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PI3Kγ is a critical immune signaling enzyme activated downstream of diverse cell surface molecules, including Ras, PKCβ activated by the IgE receptor, and Gβγ 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/Gβγ binding, but instead decreased ATP turnover. We also identified that p110γ can be activated by dual PKCβ helical domain phosphorylation leading to partial unfolding of an N-terminal region of the helical domain. PKCβ 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 PKCβ 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.

Phosphorylation is a ubiquitous mechanism by which signals are transduced in cells. Protein kinases, enzymes that catalyze the phosphotransfer reaction are, themselves, often regulated by phosphorylation. Paradoxically, however, a substantial fraction of more than 500 human protein kinases are capable of catalyzing their own activation loop phosphorylation. Commonly, these kinases perform this autophosphorylation reaction in trans , whereby transient dimerization leads to the mutual phosphorylation of the activation loop of the opposing protomer. In this study, we demonstrate that protein kinase D (PKD) is regulated by the inverse mechanism of dimerization-mediated trans -autoinhibition, followed by activation loop autophosphorylation in cis . We show that PKD forms a stable face-to-face homodimer that is incapable of either autophosphorylation or substrate phosphorylation. Dissociation of this trans -autoinhibited dimer results in activation loop autophosphorylation, which occurs exclusively in cis . Phosphorylation serves to increase PKD activity and prevent trans -autoinhibition, thereby switching PKD on. Our findings not only reveal the mechanism of PKD regulation but also have profound implications for the regulation of many other eukaryotic kinases.

Pathogenic fungi exhibit a heavy burden on medical care and new therapies are needed. Here, we develop the fungal specific enzyme sterylglucosidase 1 (Sgl1) as a therapeutic target. Sgl1 converts the immunomodulatory glycolipid ergosterol 3β-D-glucoside to ergosterol and glucose. Previously, we found that genetic deletion of Sgl1 in the pathogenic fungus Cryptococcus neoformans (Cn) results in ergosterol 3β-D-glucoside accumulation, renders Cn non-pathogenic, and immunizes mice against secondary infections by wild-type Cn, even in condition of CD4+ T cell deficiency. Here, we disclose two distinct chemical classes that inhibit Sgl1 function in vitro and in Cn cells. Pharmacological inhibition of Sgl1 phenocopies a growth defect of the Cn Δsgl1 mutant and prevents dissemination of wild-type Cn to the brain in a mouse model of infection. Crystal structures of Sgl1 alone and with inhibitors explain Sgl1’s substrate specificity and enable the rational design of antifungal agents targeting Sgl1., Sterylglucosidase 1 (Sgl1) is a virulence factor in Cryptococcus neoformans that modulates fungal pathogenesis and host response. Here, the authors characterize Sgl1 structurally, identify Sgl1 inhibitors, and demonstrate Sgl1 inhibition has efficacy in mouse models of infection.

Lipid droplets (LDs) are dynamic organelles that contain an oil core mainly composed of triglycerides (TAG) that is surrounded by a phospholipid monolayer and LD-associated proteins called perilipins (PLINs). During LD biogenesis, perilipin 3 (PLIN3) is recruited to nascent LDs as they emerge from the endoplasmic reticulum. Here, we analyzed how lipid composition affects PLIN3 recruitment to membrane bilayers and LDs, and the structural changes that occur upon membrane binding. We found the TAG precursors phosphatidic acid and diacylglycerol (DAG) recruit PLIN3 to membrane bilayers and define an expanded Perilipin-ADRP-Tip47 (PAT) domain that preferentially binds DAG enriched membranes. Membrane binding induces a disorder/order transition of alpha helices within the PAT domain and 11-mer repeats, with intramolecular distance measurements consistent with the expanded PAT domain adopting a folded but dynamic structure upon membrane binding. In cells, PLIN3 is recruited to DAG enriched ER membranes, and this requires both the PAT domain and 11-mer repeats. This provides molecular details of PLIN3 recruitment to nascent LDs and identifies a function of the PAT domain of PLIN3 in DAG binding.

