Abstract
Clan CA, family C1 cysteine peptidases (CPs) are important virulence factors and drug targets in parasites that cause neglected diseases. Natural CP inhibitors of the I42 family, known as ICP, occur in some protozoa and bacterial pathogens but are absent from metazoa. They are active against both parasite and mammalian CPs, despite having no sequence similarity with other classes of CP inhibitor. Recent data suggest that Leishmania mexicana ICP plays an important role in host-parasite interactions. We have now solved the structure of ICP from L. mexicana by NMR and shown that it adopts a type of immunoglobulin-like fold not previously reported in lower eukaryotes or bacteria. The structure places three loops containing highly conserved residues at one end of the molecule, one loop being highly mobile. Interaction studies with CPs confirm the importance of these loops for the interaction between ICP and CPs and suggest the mechanism of inhibition. Structure-guided mutagenesis of ICP has revealed that residues in the mobile loop are critical for CP inhibition. Data-driven docking models support the importance of the loops in the ICP-CP interaction. This study provides structural evidence for the convergent evolution from an immunoglobulin fold of CP inhibitors with a cystatin-like mechanism.
Highlights
Characterization of the structure and mechanisms of action of natural inhibitors of cysteine peptidases (CPs)2 has provided important insights into the functional roles of the inhibitors themselves and those of the target CPs
There are highly conserved motifs that suggest important functional regions. This has facilitated the identification of predicted ICPs from genome data and recombinant ICPs have been produced from the L. mexicana, T. brucei, Entamoeba histolytica, and P. aeruginosa genes and confirmed to have potent inhibitory activity toward CPs, notably cathepsin L homologues [7, 11, 12]
This study has shown that ICP has an Ig fold that acts as a scaffold for three interstrand loops carrying the most highly conserved residues in the chagasin family of ICP proteins
Summary
Protein Production—Recombinant L. mexicana ICP was expressed from a pET28 (Novagen)-derived plasmid in Escherichia coli BL21 (DE3) cells as described previously [11]. 15N,13C-labeled protein was produced by growing the cells in M9 medium using 15NH4Cl and [13C]glucose (Spectra Stable Isotopes) as the sole nitrogen and carbon sources. Protein Production—Recombinant L. mexicana ICP was expressed from a pET28 (Novagen)-derived plasmid in Escherichia coli BL21 (DE3) cells as described previously [11]. Distance restraints for structure calculation were derived from three-dimensional 15N and 13C HSQCNOESY spectra recorded with 100 ms mixing times recorded on an 800 MHz Bruker Avance spectrometer. Structure Calculation—Assigned, partially assigned, and ambiguous NOESY cross-peaks were used to generate distance constraints within CCPN analysis that were exported directly to CNS/XPLOR format and used as input for structure calculations using CNS v1.1 [17] using a modified version of the PARALLHDG 5.3 forcefield [18] with IUPACrecommended nomenclature [19]. Distance restraints representing hydrogen bonds were incorporated for slowly exchanging amides where corroborating NOEs existed and an acceptor atom could be unambiguously identified.
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
