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

The leading cause of malaria infections in humans is the parasite Plasmodium falciparum. The parasite contains a non-photosynthetic plastid-like organelle called the apicoplast, which is essential for its survival within the host. The apicoplast maintains its own genome, which must undergo replication and repair. The only DNA polymerase in the apicoplast (apPOL) is classified as an atypical A-family polymerase. apPOL shares no direct orthology to mammalian polymerases, making the P. falciparum apicoplast DNA polymerase an attractive anti-malarial drug target. We solved the crystal structure of P. falciparum apPOL, the first structural representative of the atypical A-family polymerases. We showed that apPOL diverges from typical members in two of three previously identified signature motifs and a region not implicated by sequence. Moreover, apPOL has an additional N-terminal subdomain that extends the exonuclease domain. This region may be involved in binding DNA, participating in protein-protein interactions, and/or stabilizing the proofreading domain. These structural variances may account for functional differences in polymerase activity. The crystal structure also provides a point of departure for structure-based anti-malarial drug design, and these atypical regions could be targeted for potent and specific inhibitors. We have developed a fluorescence-based high-throughput DNA polymerase assay to screen for compounds that inhibit apPOL activity. Analysis of validation experiments indicates that the assay is statistically robust. A pilot screen of a 2,880 compound library identified 62 possible inhibitors that cause at least 50% inhibition of polymerase activity. The simplicity and robustness of the assay provides a solid platform for screening apicoplast polymerase inhibitors that could serve as lead compounds in the efforts to discover and produce anti-malarial drugs. In addition to its potential as a potent drug target, apPOL provides structural and biochemical insight into a poorly characterized subgroup of A-family polymerases.