Preparation and crystal structure of the recombinant alpha(1)/alpha(2) catalytic heterodimer of bovine brain platelet-activating factor acetylhydrolase Ib.
The intracellular form of mammalian platelet activating factor acetylhydrolase found in brain (PAF-AH Ib) is thought to play a critical role in control in neuronal migration during cortex development. This oligomeric complex consists of a homodimer of the 45 kDa (beta) LIS1 protein, the product of the causative gene for type I lissencephaly, and, depending on the developmental stage and species, one of three possible pairs of two homologous approximately 26 kDa alpha-subunits, which harbor all of the catalytic activity. The exact composition of this complex depends on the expression patterns of the alpha(1) and alpha(2) genes, exhibiting tissue specificity and developmental control. All three possible dimers (alpha(1)/alpha(1), alpha(1)/alpha(2) and alpha(2)/alpha(2)) were identified in tissues. The alpha(1)/alpha(2) heterodimer is thought to play an important role in fetal brain. The structure of the alpha(1)/alpha(1) homodimer was solved earlier in our laboratory at 1.7 A. We report here the preparation of recombinant alpha(1)/alpha(2) heterodimers using a specially constructed bi-cistronic expression vector. The approach may be useful in studies of other systems where pure heterodimers of recombinant proteins are required. The alpha(1)/alpha(2) dimer has been crystallized and its structure was solved at 2.1 A resolution by molecular replacement. These results set the stage for a detailed characterization of the PAF-AH Ib complex.
Sheffield
P J
PJ
Department of Molecular Physiology and Biological Physics, University of Virginia Health Sciences Center, Charlottesville, VA 22906-0011, USA.
McMullen
T W
TW
Li
J
J
Ho
Y S
YS
Garrard
S M
SM
Derewenda
U
U
Derewenda
Z S
ZS
D043203:1-Alkyl-2-acetylglycerophosphocholine Esterase; D000818:Animals; D001921:Brain / Q000201:enzymology*; D002384:Catalysis; D020134:Catalytic Domain; D002417:Cattle; D003001:Cloning, Molecular; D003460:Crystallization; D019281:Dimerization; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D005796:Genes; D005822:Genetic Vectors; D006358:Hot Temperature; D008869:Microtubule-Associated Proteins / Q000737:chemistry; D010741:Phospholipases A / Q000737:chemistry*; D010957:Plasmids; D021122:Protein Subunits; D011994:Recombinant Proteins / Q000138:chemical synthesis* / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification; D014018:Tissue Distribution; D014961:X-Ray Diffraction
D008869:Microtubule-Associated Proteins; D021122:Protein Subunits; D011994:Recombinant Proteins; D010741:Phospholipases A; D043203:1-Alkyl-2-acetylglycerophosphocholine Esterase