Expression, Purification, Crystallisation and X-ray Crystallographic Analysis of a Truncated Form of Human Src Homology 2 Containing Inositol 5-Phosphatase 2

• Published in: Protein Journal 2016-06, Vol.35 (3), p.225-230
• Main Authors: Le Coq, Johanne, Heredia Gallego, Luis, Lietha, Daniel
• Format: Article
• Language: English
• Subjects: Analysis; Animal Anatomy; Biochemistry; Bioorganic Chemistry; Chemistry; Chemistry and Materials Science; Cloning, Molecular; Crystallization; Crystallography; Crystallography, X-Ray; Crystals; Enzymes; Escherichia coli - genetics; Ethylenediaminetetraacetic acid; Histology; Information management; Inositol; Morphology; Organic Chemistry; Phosphatases; Phosphatidylinositol-3,4,5-Trisphosphate 5-Phosphatases - chemistry; Phosphatidylinositol-3,4,5-Trisphosphate 5-Phosphatases - genetics; Phosphatidylinositol-3,4,5-Trisphosphate 5-Phosphatases - isolation & purification; Phosphatidylinositol-3,4,5-Trisphosphate 5-Phosphatases - metabolism; Protein Domains; Protein expression; Proteins; Signal Transduction; Type 2 diabetes
• ISSN: 1572-3887; 1573-4943; 1875-8355
• DOI: 10.1007/s10930-016-9665-y

The Src homology 2 containing inositol 5-phosphatase 2 (SHIP2) catalyses the dephosphorylation of the phospholipid phosphatidylinositol 3,4,5-triphosphate (PI(3,4,5)P 3 ) to form PI(3,4)P 2 . PI(3,4,5)P 3 is a key lipid second messenger, which can recruit signalling proteins to the plasma membrane and subsequently initiate numerous downstream signalling pathways responsible for the regulation of a plethora of cellular events such as proliferation, growth, apoptosis and cytoskeletal rearrangements. SHIP2 has been heavily implicated with several serious diseases such as cancer and type 2 diabetes but its regulation remains poorly understood. In order to gain insight into the mechanisms of SHIP2 regulation, a fragment of human SHIP2 containing the phosphatase domain and a region proposed to resemble a C2 domain was crystallized. Currently, no structural information is available on the putative C2-related domain or its relative position with respect to the phosphatase domain. Initial crystals were polycrystalline, but were optimized to obtain diffraction data to a resolution of 2.1 Å. Diffraction data analysis revealed a P2 1 2 1 2 1 space group with unit cell parameters a = 136.04 Å, b = 175.84 Å, c = 176.89 Å. The Matthews coefficient is 2.54 Å 3  Da −1 corresponding to 8 molecules in the asymmetric unit with a solvent content of 51.7 %.