A large amount of the human proteome is composed of highly dynamic regions that do not adopt a single static conformation. These regions are defined as intrinsically disordered, and they are found in a third of all eukaryotic proteins. They play instrumental roles in many aspects of protein signaling, but can be challenging to characterize by biophysical methods. Intriguingly, many of these regions can adopt stable secondary structure upon interaction with a variety of binding partners, including proteins, lipids, and ligands. This review will discuss the application of Hydrogen-deuterium exchange mass spectrometry (HDX-MS) as a powerful biophysical tool that is particularly well suited for structural and functional characterization of intrinsically disordered regions in proteins. A focus will be on the theory of hydrogen exchange, and its practical application to identify disordered regions, as well as characterize how they participate in protein–protein and protein–membrane interfaces. A particular emphasis will be on how HDX-MS data can be presented specifically tailored for analysis of intrinsically disordered regions, as well as the technical aspects that are critical to consider when designing HDX-MS experiments for proteins containing intrinsically disordered regions.

Trimer Independent of NuA4 involved in Transcription Interactions with Nucleosomes (TINTIN) is an integral module of the essential yeast lysine acetyltransferase complex NuA4 that plays key roles in transcription regulation and DNA repair. Composed of Eaf3, Eaf5, and Eaf7, TINTIN mediates targeting of NuA4 to chromatin through the chromodomain-containing subunit Eaf3 that is shared with the Rpd3S histone deacetylase complex. How Eaf3 mediates chromatin interaction in the context of TINTIN and how is it different from what has been observed in Rpd3S is unclear. Here, we reconstituted recombinant TINTIN and its subassemblies and characterized their biochemical and structural properties. Our coimmunoprecipitation, AlphaFold2 modeling, and hydrogen deuterium exchange mass spectrometry (HDX-MS) analyses revealed that the Eaf3 MRG domain contacts Eaf7 and this binding induces conformational changes throughout Eaf3. Nucleosome-binding assays showed that Eaf3 and TINTIN interact non-specifically with the DNA on nucleosomes. Furthermore, integration into TINTIN enhances the affinity of Eaf3 toward nucleosomes and this improvement is a result of allosteric activation of the Eaf3 chromodomain. Negative stain electron microscopy (EM) analysis revealed that TINTIN binds to the edge of nucleosomes with increased specificity in the presence of H3K36me3. Collectively, our work provides insights into the dynamics of TINTIN and the mechanism by which its interactions with chromatin are regulated.

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.

Summary:PIK3CA, which encodes the p110α catalytic subunit of PI3Kα, is one of the most frequently genetically activated kinases in solid tumors. In this issue of Cancer Discovery, Song and colleagues report that the related PI3Kα inhibitors taselisib and inavolisib trigger receptor tyrosine kinase (RTK)–dependent degradation of the mutant p110α protein in breast cancer cells that are positive for HER2 RTK, limiting feedback-mediated drug resistance and potentially widening the therapeutic index of PI3Kα inhibition.See related article by Song et al., p. 204.

Class IB phosphoinositide 3-kinase (PI3Kγ) is activated in immune cells by diverse stimuli and can form two distinct complexes, with the p110γ catalytic subunit binding to either p101 or p84 regulatory subunits. These two complexes are differentially activated by G-protein coupled receptors (GPCRs) and Ras, but the molecular details of this activation are still unclear. Using a combination of X-ray crystallography, HDX-MS, EM, molecular modeling, and biochemical assays we reveal molecular differences between the two p110γ-p84 and p110γ-p101 complexes that explain their differential activation. The structure of p110γ-p84 shows a similar assembly to p110γ-p101 at the p110γ interface, however the interface in p110γ-p84 is dynamic and is evolutionarily conserved to be less stable compared to p110γ-p101. The p110γ-p84 complex is only weakly recruited to membranes by Gβγ subunits alone and requires recruitment by Ras to allow for Gβγ activation through an interaction with the p110γ helical domain. The interfaces of the p101 GBD with Gβγ, and the p110γ helical domain with Gβγ were determined using computational alphafold2 modeling and HDX-MS. There are distinct differences in the C-terminal domain of p84 and p101, which allows p101 to bind Gβγ subunits, while p84 does not. The two Gβγ interfaces in p110γ and p101 are distinct, revealing how unique mutants of Gβγ cause differential disruption of PI3Kγ complex activation. Overall, our work provides key insight into the molecular basis for how different PI3Kγ complexes are activated.

