Crystallization and preliminary X-ray analysis of thePlasmodium falciparumapicoplast DNA polymerase

Abstract

Infection by the parasite Plasmodium falciparum is the leading cause of malaria in humans. The parasite has a unique and essential plastid-like organelle called the apicoplast. The apicoplast contains a genome that undergoes replication and repair through the action of a replicative polymerase (apPOL). apPOL has no direct orthologs in mammalian polymerases and is therefore an attractive antimalarial drug target. No structural information exists for apPOL, and the Klenow fragment of Escherichia coli DNA polymerase I, which is its closest structural homolog, shares only 28% sequence identity. Here, conditions for the crystallization of and preliminary X-ray diffraction data from crystals of P. falciparum apPOL are reported. Data complete to 3.5 Å resolution were collected from a single crystal (2 × 2 × 5 µm) using a 5 µm beam. The space group P6522 (unit-cell parameters a = b = 141.8, c = 149.7 Å, α = β = 90, γ = 120°) was confirmed by molecular replacement. Refinement is in progress.

Replication and maintenance of the Plasmodium falciparum apicoplast genome

“…1), which was referred to as KPom1 based on sequence similarity to the Klenow fragment of E. coli Pol I [24]. Most recently, the Nelson laboratory designed a construct based on sequence alignments (referred to as apPOL) and identified a possible polymerase boundary spanning residues 1389 to 2016 that is conserved across the Plasmodium genus [41,42] (Fig. 1).…”

Structure and function of the Plasmodium falciparum apicoplast DNA polymerase: the first look at an "atypical" A-family polymerase and its potential in antimalarial drug discovery

“…1). A 20 amino acid long targeting sequence ensures that the gene is co-translated as a single polyprotein into the ER lumen, where it is then transported as a polyprotein into the apicoplast (20)(21)(22). Prex is thought to contain the only DNA polymerase targeted to the apicoplast, and thus is responsible for both DNA replication and repair (23).…”

“…All protein constructs were purified as described (22). Mutagenesis was performed using QuickChange mutagenesis to generate exonuclease deficient mutants (D82N and E84Q), catalytic base mutants (H578Q), and finger tyrosine mutants (Y481A/485A/Y486A).…”

“…Residues in the NTR are exclusive to and highly conserved within the Plasmodium genus and may be necessary for a properly functioning polymerase (11,22). The NTR has a short helical region followed by two anti-parallel β-strands (Strand 1A and Strand 1B).…”

“…Crystallization experiments employed the method of hanging drops. N-apPOL exo− produced microcrystals(22) by the combination of equal parts protein solution (4-16 mg ml −1 N-apPOL exo− , 20 mM Tris-HCl pH 8.0, and 400 mM NaCl) and precipitant solution (0.2 M ammonium sulfate, 0.1 M MES monohydrate pH 6.5, and 35% w/v polyethylene glycol monomethyl ether 5,000). We flash froze crystals in liquid nitrogen without additional cryoprotectant.…”