title: Crystal structure of a hyperactive Escherichia coli glycerol kinase mutant Gly230 --> Asp obtained using microfluidic crystallization devices.
abstract: The crystal structure of an Escherichia coli glycerol kinase mutant Gly230 --> Asp (GKG230D) was determined to 2.0 A resolution using a microfluidics based crystallization platform. The crystallization strategy involved a suite of microfluidic devices that characterized the solubility trends of GKG230D, performed nanoliter volume free interface diffusion crystallization experiments, and produced diffraction-quality crystals for in situ data collection. GKG230D displays increased enzymatic activity and decreased allosteric regulation by the glycolytic pathway intermediate fructose 1,6-bisphosphate (FBP) compared to wild-type GK (GKWT). Structural analysis revealed that the decreased allosteric regulation is a result of the altered FBP binding loop conformations in GKG230D that interfere with the wild-type FBP binding site. The altered FBP binding loop conformations in GKG230D are supported through a series of intramolecular loop interactions. The appearance of Asp230 in the FBP binding loops also repositions the wild-type FBP binding residues away from the FBP binding site. Light scattering analysis confirmed GKG230D is a dimer and is resistant to tetramer formation in the presence of FBP, whereas GKWT dimers are converted into putatively inactive tetramers in the presence of FBP. GKG230D also provides the first structural evidence for multiple GK monomer conformations in the presence of glycerol and in the absence of a nucleotide substrate and verifies that glycerol binding is not responsible for locking GK into the closed conformation necessary for GK activity.
authors: Department of Biochemistry and Molecular Biophysics, California Institute of Technology, MS 128-95, Pasadena, California 91125, USA.
authors: Megan J
authors: MJ
authors: Anderson
authors: Byron
authors: B
authors: DeLabarre
authors: Anu
authors: A
authors: Raghunathan
authors: Bernhard O
authors: BO
authors: Palsson
authors: Axel T
authors: AT
authors: Brunger
authors: Stephen R
authors: SR
authors: Quake
mesh_terms: D019943:Amino Acid Substitution; D001665:Binding Sites; D002850:Chromatography, Gel; D003460:Crystallization; D018360:Crystallography, X-Ray; D004926:Escherichia coli / Q000201:enzymology*; D005635:Fructosediphosphates / Q000378:metabolism; D005991:Glycerol Kinase / Q000737:chemistry* / Q000235:genetics*; D007834:Lasers; D046210:Microfluidic Analytical Techniques / Q000379:methods; D008958:Models, Molecular; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D012542:Scattering, Radiation; D012995:Solubility
chemical_list: D005635:Fructosediphosphates; D005991:Glycerol Kinase; C029063:fructose-1,6-diphosphate