Allosteric activation or inhibition of PI3Kγ mediated through conformational changes in the p110γ helical domain

Abstract

PI3Kγ is a critical immune signaling enzym 31 e activated downstream of diverse cell 32 surface molecules, including Ras, PKCβ activated by the IgE receptor, and Gβγ 33 subunits released from activated GPCRs. PI3Kγ can form two distinct complexes, with 34 the p110γ catalytic subunit binding to either a p101 or p84 regulatory subunit, with these 35 complexes being differentially activated by upstream stimuli. Here using a combination 36 of cryo electron microscopy, HDX-MS, and biochemical assays we have identified novel 37 roles of the helical domain of p110γ in regulating lipid kinase activity of distinct PI3Kγ 38 complexes. We defined the molecular basis for how an allosteric inhibitory nanobody 39 potently inhibits kinase activity through rigidifying the helical domain and regulatory 40 motif of the kinase domain. The nanobody did not block either p110γ membrane 41 recruitment or Ras/Gβγ binding, but instead decreased ATP turnover. We also identified 42 that p110γ can be activated by dual PKCβ helical domain phosphorylation leading to 43 partial unfolding of an N-terminal region of the helical domain.