Preparation and crystal structure of the recombinant ₁/₂ catalytic heterodimer of bovine brain platelet-activating factor acetylhydrolase Ib

Authors: Sheffield, Peter J.; McMullen, Todd W.P.; Li, Jia; Ho, Yew-Seng; Garrard, Sarah M.; Derewenda, Urszula

Source: Protein Engineering, Volume 14, Number 7, July 2001 , pp. 513-519(7)

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Abstract:

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 ~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

<INF>1</INF> and alpha

<INF>2</INF> genes, exhibiting tissue specificity and developmental control. All three possible dimers ( alpha

<INF>1</INF>/ alpha

<INF>1</INF>, alpha

<INF>1</INF>/ alpha

<INF>2</INF> and alpha

<INF>2</INF>/ alpha

<INF>2</INF>) were identified in tissues. The alpha

<INF>1</INF>/ alpha

<INF>2</INF> heterodimer is thought to play an important role in fetal brain. The structure of the alpha

<INF>1</INF>/ alpha

<INF>1</INF> homodimer was solved earlier in our laboratory at 1.7 Å. We report here the preparation of recombinant alpha

<INF>1</INF>/ alpha

<INF>2</INF> 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

<INF>1</INF>/ alpha

<INF>2</INF> dimer has been crystallized and its structure was solved at 2.1 Å resolution by molecular replacement. These results set the stage for a detailed characterization of the PAF-AH Ib complex.

Keywords: bi-cistronic; crystal structure; expression vector; heterodimer; hydrolase

Document Type: Research article

Affiliations: 1: Department of Molecular Physiology and Biological Physics, University of Virginia, Health Sciences Center, Charlottesville, VA 22906–0011, USA

Publication date: 2001-07-01