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Crystallization and preliminary X-ray analysis of the aspartic protease plasmepsin 4 from the malarial parasite Plasmodium malariae.

ABSTRACT: Plasmepsin 4 from the malarial parasite Plasmodium malariae (PmPM4) is a member of the plasmepsins (Plasmodium pepsins), a subfamily of the pepsin-like aspartic proteases whose ortholog in the malarial parasite P. falciparum is involved in hemoglobin digestion in its digestive vacuole. Crystals of PmPM4 in complex with the small-molecule inhibitor AG1776 have been grown from a precipitant of 15% PEG 4000 and 200 mM ammonium sulfate in 100 mM sodium acetate pH 4.5. X-ray diffraction data were collected on a Rigaku rotating-anode generator from a single crystal under cryoconditions, with a maximal useful diffraction pattern to 3.3 A resolution. The crystals are shown to be orthorhombic and have been assigned to space group P2(1)2(1)2, with unit-cell parameters a = 95.88, b = 112.58, c = 90.40 A and a scaling Rsym of 0.104 for 14,334 unique reflections. Packing consideration and self-rotation function results indicate that there are two molecules per asymmetric unit. It is expected that in the near future the structure of PmPM4 will be obtained using molecular-replacement methods, obtaining phases from previously determined plasmepsin structures. Elucidation of the structure of PmPM4 in complex with inhibitors may be paramount to producing new antimalarial therapeutic agents.

SUBMITTER: Madabushi A

PROVIDER: S-EPMC1952262 | biostudies-literature | 2005 Feb

REPOSITORIES: biostudies-literature

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Crystallization and preliminary X-ray analysis of the aspartic protease plasmepsin 4 from the malarial parasite Plasmodium malariae.

Madabushi Amrita A Chakraborty Sibani S Fisher S Zoë SZ Clemente José C JC Yowell Charles C Agbandje-McKenna Mavis M Dame John B JB Dunn Ben M BM McKenna Robert R

Acta crystallographica. Section F, Structural biology and crystallization communications 20050201 Pt 2

Plasmepsin 4 from the malarial parasite Plasmodium malariae (PmPM4) is a member of the plasmepsins (Plasmodium pepsins), a subfamily of the pepsin-like aspartic proteases whose ortholog in the malarial parasite P. falciparum is involved in hemoglobin digestion in its digestive vacuole. Crystals of PmPM4 in complex with the small-molecule inhibitor AG1776 have been grown from a precipitant of 15% PEG 4000 and 200 mM ammonium sulfate in 100 mM sodium acetate pH 4.5. X-ray diffraction data were col ...[more]

PMID: 16511002

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Project description:BackgroundMolecular genotyping in Plasmodium serves many aims including providing tools for studying parasite population genetics and distinguishing recrudescence from reinfection. Microsatellite typing, insertion-deletion (INDEL) and single nucleotide polymorphisms is used for genotyping, but only limited information is available for Plasmodium malariae, an important human malaria species. This study aimed to provide a set of genetic markers to facilitate the study of P. malariae population genetics.MethodsMarkers for microsatellite genotyping and pmmsp1 gene polymorphisms were developed and validated in symptomatic P. malariae field isolates from Myanmar (N = 37). Fragment analysis was used to determine allele sizes at each locus to calculate multiplicity of infections (MOI), linkage disequilibrium, heterozygosity and construct dendrograms. Nucleotide diversity (π), number of haplotypes, and genetic diversity (Hd) were assessed and a phylogenetic tree was constructed. Genome-wide microsatellite maps with annotated regions of newly identified markers were constructed.ResultsSix microsatellite markers were developed and tested in 37 P. malariae isolates which showed sufficient heterozygosity (0.530-0.922), and absence of linkage disequilibrium (IAS=0.03, p value > 0.05) (N = 37). In addition, a tandem repeat (VNTR)-based pmmsp1 INDEL polymorphisms marker was developed and assessed in 27 P. malariae isolates showing a nucleotide diversity of 0.0976, haplotype gene diversity of 0.698 and identified 14 unique variants. The size of VNTR consensus repeat unit adopted as allele was 27 base pairs. The markers Pm12_426 and pmmsp1 showed greatest diversity with heterozygosity scores of 0.920 and 0.835, respectively. Using six microsatellites markers, the likelihood that any two parasite strains would have the same microsatellite genotypes was 8.46 × 10-4 and was further reduced to 1.66 × 10-4 when pmmsp1 polymorphisms were included.ConclusionsSix novel microsatellites genotyping markers and a set of pmmsp1 VNTR-based INDEL polymorphisms markers for P. malariae were developed and validated. Each marker could be independently or in combination employed to access genotyping of the parasite. The newly developed markers may serve as a useful tool for investigating parasite diversity, population genetics, molecular epidemiology and for distinguishing recrudescence from reinfection in drug efficacy studies.

Dataset Information

Crystallization and preliminary X-ray analysis of the aspartic protease plasmepsin 4 from the malarial parasite Plasmodium malariae.

Publications

Crystallization and preliminary X-ray analysis of the aspartic protease plasmepsin 4 from the malarial parasite Plasmodium malariae.

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