Allosteric regulation of glycogen synthase kinase 3β: a theoretical study

Abstract

Glycogen synthase kinase 3β (GSK-3β) is a serine-threonine kinase belonging to the CMGC family that plays a key role in many biological processes, such as glucose metabolism, cell cycle regulation, and proliferation. Like most protein kinases, GSK-3β is regulated via multiple pathways and sites. We performed all-atom molecular dynamics simulations on the unphosphorylated and phosphorylated unbound GSK-3β and the phosphorylated GSK-3β bound to a peptide substrate, its product, and a derived inhibitor. We found that GSK-3β autophosphorylation at residue Tyr(216) results in widening of the catalytic groove, thereby facilitating substrate access. In addition, we studied the interactions of the phosphorylated GSK-3β with a substrate and peptide inhibitor located at the active site and observed higher affinity of the inhibitor to the kinase. Furthermore, we detected a potential remote binding site which was previously identified in other kinases. In agreement with experiments we observed that binding of specific peptides at this remote site leads to stabilization of the activation loop located in the active site. We speculate that this stabilization could enhance the catalytic activity of the kinase. We point to this remote site as being structurally conserved and suggest that the allosteric phenomenon observed here may occur in the protein kinase superfamily.

Figure 1

The structure of unbound GSK-3β·ATP in ribbon display. The small N-lobe (35−138) and large C-lobe (139−386) are marked. The three regions important for the catalytic activity are highlighted in different colors: the P-loop (64−69) in green, the activation loop (200−226) in purple, and the αC-helix (96−104) in blue. The ATP appears in CPK colors inside its cavity, governed by the P-loop gatekeeper. The residues that govern the activation of GSK-3β appear in beads. These are Arg⁹⁶, Arg¹⁸⁰, and Lys²⁰⁵ which comprise the basic binding groove, Asp²⁰⁰ and Asp¹⁸¹ from the highly conserved motifs DFG (200−202) and HRD (179−181) at the catalytic site, and Glu⁹⁷, Lys⁸⁵, and phosphotyrosine Tyr²¹⁶(p) that are responsible for rendering the enzyme active.

Figure 2

Modeling and simulation flow. GSK-3β(p) denotes the phosphorylated kinase at Tyr²¹⁶. Several X-ray structures are used to model GSK-3β depending on whether it is phosphorylated at Tyr²¹⁶, and whether it is bound. These structures together with the conformational search, docking procedures, and MD simulation protocol are detailed in the Methods section.

Figure 3

The unbound structures of GSK-3β(p)·ATP and GSK-3β·ATP after 20 ns of MD simulations. (a) The final simulation snapshot of GSK-3β(p)·ATP (colored blue) superimposed on the crystal structure of PDB code 1PYX (colored red) from which the simulation originated. (b) The final simulation snapshot of GSK-3β·ATP (colored blue) superimposed on the crystal structure of PDB code 1PYX (colored red) from which the simulation originated. (a) and (b) share the same perspective. The orientation of Tyr²¹⁶ is highlighted. (c) The main structural determinants of the catalytic site of the GSK-3β(p)·ATP system shown in surface representation and colored as follows: the P-loop (residues 64−69) is in green, the activation loop (residues 200−226) is in purple, and the αC-helix (residues 96−104) is in blue. These determinants are in an “open” conformation, creating a wide groove. (d) The main structural determinants of the catalytic site of the GSK-3β·ATP system shown in surface representation and colored as in panel c. The determinants are in a “close” conformation in which the catalytic groove is nearly blocked. (e) Atomic description of the GSK-3β(p)·ATP active site key residues. The phosphotyrosine Tyr²¹⁶(p) is electrostatically bound to Arg²²⁰ and Arg²²³. (f) Atomic description of the GSK-3β·ATP active site key residues. The aromatic ring of Tyr²¹⁶ is headed outside of the cleft, allowing Arg²²⁰ to interact with the ATP γ-phosphate group and with the highly conserved Asp¹⁸¹.

