Crystallization and preliminary X-ray analysis of cyclophilin from<i>Leishmania donovani</i>

Banerjee, Rahul; Dutta, Madhuri; Sen, Malabika; Datta, Alokmay

doi:10.1107/s0907444902012611

Cited by 2 publications

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References 13 publications

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“…Modeling Studies. The crystallization and low-resolution structure of LdCyP has already been reported (44). The highresolution crystal structure of LdCyP was solved by molecular replacement (AMoRe) using the homologous molecule from Trypanosoma cruzi (PDB code 1xo7) as the search model (45).…”

Section: Methodsmentioning

confidence: 99%

Amino Acid Residues ofLeishmania donovaniCyclophilin Key to Interaction with Its Adenosine Kinase: Biological Implications

et al. 2007

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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 A 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.

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Section: Methodsmentioning

confidence: 99%

Amino Acid Residues ofLeishmania donovaniCyclophilin Key to Interaction with Its Adenosine Kinase: Biological Implications

et al. 2007

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“…As reported previously (Dutta et al, 2001;Banerjee et al, 2002), recombinant cyclophilin from L. donovani (LdCyp) was overexpressed in Escherichia coli M15 cells. The expression vector pQE32 (Qiagen) contained the LdCyP gene (residues 22-187) fused to a N-terminal His 6 tag.…”

Section: Expression and Purificationmentioning

confidence: 99%

“…The crystallization protocol reported previously (Banerjee et al, 2002) was slightly modified and optimized to obtain crystals which diffracted to higher resolution (1.97 Å ). Crystals were obtained by the hanging-drop method with 10 ml drops containing 10 mg ml À1 protein, 7.5% PEG 3350 inverted over a 1 ml reservoir of 40% PEG 3350 at 293 K. Crystals suitable for data collection appeared in about two weeks.…”

Section: Crystallization and Data Collectionmentioning

confidence: 99%

Structure of cyclophilin fromLeishmania donovaniat 1.97 Å resolution

et al. 2007

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PDB Reference: cyclophilin, 2haq, r2haqsf.The crystal structure of cyclophilin from Leishmania donovani (LdCyp) has been determined and refined at 1.97 Å 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.

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Crystallization and preliminary X-ray analysis of cyclophilin fromLeishmania donovani

Cited by 2 publications

References 13 publications

Amino Acid Residues ofLeishmania donovaniCyclophilin Key to Interaction with Its Adenosine Kinase: Biological Implications

Amino Acid Residues ofLeishmania donovaniCyclophilin Key to Interaction with Its Adenosine Kinase: Biological Implications

Structure of cyclophilin fromLeishmania donovaniat 1.97 Å resolution

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