The protein kinase Akt is a master regulator of pro-growth signalling in the cell. Akt is activated through its targeted recruitment to phosphoinositides, leading to disruption of the autoinhibitory interface between the kinase and pleckstrin homology (PH) domains. Hyper activation of Akt is common in oncogenic transformation, with multiple oncogenic activating mutants identified in Akt. This has led to the development of potent and selective ATP-competitive and allosteric inhibitors for Akt. Paradoxically, some ATP-competitive Akt inhibitors cause hyperphosphorylation of Akt. Here, using hydrogen deuterium exchange mass spectrometry (HDX-MS), we interrogated the conformational changes upon binding to the Akt active site inhibitor A-443654, and the Akt allosteric inhibitor MK-2206. We compared the conformational changes that occurred for each inhibitor under three different states of Akt: i-inactive monophosphorylated, ii-partially active tris-phosphorylated [T308, T450, S473], and iii-fully activated, tris-phosphorylated bound to PIP3 membranes. The allosteric MK-2206 inhibitor results in large scale allosteric conformational changes in all states, and restricts membrane binding through sequestration of the PH domain. Binding of the A-443654 inhibitor led to large scale allosteric conformational changes in both the monophosphorylated and phosphorylated states, leading to an alteration in the autoinhibitory PH-kinase interface. We also observed increased protection in the PH domain upon membrane binding in the presence of A-443654, suggesting that the PH domain is more accessible for membrane binding. This work provides unique insight into the autoinhibitory conformation of the PH and kinase domain and dynamic conformational changes induced by Akt inhibitors, and has important implications for the design of Akt targeted therapeutics.

Calcineurin, the conserved protein phosphatase and target of immunosuppressants, is a critical mediator of Ca2+ signaling. Here, to discover calcineurin-regulated processes we examined an understudied isoform, CNAβ1. We show that unlike canonical cytosolic calcineurin, CNAβ1 localizes to the plasma membrane and Golgi due to palmitoylation of its divergent C-terminal tail, which is reversed by the ABHD17A depalmitoylase. Palmitoylation targets CNAβ1 to a distinct set of membrane-associated interactors including the phosphatidylinositol 4-kinase (PI4KA) complex containing EFR3B, PI4KA, TTC7B and FAM126A. Hydrogen-deuterium exchange reveals multiple calcineurin-PI4KA complex contacts, including a calcineurin-binding peptide motif in the disordered tail of FAM126A, which we establish as a calcineurin substrate. Calcineurin inhibitors decrease PI4P production during Gq-coupled GPCR signaling, suggesting that calcineurin dephosphorylates and promotes PI4KA complex activity. In sum, this work discovers a calcineurin-regulated signaling pathway which highlights the PI4KA complex as a regulatory target and reveals that dynamic palmitoylation confers unique localization, substrate specificity and regulation to CNAβ1.

Covalent protein kinase inhibitors exploit currently noncatalytic cysteines in the adenosine 5′-triphosphate (ATP)-binding site via electrophiles directly appended to a reversible-inhibitor scaffold. Here, we delineate a path to target solvent-exposed cysteines at a distance >10 Å from an ATP-site-directed core module and produce potent covalent phosphoinositide 3-kinase α (PI3Kα) inhibitors. First, reactive warheads are used to reach out to Cys862 on PI3Kα, and second, enones are replaced with druglike warheads while linkers are optimized. The systematic investigation of intrinsic warhead reactivity (kchem), rate of covalent bond formation and proximity (kinactand reaction space volume Vr), and integration of structure data, kinetic and structural modeling, led to the guided identification of high-quality, covalent chemical probes. A novel stochastic approach provided direct access to the calculation of overall reaction rates as a function of kchem, kinact, Ki, and Vr, which was validated with compounds with varied linker lengths. X-ray crystallography, protein mass spectrometry (MS), and NanoBRET assays confirmed covalent bond formation of the acrylamide warhead and Cys862. In rat liver microsomes, compounds 19 and 22 outperformed the rapidly metabolized CNX-1351, the only known PI3Kα irreversible inhibitor. Washout experiments in cancer cell lines with mutated, constitutively activated PI3Kα showed a long-lasting inhibition of PI3Kα. In SKOV3 cells, compounds 19 and 22 revealed PI3Kβ-dependent signaling, which was sensitive to TGX221. Compounds 19 and 22 thus qualify as specific chemical probes to explore PI3Kα-selective signaling branches. The proposed approach is generally suited to develop covalent tools targeting distal, unexplored Cys residues in biologically active enzymes., Journal of the American Chemical Society, 144 (14), ISSN:0002-7863, ISSN:1520-5126

PIK3CAencoding 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.

