PIP2 Lipids Research Articles

Deciphering the isoform code contained in Vo a‐subunit isoforms of the V‐ATPase

V‐ATPases are highly conserved proton pumps that acidify multiple organelles, including lysosomes, endosomes, the late Golgi apparatus, synaptic vesicles, and other regulated secretory granules. Precise tuning of the luminal pH of these organelles is critical for function, but the factors governing organelle pH control are not completely understood. V‐ATPases are multisubunit complexes consisting of a peripheral subcomplex (V1) that contains sites for ATP hydrolysis and an integral membrane subcomplex (Vo) that contains the proton pore. The Vo a‐subunit is the largest subunit. It is comprised of an N‐terminal cytosolic domain and a C‐terminal domain that forms part of the proton pore. Most organisms encode multiple isoforms of the Vo a‐subunit that exhibit organelle‐specific localization. We hypothesize that the N‐terminal (NT) domains of a‐subunit isoforms participate in distinct cellular interactions that are critical for isoform‐specific V‐ATPase localization, activity, and regulation. In yeast cells, there are two organelle‐specific isoforms of the Vo a‐subunit, Vph1 and Stv1. Vph1‐containing V‐ATPases transit through the secretory pathway en route to the lysosome‐like vacuole. Their activity is regulated by reversible disassembly in response to glucose levels, and they require interaction with the yeast RAVE (regulator of acidification of vacuoles and endosomes) for both their initial biosynthetic assembly and for glucose‐dependent reassembly. The Vph1NT domain binds directly to RAVE and is responsible for glucose‐sensitive interactions with the RAVE complex. In addition, the vacuolar lipid PI(3,5)P2 promotes assembly and activity of Vph1‐containing V‐ATPases. Mutations in Vph1NT compromise PI(3,5)P2‐induced activation, suggesting direct binding to lipid. In contrast, Stv1‐containing V‐ATPases assemble and function in the absence of the RAVE complex, and Stv1NT does not bind to RAVE. However, Stv1NT binds to the Golgi‐enriched lipid PI(4)P, and mutations that abolish PI(4)P binding compromise Golgi retention of Stv1‐containing V‐ATPases. The aNT domains of the four human Vo a‐subunit isoforms exhibit differential recognition of phosphoinositide lipids. Some human aNT domains interact with the human homologue of the RAVE complex. These data indicate that NT domains of Vo a‐subunit isoforms encode information for localization and regulation of V‐ATPase activity that help determine organelle pH and protect cells from stress.Support or Funding InformationNIH R01 GM127364 and NIH R01 GM126020

Binding and Interaction of Human Beta Defensin Type 3 with Mixed PIP2 Lipid Membranes

Defensins are cationic cysteine-rich small molecules with molecular masses ranging from 3 to 5 kDa. Human β defensin type 3 (hBD-3) belongs to the human innate immune system, and is mainly secreted from the epithelial cells. It has a broad-spectrum of antimicrobial activities on virus, fungi, and both Gram-positive and Gram-negative bacteria. hBD-3 can bind to the negatively charged and even zwitterionic (but charge-neutral) lipid membrane, disrupt and then translocate through the lipid membrane. In order to understand its dynamics and functional mechanism in molecular level, all-atom molecular dynamics simulations on hBD-3 binding with and crossing mixed lipid membranes were investigated. Since PIP2 is the key minor component in cell membranes, mixed PIP2 lipid membrane represented by neutrally charged POPC lipids mixed with 10% of PIP2 lipids were studied. Simulations on hBD-3 placed inside the mixed lipid bilayer, and on the surface of the lipid bilayer were conducted. In order to consider the influence of the initial binding orientation on the hBD-3 binding structure, in total five different initial binding orientations of hBD-3 on the lipid bilayer were studied. Since hBD-3 can form a dimer, the binding of hBD-3 in both monomer and dimer forms with lipid membranes were explored. The structure of hBD-3 on lipid membranes and the key residues on the binding interface were predicted and compared. During the micro-second long simulations, the assembly of PIP2 lipids towards hBD-3 was observed in both hBD-3 on the lipid membrane surface systems and inside the lipid membrane systems. hBD-3 staying inside the mixed lipid bilayer can behave as the channel to facilitate water molecules to cross the mixed lipid bilayer. The result emphasized the importance of PIP2 in hBD-3's disruption capability with the mixed cell membrane.

