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

PI3Kγ is a critical immune signaling enzyme activated downstream of diverse cell surface molecules, including Ras, PKCß activated by the IgE receptor, and Gßγ subunits released from activated GPCRs. PI3Kγ can form two distinct complexes, with the p110γ catalytic subunit binding to either a p101 or p84 regulatory subunit, with these complexes being differentially activated by upstream stimuli. Here, using a combination of cryo electron microscopy, HDX-MS, and biochemical assays, we have identified novel roles of the helical domain of p110γ in regulating lipid kinase activity of distinct PI3Kγ complexes. We defined the molecular basis for how an allosteric inhibitory nanobody potently inhibits kinase activity through rigidifying the helical domain and regulatory motif of the kinase domain. The nanobody did not block either p110γ membrane recruitment or Ras/Gßγ binding, but instead decreased ATP turnover. We also identified that p110γ can be activated by dual PKCß helical domain phosphorylation leading to partial unfolding of an N-terminal region of the helical domain. PKCß phosphorylation is selective for p110γ-p84 compared to p110γ-p101, driven by differential dynamics of the helical domain of these different complexes. Nanobody binding prevented PKCß-mediated phosphorylation. Overall, this work shows an unexpected allosteric regulatory role of the helical domain of p110γ that is distinct between p110γ-p84 and p110γ-p101 and reveals how this can be modulated by either phosphorylation or allosteric inhibitory binding partners. This opens possibilities of future allosteric inhibitor development for therapeutic intervention.

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

PI3Kγ is a critical immune signaling enzyme activated downstream of diverse cell surface molecules, including Ras, PKCß activated by the IgE receptor, and Gßγ subunits released from activated GPCRs. PI3Kγ can form two distinct complexes, with the p110γ catalytic subunit binding to either a p101 or p84 regulatory subunit, with these complexes being differentially activated by upstream stimuli. Here using a combination of cryo electron microscopy, HDX-MS, and biochemical assays we have identified novel roles of the helical domain of p110γ in regulating lipid kinase activity of distinct PI3Kγ complexes. We defined the molecular basis for how an allosteric inhibitory nanobody potently inhibits kinase activity through rigidifying the helical domain and regulatory motif of the kinase domain. The nanobody did not block either p110γ membrane recruitment or Ras/Gßγ binding, but instead decreased ATP turnover. We also identified that p110γ can be activated by dual PKCß helical domain phosphorylation leading to partial unfolding of an N-terminal region of the helical domain. PKCß phosphorylation is selective for p110γ-p84 compared to p110γ-p101, driven by differential dynamics of the helical domain of these different complexes. Nanobody binding prevented PKCß mediated phosphorylation. Overall, this works shows an unexpected allosteric regulatory role of the helical domain of p110γ that is distinct between p110γ-p84 and p110γ-p101 and reveals how this can be modulated by either phosphorylation or allosteric inhibitory binding partners. This opens possibilities of future allosteric inhibitor development for therapeutic intervention.

Molecular basis for differential activation of p101 and p84 complexes of PI3Kγ by Ras and GPCRs.

Class IB phosphoinositide 3-kinase (PI3Kγ) is activated in immune cells and can form two distinct complexes (p110γ-p84 and p110γ-p101), which are differentially activated by G protein-coupled receptors (GPCRs) and Ras. Using a combination of X-ray crystallography, hydrogen deuterium exchange mass spectrometry (HDX-MS), electron microscopy, molecular modeling, single-molecule imaging, and activity assays, we identify molecular differences between p110γ-p84 and p110γ-p101 that explain their differential membrane recruitment and activation by Ras and GPCRs. The p110γ-p84 complex is dynamic compared with p110γ-p101. While p110γ-p101 is robustly recruited by Gßγ subunits, p110γ-p84 is weakly recruited to membranes by Gßγ subunits alone and requires recruitment by Ras to allow for Gßγ activation. We mapped two distinct Gßγ interfaces on p101 and the p110γ helical domain, with differences in the C-terminal domain of p84 and p101 conferring sensitivity of p110γ-p101 to Gßγ activation. Overall, our work provides key insight into the molecular basis for how PI3Kγ complexes are activated.