Plasmodium falciparum phosphatidylinositol 4-kinase (PfPI4K) has emerged as a promising new drug target for novel antimalarial therapeutics. In the absence of a reliable high-resolution three-dimensional structure, a homology model of PfPI4K was built as a tool for structure-based drug design. This homology model has been validated against three distinct chemical series of potent inhibitors using docking and energy minimizations to elucidate the interactions crucial for PI4K inhibition and potent antiplasmodium activity. Despite its potential as an antimalarial target, the similarity between PfPI4K and structurally related human kinases poses a risk for human off-target kinase activity and associated toxicity. Comparative docking between PfPI4K and human phosphoinositide kinases (PIKs) presents compelling evidence for the origins of selectivity. This in-depth analysis of the PfPI4K homology model, the binding modes of the inhibitors, and the interactions responsible for selectivity over human kinases provides a powerful template for future optimization of Plasmodium PI4K inhibitors.

Filtering of nonspecifically binding contaminant proteins from affinity purification mass spectrometry (AP-MS) data is a well-established strategy to improve statistical confidence in identified proteins. The CRAPome (contaminant repository for affinity purification) describes the contaminating background content present in many purification strategies. However, full contaminant lists for nickel-nitrilotriacetic acid (NiNTA) and glutathione S-transferase (GST) affinity matrices are lacking. Similarly, no Spodoptera frugiperda (Sf9) contaminants are available, and only the FLAG-purified contaminants are described for Escherichia coli. For MS experiments that use recombinant protein, such as structural mass spectrometry experiments (hydrogen-deuterium exchange mass spectrometry (HDX-MS), chemical cross-linking, and radical foot-printing), failing to include these contaminants in the search database during the initial tandem MS (MS/MS) identification stage can result in complications in peptide identification. We have created contaminant FASTA databases for Sf9 and E. coli NiNTA or GST purification strategies and show that the use of these databases can effectively improve HDX-MS protein coverage, fragment count, and confidence in peptide identification. This approach provides a robust strategy toward the design of contaminant databases for any purification approach that will expand the complexity of systems able to be interrogated by HDX-MS.

Numerous receptor tyrosine kinases and immune receptors activate phospholipase C-γ (PLC-γ) isozymes at membranes to control diverse cellular processes including phagocytosis, migration, proliferation, and differentiation. The molecular details of this process are not well understood. Using hydrogen-deuterium exchange mass spectrometry, we show that PLC-γ1 is relatively inert to lipid vesicles that contain its substrate, phosphatidylinositol 4,5-bisphosphate (PIP2), unless first bound to the kinase domain of the fibroblast growth factor receptor (FGFR1). Exchange occurs throughout PLC-γ1 and is exaggerated in PLC-γ1 containing an oncogenic substitution (D1165H) that allosterically activates the lipase. These data support a model whereby initial complex formation shifts the conformational equilibrium of PLC-γ1 to favor activation. This receptor-induced priming of PLC-γ1 also explains the capacity of a kinase-inactive fragment of FGFR1 to modestly enhance the lipase activity of PLC-γ1 operating on lipid vesicles but not a soluble analog of PIP2 and highlights potential cooperativity between receptor engagement and membrane proximity. Priming is expected to be greatly enhanced for receptors embedded in membranes and nearly universal for the myriad of receptors and co-receptors that bind the PLC-γ isozymes.

Lipid phosphoinositides are master regulators of almost all aspects of a cell's life and death and are generated by the tightly regulated activity of phosphoinositide kinases. Although extensive efforts have focused on drugging class I phosphoinositide 3-kinases (PI3Ks), recent years have revealed opportunities for targeting almost all phosphoinositide kinases in human diseases, including cancer, immunodeficiencies, viral infection and neurodegenerative disease. This has led to widespread efforts in the clinical development of potent and selective inhibitors of phosphoinositide kinases. This Review summarizes our current understanding of the molecular basis for the involvement of phosphoinositide kinases in disease and assesses the preclinical and clinical development of phosphoinositide kinase inhibitors.