Discovery of Small Molecules That Target the Phosphatidylinositol (3,4,5) Trisphosphate (PIP3)-Dependent Rac Exchanger 1 (P-Rex1) PIP3-Binding Site and Inhibit P-Rex1-Dependent Functions in Neutrophils.

Phosphatidylinositol (3,4,5) trisphosphate (PIP3)-dependent Rac exchanger 1 (P-Rex1) is a Rho guanine-nucleotide exchange factor that was originally discovered in neutrophils and is regulated by G protein βγ subunits and the lipid PIP3 in response to chemoattractants. P-Rex1 has also become increasingly recognized for its role in promoting metastasis of breast cancer, prostate cancer, and melanoma. Recent structural, biochemical, and biologic work has shown that binding of PIP3 to the pleckstrin homology (PH) domain of P-Rex1 is required for its activation in cells. Here, differential scanning fluorimetry was used in a medium-throughput screen to identify six small molecules that interact with the P-Rex1 PH domain and block binding of and activation by PIP3 Three of these compounds inhibit N-formylmethionyl-leucyl-phenylalanine induced spreading of human neutrophils as well as activation of the GTPase Rac2, both of which are downstream effects of P-Rex1 activity. Furthermore, one of these compounds reduces neutrophil velocity and inhibits neutrophil recruitment in response to inflammation in a zebrafish model. These results suggest that the PH domain of P-Rex1 is a tractable drug target and that these compounds might be useful for inhibiting P-Rex1 in other experimental contexts. SIGNIFICANCE STATEMENT: A set of small molecules identified in a thermal shift screen directed against the phosphatidylinositol (3,4,5) trisphosphate-dependent Rac exchanger 1 (P-Rex1) pleckstrin homology domain has effects consistent with P-Rex1 inhibition in neutrophils.

A Genetic Screen in Drosophila To Identify Novel Regulation of Cell Growth by Phosphoinositide Signaling.

Phosphoinositides are lipid signaling molecules that regulate several conserved sub-cellular processes in eukaryotes, including cell growth. Phosphoinositides are generated by the enzymatic activity of highly specific lipid kinases and phosphatases. For example, the lipid PIP3, the Class I PI3 kinase that generates it and the phosphatase PTEN that metabolizes it are all established regulators of growth control in metazoans. To identify additional functions for phosphoinositides in growth control, we performed a genetic screen to identify proteins which when depleted result in altered tissue growth. By using RNA-interference mediated depletion coupled with mosaic analysis in developing eyes, we identified and classified additional candidates in the developing Drosophila melanogaster eye that regulate growth either cell autonomously or via cell-cell interactions. We report three genes: Pi3K68D, Vps34 and fwd that are important for growth regulation and suggest that these are likely to act via cell-cell interactions in the developing eye. Our findings define new avenues for the understanding of growth regulation in metazoan tissue development by phosphoinositide metabolizing proteins.

Structural Basis of Human KCNQ1 Modulation and Gating

KCNQ1, also known as Kv7.1, is a voltage-dependent K+ channel that regulates gastric acid secretion, salt and glucose homeostasis, and heart rhythm. Its functional properties are regulated in a tissue-specific manner through co-assembly with beta subunits KCNE1-5. In non-excitable cells, KCNQ1 forms a complex with KCNE3, which suppresses channel closure at negative membrane voltages that otherwise would close it. Pore opening is regulated by the signaling lipid PIP2. Using cryoelectron microscopy (cryo-EM), we show that KCNE3 tucks its single-membrane-spanning helix against KCNQ1, at a location that appears to lock the voltage sensor in its depolarized conformation. Without PIP2, the pore remains closed. Upon addition, PIP2 occupies a site on KCNQ1 within the inner membrane leaflet, which triggers a large conformational change that leads to dilation of the pore's gate. It is likely that this mechanism of PIP2 activation is conserved among Kv7 channels.

Relationships between MA-RNA Binding in Cells and Suppression of HIV-1 Gag Mislocalization to Intracellular Membranes.

