Glycerol kinase from Escherichia coli and an Ala65→Thr mutant:: the crystal structures reveal conformational changes with implications for allosteric regulation

Background: Glycerol kinase (GK) from Escherichia coli is a velocity-modulated (V system) enzyme that has three allosteric effecters with independent mechanisms: fructose-1,6-bisphosphate (FBP); the phosphocarrier protein IIA(Glc); and adenosine nucleotides. The enzyme exists in solution as functional dimers that associate reversibly to form tetramers, GK is a member of a superfamily of ATPases that share a common ATPase domain and are thought to undergo a large conformational change as an intrinsic step in their catalytic cycle, Members of this family include actin, hexokinase and the heat shock protein hsc70. Results: We report here the crystal structures of GK and a mutant of GK (Ala65-->Thr) in complex with glycerol and ADP, Crystals of both enzymes contain the same 222 symmetric tetramer. The functional dimer is identical to that described previously for the IIA(Glc)-GK complex structure, The tetramer interface is significantly different, however, with a relative 22.3 degrees rotation and 6.34 Angstrom translation of one functional dimer. The overall monomer structure is unchanged except for two regions: the IIA(Glc)-binding site undergoes a structural rearrangement and residues 230-236 become ordered and bind orthophosphate at the tetramer interface. We also report the structure of a second mutant of GK (Ile474-->Asp) in complex with IIA(Glc); this complex crystallized isomorphously to the wild type IIA(Glc)-GK complex. Site-directed mutants of GK with substitutions at the IIA(Glc)-binding site show significantly altered kinetic and regulatory properties, suggesting that the conformation of the binding site is linked to the regulation of activity. Conclusions: We conclude that the new tetramer structure presented here is an inactive form of the physiologically relevant tetramer. The structure and location of the orthophosphate-binding site is consistent with it being part of the FBP-binding site, Mutational analysis and the structure of the IIA(Glc)-GK(Ile474-->Asp) complex suggest the conformational transition of the IIA(Glc)-binding site to be an essential aspect of IIA(Glc) regulation.