Probing the role of parasite-specific, distant structural regions on communication and catalysis in the bifunctional thymidylate synthase-dihydrofolate reductase from Plasmodium falciparum

Abstract

Plasmodium falciparum thymidylate synthase-dihydrofolate reductase (TS-DHFR) is an essential enzyme in nucleotide biosynthesis and a validated molecular drug target in malaria. Because P. falciparum TS and DHFR are highly homologous to their human counterparts, existing active-site antifolate drugs can have dose-limiting toxicities. In humans, TS and DHFR are two separate proteins. In P. falciparum, however, TS-DHFR is bifunctional, with both TS and DHFR active sites on a single polypeptide chain of the enzyme. Consequently, P. falciparum TS-DHFR contains unique distant or nonactive regions that might modulate catalysis: (1) an N-terminal tail and (2) a linker region tethering DHFR to TS, and encoding a crossover helix that forms critical electrostatic interactions with the DHFR active site. The role of these nonactive sites in the bifunctional P. falciparum TS-DHFR is unknown. We report the first in-depth, pre-steady-state kinetic characterization of the full-length, wild-type (WT) P. falciparum TS-DHFR enzyme and probe the role of distant, nonactive regions through mutational analysis. We show that the overall rate-limiting step in the WT P. falciparum TS-DHFR enzyme is TS catalysis. We further show that if TS is in an activated (liganded) conformation, the DHFR rate is 2-fold activated, from 60 s-1 to 130 s-1 in the WT enzyme. The TS rate is also reciprocally activated by approximately 1.5-fold if DHFR is in an activated, ligand-bound conformation. Mutations to the linker region affect neither catalytic rate nor domain-domain communication. Deletion of the N-terminal tail, although in a location remote from the active site, decreases the DHFR single rate and the bifunctional TS-DHFR rate by a factor of 2. The 2-fold activation of the DHFR rate by TS ligands remains intact, although even the activated N-terminal mutant has just half the DHFR activity of the WT enzyme. However, the reciprocal communication between TS active site and DHFR ligands is impaired in N-terminal mutants. Surprisingly, deletion of the analogous N-terminal tail in Leishmania major TS-DHFR causes a 3-fold enhancement of the DHFR rate from approximately 14 s-1 to approximately 40 s-1. In summary, our results demonstrate a complex interplay of domain-domain communication and nonactive-site modulation of catalysis in P. falciparum TS-DHFR. Furthermore, each parasitic TS-DHFR is activated by unique mechanisms, modulated by their nonactive site regions. Finally, our studies suggest the N-terminal tail of P. falciparum TS-DHFR is a highly selective, novel target for potential antifolate development in malaria.

Figure 1. The Reaction Scheme and the Non-active Site Regions of Bifunctional Parasitic TS-DHFR enzymes

a) TS catalyzes the conversion of CH₂H₄folate and dUMP to dTMP and H₂folate. H₂-folate is converted to H₄-folate at the DHFR active site while an NADPH is oxidized to NADP⁺. b) Diagrammatic representation of organism-specific differences in non-active site regions of TS-DHFR. The N-terminal tail (yellow) of the DHFR active site (blue) can be of variable length, or completely absent. This is also true of the linker or junctional region (green), which links the DHFR and TS (red) active sites.

Figure 2. The Non-active Sites Regions of P. falciparum TS-DHFR enzymes, highlighting the interactions disrupted in the Ala-FACE and D4 mutants