The HIV-1 Gag matrix (MA) domain mediates the localization of Gag to the plasma membrane (PM), the site for infectious virion assembly. The MA highly basic region (MA-HBR) interacts with phosphatidylinositol-(4,5)-bisphosphate [PI(4,5)P2], a PM-specific acidic lipid. The MA-HBR also binds RNAs. To test whether acidic lipids alone determine PM-specific localization of Gag or whether MA-RNA binding also plays a role, we compared a panel of MA-HBR mutants that contain two types of substitutions at MA residues 25 and 26 or residues 29 and 31: Lys→Arg (KR) (25/26KR and 29/31KR) and Lys→Thr (KT) (25/26KT and 29/31KT). Consistent with the importance of the HBR charge in RNA binding, both KT mutants failed to bind RNA via MA efficiently, unlike the corresponding KR mutants. Both 25/26KT Gag-yellow fluorescent protein (YFP) and 29/31KT Gag-YFP bound nonspecifically to the PM and intracellular membranes, presumably via the myristoyl moiety and remaining MA basic residues. In contrast, 25/26KR Gag-YFP bound specifically to the PM, suggesting a role for the total positive charge and/or MA-bound RNA in navigating Gag to the PM. Unlike 29/31KT Gag-YFP, 29/31KR Gag-YFP was predominantly cytosolic and showed little intracellular membrane binding despite having a higher HBR charge. Therefore, it is likely that MA-RNA binding blocks promiscuous Gag membrane binding in cells. Notably, the introduction of a heterologous multimerization domain restored PI(4,5)P2-dependent PM-specific localization for 29/31KR Gag-YFP, suggesting that the blocking of PM binding is more readily reversed than that of intracellular membrane binding. Altogether, these cell-based data support a model in which MA-RNA binding ensures PM-specific localization of Gag via suppression of nonspecific membrane binding.IMPORTANCE The PM-specific localization of HIV-1 Gag is a crucial early step in infectious progeny production. The interaction between the MA highly basic region (MA-HBR) of Gag and the PM-specific lipid PI(4,5)P2 is critical for Gag localization to the PM. Additionally, in vitro evidence has indicated that MA-RNA binding prevents nonspecific binding of Gag to non-PI(4,5)P2-containing membranes. However, cell-based evidence supporting a role for HIV-1 MA-RNA binding in PM-specific subcellular localization has been scarce; thus, it remained possible that in cells, just the high basic charge or the PI(4,5)P2 binding ability is sufficient for MA to direct Gag specifically to the PM. The present study reveals for the first time an excellent correlation between RNA binding of the MA-HBR and inhibition of promiscuous Gag localization, both within the cells, and thereby provides cell-based evidence supporting a mechanism in which HIV-1 MA binding to RNA ensures the specific localization of Gag to the PM.

Competitive Interactions between PIRT, the Cold Sensing Ion Channel TRPM8, and PIP2 Suggest a Mechanism for Regulation

TRPM8 is a member of the transient receptor potential ion channel family where it functions as a cold and pain sensor in humans and other higher organisms. Previous studies show that TRPM8 requires the signaling phosphoinositide lipid PIP2 to function. TRPM8 function is further regulated by other diverse mechanisms, including the small modulatory membrane protein PIRT (phosphoinositide regulator of TRP). Like TRPM8, PIRT also binds PIP2 and behavioral studies have shown that PIRT is required for normal TRPM8-mediated cold-sensing. To better understand the molecular mechanism of PIRT regulation of TRPM8, solution nuclear magnetic resonance (NMR) spectroscopy was used to assign the backbone resonances of full-length human PIRT and investigate the direct binding of PIRT to PIP2 and the human TRPM8 S1-S4 transmembrane domain. Microscale thermophoresis (MST) binding studies validate the NMR results and identify a competitive PIRT interaction between PIP2 and the TRPM8 S1-S4 domain. Computational PIP2 docking to a human TRPM8 comparative model was performed to help localize where PIRT may bind TRPM8. Taken together, our data suggest a mechanism where TRPM8, PIRT, and PIP2 form a regulatory complex and PIRT modulation of TRPM8 arises, at least in part, by regulating local concentrations of PIP2 accessible to TRPM8.

