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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.

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.

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 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.

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.

Serum- and glucocorticoid-regulated kinase 3 (Sgk3) is a serine/threonine protein kinase activated by the phospholipid phosphatidylinositol 3-phosphate (PI3P) downstream of growth factor signaling via class I phosphatidylinositol 3-kinase (PI3K) signaling and by class III PI3K/Vps34-mediated PI3P production on endosomes. Upregulation of Sgk3 activity has recently been linked to a number of human cancers; however, the precise mechanism of activation of Sgk3 is unknown. Here, we use a wide range of cell biological, biochemical, and biophysical techniques, including hydrogen–deuterium exchange mass spectrometry, to investigate the mechanism of activation of Sgk3 by PI3P. We show that Sgk3 is regulated by a combination of phosphorylation and allosteric activation. We demonstrate that binding of Sgk3 to PI3P via its regulatory phox homology (PX) domain induces large conformational changes in Sgk3 associated with its activation and that the PI3P-binding pocket of the PX domain of Sgk3 is sequestered in its inactive conformation. Finally, we reconstitute Sgk3 activation via Vps34-mediated PI3P synthesis on phosphatidylinositol liposomes in vitro. In addition to identifying the mechanism of Sgk3 activation by PI3P, our findings open up potential therapeutic avenues in allosteric inhibitor development to target Sgk3 in cancer.

Transport Protein Particle complexes (TRAPP) are evolutionarily conserved regulators of membrane trafficking, with this mediated by their guanine nucleotide exchange factor (GEF) activity towards Rab GTPases. In metazoans evidence suggests that two different TRAPP complexes exist, TRAPPII and TRAPPIII. These two complexes share a common core of subunits, with complex specific subunits (TRAPPC9 and TRAPPC10 in TRAPPII and TRAPPC8, TRAPPC11, TRAPPC12, TRAPPC13 in TRAPPIII). TRAPPII and TRAPPIII have distinct specificity for GEF activity towards Rabs, with TRAPPIII acting on Rab1, and TRAPPII acting on Rab1 and Rab11. The molecular basis for how these complex specific subunits alter GEF activity towards Rab GTPases is unknown. Here we have used a combination of biochemical assays, hydrogen deuterium exchange mass spectrometry (HDX-MS) and electron microscopy to examine the regulation of TRAPPII and TRAPPIIII complexes in solution and on membranes. GEF assays revealed that the TRAPPIII has GEF activity against Rab1 and Rab43, with no detectable activity against the other 18 Rabs tested. The TRAPPIII complex had significant differences in protein dynamics at the Rab binding site compared to TRAPPII, potentially indicating an important role of accessory subunits in altering the active site of TRAPP complexes. Both the TRAPPII and TRAPPIII complexes had enhanced GEF activity on lipid membranes, with HDX-MS revealing numerous conformational changes that accompany membrane association. HDX-MS also identified a membrane binding site in TRAPPC8. Collectively, our results provide insight into the functions of TRAPP complexes and how they can achieve Rab specificity.

SummarySerum- and glucocorticoid-regulated kinase 3 (Sgk3) is activated by the phospholipid phosphatidylinositol-3-phosphate (PI3P) downstream of growth factor signaling and by Vps34-mediated PI3P production on endosomes. Upregulation of Sgk3 activity has recently been linked to a number of human cancers. Here, we show that Sgk3 is regulated by a combination of phosphorylation and allosteric activation by PI3P. We demonstrate that PI3P binding induces large conformational changes in Sgk3 associated with its activation, and that the PI3P binding pocket of the PX domain of Sgk3 is sequestered in its inactive conformation. Finally, we reconstituted Sgk3 activation via Vps34-mediated PI3P synthesis on phosphatidylinositol liposomesin vitro. In addition to defining the mechanism of Sgk3 activation by PI3P, our findings open up potential therapeutic avenues in allosteric inhibitor development to target Sgk3 in cancer.

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-kinases (PI3Ks) are master regulators of cellular functions, with the p110γ subunit playing a key role in immune signalling. PI3Kγ 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 small molecule inhibitors using HDX-MS revealed that activation loop binding inhibitors induce allosteric conformational changes that mimic those seen for the R1021C mutant. Structural analysis of clinically advanced PI3K inhibitors revealed novel binding pockets that can be exploited for further therapeutic development. Overall this work provides unique insight into the regulatory mechanisms that control PI3Kγ kinase activity, and shows a framework for the design of PI3K isoform and mutant selective inhibitors.

The TRAnsport Protein Particle (TRAPP) complexes act as Guanine nucleotide exchange factors (GEFs) for Rab GTPases, which are master regulators of membrane trafficking in eukaryotic cells. In metazoans, there are two large multi-protein TRAPP complexes: TRAPPII and TRAPPIII, with the TRAPPII complex able to activate both Rab1 and Rab11. Here we present detailed biochemical characterisation of Rab-GEF specificity of the human TRAPPII complex, and molecular insight into Rab binding. GEF assays of the TRAPPII complex against a panel of 20 different Rab GTPases revealed GEF activity on Rab43 and Rab19. Electron microscopy and chemical cross-linking revealed the architecture of mammalian TRAPPII. Hydrogen deuterium exchange MS showed that Rab1, Rab11 and Rab43 share a conserved binding interface. Clinical mutations in Rab11, and phosphomimics of Rab43, showed decreased TRAPPII GEF mediated exchange. Finally, we designed a Rab11 mutation that maintained TRAPPII-mediated GEF activity while decreasing activity of the Rab11-GEF SH3BP5, providing a tool to dissect Rab11 signalling. Overall, our results provide insight into the GTPase specificity of TRAPPII, and how clinical mutations disrupt this regulation., Here the authors reveal unique structural organization of the mammalian TRAPPII complex, which is critical in regulating membrane trafficking. They find that TRAPPII serves as a guanine nucleotide exchange factor for unexpected Rab GTPases such as Rab43 and Rab19.

