Abstract
Burkholderia lethal factor 1 (BLF1), a toxin derived from Burkholderia pseudomallei, reacts with eukaryotic initiation factor (eIF) 4A to inhibit protein synthesis. eIF4A1 and eIF4A2 are involved in translation initiation and share over 90% sequence similarity. However, they exert distinct effects on cancer treatment outcomes. To understand the molecular mechanism by which BLF1 modulates eIF4A isoforms in cancer cells, we investigated its effects on eIF4A-mediated adenosine 5'-triphosphate (ATP) hydrolysis. We found that eIF4A1 has a higher ATP-binding affinity compared to eIF4A2 (K(m) = 6.55 ± 0.78 μM vs. K(m) = 11.61 ± 2.33 μM). Meanwhile, we also found that eIF4A1 is more sensitive to changes in temperature, pH, and Mg(2+) concentration. Through N-terminal swapping and single amino acid mutations, we found that leucine 98 (L98) and alanine 100 (A100) play important roles in the ATPase activities of eIF4A isoforms. Moreover, BLF1 treatment significantly enhanced eIF4A2-mediated ATP hydrolysis at all tested ATP concentrations. These differences in BLF1-regulated eIF4A isoforms may explain its selective cytotoxicity against cancer cells. Our findings provide molecular insights into the functional difference between eIF4A isoforms and suggest that BLF1 might be of promising value for anticancer therapies.