Crystal structures of Escherichia coli glucokinase and insights into phosphate binding

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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.
Funding Information
  • National Institutes of Health, National Institute of General Medical Sciences (P20GM139768, R15GM140433)
  • Arkansas Biosciences Institute

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