The formation of complexes between Rab11 and its effectors regulates multiple aspects of membrane trafficking, including recycling and ciliogenesis. WD repeat-containing protein 44 (WDR44) is a structurally uncharacterized Rab11 effector that regulates ciliogenesis by competing with prociliogenesis factors for Rab11 binding. Here, we present a detailed biochemical and biophysical characterization of the WDR44-Rab11 complex and define specific residues mediating binding. Using AlphaFold2 modeling and hydrogen/deuterium exchange mass spectrometry, we generated a molecular model of the Rab11-WDR44 complex. The Rab11-binding domain of WDR44 interacts with switch I, switch II, and the interswitch region of Rab11. Extensive mutagenesis of evolutionarily conserved residues in WDR44 at the interface identified numerous complex-disrupting mutations. Using hydrogen/deuterium exchange mass spectrometry, we found that the dynamics of the WDR44-Rab11 interface are distinct from the Rab11 effector FIP3, with WDR44 forming a more extensive interface with the switch II helix of Rab11 compared with FIP3. The WDR44 interaction was specific to Rab11 over evolutionarily similar Rabs, with mutations defining the molecular basis of Rab11 specificity. Finally, WDR44 can be phosphorylated by Sgk3, with this leading to reorganization of the Rab11-binding surface on WDR44. Overall, our results provide molecular detail on how WDR44 interacts with Rab11 and how Rab11 can form distinct effector complexes that regulate membrane trafficking events.

3-phosphoinositide-dependent kinase 1 (PDK1) is an essential serine/threonine protein kinase, which plays a crucial role in cell growth and proliferation. It is often referred to as a ‘master’ kinase due to its ability to activate at least 23 downstream protein kinases implicated in various signaling pathways. In this study, we have elucidated the mechanism of phosphoinositide-driven PDK1 auto-activation. We show that PDK1 trans-autophosphorylation is mediated by a PIP3-mediated face-to-face dimer. We report regulatory motifs in the kinase-PH interdomain linker that allosterically activate PDK1 autophosphorylation via a linker-swapped dimer mechanism. Finally, we show that PDK1 is autoinhibited by its PH domain and that positive cooperativity of PIP3 binding drives switch-like activation of PDK1. These results imply that the PDK1-mediated activation of effector kinases, including Akt, PKC, Sgk, S6K and RSK, many of whom are not directly regulated by phosphoinositides, is also likely to be dependent on PIP3 or PI(3,4)P2.

3-phosphoinositide-dependent kinase 1 (PDK1) is an essential serine/threonine protein kinase, which plays a crucial role in cell growth and proliferation. It is often referred to as a ‘master’ kinase due to its ability to activate at least 23 downstream protein kinases implicated in various signaling pathways. In this study, we have elucidated the mechanism of phosphoinositide-driven PDK1 auto-activation. We show that PDK1 trans- autophosphorylation is mediated by a PIP3-mediated face-to-face dimer. We report regulatory motifs in the kinase-PH interdomain linker that allosterically activate PDK1 autophosphorylation via a linker-swapped dimer mechanism. Finally, we show that PDK1 is autoinhibited by its PH domain and that positive cooperativity of PIP3binding drives switch- like activation of PDK1. Our work implies that the PDK1-mediated activation of effector kinases, including Akt, PKC, Sgk, S6K and RSK, many of whom are not directly regulated by phosphoinositides, is also likely to be dependent on PIP3or PI(3, 4)P2.

