Amino acid residues of Leishmania donovani cyclophilin key to interaction with its adenosine kinase: biological implications

Abstract

Cyclophilins (CyPs), by interacting with a variety of proteins, often modulate their biological activities and thus have been implicated in several cellular functions. However, mechanisms that determine such interactions are poorly understood. We earlier reported that an endoplasmic reticulum (ER)-located cyclophilin (LdCyP) from the purine auxotrophic parasitic protozoan Leishmania donovani reactivated its adenosine kinase (AdK). The AdK-reactivating property of LdCyP was however abolished at high ionic strength but not by nonionic detergents. Modeling of LdCyP, based on its crystal structure solved at 1.97 Å resolution, revealed several solvent-exposed hydrophobic and charged residues. Mutagenesis of several of such solvent-exposed residues was performed and their corresponding activities with regard to their (i) AdK reactivation property, (ii) ability to form complex with the enzyme, (iii) capacity to induce red shift in the intrinsic tryptophan fluorescence maxima of AdK, and (iv) efficiency to withdraw the ADP inhibition from the AdK-mediated reaction were compared to the wild-type protein. Results indicated that while the replacement of R147 with either A or D severely impaired all of the above characteristics displayed by the wild-type LdCyP, the effect of mutating K114 and K153 was although relatively less but nevertheless noticeable. Alteration of other exposed hydrophobic and charged residues apparently did not have any discernible effect. Under the condition of cellular stress, the ER-located LdCyP is released into the cytoplasm with concomitant increase both in the specific activity of the cytosol-resident AdK and the uptake of radiolabeled Ado into the cells. These experiments, besides demonstrating the importance of the positive charge, identified R147 as the most crucial residue in the LdCyP-AdK interaction and provide evidence for the stress-induced retrograde translocation of LdCyP from the ER to the cytoplasm. A possible implication of this interaction in the life cycle of the parasite is proposed.