Structural basis of nSH2 regulation and lipid binding in PI3Kα

// Michelle S. Miller 1,8 , Oleg Schmidt-Kittler 2,9,12 , David M. Bolduc 3,10 , Evan T. Brower 2,11 , Daniele Chaves-Moreira 4 , Marc Allaire 7 , Kenneth W. Kinzler 2 , Ian G. Jennings 1 , Philip E. Thompson 1 , Philip A. Cole 3 , L. Mario Amzel 4 , Bert Vogelstein 2 and Sandra B. Gabelli 4,5,6 1 Medicinal Chemistry, Monash Institute of Pharmaceutical Sciences, Parkville, Victoria, Australia. 2 Ludwig Center for Cancer Genetics and Therapeutics and Howard Hughes Medical Institutions, Johns Hopkins University School of Medicine, Baltimore, Maryland, USA. 3 Department of Pharmacology and Molecular Sciences, Johns Hopkins University School of Medicine, Baltimore, Maryland, USA. 4 Department of Biophysics and Biophysical Chemistry, Johns Hopkins University School of Medicine, Baltimore, Maryland, USA. 5 Department of Medicine, Johns Hopkins University School of Medicine, Baltimore, Maryland, USA. 6 Department of Oncology, Johns Hopkins University School of Medicine, Baltimore, Maryland, USA. 7 Photon Sciences, Brookhaven National Laboratory, Upton, New York, USA. 8 Present Address: Department of Oncology, Johns Hopkins University School of Medicine, Baltimore Maryland, USA. 9 Present Address: Sanofi, Cambridge, Massachusetts. 10 Present Address: Center for Neurologic Diseases, Brigham and Women’s Hospital and Harvard Medical School, Boston, Massachusetts. 11 Present Address: Paragon Bioservices, Baltimore, Maryland. 12 Present Address: Berkeley Center for Structural Biology, Physical Biosciences Division, Lawrence Berkeley National Laboratory, Berkeley, California. Correspondence: Sandra B. Gabelli, email: // Keywords : PIK3R1, p85, PIK3CA, PI3K, PIP2, PIP3 Received : June 23, 2014 Accepted : July 23, 2014 Published : July 25, 2014 Abstract We report two crystal structures of the wild-type phosphatidylinositol 3-kinase α (PI3Kα) heterodimer refined to 2.9 A and 3.4 A resolution: the first as the free enzyme, the second in complex with the lipid substrate, diC4-PIP 2 , respectively. The first structure shows key interactions of the N-terminal SH2 domain (nSH2) and iSH2 with the activation loop that suggest a mechanism by which the enzyme is inhibited in its basal state. In the second structure, the lipid substrate binds in a positively charged pocket adjacent to the ATP-binding site, bordered by the P-loop, the activation loop and the iSH2 domain. An additional lipid-binding site was identified at the interface of the ABD, iSH2 and kinase domains. The ability of PI3Kα to bind an additional PIP 2 molecule was confirmed in vitro by fluorescence quenching experiments. The crystal structures reveal key differences in the way the nSH2 domain interacts with wild-type p110α and with the oncogenic mutant p110αH1047R. Increased buried surface area and two unique salt-bridges observed only in the wild-type structure suggest tighter inhibition in the wild-type PI3Kα than in the oncogenic mutant. These differences may be partially responsible for the increased basal lipid kinase activity and increased membrane binding of the oncogenic mutant.