Crystal Structure of a Hyperactive Escherichia coli Glycerol Kinase Mutant Gly230 Asp Obtained Using Microfluidic Crystalliza
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- 作者：Megan J. Anderson ; Byron DeLaBarre ; Anu Raghunathan ; Bernhard O. Palsson ; Axel T. Brunger ; Stephen R. Quake
- 刊名：Biochemistry
- 出版年：2007
- 出版时间：May 15, 2007
- 年：2007
- 卷：46
- 期：19
- 页码：5722 - 5731
- 全文大小：400K
- 年卷期：v.46,no.19(May 15, 2007)
- ISSN：1520-4995
文摘
The crystal structure of an Escherichia coli glycerol kinase mutant Gly230 
tities/rarr.gif"> Asp (GKG230D)was determined to 2.0 Å resolution using a microfluidics based crystallization platform. The crystallizationstrategy 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 decreasedallosteric regulation by the glycolytic pathway intermediate fructose 1,6-bisphosphate (FBP) compared towild-type GK (GKWT). Structural analysis revealed that the decreased allosteric regulation is a result ofthe altered FBP binding loop conformations in GKG230D that interfere with the wild-type FBP bindingsite. The altered FBP binding loop conformations in GKG230D are supported through a series ofintramolecular loop interactions. The appearance of Asp230 in the FBP binding loops also repositions thewild-type FBP binding residues away from the FBP binding site. Light scattering analysis confirmedGKG230D is a dimer and is resistant to tetramer formation in the presence of FBP, whereas GKWT dimersare converted into putatively inactive tetramers in the presence of FBP. GKG230D also provides the firststructural evidence for multiple GK monomer conformations in the presence of glycerol and in the absenceof a nucleotide substrate and verifies that glycerol binding is not responsible for locking GK into theclosed conformation necessary for GK activity.
tities/rarr.gif"> Asp (GKG230D)was determined to 2.0 Å resolution using a microfluidics based crystallization platform. The crystallizationstrategy 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 decreasedallosteric regulation by the glycolytic pathway intermediate fructose 1,6-bisphosphate (FBP) compared towild-type GK (GKWT). Structural analysis revealed that the decreased allosteric regulation is a result ofthe altered FBP binding loop conformations in GKG230D that interfere with the wild-type FBP bindingsite. The altered FBP binding loop conformations in GKG230D are supported through a series ofintramolecular loop interactions. The appearance of Asp230 in the FBP binding loops also repositions thewild-type FBP binding residues away from the FBP binding site. Light scattering analysis confirmedGKG230D is a dimer and is resistant to tetramer formation in the presence of FBP, whereas GKWT dimersare converted into putatively inactive tetramers in the presence of FBP. GKG230D also provides the firststructural evidence for multiple GK monomer conformations in the presence of glycerol and in the absenceof a nucleotide substrate and verifies that glycerol binding is not responsible for locking GK into theclosed conformation necessary for GK activity.