Akkermansia muciniphila is key member of the human gut microbiota that impacts many features of host health. A major characteristic of this bacterium is its interaction with host mucin, which is abundant in the gut environment, and its ability to metabolize mucin as a nutrient source. The machinery deployed by A. muciniphila to enable this interaction appears to be extensive and sophisticated, yet it is incompletely defined. The uncharacterized protein AMUC_1438 is encoded by a gene that was previously shown to be upregulated when the bacterium is grown on mucin. This uncharacterized protein has features suggestive of carbohydrate-recognition and peptidase activity, which led us to hypothesize that it has a role in mucin depolymerization. Here, we provide structural and functional support for the assignment of AMUC_1438 as a unique O-glycopeptidase with mucin-degrading capacity. O-glycopeptidase enzymes recognize glycans but hydrolyze the peptide backbone and are common in host-adapted microbes that colonize or invade mucus layers. Structural, kinetic, and mutagenic analyses point to a metzincin metalloprotease catalytic motif but with an active site that specifically recognizes a GalNAc residue α-linked to serine or threonine (i.e., the Tn-antigen). The enzyme catalyzes hydrolysis of the bond immediately N-terminal to the glycosylated residue. Additional modeling analyses suggest the presence of a carbohydrate-binding module that may assist in substrate recognition. We anticipate that these results will be fundamental to a wider understanding of the O-glycopeptidase class of enzymes and how they may contribute to host adaptation.

Significance Akt is an essential protein kinase that controls cell growth, survival, and metabolism. Akt is activated by the lipid second messengers PIP3 and PI(3,4)P2 and by phosphorylation. However, the relative contributions of lipid binding and phosphorylation to Akt activity in the cell are controversial. Here, we have determined the structure of autoinhibited Akt1, which reveals how the lipid-binding PH domain maintains the kinase domain in an inactive conformation in the absence of PIP3. Despite stoichiometric phosphorylation, Akt adopts an autoinhibited conformation with low basal activity in the absence of PIP3. Our work reveals the mechanistic basis of Akt hyperactivation in cancer and overgrowth diseases and unambiguously establishes that Akt depends on lipids for activity in the cell., The protein kinase Akt is one of the primary effectors of growth factor signaling in the cell. Akt responds specifically to the lipid second messengers phosphatidylinositol-3,4,5-trisphosphate [PI(3,4,5)P3] and phosphatidylinositol-3,4-bisphosphate [PI(3,4)P2] via its PH domain, leading to phosphorylation of its activation loop and the hydrophobic motif of its kinase domain, which are critical for activity. We have now determined the crystal structure of Akt1, revealing an autoinhibitory interface between the PH and kinase domains that is often mutated in cancer and overgrowth disorders. This interface persists even after stoichiometric phosphorylation, thereby restricting maximum Akt activity to PI(3,4,5)P3- or PI(3,4)P2-containing membranes. Our work helps to resolve the roles of lipids and phosphorylation in the activation of Akt and has wide implications for the spatiotemporal control of Akt and potentially lipid-activated kinase signaling in general.

The protein kinase Akt is one of the primary effectors of growth factor signaling in the cell. Akt responds specifically to the lipid second messengers phosphatidylinositol-3,4,5-trisphosphate [PI(3,4,5)P3] and phosphatidylinositol-3,4-bisphosphate [PI(3,4)P2] via its PH domain, leading to phosphorylation of its activation loop and the hydrophobic motif of its kinase domain, which are critical for activity. We have now determined the crystal structure of Akt1, revealing an autoinhibitory interface between the PH and kinase domains that is often mutated in cancer and overgrowth disorders. This interface persists even after stoichiometric phosphorylation, thereby restricting maximum Akt activity to PI(3,4,5)P3- or PI(3,4)P2-containing membranes. Our work helps to resolve the roles of lipids and phosphorylation in the activation of Akt and has wide implications for the spatiotemporal control of Akt and potentially lipid-activated kinase signaling in general.

Phospholipid synthesis and fat storage as triglycerides are regulated by lipin phosphatidic acid phosphatases (PAPs), whose enzymatic PAP function requires association with cellular membranes. Using hydrogen deuterium exchange mass spectrometry, we find mouse lipin 1 binds membranes through an N-terminal amphipathic helix, the Ig-like domain and HAD phosphatase catalytic core, and a middle lipin (M-Lip) domain that is conserved in mammalian and mammalian-like lipins. Crystal structures of the M-Lip domain reveal a previously unrecognized protein fold that dimerizes. The isolated M-Lip domain binds membranes both in vitro and in cells through conserved basic and hydrophobic residues. Deletion of the M-Lip domain in lipin 1 reduces PAP activity, membrane association, and oligomerization, alters subcellular localization, diminishes acceleration of adipocyte differentiation, but does not affect transcriptional co-activation. This establishes the M-Lip domain as a dimeric protein fold that binds membranes and is critical for full functionality of mammalian lipins., Lipins need to bind cell membranes before they can function as phosphatidic acid phosphatases. Here, the authors elucidate the structural basis of lipin membrane-association and identify a lipin domain with a novel protein fold that is critical for membrane binding and full functionality of lipins.

