From Genome to Life:

ZHOU Cong Zhao

Lab of Structural Genomics, LEBS, CNRS-Gif, Bat 430, Orsay 91405, France

title: Crystal structure of yeast sorting nexin SNX3 an example from Yeast Structural Genomics Project in France

Sophie Cheruel1, Bruno Collinet1, Cong-Zhao Zhou1, Karine Blondeau2, Gilles Henkes2, Robert Aufrere2, Isabelle Sorel3, Ines Li de La Sierra Gallay3, Philippe Savarin3, Françoise de la Torre3, Anne Poupon 3, Joël Janin3 and Herman van Tilbeurgh

1. Institut de Biochimie et de Biophysique Moléculaire et Cellulaire (UMR 8619), Univ. Paris-Sud, Bât. 430, 91898 Orsay, France.

2. Institut de Génétique et Microbiologie (UMR 8621), Univ. Paris-Sud, Bât. 360, 91898 Orsay, France.

3. Laboratoire d’Enzymologie et Biochimie Structurale (UPR 9063), Bât. 34, 1 Av. de la Terrasse, 91198 Gif sur Yvette, France.

The Yeast Structural Genomics Project in France was launched at the middle of 2000 at Paris-Sud. It has three objectives. First is to establish a methodology for the systematic expression and purification of soluble yeast proteins and optimise these processes for large-scale work. Second is to clone and express about 250 ORFs selected within Saccharomyces cerevisiae genome, to purify at least 100 proteins and to solve somewhat 20 structures by X-ray or NMR studies. Last is prepare a detailed project for the creation of a fully fledged Centre des Biostructures at Paris-Sud. As an example of the preliminary output of this currently fully running project, here we report the crystal structure of yeast sorting nexin SNX3. Recently it was determined that phosphoinositide (PtdIns) is a module to activate Phox homology (PX) domains in many proteins with a wide range of functions. SNX3 regulates endosomal functions through its PX domain-mediated binding to PtdIns(3)P. The SNX3 gene was cloned in a pET vector, and expressed in E. coli BL21. The His-tagged protein was purified on the Ni-NTA affinity column followed by Superdex G-75 gel filtration. Crystals were grown by vapor diffusion in hanging drops obtained by mixing 1 ml protein solution with equal volume of a reservoir solution containing 15% PEG 4000 and 100 mM HEPES (pH7.5). Crystals have P6522 symmetry with unit cell dimensions of a=b=55.73 Å, c=187.55 Å, and a=b=90°, g=120°. The crystallographic structure was accomplished using the MAD method at 2.3 Å and then refined to a resolution of 2.0 Å. The overall structure of SNX3 (residues 30-101) is quite similar to the PX domain of human p40PHOX. Crystallization of the protein in complex with PtdIns(3)P is in progress.