Lipin/Pah phosphatidic acid phosphatases (PAPs) generate diacylglycerol to regulate triglyceride synthesis and cellular signaling. Inactivating mutations cause rhabdomyolysis, autoinflammatory disease, and aberrant fat storage. Disease-mutations cluster within the conserved N-Lip and C-Lip regions that are separated by 500-residues in humans. To understand how the N-Lip and C-Lip combine for PAP function, we determined crystal structures of Tetrahymena thermophila Pah2 (Tt Pah2) that directly fuses the N-Lip and C-Lip. Tt Pah2 adopts a two-domain architecture where the N-Lip combines with part of the C-Lip to form an immunoglobulin-like domain and the remaining C-Lip forms a HAD-like catalytic domain. An N-Lip C-Lip fusion of mouse lipin-2 is catalytically active, which suggests mammalian lipins function with the same domain architecture as Tt Pah2. HDX-MS identifies an N-terminal amphipathic helix essential for membrane association. Disease-mutations disrupt catalysis or destabilize the protein fold. This illustrates mechanisms for lipin/Pah PAP function, membrane association, and lipin-related pathologies., Lipin/Pah phosphatidic acid phosphatases generate diacylglycerol to regulate triglyceride synthesis and cellular signaling. Here authors determine structures of Tetrahymena thermophila Pah2 and identify an N-terminal amphipathic helix essential for membrane association.

Phosphoinositides are lipid signaling molecules that act as master regulators of cellular signaling. Recent studies have revealed novel roles of phosphoinositides in myriad cellular processes, and multiple human diseases mediated by misregulation of phosphoinositide signaling. This review will present a timely summary of recent discoveries in phosphoinositide biology, specifically their role in regulating unexpected signaling pathways, modification of signaling outcomes downstream of integral membrane proteins, and novel roles in lipid transport. This has revealed new roles of phosphoinositides in regulating membrane trafficking, immunity, cell polarity, and response to extracellular signals. A specific focus will be on novel opportunities to target phosphoinositide metabolism for treatment of human diseases, including cancer, pathogen infection, developmental disorders, and immune disorders.

The lipid kinases that generate the lipid signalling phosphoinositides have been established as fundamental signalling enzymes that control numerous aspects of how cells respond to their extracellular environment. In addition, they play critical roles in regulating membrane trafficking and lipid transport within the cell. The class I phosphoinositide kinases which generate the critical lipid signal PIP3 are hyperactivated in numerous human pathologies including cancer, overgrowth syndromes, and primary immunodeficiencies. The type III phosphatidylinositol 4-kinase beta isoform (PI4KB), which are evolutionarily similar to the class I PI3Ks, have been found to be essential host factors mediating the replication of numerous devastating pathogenic viruses. Finally, targeting the parasite variant of PI4KB has been established as one of the most promising strategies for the development of anti-malarial and anti-cryptosporidium strategies. Therefore, the development of targeted isoform selective inhibitors for these enzymes are of paramount importance. The first generation of PI3K inhibitors have recently been clinically approved for a number of different cancers, highlighting their therapeutic value. This review will examine the history of the class I PI3Ks, and the type III PI4Ks, their relevance to human disease, and the structural basis for their regulation and inhibition by potent and selective inhibitors.

Sphingosine kinase 1 (SK1) is required for production of sphingosine-1-phosphate (S1P) and thereby regulates many cellular processes, including cellular growth, immune cell trafficking, and inflammation. To produce S1P, SK1 must access sphingosine directly from membranes. However, the molecular mechanisms underlying SK1's direct membrane interactions remain unclear. We used hydrogen/deuterium exchange MS to study interactions of SK1 with membrane vesicles. Using the CRISPR/Cas9 technique to generate HCT116 cells lacking SK1, we explored the effects of membrane interface disruption and the function of the SK1 interaction site. Disrupting the interface resulted in reduced membrane association and decreased cellular SK1 activity. Moreover, SK1-dependent signaling, including cell invasion and endocytosis, was abolished upon mutation of the membrane-binding interface. Of note, we identified a positively charged motif on SK1 that is responsible for electrostatic interactions with membranes. Furthermore, we demonstrated that SK1 uses a single contiguous interface, consisting of an electrostatic site and a hydrophobic site, to interact with membrane-associated anionic phospholipids. Altogether, these results define a composite domain in SK1 that regulates its intrinsic ability to bind membranes and indicate that this binding is critical for proper SK1 function. This work will allow for a new line of thinking for targeting SK1 in disease.