Structure of the thermo-sensitive TRP channel TRP1 from the alga Chlamydomonas reinhardtii

Algae produce the largest amount of oxygen on earth and are invaluable for human nutrition and biomedicine, as well as for the chemical industry, energy production and agriculture. The mechanisms by which algae can detect and respond to changes in their environments can rely on membrane receptors, including TRP ion channels. Here we present a 3.5-Å resolution cryo-EM structure of the transient receptor potential (TRP) channel crTRP1 from the alga Chlamydomonas reinhardtii that opens in response to increased temperature and is positively regulated by the membrane lipid PIP2. The structure of crTRP1 significantly deviates from the structures of other TRP channels and has a unique 2-fold symmetrical rose-shape architecture with elbow domains and ankyrin repeat domains submerged and dipping into the membrane, respectively. Our study provides a structure of a TRP channel from a micro-organism and a structural framework for better understanding algae biology and TRP channel evolution.

PIP2 Reshapes Membranes through Asymmetric Desorption

Phosphatidylinositol-4,5-bisphosphate (PIP2) is an important signaling lipid in eukaryotic cell plasma membranes, playing an essential role in diverse cellular processes. The headgroup of PIP2 is highly negatively charged, and this lipid displays a high critical micellar concentration compared to housekeeping phospholipid analogs. Given the crucial role of PIP2, it is imperative to study its localization, interaction with proteins, and membrane-shaping properties. Biomimetic membranes have served extensively to elucidate structural and functional aspects of cell membranes including protein-lipid and lipid-lipid interactions, as well as membrane mechanics. Incorporation of PIP2 into biomimetic membranes, however, has at times resulted in discrepant findings described in the literature. With the goal to elucidate the mechanical consequences of PIP2 incorporation, we studied the desorption of PIP2 from biomimetic giant unilamellar vesicles by means of a fluorescent marker. A decrease in fluorescence intensity with the age of the vesicles suggested that PIP2 lipids were being desorbed from the outer leaflet of the membrane. To evaluate whether this desorption was asymmetric, the vesicles were systematically diluted. This resulted in an increase in the number of internally tubulated vesicles within minutes after dilution, suggesting that the desorption was asymmetric and also generated membrane curvature. By means of a saturated chain homolog of PIP2, we showed that the fast desorption of PIP2 is facilitated by presence of an arachidonic lipid tail and is possibly due to its oxidation. Through measurements of the pulling force of membrane tethers, we quantified the effect of this asymmetric desorption on the spontaneous membrane curvature. Furthermore, we found that the spontaneous curvature could be modulated by externally increasing the concentration of PIP2 micelles. Given that the local concentration of PIP2 in biological membranes is variable, spontaneous curvature generated by PIP2 may affect the formation of highly curved structures that can serve as initiators for signaling events.

Interaction of the late endo-lysosomal lipid PI(3,5)P2 with the Vph1 isoform of yeast V-ATPase increases its activity and cellular stress tolerance

The low-level endo-lysosomal signaling lipid, phosphatidylinositol 3,5-bisphosphate (PI(3,5)P2), is required for full assembly and activity of vacuolar H+-ATPases (V-ATPases) containing the vacuolar a-subunit isoform Vph1 in yeast. The cytosolic N-terminal domain of Vph1 is also recruited to membranes in vivo in a PI(3,5)P2-dependent manner, but it is not known if its interaction with PI(3,5)P2 is direct. Here, using biochemical characterization of isolated yeast vacuolar vesicles, we demonstrate that addition of exogenous short-chain PI(3,5)P2 to Vph1-containing vacuolar vesicles activates V-ATPase activity and proton pumping. Modeling of the cytosolic N-terminal domain of Vph1 identified two membrane-oriented sequences that contain clustered basic amino acids. Substitutions in one of these sequences (231KTREYKHK) abolished the PI(3,5)P2-dependent activation of V-ATPase without affecting basal V-ATPase activity. We also observed that vph1 mutants lacking PI(3,5)P2 activation have enlarged vacuoles relative to those in WT cells. These mutants exhibit a significant synthetic growth defect when combined with deletion of Hog1, a kinase important for signaling the transcriptional response to osmotic stress. The results suggest that PI(3,5)P2 interacts directly with Vph1, and that this interaction both activates V-ATPase activity and protects cells from stress.

