Metastable States in the Hinge-Bending Landscape of an Enzyme in an Atomistic Cytoplasm Simulation

Abstract

Many enzymes undergo major conformational changes to function in cells, particularly when they bind to more than one substrate. We quantify the large-amplitude hinge-bending landscape of human phosphoglycerate kinase (PGK) in a human cytoplasm. Approximately 70 μs of all-atom simulations, upon coarse graining, reveal three metastable states of PGK with different hinge angle distributions and additional substates. The "open" state was more populated than the "semi-open" or "closed" states. In addition to free energies and barriers within the landscape, we characterized the average transition state passage time of ≈0.3 μs and reversible substrate and product binding. Human PGK in a dilute solution simulation shows a transition directly from the open to closed states, in agreement with previous SAXS experiments, suggesting that the cell-like model environment promotes stability of the human PGK semi-open state. Yeast PGK also sampled three metastable states within the cytoplasm model, with the closed state favored in our simulation.

Figure 1.

Hinge-bending dynamics of hPGK in human cell cytoplasm models. (A) hPGK placed in the cytoplasm model, CellB. Protein clustering can be seen, an effect known to increase diffusivity in self-organized cytoplasm, although it may be exacerbated by the force field. (B) Hinge-bending angle trajectories θ (t) in (B) CellA and (C) CellB cluster into Open (O), Semi-Open (SO), and Closed (C) states. The 3-state STaSI model does not capture additional transiently populated substates such as at t=0 or 14 μs (see SI for fits with more states). Logistic functions (green) are applied to describe state-to-state transitions. Transition mid-points are labeled t_m1 to t_m8 as follows: t_m1=0.20 μs, t_m2=14.81 μs, t_m3=21.63 μs, t_m4=25.14 μs, t_m5=28.76 μs, t_m6=0.64 μs, t_m7=7.97 μs, and t_m8=31.91 μs. hPGK’s domains are shown as ribbons: N-terminal (blue), C-terminal (red), and other macromolecules (silver). hPGK ligands in the (A) model CellB are shown as van der Waals surfaces: 3PG (cyan), ADP (brown), and ATP (yellow). Other biomolecules are shown (silver): metabolites (licorices) and ions (spheres). Water shown as surface representation enclosed by the bounding box.

Figure 2.

Free energy landscape for hPGK hinge-bending dynamics within a human cytoplasm model. (A) Free energy diagram with quantitative free energies and transition state passage times indicated. The free energies and passage times are calculated from the STsSI-derived dwell and passage rates in SI Table S1. Errors are propagated standard errors of the mean. Values in parentheses are corrected for the low CHARMM TIP3P water model viscosity. With only a single complete dwell time for the C state observed, no error can be given for that state at ≈4.5 kJ/mol. The snapshot of hPGK (inset) is taken from CellA around the t_m4 midpoint that we predict could be a sub-state region (not quantified). (B) Histogram of the hinge-bending angle distribution ~P(θ) across all trajectories. (C) Sampled transition passage times between three major hinge-bending states.

Figure 3.

Ligand binding to hPGK in the cell cytoplasm models: (A)-(B) CellA, and (C) CellB. The hinge-bending angle data points are highlighted in corresponding colors shown in the legends when substrates bind to their respective active sites. Inset figures show binding of ligands (van der Waals surfaces are brown for ADP and cyan for 3PG) to active sites (silver van der Waals spherical surfaces) on the N- and C-terminal domains of hPGK (gray cartoons) at time 0 μs in (A), 0.28 μs in (B), and 16.72 μs in (C).

Figure 4.

Hinge-bending dynamics of hPGK in dilute solution. (A) hinge-bending angle trajectory and (B) hinge-bending angle distribution. t_m=0.57 μs was obtained from logistic function fitting to trajectory in A.

Figure 5.

Hinge-bending dynamics of yPGK in a human cytoplasm model. The angle trajectory clusters into three major ensembles, the open (O), Semi-Open (SO), and Closed (C) states of yPGK. A logistic function is applied to describe transition between ensembles. Transition midpoints are as follows: t_mi=−0.04 μs, t_mii=1.54 μs, and t_miii=33.11 μs. yPGK’s domains are shown as ribbons: N-terminal (blue), C-terminal and hinge region (silver).