title: Cyclophilin-mediated reactivation pathway of inactive adenosine kinase aggregates.
abstract: Monomeric adenosine kinase (AdK), a pivotal salvage enzyme of the purine auxotrophic parasite, Leishmania donovani, tends to aggregate naturally or selectively in presence of ADP, leading to inactivation. A cyclophilin (LdCyP) from the parasite reactivated the enzyme by disaggregating it. We studied the aggregation pathway of AdK with or without ADP. Transmission electron microscopy revealed that ADP-induced aggregates, as opposed to annular or torus-shaped natural aggregates, were mostly amorphous with protofibril-like structures. Interestingly, only the natural aggregates bound thioflavin T with a KD of 3.33 μM, indicating cross β-sheet structure. Dynamic light scattering experiments indicated that monomers formed aggregates either upon prolonged storage or ADP exposure. ADP-aggregates were disaggregated by LdCyP with concomitant reactivation of the enzyme. The activity revived with decrease in the aggregate size. Displacement of ADP from the ADP-aggregated enzyme by LdCyP resulted in reactivation. CD-spectral studies suggested that, like the natural aggregates, ADP induced formation of β-sheet structure in the ADP-aggregates. However, unlike the natural aggregate, it could be reconverted to α-helical conformation upon addition of LdCyP. Based on the results, a regulatory mechanism through interplay of ADP and/or LdCyP interaction with the enzyme is envisaged and a pathway of AdK reactivation by LdCyP-chaperone is proposed.
authors.affiliation: Division of Structural Biology and Bioinformatics, CSIR-Indian Institute of Chemical Biology, Kolkata, India.
authors.forename: Debalina
authors.initials: D
authors.lastname: Mukherjee
authors.forename: Hirak
authors.initials: H
authors.lastname: Patra
authors.forename: Aparna
authors.initials: A
authors.lastname: Laskar
authors.forename: Anjan
authors.initials: A
authors.lastname: Dasgupta
authors.forename: Nakul C
authors.initials: NC
authors.lastname: Maiti
authors.forename: Alok K
authors.initials: AK
authors.lastname: Datta
mesh_terms: D000248:Adenosine Kinase / Q000737:chemistry*; D021983:Cyclophilins / Q000737:chemistry*; D019281:Dimerization; D004789:Enzyme Activation; D007893:Leishmania donovani / Q000201:enzymology*
chemical_list: D000248:Adenosine Kinase; D021983:Cyclophilins
keywords: Chaperone; Cyclophilin; Oligomer; Protein–protein interaction; Structural transition