The biological function of PIP2 clustering in enzyme activation and ion channel regulation

The signaling lipid phosphatidylcholine 4,5 bisphosphate (PIP2) clusters with proteins in the plasma membrane away from saturated domains formed by GM1 lipids and cholesterol (GM1 domains or ‘lipid rafts’). The lipid metabolizing enzyme phospholipase D2 (PLD2) localizes to both lipid raft and PIP2 clusters creating a competition for localization. We show that disruption of GM1 domains by mechanical force or inhaled anesthetics causes PLD2 to translocate to PIP2 clusters where it activates twik related potassium (TREK‐1) channels giving rise to the channel's mechano and anesthetic sensitivity. We conclude cells have evolved to (i.) sense and respond to disruption of lipid order and (ii.) utilize lipid order to dynamically regulate enzymes and ion channels through substrate presentation.Support or Funding InformationThis work was supported by a Director's New Innovator Award (1DP2NS087943‐01 to S.B.H.) from the NIH, a graduate fellowship from the Joseph B. Scheller & Rita P. Scheller Charitable Foundation to E.N.P. We are grateful to the Iris and Junming Le Foundation for funds to purchase a super‐resolution microscope, making this study possible.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

K-Ras G-domain binding with signaling lipid phosphatidylinositol (4,5)-phosphate (PIP2): membrane association, protein orientation, and function

Ras genes potently drive human cancers, with mutated proto-oncogene GTPase KRAS4B (K-Ras4B) being the most abundant isoform. Targeted inhibition of oncogenic gene products is considered the "holy grail" of present-day cancer therapy, and recent discoveries of small-molecule KRas4B inhibitors were made thanks to a deeper understanding of the structure and dynamics of this GTPase. Because interactions with biological membranes are key for Ras function, Ras-lipid interactions have become a major focus, especially because such interactions evidently involve both the Ras C terminus for lipid anchoring and its G-protein domain. Here, using NMR spectroscopy and molecular dynamics simulations complemented by biophysical- and cell-biology assays, we investigated the interaction between K-Ras4B with the signaling lipid phosphatidylinositol (4,5)-phosphate (PIP2). We discovered that the β2 and β3 strands as well as helices 4 and 5 of the GTPase G-domain bind to PIP2 and identified the specific residues in these structural elements employed in these interactions, likely occurring in two K-Ras4B orientation states relative to the membrane. Importantly, we found that some of these residues known to be oncogenic when mutated (D47K, D92N, K104M, and D126N) are critical for K-Ras-mediated transformation of fibroblast cells, but do not substantially affect basal and assisted nucleotide hydrolysis and exchange. Moreover, the K104M substitution abolished localization of K-Ras to the plasma membrane. The findings suggest that specific G-domain residues can critically regulate Ras function by mediating interactions with membrane-associated PIP2 lipids; these insights that may inform the future design of therapeutic reagents targeting Ras activity.

PLC\u03b22 negatively regulates the inflammatory response to virus infection by inhibiting phosphoinositide-mediated activation of TAK1

Excessive or uncontrolled release of proinflammatory cytokines caused by severe viral infections often results in host tissue injury or even death. Phospholipase C (PLC)s degrade phosphatidylinositol-4, 5-bisphosphate (PI(4,5)P2) lipids and regulate multiple cellular events. Here, we report that PLCβ2 inhibits the virus-induced expression of pro-inflammatory cytokines by interacting with and inhibiting transforming growth factor-β-activated kinase 1 (TAK1) activation. Mechanistically, PI(4,5)P2 lipids directly interact with TAK1 at W241 and N245, and promote its activation. Impairing of PI(4,5)P2’s binding affinity or mutation of PIP2-binding sites on TAK1 abolish its activation and the subsequent production of pro-inflammatory cytokines. Moreover, PLCβ2-deficient mice exhibit increased expression of proinflammatory cytokines and a higher frequency of death in response to virus infection, while the PLCβ2 activator, m-3M3FBS, protects mice from severe Coxsackie virus A 16 (CVA16) infection. Thus, our findings suggest that PLCβ2 negatively regulates virus-induced pro-inflammatory responses by inhibiting phosphoinositide-mediated activation of TAK1.