Oncogenic mutations of PIK3CA lead to increased membrane recruitment driven by reorientation of the ABD, p85 and C-terminus.

PIK3CA encoding the phosphoinositide 3-kinase (PI3K) p110α catalytic subunit is frequently mutated in cancer, with mutations occurring widely throughout the primary sequence. The full set of mechanisms underlying how PI3Ks are activated by all oncogenic mutations on membranes are unclear. Using a synergy of biochemical assays and hydrogen deuterium exchange mass spectrometry (HDX-MS), we reveal unique regulatory mechanisms underlying PI3K activation. Engagement of p110α on membranes leads to disengagement of the ABD of p110α from the catalytic core, and the C2 domain from the iSH2 domain of the p85 regulatory subunit. PI3K activation also requires reorientation of the p110α C-terminus, with mutations that alter the inhibited conformation of the C-terminus increasing membrane binding. Mutations at the C-terminus (M1043I/L, H1047R, G1049R, and N1068KLKR) activate p110α through distinct mechanisms, with this having important implications for mutant selective inhibitor development. This work reveals unique mechanisms underlying how PI3K is activated by oncogenic mutations, and explains how double mutants can synergistically increase PI3K activity.

Structure of the phosphoinositide 3-kinase (PI3K) p110γ-p101 complex reveals molecular mechanism of GPCR activation.

The class IB phosphoinositide 3-kinase (PI3K), PI3Kγ, is a master regulator of immune cell function and a promising drug target for both cancer and inflammatory diseases. Critical to PI3Kγ function is the association of the p110γ catalytic subunit to either a p101 or p84 regulatory subunit, which mediates activation by G protein-coupled receptors. Here, we report the cryo-electron microscopy structure of a heterodimeric PI3Kγ complex, p110γ-p101. This structure reveals a unique assembly of catalytic and regulatory subunits that is distinct from other class I PI3K complexes. p101 mediates activation through its Gßγ-binding domain, recruiting the heterodimer to the membrane and allowing for engagement of a secondary Gßγ-binding site in p110γ. Mutations at the p110γ-p101 and p110γ-adaptor binding domain interfaces enhanced Gßγ activation. A nanobody that specifically binds to the p101-Gßγ interface blocks activation, providing a novel tool to study and target p110γ-p101-specific signaling events in vivo.

HDX-MS-optimized approach to characterize nanobodies as tools for biochemical and structural studies of class IB phosphoinositide 3-kinases.

There is considerable interest in developing antibodies as modulators of signaling pathways. One of the most important signaling pathways in higher eukaryotes is the phosphoinositide 3-kinase (PI3K) pathway, which plays fundamental roles in growth, metabolism, and immunity. The class IB PI3K, PI3Kγ, is a heterodimeric complex composed of a catalytic p110γ subunit bound to a p101 or p84 regulatory subunit. PI3Kγ is a critical component in multiple immune signaling processes and is dependent on activation by Ras and G protein-coupled receptors (GPCRs) to mediate its cellular roles. Here we describe the rapid and efficient characterization of multiple PI3Kγ binding single-chain camelid nanobodies using hydrogen-deuterium exchange (HDX) mass spectrometry (MS) for structural and biochemical studies. We identify nanobodies that stimulated lipid kinase activity, block Ras activation, and specifically inhibited p101-mediated GPCR activation. Overall, our work reveals insight into PI3Kγ regulation and identifies sites that may be exploited for therapeutic development.

Accurate prediction of protein structures and interactions using a three-track neural network.

DeepMind presented notably accurate predictions at the recent 14th Critical Assessment of Structure Prediction (CASP14) conference. We explored network architectures that incorporate related ideas and obtained the best performance with a three-track network in which information at the one-dimensional (1D) sequence level, the 2D distance map level, and the 3D coordinate level is successively transformed and integrated. The three-track network produces structure predictions with accuracies approaching those of DeepMind in CASP14, enables the rapid solution of challenging x-ray crystallography and cryo-electron microscopy structure modeling problems, and provides insights into the functions of proteins of currently unknown structure. The network also enables rapid generation of accurate protein-protein complex models from sequence information alone, short-circuiting traditional approaches that require modeling of individual subunits followed by docking. We make the method available to the scientific community to speed biological research.

