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Resistance mutations at the lipid substrate binding site of Plasmodium falciparum protein farnesyltransferase.

ABSTRACT: The post-translational farnesylation of proteins serves to anchor a subset of intracellular proteins to membranes in eukaryotic organisms and also promotes protein-protein interactions. This enzymatic reaction is carried out by protein farnesyltransferase (PFT), which catalyzes the transfer of a 15-carbon isoprenoid lipid unit, a farnesyl group, from farnesyl pyrophosphate to the C-termini of proteins containing a CaaX motif. Inhibition of PFT is lethal to the pathogenic protozoa Plasmodium falciparum. Previously, we have shown that parasites resistant to a tetrahydroquinoline (THQ)-based PFT inhibitor BMS-388891 have mutations leading to amino acid substitutions in PFT that map to the peptide substrate binding domain. We now report the selection of parasites resistant to another THQ PFT inhibitor BMS-339941. In whole cell assays sensitivity to BMS-339941 was reduced by 33-fold in a resistant clone, and biochemical analysis demonstrated a corresponding 33-fold increase in the BMS-339941 K(i) for the mutant PFT enzyme. More detailed kinetic analysis revealed that the mutant enzyme required higher concentration of peptide and farnesyl pyrophosphate substrates for optimum catalysis. Unlike previously characterized parasites resistant to BMS-388891, the resistant parasites have a mutation which is predicted to be in a distinct location of the enzymatic pocket, near the farnesyl pyrophosphate binding pocket. This is the first description of a mutation from any species affecting the farnesyl pyrophosphate binding pocket with reduced efficacy of PFT inhibitors. These data provide further support that PFT is the target of THQ inhibitors in P. falciparum and suggest that PFT inhibitors should be combined with other antimalarial agents to minimize the development of resistant parasites.

SUBMITTER: Eastman RT

PROVIDER: S-EPMC2875941 | biostudies-literature | 2007 Mar

REPOSITORIES: biostudies-literature

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Resistance mutations at the lipid substrate binding site of Plasmodium falciparum protein farnesyltransferase.

Eastman Richard T RT White John J Hucke Oliver O Yokoyama Kohei K Verlinde Christophe L M J CL Hast Michael A MA Beese Lorena S LS Gelb Michael H MH Rathod Pradipsinh K PK Van Voorhis Wesley C WC

Molecular and biochemical parasitology 20061222 1

The post-translational farnesylation of proteins serves to anchor a subset of intracellular proteins to membranes in eukaryotic organisms and also promotes protein-protein interactions. This enzymatic reaction is carried out by protein farnesyltransferase (PFT), which catalyzes the transfer of a 15-carbon isoprenoid lipid unit, a farnesyl group, from farnesyl pyrophosphate to the C-termini of proteins containing a CaaX motif. Inhibition of PFT is lethal to the pathogenic protozoa Plasmodium falc ...[more]

PMID: 17208314

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Project description:BackgroundPlasmodium species are entirely dependent upon their host as a source of essential iron. Although it is an indispensable micronutrient, oxidation of excess ferrous iron to the ferric state in the cell cytoplasm can produce reactive oxygen species that are cytotoxic. The malaria parasite must therefore carefully regulate the processes involved in iron acquisition and storage. A 273 amino acid membrane transporter that is a member of the vacuolar iron transporter (VIT) family and an orthologue of the yeast Ca2+-sensitive cross complementer (CCC1) protein plays a major role in cytosolic iron detoxification of Plasmodium species and functions in transport of ferrous iron ions into the endoplasmic reticulum for storage. While this transporter, termed PfVIT, is not critical for viability of the parasite evidence from studies of mice infected with VIT-deficient Plasmodium suggests it could still provide an efficient target for chemoprophylactic treatment of malaria. Individual amino acid residues that constitute the Fe2+ binding site of the protein were identified to better understand the structural basis of substrate recognition and binding by PfVIT.MethodsUsing the crystal structure of a recently published plant VIT as a template, a high-quality homology model of PfVIT was constructed to identify the amino acid composition of the transporter's substrate binding site and to act as a guide for subsequent mutagenesis studies. To test the effect of mutation of the substrate binding-site residues on PfVIT function a yeast complementation assay assessed the ability of overexpressed, recombinant wild type and mutant PfVIT to rescue an iron-sensitive deletion strain (ccc1∆) of Saccharomyces cerevisiae yeast from the toxic effects of a high concentration of extracellular iron.ResultsThe combined in silico and mutagenesis approach identified a methionine residue located within the cytoplasmic metal binding domain of the transporter as essential for PfVIT function and provided insight into the structural basis for the Fe2+-selectivity of the protein.ConclusionThe structural model of the metal binding site of PfVIT opens the door for rational design of therapeutics to interfere with iron homeostasis within the malaria parasite.

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Resistance mutations at the lipid substrate binding site of Plasmodium falciparum protein farnesyltransferase.

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Resistance mutations at the lipid substrate binding site of Plasmodium falciparum protein farnesyltransferase.

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