Steered molecular dynamics simulations reveal critical residues for (un)binding of substrates, inhibitors and a product to the malarial M1 aminopeptidase

Abstract

Malaria is a life-threatening disease spread by mosquitoes. Plasmodium falciparum M1 alanyl aminopeptidase (PfM1-AAP) is a promising target for the treatment of malaria. The recently solved crystal structures of PfM1-AAP revealed that the buried active site can be accessed through two channel openings: a short N-terminal channel with the length of 8 Å and a long C-terminal channel with the length of 30 Å. It is unclear, however, how substrates and inhibitors migrate to the active site and a product of cleavage leaves. Here, we study the molecular mechanism of substrate and inhibitor migration to the active site and the product release using steered molecular dynamics simulations. We identified a stepwise passage of substrates and inhibitors in the C-terminal channel of PfM1-AAP, involving (I) ligand recognition at the opening of the channel, (II) ionic translation to the 'water reservoir', (III) ligand reorientation in the 'water reservoir' and (IV) passage in a suitable conformation into the active site. Endorsed by enzymatic analysis of functional recombinant PfM1-AAP and mutagenesis studies, our novel ligand-residue binding network analysis has identified the functional residues controlling ligand migration within the C-terminal channel of PfM1-AAP. Furthermore, from unbinding simulations of the Arg product we propose a charge repulsion as the driving force to expel the product out from the N-terminal channel of PfM1-AAP. Our work paves the way towards the design of a novel class of PfM1-AAP inhibitors based on preventing substrate entry to the active site.

Fig 1. The Plasmodium falciparum M1 alanyl aminopeptidase (PfM1-AAP) with the substrates (Arg-Ala and Met-Phe), inhibitors (Bestatin and R5X) and Arg product.

The PfM1-AAP domains are coloured and labelled. The long C-terminal and short N-terminal channels and the position of residues selected for calculation of R_gyr are shown.

Fig 2. Work values from the six replicate 30 ns and single 100 ns sMD simulations indicate the preference of the substrate and inhibitors to migrate through the C-terminal channel and for the product though the N-terminal channel.

Fig 3. Ligand migration through the C-terminal channel.

A: Definition of 2D projection to create a ligand occupancy map. The plane defined by the cross section through the centre of the C-terminal channel was used to map the 3D coordinates of the centre of mass (COM) of all the ligands into the X and Y projections. The plane is in blue and the protein slice in this plane is in red. The ligand occupancy map was calculated using a 75x75 grid matrix. For each frame of each simulation, a 2D coordinates of the ligand COM is counted and located into one of the grid cell. B: The 2D ligand occupancy map from the all 30-ns sMD trajectories for each ligand. The entrance of the C-terminal channel and the active site are labelled. The transient stable states corresponding to interactions with Glu850 and Lys907 (I); Lys849 and Asp830 (II); Arg969 and water reservoir (III) and Arg489 and Lys965 (IV). Red indicates high occupancy and blue low occupancy. Colouring is linear and values in the bar in counts. C: The ligand-residue binding network (LRBN) derived from the ligand-residue interaction energy of the all sMD trajectories. Nodes are residues interacting with the ligand during the migration. The size and colour of the nodes correspond to the strength of interaction. The 25% and 50% strongest interactions and the remaining frequently occurring interactions are in red, yellow and grey, respectively. Edges reflect the period of occurrence of a ligand-residue interaction; thicker edges show prolong interactions. The average ligand path is shown as a thick light-to-dark-blue-changing colour line. The active site, positively-charged and negatively-charged residues are circled in black, blue and red, respectively.

Fig 4. The C-terminal channel opening is made up of positively and negatively charged residues.

A: The overall structure of PfM1-AAP with the visualized charged residues of the channel entrance that coordinate the initial migration of the substrates and inhibitors. B: The zoomed view of the channel entrance. The flexibility of the Lys907 side chain is shown in the circle; conformations captured in sMD are in cyan and conformations observed in the crystal structures are in purple. C: Transient stable binding of Bestatin with Glu850 and Lys907. D: Transient stable binding of Bestatin with Lys849 and Asp830. The salt bridges are shown in a black-dotted line.