Membrane Curvature Generation through Asymmetric Desorption of Pi(4,5)P2

Phosphatidylinositol-4,5-bisphosphate [PIP2] is an important signaling lipid in the eukaryotic cell plasma membrane, playing an important role in diverse signaling processes. The headgroup of PIP2 is highly negatively charged with a high estimated critical micellar concentration compared to housekeeping phospholipid analogs. Given the crucial role played by PIP2 in cellular processes, it is imperative to study its localization, interaction with proteins, and membrane shaping properties. Biomimetic membranes have served extensively to elucidate aspects including protein-lipid interactions, lipid lipid interactions, and membrane mechanics. Incorporation of PIP2 in the biomimetic membranes, however, has at times resulted in discrepant findings described in the current literature. With the goal to elucidate the mechanical consequences of PIP2 incorporation, we have studied the desorption of PIP2 from biomimetic giant unilamellar vesicles [GUVs] by means of a fluorescent marker. The decrease in fluorescence intensity with the age of the vesicles suggested that PIP2 lipids were being desorbed from the outer leaflet of the membrane. To ask the question whether this desorption was symmetric, the vesicles were systematically diluted. This resulted in an increase in the number of internally tubulated vesicles suggesting that the desorption was asymmetric. In order to quantify this effect, we obtained the spontaneous curvature that was being generated due to this asymmetric desorption. The deduction was made by pulling narrow membrane tethers from aspirated vesicles using optically trapped beads. The effect on the tether pulling force resulting from a variable membrane tension allowed us to quantify the generated spontaneous curvature. Given that the local concentration of PIP2 in biological membranes is variable, their spontaneous desorption can result in generating highly curved structures which can serve as an initiator for signaling events.

PIP2 Lipids as Regulators of Membrane Curvature Sensing by Enth Domains

PIP2 Lipids as Regulators of Membrane Curvature Sensing by Enth Domains

Molecular Mechanisms of the Interaction between Arf1 and ASAP1 pH Domain at the Membrane Interface

Pleckstrin Homology (PH) domains have dual functions: localizing proteins to specific membrane regions and modulating membrane protein function through interdomain interactions. Here, we focused on the PH domain of ASAP1, which is necessary for phosphatidylinositol 4, 5- bisphosphate (PIP2)- dependent conversion of Arf1, a member of the RAS superfamily, from its GTP to GDP form. The interaction of the PH domain with the lipid surface was studied by 15N, 13C methyl NMR and SPR, using PIP2 and phosphatidylserine (PS) - containing nanodiscs as mimics of the membrane surface. Comparison of the chemical shift perturbations within PH upon interaction with nanodiscs- containing a single PIP2, multiple PIP2 lipids or a single PIP2 and increasing PS mole fraction showed that (i) the two loops between β sheets 1, 2 and 3, 4 delineate two, equivalent and independent PIP2 lipid binding sites and (ii) the PH domain is attracted to the membrane surface predominantly by non- specific electrostatic interactions with the background anionic lipids, before docking to the target lipid PIP2. Constraints obtained from neutron reflectometry and Paramagnetic Relaxation NMR experiments suggest that the PH domain is oriented with its long axis parallel to the membrane normal. Interestingly, deletion of 14 amino acids immediately N term of the first beta strand of the PH domain strongly reduces ASAP1 catalytic efficiency even if the deletion has no effect on ASAP1 equilibrium binding to PIP2. Taken together, this suggests that simultaneous binding to two PIP2 strictly orients the PH domain at the membrane interface, promoting functionally critical contacts between Arf1 and the N-term of ASAP1.

Membrane Binding by Synaptotagmin-Like Protein 4: Site Directed Mutagenesis of the Lipid Interaction Surface

Membrane-binding proteins play crucial roles in many biological process such as vesicle trafficking and exocytosis. Synaptotagmin-like protein 4 (Slp-4), also known as granuphilin, is a Rab effector protein whose overexpression is known to suppress insulin secretion despite increasing the number of secretory vesicles docked to the plasma membrane. Therefore, its structural mechanism may hold clues for understanding the regulation of efficiency in exocytosis signaling. Slp-4 binds to vesicular Rab effector proteins through its N-terminal Slp homology domain, to SNARE complexes via its central linker region, and contains C-terminal tandem C2A and C2B domains that interact with plasma membrane lipids. We have previously shown that the Slp-4 C2A domain binds with high affinity to liposomes approximating the lipid composition of the plasma membrane interior leaflet. The C2A domain has a lysine cluster that binds to phosphatidylinositol-(4, 5)-bisphosphate (PIP2) and soluble inositol phosphates with micromolar affinity, but much more strongly to membranes containing both PIP2 and anionic background lipids such as phosphatidylserine (PS). We are combining computational and experimental approaches to reveal which residues of the Slp-4 C2A domain participate in binding PS and PIP2. Using mutational analysis and protein binding assays, here we show that mutation of specific individual amino acid residues tends to have modest effects on the domain's membrane affinity; however, combined mutations at multiple predicted binding sites have greater effects. This finding indicates that the C2A domain interacts with plasma membrane lipids through multiple binding sites that act in a concerted manner to produce its high membrane affinity.