Disease-related mutations in PI3Kγ disrupt regulatory C-terminal dynamics and reveal a path to selective inhibitors.

Class I Phosphoinositide 3-kinases (PI3Ks) are master regulators of cellular functions, with the class IB PI3K catalytic subunit (p110γ) playing key roles in immune signalling. p110γ is a key factor in inflammatory diseases and has been identified as a therapeutic target for cancers due to its immunomodulatory role. Using a combined biochemical/biophysical approach, we have revealed insight into regulation of kinase activity, specifically defining how immunodeficiency and oncogenic mutations of R1021 in the C-terminus can inactivate or activate enzyme activity. Screening of inhibitors using HDX-MS revealed that activation loop-binding inhibitors induce allosteric conformational changes that mimic those in the R1021C mutant. Structural analysis of advanced PI3K inhibitors in clinical development revealed novel binding pockets that can be exploited for further therapeutic development. Overall, this work provides unique insights into regulatory mechanisms that control PI3Kγ kinase activity and shows a framework for the design of PI3K isoform and mutant selective inhibitors.

Class I phosphoinositide 3-kinase (PI3K) regulatory subunits and their roles in signaling and disease.

The Class I phosphoinositide 3-kinases (PI3Ks) are a group of heterodimeric lipid kinases that regulate crucial cellular processes including proliferation, survival, growth, and metabolism. The diversity in functions controlled by the various catalytic isoforms (p110α, p110ß, p110δ, and p110γ) depends on their abilities to be activated by distinct stimuli such as receptor tyrosine kinases (RTKs), G-protein coupled receptors (GPCRs), and the Ras family of small G-proteins. A major factor determining the ability of each p110 enzyme to be activated is the presence of regulatory binding partners. Given the overwhelming evidence for the involvement of PI3Ks in diseases such as cancer, inflammation, immunodeficiency and diabetes, an understanding of how these regulatory proteins influence PI3K function is essential. This article highlights research deciphering the role of regulatory subunits in PI3K signaling and their involvement in human disease.

Defining How Oncogenic and Developmental Mutations of PIK3R1 Alter the Regulation of Class IA Phosphoinositide 3-Kinases.

The class I phosphoinositide 3-kinases (PI3Ks) are key signaling enzymes composed of a heterodimer of a p110 catalytic subunit and a p85 regulatory subunit, with PI3K mutations being causative of multiple human diseases including cancer, primary immunodeficiencies, and developmental disorders. Mutations in the p85α regulatory subunit encoded by PIK3R1 can both activate PI3K through oncogenic truncations in the iSH2 domain, or inhibit PI3K through developmental disorder mutations in the cSH2 domain. Using a combined biochemical and hydrogen deuterium exchange mass spectrometry approach we have defined the molecular basis for how these mutations alter the activity of p110α/p110δ catalytic subunits. We find that the oncogenic Q572∗ truncation of PIK3R1 disrupts all p85-inhibitory inputs, with p110α being hyper-activated compared with p110δ. In addition, we find that the R649W mutation in the cSH2 of PIK3R1 decreases sensitivity to activation by receptor tyrosine kinases. This work reveals unique insight into isoform-specific regulation of p110s by p85α.

Neolymphostin A Is a Covalent Phosphoinositide 3-Kinase (PI3K)/Mammalian Target of Rapamycin (mTOR) Dual Inhibitor That Employs an Unusual Electrophilic Vinylogous Ester.

Using a novel chemistry-based assay for identifying electrophilic natural products in unprocessed extracts, we identified the PI3-kinase/mTOR dual inhibitor neolymphostin A from Salinispora arenicola CNY-486. The method further showed that the vinylogous ester substituent on the neolymphostin core was the exact site for enzyme conjugation. Tandem MS/MS experiments on PI3Kα treated with the inhibitor revealed that neolymphostin covalently modified Lys802 with a shift in mass of +306 amu, corresponding to addition of the inhibitor and elimination of methanol. The binding pose of the inhibitor bound to PI3Kα was modeled, and hydrogen-deuterium exchange mass spectrometry experiments supported this model. Against a panel of kinases, neolymphostin showed good selectivity for PI3-kinase and mTOR. In addition, the natural product blocked AKT phosphorylation in live cells with an IC50 of ∼3 nM. Taken together, neolymphostin is the first reported example of a covalent kinase inhibitor from the bacterial domain of life.

