PIP2 Molecules Research Articles

Cryo-EM architecture of a near-native stretch-sensitive membrane microdomain

Biological membranes are partitioned into functional zones termed membrane microdomains, which contain specific lipids and proteins1–3. The composition and organization of membrane microdomains remain controversial because few techniques are available that allow the visualization of lipids in situ without disrupting their native behaviour3,4. The yeast eisosome, composed of the BAR-domain proteins Pil1 and Lsp1 (hereafter, Pil1/Lsp1), scaffolds a membrane compartment that senses and responds to mechanical stress by flattening and releasing sequestered factors5–9. Here we isolated near-native eisosomes as helical tubules made up of a lattice of Pil1/Lsp1 bound to plasma membrane lipids, and solved their structures by helical reconstruction. Our structures reveal a striking organization of membrane lipids, and, using in vitro reconstitutions and molecular dynamics simulations, we confirmed the positioning of individual PI(4,5)P2, phosphatidylserine and sterol molecules sequestered beneath the Pil1/Lsp1 coat. Three-dimensional variability analysis of the native-source eisosomes revealed a dynamic stretching of the Pil1/Lsp1 lattice that affects the sequestration of these lipids. Collectively, our results support a mechanism in which stretching of the Pil1/Lsp1 lattice liberates lipids that would otherwise be anchored by the Pil1/Lsp1 coat, and thus provide mechanistic insight into how eisosome BAR-domain proteins create a mechanosensitive membrane microdomain.

Abstract 1510: Cell-cell contacts regulate HER2 cellular localization and the transitions between migration and proliferation in breast cancer cells

Abstract Introduction: Epithelial-to-mesenchymal transition (EMT) is widely recognized as the primary mechanism regulating invasion and metastatic dissemination in carcinomas. In contrast, the mechanisms triggering single disseminated cancer cells (DCC) to reacquire their epithelial identity at ectopic sites, initiating metastasis formation, remain unclear. Since we had previously noted that HER2 expression levels and cellular density impact on migration and proliferation, we sought to determine whether they are involved in molecular mechanisms regulating transitions between proliferation and migration. Methods: To control HER2 expression levels we generated inducible cell models. Cell density was assessed by the number of cell-cell contacts or by the number of cells per area. Pathway analyses were performed by gene expression analysis, protein interactions by western blotting and mass spectrometry. Migration and proliferation were analyzed by functional assays including in vitro live cell imaging. Results: Migrating epithelial cells undergo reduced protein trafficking, resulting in a perinuclear deposition of HER2. This was accompanied by increased calcium ion regulation via phospholipase-C in the endoplasmic reticulum (ER). Upon the establishment of cell-cell contacts, particularly between cells expressing desmosome cadherin proteins, HER2 is redirected to the cell surface through direct physical binding with desmoplakin, facilitating proliferation signals. Then, PIP2 to PIP3 is converted through the activation of the PI3K pathway, concomitant with the upregulation of PDPK1. The PH domain of PDPK1 exhibits a high binding affinity to PIP2 molecules. Consequently, membrane localization of HER2 significantly diminishes PIP2 availability for the PLC pathway, redirecting cells towards proliferation. Patient sample analysis confirmed the loss of membrane-bound HER2 in solitary circulating tumor cells (CTCs), which was paralleled by increased plakoglobin, elevated ZEB1, and reduced KI67 protein levels. Conclusion: Cell-cell contacts determine the cellular localization and function of growth factor receptors like HER2. Single disseminating cancer cells therefore need to establish such contacts at metastatic sites to activate proliferation programs. Our data are in line with findings that circulating tumor cell clusters are associated with higher metastatic rates, the impact of specific metastatic niches for outgrowth and have implementation for the targeting of single DCC. Citation Format: Hedayatollah Hosseini, Saba Sameri, Shabnam Shahrivari, Carolina Fisch, Miriam Kokal, Michael Schletter, Paul Sylvester Hahn, Lisa Seider, Marisa Schübel, Durdam Das, Martin Hoffmann, Christian Werno, Kathrin Weidele, Vladan Milosevic, Arne Östman, Shamim Sarhadi, Joachim Wegener, Stefanie Michaelis, Florian Weber, Astrid Bruckmann, Ernst R. Tamm, Yuting Li, Christoph A. Klein. Cell-cell contacts regulate HER2 cellular localization and the transitions between migration and proliferation in breast cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1510.

