Homology modeling and molecular dynamics study of GSK3/SHAGGY-like kinase

Abstract

Although the GSK3/SHAGGY-like kinase is a highly conserved serine/threonine kinase implicated in many signaling pathways in eukaryotes, the lack of knowledge of its three-dimensional (3D) structure has hindered efforts to understand the binding specificities of substrate and catalytic mechanism. To understand the structure–activity relationships, the protein 3D structure was built by using homology modeling based on the known X-ray diffraction structure of Glycogen synthase kinase-3β (Gsk3β) and the model structure was further refined using unrestrained molecular dynamics simulations. The research indicates that the general 3D organization of the GSK3/SHAGGY-like kinase is a typical kinase family and comprises an N-terminal domain of β-sheet and a larger C-terminal domain mainly constituted by α-helix. In order to understand the molecular interactions between the natural substrate-ATP and GSK3/SHAGGY-like kinase, a 3D model of the complex ATP–GSK3/SHAGGY-like kinase is developed by molecular docking program, which is helpful to guide the experimental realization and the new mutant designs as well. One important finding is that the identification of the key binding-site residue of Lys69 which plays an important role in the catalysis of GSK3/SHAGGY-like kinase and this is in consistent with experimental observation.

Introduction

The GSK3/SHAGGY-like kinase is a highly conserved serine/threonine kinase implicated in many signaling pathways in eukaryotes. The main mechanism of signal transduction in cells is reversible phosphorylation of proteins represents. The results given by Li and Nam (2002) indicated that this kinase plays an important role in a brassinosteroid (BR) signal transduction pathway. BRs are unique class of plant steroids that play important roles throughout the plant life cycle (Clouse and Sasse, 1998). And some molecular and biological experimental studies have suggested possible roles for GSK3/SHAGGY-like kinase in osmotic stress response (Piao et al., 1999), wound signaling (Jonak et al., 2000), and flower meristem patterning (Dornelas et al., 2000). The GSK3/SHAGGY-like kinase displays large sequence identity to those of the mammalian Glycogen synthase kinase-3β (Woodgett, 1990) and the Drosophila SHAGGY protein kinase (Siegfried et al., 1990). The site mutation in which Lys69 was replaced by Arg (K69R) to destroy the GSK3/SHAGGY kinase activity indicates that the Lys69 may play an important role in the catalysis of GSK3/SHAGGY-like kinase (Pierce and Kimelman, 1995).

Despite these experimental efforts, the detailed structures of GSK3/SHAGGY kinase and of the enzyme–substrate complex remain unknown. The function of protein is determined essentially by its corresponding three-dimensional (3D) structure (Lai, 1993). One of the major obstacles to further elucidating the molecular origin of the observed metabolic profiles of these enzymes is lack of the corresponding 3D structures. Obtaining and analyzing the protein structure may be an important task for modern molecular biology. In the present investigation, we constructed a 3D model of the title enzyme and searched for the binding site of substrate. The 3D features of the model were obtained by a homology modeling procedure based on the X-ray diffraction structure of Glycogen synthase kinase-3β (PDB code: 1I09) (Ter Haar et al., 2001) and it is known that the homology model is effective for the 3D structure construction of protein (Eisenhaber et al., 1995). The obtained result can be used to explain substrate specificity and relate enzyme function to its structure.

The complex of ATP–GSK3/SHAGGY-like kinase obtained by docking program can be used to identify the key residues involved in the substrate reaction mechanism. In order to design novel and higher affinity ligands, an understanding of the interaction between adenosine 5′-triphosphate (ATP) and GSK3/SHAGGY-like kinase at the molecular level would be invaluable.