Membrane Binding of Synaptotagmin-Like Protein 4: Insight from Molecular Dynamics Simulations

Synaptotagmin-like proteins (Slps) are a family of Rab GTPase effectors involved in secretory vesicle trafficking and membrane docking. Slps possess an N-terminal domain that binds Rab27 on large dense-core secretory vesicles and one or two C-terminal C2 domains that dock to plasma membranes via interaction with anionic lipid molecules such as phosphatidylinositol-(4,5)-bisphosphate (PIP2) and phosphatidylserine (PS). In particular, Slp-4, also known as granuphilin, docks secretory vesicles to the plasma membrane primarily via its C2A domain, which has a high affinity for anionic lipid bilayers. Although the structure of the Slp-4 C2A domain is known and some of its key PIP2-binding residues have been identified, the full structural mechanism of its strong membrane binding remains unclear. Here, we employ molecular dynamics simulations of the Slp-4 C2A domain to determine contributions of particular residues in binding to a lipid bilayer containing physiologically relevant levels of the anionic target lipids PS and PIP2. Simulations include mutations of two important residues: Lys398, which is known to participate in PIP2 coordination, and Phe452, which is suspected to insert into target membranes hydrophobically. Preliminary results support the experimental finding that both the K398A and F452A mutants are capable of binding membranes but suggest a different balance of lipid interactions in the two mutants. Overall, experimental and computational results indicate that high-affinity membrane binding of the Slp-4 C2A domain is sustained via many lipid-interacting residues distributed over a large electropositive protein surface.

The Mechanism of PI3Kα Activation at the Atomic Level

PI3K lipid kinases phosphorylate PIP2 to PIP3 in the PI3K/Akt/mTOR pathway to regulate cellular processes. They are frequently mutated in cancer. Here we determine the PI3Kα activation mechanism at the atomic level. Unlike protein kinases where the substrate abuts the ATP, crystal structures indicate that in PI3Kα, the distance between the γ phosphate of the ATP and the PIP2 lipid substrate is over 6 A, much too far for the phosphoryl transfer, raising the question of how catalysis is executed. PI3Kα has two subunits, the catalytic p110α and the regulatory p85α. Our simulations show that release of the autoinhibition exerted by the nSH2 domain of the p85α triggers significant conformational change in p110α, leading to the exposure of the kinase domain for membrane interaction. Structural rearrangement in the C-lobe of the kinase domain reduces the distance between the ATP γ-phosphate and the substrate, offering an explanation as to how phosphoryl transfer is executed. An alternative mechanism may involve ATP relocation. This mechanism not only explains how oncogenic mutations promote PI3Kα activation by facilitating nSH2 release, or nSH2-release-induced, allosteric motions; it also offers an innovative, PI3K isoform-specific drug discovery principle. Rather than competing with nanomolar range ATP in the ATP-binding pocket and contending with ATP pocket conservation and massive binding targets, this mechanism suggests blocking the PI3Kα sequence-specific cavity between the ATP-binding pocket and the substrate binding site. Targeting isoform-specific residues in the cavity may prevent PIP2 phosphorylation.

PIP2 Phospholipid-Induced Aggregation of Tau Filaments Probed by Tip-Enhanced Raman Spectroscopy.

The morphology and secondary structure of peptide fibers formed by aggregation of tubulin-associated unit (Tau) fragments (K18), in the presence of the inner cytoplasmic membrane phosphatidylinositol component (PIP2 ) or heparin sodium (HS) as cofactors, are determined with nanoscale (<10 nm) spatial resolution. By means of tip-enhanced Raman spectroscopy (TERS), the inclusion of PIP2 lipids in fibers is determined based on the observation of specific C=O ester vibration modes. Moreover, analysis of amide I and amide III bands suggests that the parallel β-sheet secondary structure content is lower and the random coil content is higher for fibers grown from the PIP2 cofactor instead of HS. These observations highlight the occurrence of some local structural differences between these fibers. This study constitutes the first nanoscale structural characterization of Tau/phospholipid aggregates, which are implicated in deleterious mechanisms on neural membranes in Alzheimer's disease.