The structure of class IB PI3K (p110γ-p101) reveals mechanism of GPCR activation and differences from class IA PI3Ks., 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 Gβγ-binding domain, recruiting the heterodimer to the membrane and allowing for engagement of a secondary Gβγ-binding site in p110γ. Mutations at the p110γ-p101 and p110γ–adaptor binding domain interfaces enhanced Gβγ activation. A nanobody that specifically binds to the p101-Gβγ interface blocks activation, providing a novel tool to study and target p110γ-p101–specific signaling events in vivo.

Calcineurin, the conserved protein phosphatase and target of immunosuppressants, is a critical mediator of Ca2+ signaling. To discover novel calcineurin-regulated processes we examined an understudied isoform, CNAβ1. We show that unlike canonical cytosolic calcineurin, CNAβ1 localizes to the plasma membrane and Golgi due to palmitoylation of its divergent C-terminal tail, which is reversed by the ABHD17A depalmitoylase. Palmitoylation targets CNAβ1 to a distinct set of membrane-associated interactors including the phosphatidylinositol 4-kinase (PI4KA) complex containing EFR3B, PI4KA, TTC7B and FAM126A. Hydrogen-deuterium exchange reveals multiple calcineurin-PI4KA complex contacts, including a calcineurin-binding peptide motif in the disordered tail of FAM126A, which we establish as a calcineurin substrate. Calcineurin inhibitors decrease PI4P production during Gq-coupled GPCR signaling, suggesting that calcineurin dephosphorylates and promotes PI4KA complex activity. In sum, this work discovers a new calcineurin-regulated signaling pathway highlighting the PI4KA complex as a regulatory target and revealing that dynamic palmitoylation confers unique localization, substrate specificity and regulation to CNAβ1.

CNNM/CorB proteins are a broadly conserved family of integral membrane proteins with close to 90,000 protein sequences known. They are associated with Mg2+ transport but it is not known if they mediate transport themselves or regulate other transporters. Here, we determine the crystal structure of an archaeal CorB protein in two conformations (apo and Mg2+-ATP bound). The transmembrane DUF21 domain exists in an inward-facing conformation with a Mg2+ ion coordinated by a conserved π-helix. In the absence of Mg2+-ATP, the CBS-pair domain adopts an elongated dimeric configuration with previously unobserved domain-domain contacts. Hydrogen-deuterium exchange mass spectrometry, analytical ultracentrifugation, and molecular dynamics experiments support a role of the structural rearrangements in mediating Mg2+-ATP sensing. Lastly, we use an in vitro, liposome-based assay to demonstrate direct Mg2+ transport by CorB proteins. These structural and functional insights provide a framework for understanding function of CNNMs in Mg2+ transport and associated diseases., CNNM/CorB proteins are a conserved family of membrane proteins associated with Mg2+ transport. Here, structures of an archaeal CorB protein in apo state and with Mg2+-ATP bound and accompanying biophysical experiments suggest direct Mg2+ transport by CorB proteins.

DeepMind presented remarkably accurate protein structure predictions at the CASP14 conference. We explored network architectures incorporating related ideas and obtained the best performance with a 3-track network in which information at the 1D sequence level, the 2D distance map level, and the 3D coordinate level is successively transformed and integrated. The 3-track network produces structure predictions with accuracies approaching those of DeepMind in CASP14, enables rapid solution of challenging X-ray crystallography and cryo-EM structure modeling problems, and provides insights into the functions of proteins of currently unknown structure. The network also enables rapid generation of accurate models of protein-protein complexes from sequence information alone, short circuiting traditional approaches which require modeling of individual subunits followed by docking. We make the method available to the scientific community to speed biological research.One-Sentence SummaryAccurate protein structure modeling enables rapid solution of structure determination problems and provides insights into biological function.