Structure, function, and folding of phosphoglycerate kinase are strongly perturbed by macromolecular crowding

Abstract

We combine experiment and computer simulation to show how macromolecular crowding dramatically affects the structure, function, and folding landscape of phosphoglycerate kinase (PGK). Fluorescence labeling shows that compact states of yeast PGK are populated as the amount of crowding agents (Ficoll 70) increases. Coarse-grained molecular simulations reveal three compact ensembles: C (crystal structure), CC (collapsed crystal), and Sph (spherical compact). With an adjustment for viscosity, crowded wild-type PGK and fluorescent PGK are about 15 times or more active in 200 mg/ml Ficoll than in aqueous solution. Our results suggest a previously undescribed solution to the classic problem of how the ADP and diphosphoglycerate binding sites of PGK come together to make ATP: Rather than undergoing a hinge motion, the ADP and substrate sites are already located in proximity under crowded conditions that mimic the in vivo conditions under which the enzyme actually operates. We also examine T-jump unfolding of PGK as a function of crowding experimentally. We uncover a nonmonotonic folding relaxation time vs. Ficoll concentration. Theory and modeling explain why an optimum concentration exists for fastest folding. Below the optimum, folding slows down because the unfolded state is stabilized relative to the transition state. Above the optimum, folding slows down because of increased viscosity.

Fig. 1.

Programmable T-jump setup. A computer-controlled diode laser delivers a shaped heating pulse that induces a sustained step function in temperature for the duration of the measurement. A blue LED excites the donor label (green barrel on the PGK model). A microscope objective images donor and acceptor (red barrel) fluorescence from the very center of the heated area onto a single CCD camera, which collects successive snapshots of green and red fluorescence.

Fig. 2.

Conformational, thermal stability and kinetics trends of the PGK construct as a function of Ficoll concentration. (A) Donor–acceptor ratio at 22.3 ± 0.5 °C (filled circles). Open triangles show the intrinsic fluorophore response of AcGFP1 and mCherry as a function of Ficoll concentration. (B) “Inverse chevron” plot of the observed T-jump relaxation time. Red squares show the kinetic trend obtained from simulations in ref.  at volume fraction . was computed using the size of the crowding agents equal to the native state of the protein. The simulations were scaled to match up the fastest folding rate observed in our experiments. (C) Melting temperature. (D) Cooperativity parameter Δg₁ (free energy derivative) from Eq. 1. All error bars are ± 1 standard deviation.

Fig. 3.

Two-dimensional folding free energy landscape of PGK in the bulk (A–C) and under the crowded conditions at ϕ_c = 25% (D–F) and ϕ_c = 40% (G–I) as a function of the radius of gyration R_g and the overlap function χ at different temperatures (in units of k_BT/ε). The color is scaled by k_BT where k_B is the Boltzmann constant and ε is 0.6 kcal/mol. The crystal structure is labeled C, the collapsed crystal structure CC, the spherical state Sph, and the unfolded state U.

Fig. 4.

Structural characteristics of the dominant compact ensemble structures of PGK in cartoon representation. (A) Crystal state C, (B) collapsed crystal state CC, and (C) spherical state Sph. The coloring of each protein model ranges from N terminus (red) to C terminus (blue). The N and C termini are represented with van der Waals spheres. The schematic representation at the bottom left of each panel is to address a simplistic view of the structural arrangement of the N- and C-lobes in each conformation. (D) The probability distribution of the distance between N and C termini (D_N-C) of the three dominant structures of PGK under the condition in which each prevails in the simulations. C state (solid black), CC (dashed red), and Sph (dotted blue). Atomistic structures were reconstructed from coarse-grained protein models using SCAAL.

Fig. 5.

The probability distribution of the distance between the centers of mass of the Mg-ATP and 3PG binding sites (D_b) calculated for the three dominant structures of PGK under the condition in which each prevails in the simulations. (D_b) of C state (solid black), CC (dotted red), and Sph (dotted blue) ensembles are plotted. The purple solid line represents the convolution of the two profiles from CC and Sph states. The three characteristic structures are illustrated in cartoon representation. The coloring of each protein model ranges from N terminus (red) to C terminus (blue). The Mg-ATP and 3PG binding sites are colored in yellow and green representations of van der Waals spheres, respectively. Atomistic structures were reconstructed from coarse-grained protein models using SCAAL.