PO-01-206 MOLECULAR MODELING OF CLINICAL KCNJ2 MUTATIONS REVEALS STRUCTURE-FUNCTION IMPACT ON PIP2 BINDING SITE

Binding phosphatidylinositol 4,5-bisphosphate (PIP2) is essential for Kir2.1(encoded by KCNJ2), function. Disease associated KCNJ2 mutations, such as those associated with Andersen-Tawil Syndrome (ATS), may exert molecular dysfunction by interrupting PIP2 binding, however a full-length molecular model of Kir2.1 has not been developed to explore this hypothesis. Elucidate the molecular structure of Kir2.1 and determine the functional impact of clinical KCNJ2 mutations on PIP2 binding using experimental and improved molecular dynamic (MD) method. Whole-cell patch clamp experiments of WT-Kir2.1 and selected mutations were performed on stably transfected HEK293 cells using a standard protocol. A full-length Kir2.1 tetramer model (open and closed conformational states) based on Kir2.2 cryo-EM structure using MOE was created. Mutations (R67Q, G300D, R218L) were individually introduced to the channel, and a mutagenesis analysis was performed. Initially, MD simulation for 100ns was performed for wild-type (WT) and mutated structures (GROMACS software). We expanded the model to include +/- bound PIP2. Due to > 77% degree of homology of Kir2.2 (6m84) with Kir2.1, we developed a MD protocol with Kir2.2 as test system. The 2.5% free PIP2 molecules were released into the membrane and MD simulation was performed for 700ns using Amber software. Homomeric WT demonstrated normal Kir2.1 function while mutations G300D, R67Q, and R218L showed complete loss of function. Mutational analysis revealed that residue changes result in disruption of hydrogen bonding and affects the structural conformation at the residue sight and caused fluctuation of residues in other regions, i.e., selectivity filter and pore domain. The improved MD protocol validated Kir2.2 PIP2 binding sites. Moreover, we found mutation disruption of hydrogen bonds and hydrophobic interaction analysis between the channel and PIP2 molecules revealed significant structural changes. We developed the first full length MD model of Kir2.1 and found that ATS KCNJ2 mutations disrupt normal conformation and hydrogen bonding. Further, we validated PIP2 binding sites which illustrate the robustness of our method, and this technique can be applied to delineate Kir2.1 molecular PIP2 binding sites. Combined with experimental validation, our modeling will increase our understanding of structure-function relationships of KCNJ2 clinical mutations.

Phosphatidylinositol 4,5-Bisphosphate Sensing Lipid Raft via Inter-Leaflet Coupling Regulated by Acyl Chain Length of Sphingomyelin

Phosphatidylinositol 4,5-bisphosphate (PIP2) is an important molecule located at the inner leaflet of cell membrane, where it serves as anchoring sites for a cohort of membrane-associated molecules and as a broad-reaching signaling intermediate. The lipid raft is thought as the major platform recruiting proteins for signal transduction and also known to mediate PIP2 accumulation across the membrane. While the significance of this cross-membrane coupling is increasingly appreciated, it remains unclear whether and how PIP2 senses the dynamic change of the ordered lipid domains over the packed hydrophobic core of the bilayer. Herein, by means of molecular dynamic simulation, we reveal that inner PIP2 molecules can sense the outer lipid domain via inter-leaflet coupling, and the coupling manner is dictated by the acyl chain length of sphingomyelin (SM) partitioned to the lipid domain. Shorter SM promotes membrane domain registration, whereby PIP2 accumulates beneath the domain across the membrane. In contrast, the anti-registration is thermodynamically preferred if the lipid domain has longer SM due to the hydrophobic mismatch between the corresponding acyl chains in SM and PIP2. In this case, PIP2 is expelled by the domain with a higher diffusivity. These results provide molecular insights into the regulatory mechanism of correlation between the outer lipid domain and inner PIP2, both of which are critical components for cell signal transduction.

