Crystallization and preliminary X-ray diffraction analysis of the catalytic domain of recombinant human phosphodiesterase 3B.
The catalytic domain of human phosphodiesterase 3B has been cloned, expressed in Escherichia coli and purified in the presence of the PDE3 inhibitors IBMX (3-isobutylmethylxanthine) or MERCK1 by affinity chromatography. Initial screening of crystallization conditions for these complexes in the hanging-drop vapor-diffusion mode resulted in three different crystal forms, all characterized by quite large unit-cell parameters, elevated solvent content and poor diffraction quality. Subsequent optimization of these conditions led to crystals that diffract to 2.4 A and belong to space group C2, with unit-cell parameters a = 146.7, b = 121.5, c = 126.3 A, beta = 100.6 degrees. Rotation-function analysis indicates that the asymmetric unit contains four copies of the monomeric enzyme, corresponding to a solvent content of 64%. To solve the structure of the PDE3B catalytic domain, molecular replacement as well as multiple isomorphous replacement methods are currently being utilized.
Patel
Sangita B
SB
Department of Medicinal Chemistry, Merck and Co., Rahway, NJ 07065, USA.
Varnerin
Jeffrey P
JP
Tota
Michael R
MR
Edmondson
Scott D
SD
Parmee
Emma R
ER
Becker
Joseph W
JW
Scapin
Giovanna
G
D015056:1-Methyl-3-isobutylxanthine / Q000737:chemistry; D015105:3',5'-Cyclic-AMP Phosphodiesterases / Q000737:chemistry*; D020134:Catalytic Domain; D002846:Chromatography, Affinity; D003460:Crystallization; D018360:Crystallography, X-Ray; D054684:Cyclic Nucleotide Phosphodiesterases, Type 3; D006801:Humans; D010726:Phosphodiesterase Inhibitors / Q000737:chemistry; D011994:Recombinant Proteins / Q000737:chemistry
D010726:Phosphodiesterase Inhibitors; D011994:Recombinant Proteins; D015105:3',5'-Cyclic-AMP Phosphodiesterases; D054684:Cyclic Nucleotide Phosphodiesterases, Type 3; C517291:PDE3B protein, human; D015056:1-Methyl-3-isobutylxanthine