linking Reagents; D010455:Peptides; D016572:Cyclosporine; D000248:Adenosine Kinase; D021983:Cyclophilins; D020104:Immunophilins