Conformational sampling of membranes by Akt controls its activation and inactivation.

The protein kinase Akt controls myriad signaling processes in cells, ranging from growth and proliferation to differentiation and metabolism. Akt is activated by a combination of binding to the lipid second messenger PI(3,4,5)P3 and its subsequent phosphorylation by phosphoinositide-dependent kinase 1 and mechanistic target of rapamycin complex 2. The relative contributions of these mechanisms to Akt activity and signaling have hitherto not been understood. Here, we show that phosphorylation and activation by membrane binding are mutually interdependent. Moreover, the converse is also true: Akt is more rapidly dephosphorylated in the absence of PIP3, an autoinhibitory process driven by the interaction of its PH and kinase domains. We present biophysical evidence for the conformational changes in Akt that accompany its activation on membranes, show that Akt is robustly autoinhibited in the absence of PIP3 irrespective of its phosphorylation, and map the autoinhibitory PH-kinase interface. Finally, we present a model for the activation and inactivation of Akt by an ordered series of membrane binding, phosphorylation, dissociation, and dephosphorylation events.

Ras Binder Induces a Modified Switch-II Pocket in GTP and GDP States.

Covalent inhibitors of K-Ras(G12C) have been reported that exclusively recognize the GDP state. Here, we utilize disulfide tethering of a non-natural cysteine (K-Ras(M72C)) to identify a new switch-II pocket (S-IIP) binding ligand (2C07) that engages the active GTP state. Co-crystal structures of 2C07 bound to H-Ras(M72C) reveal binding in a cryptic groove we term S-IIG. In the GppNHp state, 2C07 binding to a modified S-IIP pushes switch I away from the nucleotide, breaking the network of polar contacts essential for adopting the canonical GTP state. Biochemical studies show that 2C07 alters nucleotide preference and inhibits SOS binding and catalyzed nucleotide exchange. 2C07 was converted to irreversible covalent analogs, which target both nucleotide states, inhibit PI3K activation in vitro, and function as occupancy probes to detect reversible engagement in competition assays. Targeting both nucleotide states opens the possibility of inhibiting oncogenic mutants of Ras, which exist predominantly in the GTP state in cells.

Molecular mechanism of activation of class IA phosphoinositide 3-kinases (PI3Ks) by membrane-localized HRas.

Class IA PI3Ks are involved in the generation of the key lipid signaling molecule phosphatidylinositol 3,4,5-trisphosphate (PIP3), and inappropriate activation of this pathway is implicated in a multitude of human diseases, including cancer, inflammation, and primary immunodeficiencies. Class IA PI3Ks are activated downstream of the Ras superfamily of GTPases, and Ras-PI3K interaction plays a key role in promoting tumor formation and maintenance in Ras-driven tumors. Investigating the detailed molecular events in the Ras-PI3K interaction has been challenging because it occurs on a membrane surface. Here, using maleimide-functionalized lipid vesicles, we successfully generated membrane-resident HRas and evaluated its effect on PI3K signaling in lipid kinase assays and through analysis with hydrogen-deuterium exchange MS. We screened all class IA PI3K isoforms and found that HRas activates both p110α and p110δ isoforms but does not activate p110ß. The p110α and p110δ activation by Ras was synergistic with activation by a soluble phosphopeptide derived from receptor tyrosine kinases. Hydrogen-deuterium exchange MS revealed that membrane-resident HRas, but not soluble HRas, enhances conformational changes associated with membrane binding by increasing membrane recruitment of both p110α and p110δ. Together, these results afford detailed molecular insight into the Ras-PI3K signaling complex, provide a framework for screening Ras inhibitors, and shed light on the isoform specificity of Ras-PI3K interactions in a native membrane context.