TRP434 and TRP435 residues are crucial for sensing calcium ions and PI(4,5)P2 molecules in TRPC5 channel.

Transient receptor potential canonical (TRPC) channels are non-selective calcium-permeable cation channels. Among 7 subunits, TRPC4, 5 channels are well known to be potentiated by direct bindings of calcium ion and phosphatidylinositol 4,5-biphosphate. Both calcium and PIP2 were therefore also implicated in activation and desensitization of the channel. The binding site for calcium ion was revealed but PIP2 binding site remains controversial. Previously, we found two tryptophan residues in TRPC4 channel as a candidate by which calcium sensing in S2-S3 linker is delivered to channel gating. Here, we suggest the WW site in TRPC5 channel conducts delivering of calcium sensing like in TRPC4, and also acts as a potential PIP2 binding site. We made two single mutants (W434A, W435A) and a double mutant (WW/AA) from human TRPC5 channel. Wild-type and mutant TRPC5 channels were expressed in HEK293 cells for macroscopic current recordings in whole-cell or inside-out mode. Generally, our data demonstrate W434 and W435 residues participate in PIP2 binding and calcium sensing. Interestingly, when co-expressed with human muscarinic acetylcholine receptor type 3 (mAChR3) and stimulated by carbachol, W434A channel didn’t show desensitization characteristically observed in wild-type TRPC5 channel. Loss of desensitization in W434A channel implies that it has lost the ability of sensing PIP2 hydrolysis by PLC or the ability of phosphorylation by PKC. The latter possibility is ruled out because the C-terminus of the channels we used were truncated so they do not contain the phosphorylation site. Recently, PIP2-bound ion channel structures were revealed including TRPs. Many channels had PIP2 molecules near S3, S4, and S5 helices. Our electrophysiological data also indicate that PIP2 binds near S3 helix in TRPC5 channel, and the WW site is a specific candidate for sensing PIP2 and calcium.

Structural insights into the complex between Arf1 and ASAP1 at the membrane surface by NMR.

ADP-ribosylation factor (Arf) GTPase-activating proteins (GAPs) are enzymes that need to bind to membranes to catalyze the hydrolysis of GTP bound to the small GTP-binding protein Arf1. Simultaneous binding of the Pleckstrin homology (PH) domain of the ArfGAP ASAP1 to membranes containing the phosphatidylinositol phosphate PIP(4,5)P2 and to Arf1.GTP is key for maximum GTP hydrolysis, but is not fully understood. Recently, we showed that binding of multiple PI(4,5)P2 molecules to the ASAP1 PH domain triggers an allosteric conformational switch (PIP switch) involving regions distant from the membrane interface. Here, we examined the interface between Arf1 and ASAP1 PH at the membrane surface of nanodiscs by Nuclear Magnetic Resonance (NMR) methods. Chemical Shift Perturbations (CSPs) observed upon complex formation and distance restraints obtained from Paramagnetic Relaxation Enhancement (PRE) NMR experiments between multiple MTSL-tagged Arf1 mutants and isotopically labeled ASAP1 PH were combined to build a model of the Arf1:ASAP1 PH complex at the membrane. Interestingly, the PIP switch resides at the interface between Arf1 and ASAP1 PH, shedding light on how PIP binding to ASAP1 PH may affect GTP hydrolysis.

Imaging PIP2 and BCR microclusters in B cell immunological synapse.

The humoral immune response is dependent on B cell activation and differentiation, which is typically triggered by the formation of immunological synapses at the interface between B cells and the antigen presenting surfaces. However, due to the highly dynamic and transient feature of immunological synapses, it has been difficult to capture and investigate the molecular events that occur within them. The planar lipids bilayer (PLB) supported antigen presenting surface combined with high-resolution high-speed total internal reflection fluorescence microscope (TIRFM) live cell imaging system has been proved to be a powerful tool that allows us to visualize the dynamic events in immunological synapse. In addition, the phospholipid phosphatidylinositol-(4,5)-biphosphate (PIP2) plays a unique role in B cell activation, and it is difficult to investigate the synaptic dynamics of PIP2 molecules. Hence, we describe here the general procedures for the utilization of a PLB based antigen presenting system combining TIRFM based imaging methods to visualize the spatial-temporal co-distribution of PIP2 and BCR microcluster within the B cell immunological synapse.