Significance Akt is a paradigmatic lipid-activated kinase, which is frequently hyperactivated in human cancer. In the absence of PI(3,4,5)P3 or PI(3,4)P2, Akt is maintained in an inactive conformation by an inhibitory interaction between its membrane-binding PH domain and its kinase domain. Here, we describe the conformational changes associated with its binding to PI(3,4,5)P3, leading to disruption of the inhibitory PH−kinase interface, and its consequent activation by protein kinases. Intriguingly, we find that reversal of those conformational changes promotes its inactivation by protein phosphatases. The activation of Akt is thereby restricted to discrete membrane locations, and it is rapidly inactivated upon dissociation. We propose a model in which activation, substrate phosphorylation, and inactivation of Akt are tightly coupled to the membrane., 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.

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.

Apicomplexan parasites such as Toxoplasma gondii rely on a unique form of locomotion known as gliding motility. Generating the mechanical forces to support motility are divergent class XIV myosins (MyoA) coordinated by accessory proteins known as light chains. Although the importance of the MyoA–light chain complex is well-established, the detailed mechanisms governing its assembly and regulation are relatively unknown. To establish a molecular blueprint of this dynamic complex, we first mapped the adjacent binding sites of light chains MLC1 and ELC1 on the MyoA neck (residues 775–818) using a combination of hydrogen–deuterium exchange mass spectrometry and isothermal titration calorimetry. We then determined the 1.85 Å resolution crystal structure of MLC1 in complex with its cognate MyoA peptide. Structural analysis revealed a bilobed architecture with MLC1 clamping tightly around the helical MyoA peptide, consistent with the stable 10 nm Kd measured by isothermal titration calorimetry. We next showed that coordination of calcium by an EF-hand in ELC1 and prebinding of MLC1 to the MyoA neck enhanced the affinity of ELC1 for the MyoA neck 7- and 8-fold, respectively. When combined, these factors enhanced ELC1 binding 49-fold (to a Kd of 12 nm). Using the full-length MyoA motor (residues 1–831), we then showed that, in addition to coordinating the neck region, ELC1 appears to engage the MyoA converter subdomain, which couples the motor domain to the neck. These data support an assembly model where staged binding events cooperate to yield high-affinity complexes that are able to maximize force transduction.

Phospholipase C (PLC) enzymes hydrolyse phosphoinositide lipids to inositol phosphates and diacylglycerol. Direct activation of PLCβ by Gαqand/or Gβγ subunits mediates signalling by Gq and some Gi coupled G protein-coupled receptors (GPCRs), respectively. PLCβ isoforms contain a unique C-terminal extension, consisting of proximal and distal C-terminal domains (CTD) separated by a flexible linker. The structure of PLCβ3 bound to Gαqis known, however, for both Gαqand Gβγ, the mechanism for PLCβ activation on membranes is unknown. We examined PLCβ2 dynamics on membranes using hydrogen deuterium exchange mass spectrometry (HDX-MS). Gβγ caused a robust increase in dynamics of the distal C-terminal domain (CTD). Gαqshowed decreased deuterium incorporation at the Gαqbinding site on PLCβ.In vitroGβγ-dependent activation of PLC is inhibited by the distal CTD. The results suggest that disruption of auto-inhibitory interactions with the CTD, respectively, leads to increased PLCβ hydrolase activity.

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, p110β, 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.

SummaryThe class IA PI3Ks are key signalling 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 byPIK3R1can 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 activate both the p110α/p110δ catalytic subunits. We find that the oncogenic Q572* truncation ofPIK3R1disrupts all inhibitory inputs, with p110α being hyper-activated compared to p110δ. In addition, we find that the R649W mutation in the cSH2 ofPIK3R1decreases sensitivity to activation by receptor tyrosine kinases. This work reveals novel insight into isoform specific regulation of p110s by p85α.Highlights- An oncogenic variant of p85α, Q572*, leads to hyper-activation of p110α compared to p110δ- HDX-MS revealed that Q572* leads to disruption of all inhibitory interfaces in p110α- A SHORT syndrome mutation in p85α leads to decreased sensitivity to RTKs for p110α/δ

SummaryThe lipid kinase PI4KB, which generates phosphatidylinositol 4-phosphate (PI4P), is a key enzyme in regulating membrane transport and is also hijacked by multiple picornaviruses to mediate viral replication. PI4KB can interact with multiple protein binding partners, which are differentially manipulated by picornaviruses to facilitate replication. The protein c10orf76 is a PI4KB-associated protein that increases PI4P levels at the Golgi, and is essential for the viral replication of specific enteroviruses. We used hydrogen deuterium exchange mass spectrometry to characterize the c10orf76-PI4KB complex and reveal that binding is mediated by the kinase linker of PI4KB, with formation of the heterodimeric complex modulated by PKA-dependent phosphorylation. Complex-disrupting mutations demonstrate that PI4KB is required for membrane recruitment of c10orf76 to the Golgi, and that an intact c10orf76-PI4KB complex is required for the replication of c10orf76-dependent enteroviruses. Intriguingly, c10orf76 was also required for proper Arf1 activation at the Golgi, providing a putative mechanism for the c10orf76-dependent increase in PI4P levels at the Golgi.Highlightsc10orf76 forms a direct complex with PI4KB, with the interface formed by a disorder-to-order transition in the kinase linker of PI4KBThe c10orf76 binding site of PI4KB can be phosphorylated by PKA, with phosphorylation leading to decreased affinity for c10orf76Complex-disrupting mutants of PI4KB and c10orf76 reveal that PI4KB recruits c10orf76 to the Golgi/TGNDepletion of c10orf76 leads to decreases in both active Arf1 and Golgi PI4P levelsEnteroviruses that rely on c10orf76 for replication depend on formation of the c10orf76-PI4KB complex

