Fig 1.
Protein structure prediction of PfCCT MΔKWT and PfCCT MΔKR681H.
A) Alignment of rat CCT (PDB ID: 4MVC) and PfCCT MΔKWT sequences used for modeling and MD stimulations. Numbering is according to PfCCT MΔKWT. Secondary structure elements are represented by squiggles (α-helices), arrows (β-strands) and lines (turns). In the aligned sequences, red box with white character indicates strict identity and red character means similarity in groups. R681 corresponding to R140 in rat CCT is indicated by a green star. Hydropathy (pink—hydrophobic, grey—intermediate, cyan—hydrophilic) and accessibility (blue—accessible, cyan—intermediate, white—buried) are also presented below the sequences. The layout with secondary structure elements was generated with ESPript 3.0 [66], supplemented with visual inspection of structures. B) Conservation of the RYVD signature sequence in CCTs, shown by Weblogo. Numbering is according to the Plasmodium falciparum sequence, where the 681RWVD684 corresponds to the 140RYVD143 in the rat sequence. C) Dimer structure of MΔKWT homology model. Chain A is coloured in red and chain B is coloured in yellow. Important secondary structure elements are indicated.
Table 1.
Polar inter-chain interactions between L3, αA and N-terminal regions in homology models of both enzyme variants.
Fig 2.
Polar interactions at the dimer interface of PfCCT MΔKWT and PfCCT MΔKR681H involving 681RWVD684.
A) Direct interactions harbouring 681RWVD684 signature sequence motif in PfCCT MΔKWT. The residues involved in the inter-chain interaction are shown in stick representation, and interactions are indicated by pink dashed lines. Characteristic dimer interface distances d(R681, CA - H679’, O) and d(I680, CA - I680’, N) are denoted by blue double-headed arrows. Residues in chain B are marked with apostrophes. B) Direct interactions harbouring the 681HWVD684 mutated signature sequence motif PfCCT MΔKR681H. The residues involved in the inter-chain interaction are shown in stick representation, and interactions are indicated by pink dashed lines. Characteristic dimer interface distances d(H681, CA - H679’, O) and d(I680, CA - I680’, N) are denoted by blue double-headed arrows. Residues in chain B are marked with apostrophes.
Fig 3.
Steady-state kinetic analysis of PfCCT MΔKWT and PfCCT MΔKR681H.
A) CTP titration of the activity of the CCTs at a fixed ChoP concentration of 5 mM. The plot shows one representative experiment. Titration data are fitted with the Michaelis–Menten kinetic model assuming no cooperativity. B) ChoP titration of the activity of the CCTs at a fixed CTP concentration of 1 mM. The plot shows one representative experiment. Titration data are fitted with a kinetic model assuming substrate inhibition without cooperativity. Note the substrate inhibition effect of ChoP as an initial rate decrease is observed at higher substrate concentrations.
Table 2.
Kinetic parameters of PfCCT MΔKWT and PfCCT MΔKR681H catalysis.
Fig 4.
Kinetics of thermal inactivation of PfCCT MΔKWT and PfCCT MΔKR681H.
Protein samples were incubated for 15 min in buffer A at different temperatures prior to the measurement performed at 20°C. Inactivation is shown as the fraction of remaining CCT activity. One representative is shown for each temperature and each protein.
Fig 5.
Mass spectra of PfCCT MΔKWT and PfCCT MΔKR681H proteins under native electrospray conditions.
M and D indicate signals contributing monomers and dimers, respectively, while numbers denote the charge states. A) Mass spectrum of PfCCT MΔKWT measured in the present study for direct comparison (cf. also [10]). B) Mass spectrum of PfCCT MΔKR681H. In the inset the 10-times enlarged graph of dimer regions (3150–4000 m/z) is shown.
Table 3.
Overall effective interaction energy () and the contribution of non-bonded van der Waals (
) and Coulomb (
) interaction energy terms and of the solvation free energy change upon dimerization (
) averaged over all frames of the equilibrated phase of the productive MD simulations.
Fig 6.
Inter-subunit interaction energies during molecular dynamics simulations.
Interaction energies are calculated as Eq 1 and represented by black line in case of PfCCT MΔKWT and by grey line in case of PfCCT MΔKR681H, respectively.
Fig 7.
Inter-subunit interaction distances during molecular dynamics simulations.
A) Variation of the atomic distance d(R/H681, CA - H679’, O) as a characteristic proximal inter-subunit contact of the catalytic domains (cf. Fig 2A and 2B). Distances are represented by black line in case of PfCCT MΔKWT and by grey line in case of PfCCT MΔKR681H, respectively. B) Variation of the atomic distance d(I680, CA - I680’, N) as a characteristic proximal inter-subunit distance of the catalytic domains (cf. Fig 2A and 2B). Distances are represented by black line in case of PfCCT MΔKWT and by grey line in case of PfCCT MΔKR681H.