Purification, crystallization and preliminary X-ray diffraction of a proteolytic fragment of PDK1 containing the pleckstrin homology domain

• Published in: Acta crystallographica. Section D, Biological crystallography. Biological crystallography., 2004-02, Vol.60 (2), p.314-316
• Main Authors: Komander, David, Deak, Maria, Morrice, Nick, Van Aalten, Daan M. F.
• Format: Article
• Language: English
• Subjects: 3-phosphoinositide-dependent protein kinase-1; 3-Phosphoinositide-Dependent Protein Kinases; Blood Proteins - chemistry; Cloning, Molecular; Crystallization; DNA, Complementary - metabolism; Electrophoresis, Polyacrylamide Gel; Glutathione Transferase - metabolism; Humans; Phosphoproteins - chemistry; pleckstrin homology domains; Protein Structure, Tertiary; Protein-Serine-Threonine Kinases - chemistry; Protein-Serine-Threonine Kinases - genetics; Protein-Serine-Threonine Kinases - isolation & purification; Recombinant Fusion Proteins - metabolism; Time Factors; X-Ray Diffraction
• ISSN: 1399-0047; 0907-4449; 1399-0047
• DOI: 10.1107/S0907444903028518

3‐Phosphoinositide‐dependent protein kinase‐1 (PDK1) is a Ser/Thr kinase with an essential role in insulin and growth‐factor signalling. PDK1 activity towards protein kinase B (PKB) is partially regulated by its pleckstrin homology (PH) domain, which preferentially binds to 3‐phosphoinositides. However, the precise molecular mechanism of this regulation is not well understood. Here, the cloning, purification and crystallization of a 150‐amino‐acid C‐terminal region of PDK1 containing the PH domain is reported. A crystal of the PDK1 PH domain grown in the presence of inositol 1,3,4,5‐tetrakisphosphate and derivatized with AuCN diffracted to 1.5 Å at a synchrotron source. Diffraction data collected near the Au edge resulted in an anomalous Patterson map with a 30σ peak.