Alok K. Datta

Leishmania major encodes an unusual peroxidase that is a close homologue of plant ascorbate peroxidase: a novel role of the transmembrane domain

Haem-containing enzymes (peroxidase and catalase) are widely distributed among prokaryotes and eukaryotes and play a vital role in H2O2 detoxification. But, to date, no haem-containing enzymatic defence against toxic H2O2 has been discovered in Leishmania species. We cloned, expressed and purified an unusual plant-like APX (ascorbate peroxidase) from Leishmania major (LmAPX) and characterized its catalytic parameters under steady-state conditions. Examination of its protein sequence indicated approx. 30-60% identity with other APXs. The N-terminal extension of LmAPX is characterized by a charged region followed by a stretch of 22 amino acids containing a transmembrane domain. To understand how the transmembrane domain influences the structure-function of LmAPX, we generated, purified and extensively characterized a variant that lacked the transmembrane domain. Eliminating the transmembrane domain had no impact on substrate-binding affinity but slowed down ascorbate oxidation and increased resistance to H2O2-dependent inactivation in the absence of electron donor by 480-fold. Spectral studies show that H2O2 can quickly oxidize the native enzyme to compound (II), which subsequently is reduced back to the native enzyme by an electron donor. In contrast, ascorbate-free transmembrane domain-containing enzyme did not react with H2O2, as revealed by the absence of compound (II) formation. Our findings suggest that the single copy LmAPX gene may play an important role in detoxification of H2O2 that is generated by endogenous processes and as a result of external influences such as the oxidative burst of infected host macrophages or during drug metabolism by Leishmania.

Structure of Cyclophilin from Leishmania Donovani Bound to Cyclosporin at 2.6 A Resolution: Correlation between Structure and Thermodynamic Data.

Drug development against Leishmania donovani, the pathogen that causes visceral leishmaniasis in humans, is currently an active area of research given the widespread prevalence of the disease and the emergence of resistant strains. The immunosuppressive drug cyclosporin is known to have antiparasitic activity against a variety of pathogens. The receptor for cyclosporin is the protein cyclophilin, which is a ubiquitous peptidylprolyl isomerase. The crystal structure of cyclophilin from L. donovani complexed with cyclosporin has been solved at 2.6 A resolution. The thermodynamic parameters of the interaction have been determined using spectroscopic and calorimetric techniques. A detailed effort has been made to predict the thermodynamic parameters of binding from computations based on the three-dimensional crystal structure. These results were in good agreement with the corresponding experimental values. Furthermore, the structural and biophysical results have been discussed in the context of leishmanial drug resistance and could also set the stage for the design of potent non-immunosuppressive antileishmanials.

Structure of cyclophilin from Leishmania donovani at 1.97 A resolution.

The crystal structure of cyclophilin from Leishmania donovani (LdCyp) has been determined and refined at 1.97 A resolution to a crystallographic R factor of 0.178 (R(free) = 0.197). The structure was solved by molecular replacement using cyclophilin from Trypanosoma cruzi as the search model. LdCyp exhibits complete structural conservation of the cyclosporin-binding site with respect to the homologous human protein, as anticipated from LdCyp-cyclosporin binding studies. Comparisons with other cyclophilins show deviations primarily in the loop regions. The solvent structure encompassing the molecule has also been analyzed in some detail.

Equilibrium unfolding of cyclophilin from Leishmania donovani: characterization of intermediate states.

Cyclophilin from Leishmania donovani (LdCyp) is a ubiquitous peptidyl-prolyl cis-trans isomerase involved in a host of important cellular activities, such as signaling, heat shock response, chaperone activity, mitochondrial pore maintenance and regulation of HIV-1 infectivity. It also acts as the prime cellular target for the auto-immune drug cyclosporine A (CsA). LdCyp is composed of a beta barrel encompassing the unique hydrophobic core of the molecule and is flanked by two helices (H1, H2) on either end of the barrel. The protein contains a lone partially exposed tryptophan. In the present work the equilibrium unfolding of LdCyp has been studied by fluorescence, circular dichroism and the non-coincidence of their respective Cms, indicates a non-two state transition. This fact was further corroborated by binding studies of the protein with bis-ANS and the lack of an isochromatic point in far UV CD. The thermal stability of the possible intermediates was characterized by differential scanning calorimetry. Further, MD simulations performed at 310, 400 and 450K exhibited the tendency of both helices to partially unwind and adopt non-native geometries with respect to the core, quite early in the unfolding process, in contrast to the relatively stable beta barrel.

Cyclophilin-mediated reactivation pathway of inactive adenosine kinase aggregates.

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.

Crystallization and preliminary X-ray analysis of cyclophilin from Leishmania donovani

Cyclophilin from the parasite Leishmania donovani is a protein with peptidylprolyl cis-trans isomerase activity, in addition to being a receptor for the drug cyclosporin. Crystals of the enzyme have been obtained in space group P4(3)2(1)2, with unit-cell parameters a = b = 48.73, c = 140.93 A, and diffract to 3.5 A resolution. One molecule per asymmetric unit gives a solvent content and Matthews coefficient of 46% and 2.3 A(3) Da(-1), respectively. Molecular-replacement calculations with human cyclophilin A as the search model give an unambiguous solution in rotation and translation functions.