Fig 5. The regulatory Arg969.

A: The overall structure of PfM1-AAP with the visualized Asp830, Lys849 and Arg969. B: Lys849 passes the Met-Phe substrate to Arg969. C: The migration of the Met-Phe substrate to the deeper cavity of the C-terminal channel with the help of Arg969. D: The various conformations of Arg969 from the PfM1-AAP crystal structures.

Fig 6. Ligand migration in the water reservoir.

A: Root-mean-square fluctuation (RMSF) of the ligand (the Met-Phe substrate as an example) along the sMD trajectories. B: The various orientation of the Met-Phe substrate in the water reservoir. The part of the C-terminal channel involving the water reservoir is shown in the surface-like representation.

Fig 7. Enzymatic analysis of PfM1-AAP and the variant PfM1-AAP Arg969Ala.

A. Peptides EEKSAVTA and LSFPTTK (50 μM) were incubated with functionally-active recombinant PfM1-AAP for 10 mins before the addition of the fluorogenic aminopeptidase substrate L-Arg-NHMec (5 μM). The release of the fluorescent moiety (-NHMec) was monitored over time in a fluorimetre (excitation 370nm, emission 460 nm). Relative fluorescent units within the linear phase of the reactions were calculated and no inhibitor controls taken as 100%. LSFPTTK inhibits the cleavage of the fluorogenic substrate, while the E-peptide does not. B. Concentration dependent competitive inhibition of PfM1-AAP and the variant PfM1-AAP Arg969Ala is observed the peptide LSFPTTK but this is less effective for the variant enzyme. C. Competitive binding assays performed by adding the fluorogenic substrate L-Arg-NHMec at various time-points (5, 10, 30 and 60 mins) after the addition of the peptide LSFPTTK (50 μM) to PfM1-AAP reveals a constant level of inhibition suggesting that the L-peptide can enter the C-terminal channel but is not cleaved by the enzyme.

Fig 8. Migration of the ligand from the water reservoir to the active site of PfM1-AAP.

A: Met-Phe approaches the positively charged Lys965-Glu962-Arg489 cluster to form a salt bridge with Arg489 that brings Met-Phe to the active site. Rotation of Arg489 to the active site is shown with two transparent Arg489 side chains. B: The final position of Met-Phe in the active site. The salt bridges and hydrogen bonds are shown in a black-dotted line.

Fig 9. Ligand migration through the N-terminal channel.

A: The 2D ligand occupancy map from the all 30-ns sMD trajectories of the Arg product migration plotted on the cross section through the channel centre (See in the Supporting Information, S1 Fig). The exit from the N-terminal channel and the active site are labelled. Red means high occupancy and blue low occupancy. Colouring is linear. B: The ligand-residue binding network (LRBN) derived from the ligand-residue interaction energy of the all sMD trajectories of the Arg product migration. Nodes are residues interacting with the ligand during the migration. The size and colour of the nodes correspond to the strength of interaction. The 25%, 50% strongest interactions and the remaining frequently occurring interactions are in red, yellow and grey, respectively. Edges reflect the interval of occurrence of a ligand-residue interaction; thicker edges show prolonged interactions. The average ligand path is shown as a thick light-to-dark-blue-changing colour line. The active site, positively-charged and negatively-charged residues are circled in black, blue and red, respectively.

Fig 10. The non-bonded interaction energy between the carboxyl group of the Arg product and the glutamic acid bundle, involving Glu319, Glu463, Glu497 and Glu519 along the representative sMD trajectory.

Fig 11. The opening of the N-terminal channel, involving loops D1 and P1.

A: the overall structure of PfM1-AAP with highlighted loops; B: the Arg product bound to the active site; C: The interaction of Arg with Arg325 and Glu572 during the release from the active site. The salt bridges and hydrogen bonds are shown in a black-dotted line. D: The distance between the centre of mass of P1 and D1 loops in the1μs cMD simulations of the PfM1-AAP the ligand-unbound (red) and ligand-bound forms (black).