Figure 4

Peptide−ligand binding at the substrate binding site (SBS). The snapshots of two ligands bound at the SBS were taken from the most populated cluster of conformations. GSK-3β(p) appears in surface representation, and the ligands appear in sticks. The key interacting residues of the ligands are highlighted. (a) HSF-1 regulatory domain (the substrate) bound at the SBS. The substrate Ser⁶ is the residue due to be phosphorylated by the kinase. (b) The inhibitor L803 bound at the SBS. Alanine replaces Ser⁶ of the substrate. The different surfaces in panels a and b reflect bound and unbound regions. (c) The Cα RMSD of both substrate and inhibitor measured along the 40 ns of simulations.

Figure 5

Ligand binding at the allosteric binding site (ABS). All complexes were first superimposed, to enable a uniform perspective. (a) GSK-3β(p) in a surface representation, with the substrate (in orange ribbon) bound at the ABS, in the groove between the N-lobe and C-lobe. The positively and negatively charged key interacting residues appear as blue and red spots, respectively. The inset magnifies the interacting residues of the ABS and the substrate and displays the main secondary structures at the N-lobe. The key interacting residues Lys⁹⁴, Arg¹⁰², and Lys¹⁰³ appear from both sides of the αC-helix. (b) Magnification of the interaction between the phosphorylated substrate (the product) and the ABS. The same key interacting residues Lys⁹⁴, Arg¹⁰², and Lys¹⁰³ bind to two negative centers at the p-substrate: S⁶(p) and S¹⁰(p). (c) The phosphorylated substrate (product), as it appears in panel b, being projected on the structural alignment of CDK2 and GSK-3β(p). CDK2 is in surface representation (colored gray), and its residues which are located within 4 Å of the activator protein cyclinA (PDB code 1FIN) are highlighted in pink(33). This perspective exhibits the high overlap between the interface product, GSK-3β(p), and the interface cyclinA, CDK2. The structure of GSK-3β(p) is not shown, for clarity.

Figure 6

The Cα root-mean-square fluctuations (RMSF) of three catalytically important regions measured in all bound complexes at the allosteric binding site. The results were compared to the fluctuations at apo GSK-3β(p). (a) The activation loop. (b) The αC-helix. (c) The P-loop.

Figure 7

Structural alignment between bound PKA (in orange) and GSK-3β(p) (in silver) with highlighted residues of GSK-3β(p) that contribute to the kinase activation state (27). (a) The spine residues His¹⁷⁹, Phe²⁰¹, Met¹⁰¹, and Leu¹¹². (b) The salt bridge between Lys⁸⁵ and Glu⁹⁷ (on top), marked in black, and the hydrogen bond between Asp²⁰⁰ and the backbone nitrogen of Gly²⁰², marked in green. For clarity, both structures are displayed without hydrogens.

Figure 8

Structural analyses of functional residues in the active site along 20 ns of simulations. The trajectories of apo GSK-3β(p) and the bound complexes were compared to the bound GSK-3β(p) crystal structure (PDB code 1H8F). In this bound structure, the spine hydrophobic residues are aligned; there is a salt bridge between Lys⁸⁵ and Glu⁹⁷ and a hydrogen bond between Asp²⁰⁰ and Gly²⁰². These indicate an active kinase conformation. (a) All-atom RMSD of the four hydrophobic spine residues Met¹⁰¹, Leu¹¹², His¹⁷⁹, and Phe²⁰¹. A high RMSD indicates an inactive kinase conformation. (b) The distance Lys⁸⁵−Glu⁹⁷. A salt bridge is formed at a distance in range (4 Å, 5 Å). Both substrate bound at the SBS and phosphorylated substrate bound at the ABS exhibit a salt bridge throughout the simulation. (c) The distance Asp²⁰⁰−Gly²⁰². A hydrogen bond cutoff is typically less than 3.2 Å. This distance is seen when the substrate was bound at the SBS and at the ABS.