Insights into the role of the junctional region of Plasmodium falciparum dihydrofolate reductase-thymidylate synthase

Abstract

Abstract Background Plasmodium falciparum dihydrofolate reductase-thymidylate synthase (pf DHFR-TS) is a well-defined target of anti-malarial drug, such as pyrimethamine and cycloguanil. Emergence of malaria parasites resistant to these drugs has been shown to be associated with point mutations of the gene coding for the target enzymes. Although the 3D-structure of P. falciparum bifunctional pf DHFR-TS has been reported previously, relatively little is known about the interactions between the pf DHFR and pf TS domains and the roles of the junctional region that links the two domains together. Therefore, a thorough understanding of the interaction of the two domains and the role of the junctional region of this target is important as the knowledge could assist the development of new effective anti-malarial drugs aimed at overcoming drug-resistant malaria. Methods A system was developed to investigate the interaction between pf DHFR and pf TS domains and the role of the junctional region on the activity of the recombinant pf TS. Based on the ability of co-transformed plasmids coding for pf DHFR and pf TS with truncated junctional region to complement the growth of TS-deficient Escherichia coli strain χ2913recA(DE3) on minimum media without thymidine supplementation, active pf TS mutants with minimal length of junctional region were identified. Interactions between active pf DHFR and the pf TS domains were demonstrated by using a bacterial two-hybrid system. Results Using TS-deficient E. coli strain χ2913recA(DE3), the authors have shown for the first time that in P. falciparum a junctional region of at least 44 amino acids or longer was necessary for the pf TS domain to be active for the synthesis of thymidylate for the cells. Truncation of the junctional region of the bifunctional pf DHFR-TS further confirmed the above results, and suggested that a critical length of the junctional peptide of pf DHFR-TS would be essential for the activity of TS to catalyze the synthesis of thymidylate. Conclusion The present study demonstrated the interactions between the pf DHFR and pf TS domains of the bifunctional pf DHFR-TS, and revealed that the junctional region linking the two protein domains is essential for the expression of catalytically active pf TS domain. The findings could be useful since inhibition of the pf DHFR-TS domain-domain interaction could form a basis for the development of new anti-malarial drugs based on targeting the non-active site region of this important enzyme.