Cholesterol promotes clustering of PI(4,5)P2 driving unconventional secretion of FGF2.

FGF2 is a cell survival factor involved in tumor-induced angiogenesis that is secreted through an unconventional secretory pathway based upon direct protein translocation across the plasma membrane. Here, we demonstrate that both PI(4,5)P2-dependent FGF2 recruitment at the inner plasma membrane leaflet and FGF2 membrane translocation into the extracellular space are positively modulated by cholesterol in living cells. We further revealed cholesterol to enhance FGF2 binding to PI(4,5)P2-containing lipid bilayers. Based on extensive atomistic molecular dynamics (MD) simulations and membrane tension experiments, we proposed cholesterol to modulate FGF2 binding to PI(4,5)P2 by (i) increasing head group visibility of PI(4,5)P2 on the membrane surface, (ii) increasing avidity by cholesterol-induced clustering of PI(4,5)P2 molecules triggering FGF2 oligomerization, and (iii) increasing membrane tension facilitating the formation of lipidic membrane pores. Our findings have general implications for phosphoinositide-dependent protein recruitment to membranes and explain the highly selective targeting of FGF2 toward the plasma membrane, the subcellular site of FGF2 membrane translocation during unconventional secretion of FGF2.

Structural insights into the lipid and ligand regulation of a human neuronal KCNQ channel

The KCNQ family (KCNQ1-KCNQ5) of voltage-gated potassium channels plays critical roles in many physiological and pathological processes. It is known that the channel opening of all KCNQs relies on the signaling lipid molecule phosphatidylinositol 4,5-bisphosphate (PIP2). However, the molecular mechanism of PIP2 in modulating the opening of the four neuronal KCNQ channels (KCNQ2-KCNQ5), which are essential for regulating neuronal excitability, remains largely elusive. Here, we report the cryoelectron microscopy (cryo-EM) structures of human KCNQ4 determined in complex with the activator ML213 in the absence or presence of PIP2. Two PIP2 molecules are identified in the open-state structure of KCNQ4, which act as a bridge to couple the voltage-sensing domain (VSD) and pore domain (PD) of KCNQ4 leading to the channel opening. Our findings reveal the binding sites and activation mechanisms of ML213 and PIP2 for neuronal KCNQ channels, providing a framework for therapeutic intervention targeting on these important channels.

Structure of the endocytic adaptor complex reveals the basis for efficient membrane anchoring during clathrin-mediated endocytosis

During clathrin-mediated endocytosis, a complex and dynamic network of protein-membrane interactions cooperate to achieve membrane invagination. Throughout this process in yeast, endocytic coat adaptors, Sla2 and Ent1, must remain attached to the plasma membrane to transmit force from the actin cytoskeleton required for successful membrane invagination. Here, we present a cryo-EM structure of a 16-mer complex of the ANTH and ENTH membrane-binding domains from Sla2 and Ent1 bound to PIP2 that constitutes the anchor to the plasma membrane. Detailed in vitro and in vivo mutagenesis of the complex interfaces delineate the key interactions for complex formation and deficient cell growth phenotypes demonstrate its biological relevance. A hetero-tetrameric unit binds PIP2 molecules at the ANTH-ENTH interfaces and can form larger assemblies to contribute to membrane remodeling. Finally, a time-resolved small-angle X-ray scattering study of the interaction of these adaptor domains in vitro suggests that ANTH and ENTH domains have evolved to achieve a fast subsecond timescale assembly in the presence of PIP2 and do not require further proteins to form a stable complex. Together, these findings provide a molecular understanding of an essential piece in the molecular puzzle of clathrin-coated endocytic sites.

