Using a baculovirus system the authors were able to produce stable levels of p110α only when coexpressed with p85α. However, this led to protein aggregation, so the authors determined whether different truncated versions of p85α would improve solubility. Expression of residues 322–600 of p85α, which contain the inter-SH2 domain (iSH2) that binds p110α and the amino-terminal SH2 domain (nSH2), gave the highest protein yield. The complex was catalytically active and the resulting p110α–niSH2 crystals were used to determine the structure to 3.05 Å resolution.
Mutations in PIK3CA have been found in the N-terminal adaptor binding domain (ABD), the C2 domain thought to interact with membranes, the helical domain of unknown function and the kinase domain. By examining the crystal structure the authors were able to more fully understand how these mutations disrupt the function of p110α. For example, Arg 38 and Arg 88 in the ABD are often mutated to cysteine, histidine or glutamine and the new structure indicates that these mutations alter the interaction of the ABD with the kinase domain, changing the activity of the enzyme. Mutations in the C2 domain were thought to alter the interaction of p110α with the cell membrane. However, the crystal structure data indicate that mutation of Asn 345 in the C2 domain disrupts the interaction with the iSH2 domain of p85α, leading to p110α activation. Moreover, although less common, a mutation in p85α that leads to truncation of the protein at residue 571 was thought to constrain the function of the p85α inhibitory domain. However, results from the structure indicate that the truncation destabilizes the coiled-coil part of iSH2, disrupting the interaction with Asn 345 in p110α.
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