Neutral sphingomyelinase 2 (nSMase2) produces the bioactive lipid ceramide and has important roles in neurodegeneration, cancer, and exosome formation. Although nSMase2 has low basal activity, it is fully activated by phosphatidylserine (PS). Previous work showed that interdomain interactions within nSMase2 are needed for PS activation. Here, we use multiple approaches, including small angle X-ray scattering, hydrogen–deuterium exchange–MS, circular dichroism and thermal shift assays, and membrane yeast two-hybrid assays, to define the mechanism mediating this interdomain interactions within nSMase2. In contrast to what we previously assumed, we demonstrate that PS binding at the N-terminal and juxtamembrane regions of nSMase2 rather acts as a conformational switch leading to interdomain interactions that are critical to enzyme activation. Our work assigns a unique function for a class of linkers of lipid-activated, membrane-associated proteins. It indicates that the linker actively participates in the activation mechanism via intramolecular interactions, unlike the canonical linkers that typically aid protein dimerization or localization.

Since I started doing scientific research, I've been fascinated by the interplay of protein structure and dynamics and how they together mediate protein function. A particular area of interest has been in understanding the mechanistic basis of how lipid-signaling enzymes function on membrane surfaces. In this award lecture article, I will describe my laboratory's studies on the structure and dynamics of lipid-signaling enzymes on membrane surfaces. This is important, as many lipid-signaling enzymes are regulated through dynamic regulatory mechanisms that control their enzymatic activity. This article will discuss my continued enthusiasm in using a synergistic application of hydrogen–deuterium exchange MS (HDX–MS) with other structural biology techniques to probe the mechanistic basis for how membrane-localized signaling enzymes are regulated and how these approaches can be used to understand how they are misregulated in disease. I will discuss specific examples of how we have used HDX–MS to study phosphoinositide kinases and the protein kinase Akt. An important focus will be on a description of how HDX–MS can be used as a powerful tool to optimize the design of constructs for X-ray crystallography and EM. The use of a diverse toolbox of biophysical methods has revealed novel insight into the complex and varied regulatory networks that control the function of lipid-signaling enzymes and enabled unique insight into the mechanics of membrane recruitment.

Phosphoinositide 3-kinase β (PI3Kβ) is regulated by receptor tyrosine kinases (RTKs), G protein–coupled receptors (GPCRs), and small GTPases such as Rac1 and Rab5. Our lab previously identified two residues (Gln(596) and Ile(597)) in the helical domain of the catalytic subunit (p110β) of PI3Kβ whose mutation disrupts binding to Rab5. To better define the Rab5–p110β interface, we performed alanine-scanning mutagenesis and analyzed Rab5 binding with an in vitro pulldown assay with GST-Rab5(GTP). Of the 35 p110β helical domain mutants assayed, 11 disrupted binding to Rab5 without affecting Rac1 binding, basal lipid kinase activity, or Gβγ-stimulated kinase activity. These mutants defined the Rab5-binding interface within p110β as consisting of two perpendicular α-helices in the helical domain that are adjacent to the initially identified Gln(596) and Ile(597) residues. Analysis of the Rab5–PI3Kβ interaction by hydrogen-deuterium exchange MS identified p110β peptides that overlap with these helices; no interactions were detected between Rab5 and other regions of p110β or p85α. Similarly, the binding of Rab5 to isolated p85α could not be detected, and mutations in the Ras-binding domain (RBD) of p110β had no effect on Rab5 binding. Whereas soluble Rab5 did not affect PI3Kβ activity in vitro, the interaction of these two proteins was critical for chemotaxis, invasion, and gelatin degradation by breast cancer cells. Our results define a single, discrete Rab5-binding site in the p110β helical domain, which may be useful for generating inhibitors to better define the physiological role of Rab5–PI3Kβ coupling in vivo.

The lipid kinase PI4KB, which generates phosphatidylinositol 4-phosphate (PI4P), is a key enzyme in regulating membrane transport and is also hijacked by multiple picornaviruses to mediate viral replication. PI4KB can interact with multiple protein binding partners, which are differentially manipulated by picornaviruses to facilitate replication. The protein c10orf76 is a PI4KB-associated protein that increases PI4P levels at the Golgi and is essential for the viral replication of specific enteroviruses. We used hydrogen-deuterium exchange mass spectrometry to characterize the c10orf76-PI4KB complex and reveal that binding is mediated by the kinase linker of PI4KB, with formation of the heterodimeric complex modulated by PKA-dependent phosphorylation. Complex-disrupting mutations demonstrate that PI4KB is required for membrane recruitment of c10orf76 to the Golgi, and that an intact c10orf76-PI4KB complex is required for the replication of c10orf76-dependent enteroviruses. Intriguingly, c10orf76 also contributed to proper Arf1 activation at the Golgi, providing a putative mechanism for the c10orf76-dependent increase in PI4P levels at the Golgi.

