Assembly and Molecular Architecture of the Phosphoinositide 3-Kinase p85α Homodimer

Abstract

Phosphoinositide 3-kinases (PI3Ks) are a family of lipid kinases that are activated by growth factor and G-protein-coupled receptors and propagate intracellular signals for growth, survival, proliferation, and metabolism. p85α, a modular protein consisting of five domains, binds and inhibits the enzymatic activity of class IA PI3K catalytic subunits. Here, we describe the structural states of the p85α dimer, based on data from in vivo and in vitro solution characterization. Our in vitro assembly and structural analyses have been enabled by the creation of cysteine-free p85α that is functionally equivalent to native p85α. Analytical ultracentrifugation studies showed that p85α undergoes rapidly reversible monomer-dimer assembly that is highly exothermic in nature. In addition to the documented SH3-PR1 dimerization interaction, we identified a second intermolecular interaction mediated by cSH2 domains at the C-terminal end of the polypeptide. We have demonstrated in vivo concentration-dependent dimerization of p85α using fluorescence fluctuation spectroscopy. Finally, we have defined solution conditions under which the protein is predominantly monomeric or dimeric, providing the basis for small angle x-ray scattering and chemical cross-linking structural analysis of the discrete dimer. These experimental data have been used for the integrative structure determination of the p85α dimer. Our study provides new insight into the structure and assembly of the p85α homodimer and suggests that this protein is a highly dynamic molecule whose conformational flexibility allows it to transiently associate with multiple binding proteins.

Keywords: analytical ultracentrifugation; molecular modeling; phosphatidylinositide 3-kinase (PI 3-kinase); phosphatidylinositol signaling; small-angle X-ray scattering (SAXS); structural model.

FIGURE 1.

Cysteine-free p85α and fragments. A, the six cysteines of wild-type human p85α were mutated (C146S, C167S, C498S, C656S, C659V, and C670L) to generate cysteine-free recombinant p85α, herein referred to as p85α. Four 85α truncations were also prepared. B, Coomassie-stained SDS-polyacrylamide gel of purified p85α and fragments.

FIGURE 2.

Characterization of cysteine-free p85α. A, immunoblot analysis of Myc-p110α binding to GST alone, GST-p85α, or native GST-p85α. B, kinase activity of p110α purified from HEK293T cells in the presence of GST-p85α or native GST-p85α. Shown are the average ± S.E. (error bars) from three independent experiments. ns, not significant. C, binding of GST-cSH2 domains from p85α or native p85α to a photoactivatable ¹²⁵I-labeled tyrosine phosphopeptide in the absence or presence of unlabeled phosphopeptide. D, immunoblot analysis of GST-p85α(1–432) binding to recombinant Rac1 loaded without or with GTPγS.

FIGURE 3.

p85α dimerization in vitro measured by AUC. A, sedimentation coefficient distribution, g(s*), obtained from sedimentation velocity analysis of p85α at three concentrations (1.2, 3.6, and 9.5 μm) in 20 mm NaCl-containing buffer at 4 °C. All sedimentation coefficient (S, in Svedberg units) values presented in the paper have been corrected to 20 °C and the density of water (s_20,w) to allow comparison of data from experiments performed in different buffers and temperatures. B, plot of sedimentation velocity data showing S as a function of p85α concentration (absorbance at 280 nm) at 4, 15, 37, and 15 °C in 300 mm NaCl-containing buffer. C and D, van't Hoff plots relating the association constant (K_a) determined by sedimentation equilibrium analysis as a function of temperature for full-length p85α (C) and p85α(1–333) (D) in 20 and 500 mm NaCl-containing buffer. E, weight average molecular weights obtained from sedimentation equilibrium analysis of p85α and corresponding fragments in buffer containing either 20 mm NaCl or 500 mm NaCl and at 500 mm NaCl in the presence of 250 μm SH3-binding peptide. Solid and dashed lines, dimer and monomer molecular weights calculated from sequence, respectively. F, dissociation constant (K_d) values for each p85α construct in low and high salt, derived from fitting sedimentation equilibrium data to a monomer-dimer association model. Values are represented with joint confidence intervals of one S.D.

FIGURE 4.