a) The Pf TS-DHFR (PDB entry: 1J3I) structure is colored with DHFR domains in blue, TS in red, with the linker region highlighted in green, and the N-terminal tail in yellow. b) The interactions between the crossover helix (green) and the backside DHFR active site (burgundy) disrupted by the Ala-FACE mutant. The residues shown are Asp 284, Glu 285, Asp 288, Asp 289 and Tyr 292 in the crossover helix, and Lys 69, Lys 72, Tyr 158, Lys 160, Lys 180, Lys 181 and Tyr 183 in the DHFR backbone. The substrates WR99210 and NADPH are shown in navy blue to mark the active site. For clarity, only a single DHFR monomer and its adjacent TS domain are shown, and also the residues Asn 231 to Asp 222 and Lys 227 are not shown. c) Interactions of the residues of the N-terminal tail (shown in yellow) with Insert II (shown in orange) and with the αE-βE loop (shown in purple). The residues highlighted are Glu 3 and Val 5 in the N-terminal tail, Tyr 90 in Insert II and Ile 50 in the αE-βE loop. The substrates WR99210 [23] and NADPH are shown in navy blue to mark the active site. Only a single DHFR domain is shown for clarity.

Figure 3. Single turnover experiments for WT, Ala-FACE and D4 mutants of P. falciparum TS-DHFR show the N-terminal tail mutant slows the all three reactions, TS, DHFR and TS-DHFR

The data for WT enzyme are (red, circle), for Ala-FACE enzyme (blue, square) and for the D4 N-terminal mutant (green, diamond) are plotted by reaction. The reactions were conducted using rapid chemical quench apparatus under single turnover conditions at 25°C. a) The DHFR experiment was conducted with 80 µM enzyme and 500 µM NADPH mixed with 6.5 µM tritiated H₂-folate. b) The TS experiment was conducted with 80 µM enzyme and 500 µM dUMP mixed with 6.5 µM tritiated CH₂H₄-folate. c) The bifunctional TS-DHFR experiment was conducted with 80 µM enzyme, 500 µM NADPH, and 500 µM dUMP mixed with 6.5 µM tritiated H₂-folate. In all cases, the data were fit to a single exponential equation, and the rate constants from these are summarized in Table 1.

Figure 4. DHFR rate in P. falciparum TS-DHFR is activated in the presence of TS ligands, dUMP and CH₂H₄-folate

DHFR burst experiments for WT, Ala-FACE and D4 N-terminal mutants conducted in the presence or absence of TS ligands. Using stopped flow fluorescence, we performed these experiments under burst conditions, with fluorescence excitation at 287 nm and the emission at 450 nm. Column a) shows data from DHFR burst experiments conducted in the absence of TS ligands. The conditions were used were 7.5 µM enzyme and 500 µM NADPH, mixed with 50 µM H₂-folate. Column b) shows data from DHFR burst experiments conducted in the presence of TS ligands, FdUMP and CH₂H₄-folate. The conditions were used were 7.5 µM enzyme, 500 µM NADPH, 100 µM FdUMP, and 25 µM CH₂H₄folate, mixed with 50 µM H₂-folate. WT data points are shown in red, Ala-FACE data points, in blue and D4 data points, in green. In all cases, the data were fit to a single exponential equation, and the rate constants from these are summarized in Table 2.

Figure 5. DHFR reactions of the L. major N-terminal deletion mutant, N22, show that the N-terminal tail is autoinhibitory in the L. major TS-DHFR enzyme

a) Single turnover, rapid chemical quench experiments for N22 conducted using 45 µM enzyme and 500 µM NADPH mixed with 5.5 µM tritiated H₂-folate. The data were fit to a single exponential equation to provide a rate constant of 40.2 ± 4.6 s⁻¹. In comparison, the DHFR single turnover rate for WT L. major TS-DHFR had a rate constant of 14.5 ± 1.37 s⁻¹ (data not shown). b) N22 mutant DHFR burst experiment, performed on the stopped flow, was conducted using 7.5 µM enzyme and 500 µM NADPH, mixed with 50 µM H₂-folate. The fluorescence excitation was at 287 nm and the emission was at 450 nm. The data were fit to a single exponential burst equation to provide a rate constant of 45.21 ± 0.853 s⁻¹. c) The L. major TS-DHFR structure is colored with DHFR domains in blue, TS in red, and the N-terminal tail in yellow, which wraps around and makes extensive contacts with the TS domain. The substrates, methotrexate, NADPH, FdUMP and 10-propargyl-5,8-dideazafolate (PDDF) are shown in gray.