Aberrant glycosylation and the overexpression of specific carbohydrate epitopes is a hallmark of many cancers, and tumor-associated oligosaccharides are actively investigated as targets for immunotherapy and diagnostics. Wisteria floribunda agglutinin (WFA) is a legume lectin that recognizes terminal N-acetylgalactosaminides with high affinity. WFA preferentially binds the disaccharide LacdiNAc (β-d-GalNAc-[1→4]-d-GlcNAc), which is associated with tumor malignancy in leukemia, prostate, pancreatic, ovarian, and liver cancers and has shown promise in cancer glycobiomarker detection. The mechanism of specificity for WFA recognition of LacdiNAc is not fully understood. To address this problem, we have determined affinities and structure of WFA in complex with GalNAc and LacdiNAc. Affinities toward Gal, GalNAc, and LacdiNAc were measured via surface plasmon resonance, yielding KD values of 4.67 × 10−4 m, 9.24 × 10−5 m, and 5.45 × 10−6 m, respectively. Structures of WFA in complex with LacdiNAc and GalNAc have been determined to 1.80–2.32 Å resolution. These high resolution structures revealed a hydrophobic groove complementary to the GalNAc and, to a minor extent, to the back-face of the GlcNAc sugar ring. Remarkably, the contribution of this small hydrophobic surface significantly increases the observed affinity for LacdiNAc over GalNAc. Tandem MS sequencing confirmed the presence of two isolectin forms in commercially available WFA differing only in the identities of two amino acids. Finally, the WFA carbohydrate binding site is similar to a homologous lectin isolated from Vatairea macrocarpa in complex with GalNAc, which, unlike WFA, binds not only αGalNAc but also terminal Ser/Thr O-linked αGalNAc (Tn antigen).

In chemotaxing ameboid cells, a complex leading-edge signaling circuit forms on the cytoplasmic leaflet of the plasma membrane and directs both actin and membrane remodeling to propel the leading edge up an attractant gradient. This leading-edge circuit includes a putative amplification module in which Ca2+-protein kinase C (Ca2+-PKC) is hypothesized to phosphorylate myristoylated alanine-rich C kinase substrate (MARCKS) and release phosphatidylinositol-4,5-bisphosphate (PIP2), thereby stimulating production of the signaling lipid phosphatidylinositol-3,4,5-trisphosphate (PIP3) by the lipid kinase phosphoinositide-3-kinase (PI3K). We investigated this hypothesized Ca2+-PKC-MARCKS-PIP2-PI3K-PIP3 amplification module and tested its key predictions using single-molecule fluorescence to measure the surface densities and activities of its protein components. Our findings demonstrate that together Ca2+-PKC and the PIP2-binding peptide of MARCKS modulate the level of free PIP2, which serves as both a docking target and substrate lipid for PI3K. In the off state of the amplification module, the MARCKS peptide sequesters PIP2 and thereby inhibits PI3K binding to the membrane. In the on state, Ca2+-PKC phosphorylation of the MARCKS peptide reverses the PIP2 sequestration, thereby releasing multiple PIP2 molecules that recruit multiple active PI3K molecules to the membrane surface. These findings 1) show that the Ca2+-PKC-MARCKS-PIP2-PI3K-PIP3 system functions as an activation module in vitro, 2) reveal the molecular mechanism of activation, 3) are consistent with available in vivo data, and 4) yield additional predictions that are testable in live cells. More broadly, the Ca2+-PKC-stimulated release of free PIP2 may well regulate the membrane association of other PIP2-binding proteins, and the findings illustrate the power of single-molecule analysis to elucidate key dynamic and mechanistic features of multiprotein signaling pathways on membrane surfaces.

Type III Phosphatidylinositol 4-kinase (PI4KIIIβ) is an essential enzyme in mediating membrane trafficking, and is implicated in a variety of pathogenic processes. It is a key host factor mediating replication of RNA viruses. The design of potent and specific inhibitors of this enzyme will be essential to define its cellular roles, and may lead to novel anti-viral therapeutics. We previously reported the PI4K inhibitor PIK93, and this compound has defined key functions of PI4KIIIβ. However, this compound showed high cross reactivity with class I and III PI3Ks. Using structure-based drug design we have designed novel potent and selective (>1000 fold over class I and class III PI3Ks) PI4KIIIβ inhibitors. These compounds showed anti-viral activity against Hepatitis C Virus. The co-crystal structure of PI4KIIIβ bound to one of the most potent compounds reveals the molecular basis of specificity. This work will be vital in the design of novel PI4KIIIβ inhibitors, which may play significant roles as anti-viral therapeutics.

Many important cellular functions are regulated by the selective recruitment of proteins to intracellular membranes mediated by specific interactions with lipid phosphoinositides. The enzymes that generate lipid phosphoinositides therefore must be properly positioned and regulated at their correct cellular locations. Phosphatidylinositol 4 kinases (PI4Ks) are key lipid signalling enzymes, and they generate the lipid species phosphatidylinositol 4-phosphate (PI4P), which plays important roles in regulating physiological processes including membrane trafficking, cytokinesis and organelle identity. PI4P also acts as the substrate for the generation of the signalling phosphoinositides phosphatidylinositol 4,5-bisphosphate (PIP2) and phosphatidylinositol 3,4,5-trisphosphate (PIP3). PI4Ks also play critical roles in a number of pathological processes including mediating replication of a number of pathogenic RNA viruses, and in the development of the parasite responsible for malaria. Key to the regulation of PI4Ks is their regulation by a variety of both host and viral protein-binding partners. We review herein our current understanding of the structure, regulatory interactions and role in disease of the type III PI4Ks. * ACBD3, : acyl-CoA-binding domain containing protein 3; NCS-1, : neuronal calcium sensor 1; NS5A, : non-structural protein 5A; PI4K, : phosphatidylinositol 4 kinase; PI4KIIIα/β, : type III phosphatidylinositol 4 kinase α/β; PI4P, : phosphatidylinositol 4-phosphate; PIP2, : phosphatidylinositol 4,5-bisphosphate; PIP3, : phosphatidylinositol 3,4,5-trisphosphate; TGN, : trans -Golgi network; TTC7, : tetratricopeptide repeat protein 7

