Crystal structures of Escherichia coli glucokinase and insights into phosphate binding

Abstract

Here, we report the crystal structure of Escherichia coli glucokinase (GLK), which has phosphate bound in the cleft between the α and β domains adjacent to the active site. A ternary complex consisting of GLK, glucose and phosphate is also reported in this work. Diffraction data were collected at 2.63 Å resolution for the phospate-bound form (R_work/R_free = 0.191/0.230) and at 2.54 Å resolution for the ternary complex (R_work/R_free = 0.202/0.258), both at 297 K. A B-factor analysis of the phosphate-bound GLK structure revealed consistently lower values for phosphate-interacting basic residues in the α4, α5 and α9 helices, while significant root-mean-square deviation (r.m.s.d.) spikes indicated flexibility in regions preceding β1 and within the loop between the β5 and β6 sheets of the α domain. In the ternary complex, phosphate is bound adjacent to glucose, and the B factors for the α4, α5 and α9 helices were further reduced, while r.m.s.d. spikes were observed at the end of the β10 sheet and within the α6 helix of the β-domain. This structural characterization suggests that phosphate could influence the activity of GLK by altering glucose binding and modulating interactions with a loop-interacting regulatory protein.

Keywords: glucokinases; hexokinases; phosphate binding; sulfate binding.

Figure 1

(a) Secondary-structure mapping of the ecGLK monomer. The model uses a blue color for α-helices and a red color for β-sheets. β6 is hidden behind because the model is placed in the same direction as in (b) for easy identification. (b) Comparison of the structures of phosphate-bound ecGLK (PDB entry 9duc, cyan) and apo ecGLK (PDB entry 1q18, tan). Sections with the highest observed differences from the B-factor graphing and the r.m.s.d. graphing are highlighted in red.

Figure 2

The structure of the phosphate-binding site. (a) Amino-acid residues in the phosphate-binding site. (b) Superimposition of the residues in apo-form ecGLK (PDB entry 1q18, tan) and phosphate-bound ecGLK (PDB entry 9duc, cyan) at the phosphate-binding site.

Figure 3

Comparison of the structures of the phosphate-bound glucose–ecGLK complex (PDB entry 9dvz, cyan) and the glucose–GLK complex (PDB entry 1q18, tan). Sections with the highest observed differences from the B-factor graphing and the r.m.s.d. graphing are highlighted in red.

Figure 4

The B factors of the structures of the glucose–ecGLK complex (PDB entry 1sz2) and the phosphate-bound glucose–ecGLK complex (PDB entry 9dvz).

Figure 5

The structure of the phosphate-binding site. (a) Amino-acid residues in the phosphate-binding site. (b) Superimposition of the residues in the glucose–ecGLK complex (PDB entry 1sz2, tan) and the phosphate-bound glucose–ecGLK complex (PDB entry 9dvz, cyan) at the phosphate-binding site.

Figure 6

The structure of the glucose- and phosphate-binding sites. (a) Amino-acid residues in the glucose- and phosphate-binding sites. (b) Superimposition of the residues in the glucose–ecGLK complex (PDB entry 1sz2, tan) and the phosphate-bound glucose–ecGLK complex (PDB entry 9dvz, cyan) at the glucose- and phosphate-binding sites.