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Structural basis of nSH2 regulation and lipid binding in PI3Kα
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Michelle S. Miller1,8, Oleg Schmidt-Kittler2,9,12, David M. Bolduc3,10, Evan T. Brower2,11, Daniele Chaves-Moreira4, Marc Allaire7, Kenneth W. Kinzler2, Ian G. Jennings1, Philip E. Thompson1, Philip A. Cole3, L. Mario Amzel4, Bert Vogelstein2 and Sandra B. Gabelli4,5,6
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.
Sandra B. Gabelli, email:
Keywords: PIK3R1, p85, PIK3CA, PI3K, PIP2, PIP3
Received: June 23, 2014 Accepted: July 23, 2014 Published: July 25, 2014
We report two crystal structures of the wild-type phosphatidylinositol 3-kinase α (PI3Kα) heterodimer refined to 2.9 Å and 3.4 Å resolution: the first as the free enzyme, the second in complex with the lipid substrate, diC4-PIP2, 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 PIP2 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.
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