The phosphatidylinositol 3-kinase Vps34 is part of several protein complexes. The structural organization of heterotetrameric complexes is starting to emerge, but little is known about organization of additional accessory subunits that interact with these assemblies. Combining hydrogen-deuterium exchange mass spectrometry (HDX-MS), X-ray crystallography and electron microscopy (EM), we have characterized Atg38 and its human ortholog NRBF2, accessory components of complex I consisting of Vps15-Vps34-Vps30/Atg6-Atg14 (yeast) and PIK3R4/VPS15-PIK3C3/VPS34-BECN1/Beclin 1-ATG14 (human). HDX-MS shows that Atg38 binds the Vps30-Atg14 subcomplex of complex I, using mainly its N-terminal MIT domain and bridges the coiled-coil I regions of Atg14 and Vps30 in the base of complex I. The Atg38 C-terminal domain is important for localization to the phagophore assembly site (PAS) and homodimerization. Our 2.2 Å resolution crystal structure of the Atg38 C-terminal homodimerization domain shows 2 segments of α-helices assembling into a mushroom-like asymmetric homodimer with a 4-helix cap and a parallel coiled-coil stalk. One Atg38 homodimer engages a single complex I. This is in sharp contrast to human NRBF2, which also forms a homodimer, but this homodimer can bridge 2 complex I assemblies.

Parasites of the phylum Apicomplexa are responsible for significant morbidity and mortality on a global scale. Central to the virulence of these pathogens are the phylum-specific, unconventional class XIV myosins that power the essential processes of parasite motility and host cell invasion. Notably, class XIV myosins differ from human myosins in key functional regions, yet they are capable of fast movement along actin filaments with kinetics rivaling previously studied myosins. Toward establishing a detailed molecular mechanism of class XIV motility, we determined the 2.6-Å resolution crystal structure of the Toxoplasma gondii MyoA (TgMyoA) motor domain. Structural analysis reveals intriguing strategies for force transduction and chemomechanical coupling that rely on a divergent SH1/SH2 region, the class-defining “HYAG”-site polymorphism, and the actin-binding surface. In vitro motility assays and hydrogen–deuterium exchange coupled with MS further reveal the mechanistic underpinnings of phosphorylation-dependent modulation of TgMyoA motility whereby localized regions of increased stability and order correlate with enhanced motility. Analysis of solvent-accessible pockets reveals striking differences between apicomplexan class XIV and human myosins. Extending these analyses to high-confidence homology models of Plasmodium and Cryptosporidium MyoA motor domains supports the intriguing potential of designing class-specific, yet broadly active, apicomplexan myosin inhibitors. The successful expression of the functional TgMyoA complex combined with our crystal structure of the motor domain provides a strong foundation in support of detailed structure–function studies and enables the development of small-molecule inhibitors targeting these devastating global pathogens.

Summary Phosphoinositides, diacylglycerolpyrophosphate, ceramide-1-phosphate, and phosphatidic acid belong to a unique class of membrane signaling lipids that contain phosphomonoesters in their headgroups having pKa values in the physiological range. The phosphomonoester headgroup of phosphatidic acid enables this lipid to act as a pH biosensor as changes in its protonation state with intracellular pH regulate binding to effector proteins. Here, we demonstrate that binding of pleckstrin homology (PH) domains to phosphatidylinositol 4-phosphate (PI4P) in the yeast trans-Golgi network (TGN) is dependent on intracellular pH, indicating PI4P is a pH biosensor. pH biosensing by TGN PI4P in response to nutrient availability governs protein sorting at the TGN, likely by regulating sterol transfer to the TGN by Osh1, a member of the conserved oxysterol-binding protein (OSBP) family of lipid transfer proteins. Thus, pH biosensing by TGN PI4P allows for direct metabolic regulation of protein trafficking and cell growth.

Fucoidans are chemically complex and highly heterogeneous sulfated marine fucans from brown macro algae. Possessing a variety of physicochemical and biological activities, fucoidans are used as gelling and thickening agents in the food industry and have anticoagulant, antiviral, antitumor, antibacterial, and immune activities. Although fucoidan-depolymerizing enzymes have been identified, the molecular basis of their activity on these chemically complex polysaccharides remains largely uninvestigated. In this study, we focused on three glycoside hydrolase family 107 (GH107) enzymes: MfFcnA and two newly identified members, P5AFcnA and P19DFcnA, from a bacterial species of the genus Psychromonas. Using carbohydrate-PAGE, we show that P5AFcnA and P19DFcnA are active on fucoidans that differ from those depolymerized by MfFcnA, revealing differential substrate specificity within the GH107 family. Using a combination of X-ray crystallography and NMR analyses, we further show that GH107 family enzymes share features of their structures and catalytic mechanisms with GH29 α-l-fucosidases. However, we found that GH107 enzymes have the distinction of utilizing a histidine side chain as the proposed acid/base catalyst in its retaining mechanism. Further interpretation of the structural data indicated that the active-site architectures within this family are highly variable, likely reflecting the specificity of GH107 enzymes for different fucoidan substructures. Together, these findings begin to illuminate the molecular details underpinning the biological processing of fucoidans.