Sedimentation equilibrium analysis of p85α at 10 °C. The graphs show the distribution of p85α protein concentration during sedimentation equilibrium analysis at 10 °C obtained by measuring the absorption at 280 nm versus the radial distance from the center of the rotor. Three concentrations of full-length p85α were analyzed (1.2, 3.6, and 9.6 μm). The equilibrium protein concentration distributions measured at each of the two rotor speeds are shown (top curve, 8,000 rpm; bottom curve, 16,000 rpm) with the best global fit to a monomer-dimer association model shown as the solid line in 20 mm NaCl (A) and 500 mm NaCl (B) and fit to the weight average molecular weight of a single species in 500 mm NaCl in the presence of proline-rich peptide (C). The residuals for the fit are shown by the symbols along the dotted line at 0.0.

FIGURE 5.

p85α dimerization in vivo measured by FFS. A, U2OS cells were transfected with GFP-p85α or native GFP-p85α. Brightness was measured as a function of concentration and compared with a monomeric GFP control. The data sets were not significantly different, as indicated by their common regression line. B, U201S cells were transfected with native GFP-p85α or native GFP-p85α PR1/M176A. Brightness was measured as a function of concentration and compared with a monomeric GFP control.

FIGURE 6.

Structure and dynamics of the p85α(1–333) homodimer revealed through an integrative modeling approach. A, the best scoring multistate model is composed of three major states with population weights of 40.3% (blue), 27.7% (red), and 16.6% (yellow) and two minor states with population weights of 8.0% (green) and 7.4% (purple). The most populated state (blue) was used as a reference for rigid body least-squares superposition of the remaining four states. The ab initio shape (represented as a gray envelope) computed from the experimental SAXS profile was also superposed for comparison. B, comparison of the experimental SAXS profile (black, in arbitrary units (a.u.)) of the p85α(1–333) dimer with the calculated SAXS profiles from the single-state (χ = 13.90, red) and the five-state (χ = 2.623, blue) models. The bottom plot shows the residuals (calculated intensity/experimental intensity) of each calculated SAXS profile. The top inset shows the SAXS profiles in the Guinier plot (in arbitrary units) with an R_g fit of 44.1 ± 0.62 Å. The maximum particle size (D_max) was ∼150 Å (determined experimentally; Table 1). C, each of the five states in the multistate model along with population weights and domain labels is shown. Colors were adjusted to distinguish individual domains in the dimer. The conformational dynamics of p85α(1–333) dimer appear to be dominated by the relative intramolecular motions of the SH3 and BCR domains, connected by the PR1 motif linkers. D, consistency between the 25 DST chemical cross-links and the multistate model of the p85α(1–333) dimer. The green dots represent cross-linked residue pairs satisfied by the multistate model within the distance threshold of 35 Å. The multistate model of the p85α(1–333) dimer satisfied all 25 DST chemical cross-links.

FIGURE 7.

Structure and dynamics of the full-length p85α homodimer revealed through an integrative modeling approach. A, the best scoring multistate model is composed of three major states with population weights of 33.2% (blue), 27.4% (red), and 18.2% (yellow) and two minor states with population weights of 13.4% (green) and 7.9% (purple). The most populated state (blue) was used as a reference for rigid body least-squares superposition of the remaining four states. The ab initio shape (represented as a gray envelope) computed from the experimental SAXS profile was also superposed for comparison. B, comparison of the experimental SAXS profile (black, in arbitrary units (a.u.)) of the p85α dimer with the calculated SAXS profiles from the single-state (χ = 1.671, red) and the five-state (χ = 1.275, blue) models. The bottom plot shows the residuals (calculated intensity/experimental intensity) of each calculated SAXS profile. The top inset shows the SAXS profiles in the Guinier plot (in arbitrary units) with an R_g fit of 57.7 ± 0.9 Å. The maximum particle size (D_max) was ∼200 Å (determined experimentally; Table 1). C, each of the five states in the multistate model, along with population weights and domain labels, is shown. Colors were adjusted to distinguish individual domains in the dimer. A large degree of heterogeneity was observed in the full-length p85α dimer, particularly in the coiled-coil (iSH2) domains as well as the neighboring nSH2 and cSH2 domains. The two iSH2 domains are oriented in multiple conformations relative to one another (e.g. parallel, anti-parallel, and perpendicular). D, consistency between the combined 256 (25 DST and 231 DSS) chemical cross-links and the multistate model of the full-length p85α dimer. Green dots, cross-linked residue pairs satisfied by the multistate model within the distance threshold of 35 Å. Red triangles, cross-linked residue pairs that violated the distance threshold of 35 Å. Blue dots, five homodimer chemical cross-links identified on the same residues between two subunits in the dimer. The multistate model of p85α dimer satisfied 244 (95%) of the combined 256 chemical cross-links.