Allosteric mechanism of pyruvate kinase from Leishmania mexicana uses a rock and lock model

Abstract

Allosteric regulation provides a rate management system for enzymes involved in many cellular processes. Ligand-controlled regulation is easily recognizable, but the underlying molecular mechanisms have remained elusive. We have obtained the first complete series of allosteric structures, in all possible ligated states, for the tetrameric enzyme, pyruvate kinase, from Leishmania mexicana. The transition between inactive T-state and active R-state is accompanied by a simple symmetrical 6 degrees rigid body rocking motion of the A- and C-domain cores in each of the four subunits. However, formation of the R-state in this way is only part of the mechanism; eight essential salt bridge locks that form across the C-C interface provide tetramer rigidity with a coupled 7-fold increase in rate. The results presented here illustrate how conformational changes coupled with effector binding correlate with loss of flexibility and increase in thermal stability providing a general mechanism for allosteric control.

FIGURE 1.

Structure of the LmPYK·ATP·OX·Fru-2,6-BP complex showing domain boundaries and tetramer architecture. a, LmPYK·ATP·OX·Fru-2,6-BP tetramer highlighting the active, effector, and potassium (K⁺) sites. Each monomer has been colored to aid the identification of subunit interfaces. The large (A-A) and small (C-C) interfaces between monomers are shown as dashed lines. b, LmPYK·ATP·OX·Fru-2,6-BP tetramer in which one subunit has been colored to show domains; N (green, residues 1–17), A (yellow, residues 18–88 and 187–356), B (blue, residues 89–186), and C (red, residues 358–498).

FIGURE 2.

LmPYK crystal structures reveal the allosteric mechanism. a, schematic representation of the inactive T-state LmPYK structure. The relative positions of the AC core, pivot point (gray circle), active site, effector site, Arg³¹⁰ (R310), Aα6′, and Aα7 helices are shown for the LmPYK tetramer. b, binding of ATP and oxalate (red lozenges) to the apoenzyme, causing Arg³¹⁰ to move into the R-state position, forming hydrogen bonds (shown as dashed red lines) with the active site Aα6′ helix (for details, see supplemental Fig. S7) of the adjacent chain and a stabilizing bridge at the A-A interface, stabilizing the AC core in an R-conformation. The LmPYK·ATP·OX tetramer consists of two different conformers; conformer 1 has a partially closed B-domain (yellow), and conformer 2 has a fully closed B-domain (red). c, active R-state LmPYK·ATP·OX·Fru-2,6-BP structure. All monomers have rotated AC cores, fully closed B domains, Fru-2,6-BP-induced salt bridges across the C-C interface, and stabilizing Arg³¹⁰-Aα6′ hydrogen bonds across the A-A interface. d, binding of Fru-2,6-BP (green rectangles) to the apoenzyme, resulting in the formation of four pairs of stabilizing salt bridges (shown as red lines) formed across each C-C interface, locking the AC core in an R-conformation. The LmPYK·Fru-2,6-BP structure has fully open B-domains. e, superposition of the inactive T-state AC core structure onto the R-state AC core structure (all B-domains have been removed). The 6° rigid body rotations of the AC cores occurring on the T- to R-state transition have been highlighted using arrows to show the direction of movement.

FIGURE 3.

Stabilization of the LmPYK tetramer by binding of Fru-2,6-BP. a, enlargement of the C-C interface showing four salt bridges formed as a result of Fru-2,6-BP binding. Residues along the stabilized effector loops (shown in green) are pushed toward adjacent chains forming two pairs of conserved salt bridge interactions between Lys⁴⁸⁴-Glu⁴⁹⁸ and Asp⁴⁸²-Arg⁴⁹³ at each of the C-C interfaces. Conserved salt bridges are shown as dashed lines. b, thermal shift assay results for the four LmPYK complexes. A two-state unfolding process was observed for LmPYK complexes in the absence of Fru-2,6-BP (yellow and magenta traces). The four stages of the unfolding process are shown: 1, LmPYK below 30 °C is stable; 2, LmPYK in complex with active site ligands begins to dissociate into monomeric or dimeric species (T_m = ∼40 °C); 3, LmPYK in complex with active site ligands only, fully denatured; 4, LmPYK in complex with Fru-2,6-BP in the absence (blue) or presence (green) of active site ligands unfolds as a single, highly stable species, presumably due to additional salt bridges formed at the C-C interface. The presence of active site ligands conferred only a very modest increase in stability compared with the stabilization by Fru-2,6-BP. c, schematic representations of LmPYK crystal structures and their experimentally determined melting temperatures (T_m).