Lipid phosphoinositides play fundamental roles in virtually all pathways that control a cell's decision to grow, move, divide, and die. Because of this, kinases that phosphorylate phosphoinositide lipids are critically involved in myriad essential functions including growth, development, and membrane trafficking. The misregulation of phosphoinositide kinases is critical in human diseases, including cancer, primary immunodeficiencies, and developmental disorders. Phosphoinositide kinases also play a role in mediating bacterial and viral infections for many potent human pathogens. Furthermore, inhibitors of parasite phosphoinositide kinases are in development as therapies for both malaria and cryptosporidiosis. Therefore, understanding how phosphoinositide kinases are regulated has implications for the treatment of many devastating human diseases. Recent structures of phosphoinositide kinases have revealed unique molecular insight into their regulation. This review will summarize our current molecular knowledge on phosphoinositide kinase regulation, and how this information is being used to generate novel small molecule inhibitors as potential therapeutics.

The signaling lipid phosphatidylinositol 3,4,5, trisphosphate (PIP3) is an essential mediator of many vital cellular processes, including growth, survival, and metabolism. PIP3 is generated through the action of the class I phosphoinositide 3-kinases (PI3K), and their activity is tightly controlled through interactions with regulatory proteins and activating stimuli. The class IA PI3Ks are composed of three distinct p110 catalytic subunits (p110α, p110β, and p110δ), and they play different roles in specific tissues due to disparities in both expression and engagement downstream of cell-surface receptors. Disruption of PI3K regulation is a frequent driver of numerous human diseases. Activating mutations in the PIK3CA gene encoding the p110α catalytic subunit of class IA PI3K are frequently mutated in several cancer types, and mutations in the PIK3CD gene encoding the p110δ catalytic subunit have been identified in primary immunodeficiency patients. All class IA p110 subunits interact with p85 regulatory subunits, and mutations/deletions in different p85 regulatory subunits have been identified in both cancer and primary immunodeficiencies. In this review, we will summarize our current understanding for the molecular basis of how class IA PI3K catalytic activity is regulated by p85 regulatory subunits, and how activating mutations in the PI3K catalytic subunits PIK3CA and PIK3CD (p110α, p110δ) and regulatory subunits PIK3R1 (p85α) mediate PI3K activation and human disease.

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 p110β. 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.

Group IVA cytosolic phospholipase A(2) (cPLA(2)alpha) catalyzes release of arachidonic acid from glycerophospholipids, leading to thromboxane A(2) (TxA(2)) production. Some platelet agonists stimulate cPLA(2)alpha, but others require fibrinogen binding to alphaIIbbeta3 to elicit TxA(2). Therefore, relationships between cPLA(2)alpha and alphaIIbbeta3 were examined. cPLA(2)alpha and a cPLA(2)alpha binding partner, vimentin, coimmunoprecipitated with alphaIIbbeta3 from platelets, independent of fibrinogen binding. Studies with purified proteins and with recombinant proteins expressed in CHO cells determined that the interaction between cPLA(2)alpha and alphaIIbbeta3 was indirect and was dependent on the alphaIIb and beta3 cytoplasmic tails. Fibrinogen binding to alphaIIbbeta3 caused an increase in integrin-associated cPLA(2)alpha activity in normal platelets, but not in cPLA(2)alpha-deficient mouse platelets or in human platelets treated with pyrrophenone, a cPLA(2)alpha inhibitor. cPLA(2)alpha activation downstream of alphaIIbbeta3 had functional consequences for platelets in that it was required for fibrinogen-dependent recruitment of activated protein kinase Cbeta to the alphaIIbbeta3 complex and for platelet spreading. Thus, cPLA(2)alpha and alphaIIbbeta3 interact to reinforce each other's functions during alphaIIbbeta3 signaling. This provides a plausible explanation for the role of alphaIIbbeta3 in TxA(2) formation and in the defective hemostatic function of mouse or human platelets deficient in cPLA(2)alpha.

The Group IVA (GIVA) phospholipase A2 associates with natural membranes in response to an increase in intracellular Ca2+ along with increases in certain lipid mediators. This enzyme associates with the membrane surface as well as binding a single phospholipid molecule in the active site for catalysis. Employing deuterium exchange mass spectrometry, we have identified the regions of the protein binding the lipid surface and conformational changes upon a single phospholipid binding in the absence of a lipid surface. Experiments were carried out using natural palmitoyl arachidonyl phosphatidylcholine vesicles with the intact GIVA enzyme as well as the isolated C2 and catalytic domains. Lipid binding produced changes in deuterium exchange in eight different regions of the protein. The regions with decreased exchange included Ca2+ binding loop one, which has been proposed to penetrate the membrane surface, and a charged patch of residues, which may be important in interacting with the polar head groups of phospholipids. The regions with an increase in exchange are all located either in the hydrophobic core underneath the lid region or near the lid and hinge regions from 403 to 457. Using the GIVA phospholipase A2 irreversible inhibitor methyl-arachidonyl fluorophosphonate, we were able to isolate structural changes caused only by pseudo-substrate binding. This produced results that were very similar to natural lipid binding in the presence of a lipid interface with the exception of the C2 domain and region 466-470. This implies that most of the changes seen in the catalytic domain are due to a substrate-mediated, not interface-mediated, lid opening, which exposes the active site to water. Finally experiments carried out with inhibitor plus phospholipid vesicles showed decreases at the C2 domain as well as charged residues on the putative membrane binding surface of the catalytic domain revealing the binding sites of the enzyme to the lipid surface.

Many cellular signalling events are controlled by the selective recruitment of protein complexes to membranes. Determining the molecular basis for how lipid signalling complexes are recruited, assembled and regulated on specific membrane compartments has remained challenging due to the difficulty of working in conditions mimicking native biological membrane environments. Enzyme recruitment to membranes is controlled by a variety of regulatory mechanisms, including binding to specific lipid species, protein–protein interactions, membrane curvature, as well as post-translational modifications. A powerful tool to study the regulation of membrane signalling enzymes and complexes is hydrogen deuterium exchange–MS (HDX–MS), a technique that allows for the interrogation of protein dynamics upon membrane binding and recruitment. This review will highlight the theory and development of HDX–MS and its application to examine the molecular basis of lipid signalling enzymes, specifically the regulation and activation of phosphoinositide 3-kinases (PI3Ks). * ABD, : adaptor-binding domain; ECD, : electron capture dissociation; ETD, : electron transfer dissociation; Gβγ, : G protein βγ heterodimers; GPCR, : G-protein-coupled receptor; HDX–MS, : hydrogen deuterium exchange–MS; PI3K, : phosphoinositide 3 kinases; PIK3CA, : gene encoding for the p110α catalytic subunit of PI3K; PIP3, : phosphatidylinositol 3,4,5 trisphosphate; PLA2, : phospholipase A2; RTK, : receptor tyrosine kinase; SH2, : Src-homology 2, inter SH2 coiled coil (iSH2), n-terminal SH2 (nSH2), c-terminal SH2, phosphatase and tensin homolog (PTEN)

Opening up Vps34 protein complexes During intracellular membrane trafficking, large protein complexes regulate and adapt the activity of signal transducer enzymes such as the class III phosphatidylinositol 3-kinase Vps34. These large enzyme complexes are present in all eukaryotic cells, having widespread importance in neurodegeneration, aging, and cancer; however, a structural understanding has been lacking. Rostislavleva et al. provide atomic-resolution insights into the structures of the Vps34-containing protein complexes required for autophagy, endocytic sorting, and cytokinesis. The V-shaped complexes can undergo opening motions, which allows them to adapt to and phosphorylate membranes. Science , this issue p. 10.1126/science.aac7365

The unstructured regions found at the C-terminus of the tumour suppressor PTEN and the N-terminus PTEN-L can switch the enzymes' substrate specificity from soluble to membrane-embedded, and can also dramatically alter the enzymes' affinity for membranes., Phosphatase and tensin homologue deleted on chromosome 10 (PTEN) is a lipid and protein phosphatase, and both activities are necessary for its role as a tumour suppressor. PTEN activity is controlled by phosphorylation of its intrinsically disordered C-terminal tail. A recently discovered variant of PTEN, PTEN-long (PTEN-L), has a 173-residue N-terminal extension that causes PTEN-L to exhibit unique behaviour, such as movement from one cell to another. Using hydrogen/deuterium exchange mass spectrometry (HDX–MS) and biophysical assays, we show that both the N-terminal extension of PTEN-L and C-terminal tail of PTEN affect the phosphatase activity using unique mechanisms. Phosphorylation of six residues in the C-terminal tail of PTEN results in auto-inhibitory interactions with the phosphatase and C2 domains, effectively blocking both the active site and the membrane-binding interface of PTEN. Partially dephosphorylating PTEN on pThr366/pSer370 results in sufficient exposure of the active site to allow a selective activation for soluble substrates. Using HDX–MS, we identified a membrane-binding element in the N-terminal extension of PTEN-L, termed the membrane-binding helix (MBH). The MBH radically alters the membrane binding mechanism of PTEN-L compared with PTEN, switching PTEN-L to a ‘scooting’ mode of catalysis from the ‘hopping’ mode that is characteristic of PTEN.

The lipid-anchored GTPase Ras is well known for its role in oncogenesis – about one quarter of all human tumors show mutations in a Ras family member. One of the best characterized Ras targets is the lipid kinase Phosphatidylinositol 3-Kinase (PI3K) which, like Ras, is highly oncogenic. PI3K phosphorylates the constitutive plasma membrane lipid phosphatidyl-inositol-(4,5)-bisphosphate (PIP2) yielding the essential signaling lipid phosphatidylinositol-(3,4,5)-bisphosphate (PIP3). The resulting PIP3 lipid signal regulates an array of cell processes including migration and growth.The underlying mechanism by which Ras activates PI3K is unclear - this project aims to better elucidate the activation mechanism using single molecule TIRF microscopy to probe the interactions and activities of single proteins on a target membrane surface. One hypothesis is that the lipid-anchored, membrane-bound Ras protein increases the surface density of PI3K on the membrane by increasing kon and/or decreasing koff. An alternative hypothesis proposes that Ras binding increases the PI3K kcat and/or affinity for substrate lipid PI(4,5)P2 (PIP2) and/or ATP. Single-molecule fluorescence methods will be used to distinguish between these possibilities by observing the number of single particle diffusion tracks, track length, diffusion speed, specific activity, kcat, and Km of PI3K in the presence and absence of Ras. Our poster will present the latest findings of these studies.