Membrane Surface Recognition by the ASAP1 Ph Domain and Consequences for Interactions with the Small GTPASE ARF1

ADP-ribosylation factor (Arf) GTPase-activating proteins (GAPs) are enzymes that need to bind to membranes to catalyze the hydrolysis of GTP bound to the small GTP-binding protein Arf. Binding of the Pleckstrin homology (PH) domain of the ArfGAP ASAP1 to membranes containing the phosphatidylinositol phosphate PIP(4,5)P2 is key for maximum GTP hydrolysis, but not fully understood. By combining Nuclear Magnetic Resonance, Neutron Reflectometry and Molecular Dynamics simulation, we show that binding of multiple PI(4,5)P2 molecules to the ASAP1 PH domain (i) triggers a functionally relevant allosteric conformational switch involving regions distant from the membrane interface, and (ii) maintains the PH domain in a well-defined orientation, allowing critical contacts between the newly exposed segments and an Arf1 mimic to occur. Our model provides a framework to understand how the interaction between PI(4,5)P2 and the ASAP1 PH domain at the membrane may play a role in the regulation of ASAP1.

Immobilization of Phosphatidylinositides Revealed by Bilayer Leaflet Decoupling.

Phosphatidylinositol 4,5-bisphosphate (PIP2) has a significantly lower mobile fraction than most other lipids in supported lipid bilayers (SLBs). Moreover, the fraction of mobile PIP2 continuously decreases with time. To explore this, a bilayer unzipping technique was designed to uncouple the two leaflets of the SLB. The results demonstrate that PIP2 molecules in the top leaflet are fully mobile, while the PIP2 molecules in the lower leaflet are immobilized on the oxide support. Over time, mobile PIP2 species flip from the top leaflet to the bottom leaflet and become trapped. It was found that PIP2 flipped between the leaflets through a defect-mediated process. The flipping could be completely inhibited when holes in the bilayer were backfilled with bovine serum albumin (BSA). Moreover, by switching from H2O to D2O, it was shown that the primary interaction between PIP2 and the underlying substrate was due to hydrogen bond formation, which outcompeted electrostatic repulsion. Using substrates with fewer surface silanol groups, like oxidized polydimethylsiloxane, led to a large increase in the mobile fraction of PIP2. Moreover, PIP2 immobilization also occurred when the bilayer was supported on a protein surface rather than glass. These results may help to explain the behavior of PIP2 on the inner leaflet of the plasma membrane, where it is involved in attaching the membrane to the underlying cytoskeleton.

Quantification of phosphoinositides reveals strong enrichment of PIP2 in HIV-1 compared to producer cell membranes

Human immunodeficiency virus type 1 (HIV-1) acquires its lipid envelope during budding from the plasma membrane of the host cell. Various studies indicated that HIV-1 membranes differ from producer cell plasma membranes, suggesting budding from specialized membrane microdomains. The phosphoinositide PI(4,5)P2 has been of particular interest since PI(4,5)P2 is needed to recruit the viral structural polyprotein Gag to the plasma membrane and thus facilitates viral morphogenesis. While there is evidence for an enrichment of PIP2 in HIV-1, fully quantitative analysis of all phosphoinositides remains technically challenging and therefore has not been reported, yet. Here, we present a comprehensive analysis of the lipid content of HIV-1 and of plasma membranes from infected and non-infected producer cells, resulting in a total of 478 quantified lipid compounds, including molecular species distribution of 25 different lipid classes. Quantitative analyses of phosphoinositides revealed strong enrichment of PIP2, but also of PIP3, in the viral compared to the producer cell plasma membrane. We calculated an average of ca. 8,000 PIP2 molecules per HIV-1 particle, three times more than Gag. We speculate that the high density of PIP2 at the HIV-1 assembly site is mediated by transient interactions with viral Gag polyproteins, facilitating PIP2 concentration in this microdomain. These results are consistent with our previous observation that PIP2 is not only required for recruiting, but also for stably maintaining Gag at the plasma membrane. We believe that this quantitative analysis of the molecular anatomy of the HIV-1 lipid envelope may serve as standard reference for future investigations.

Evidence for an Interaction between Influenza Hemagglutinin and PIP2

The influenza viral protein hemagglutinin (HA) forms dense nanoscale clusters on the plasma membrane of infected cells. HA clusters mediate fusion, which is crucial to infectivity. Previous studies have shown a connection between influenza infection and phosphoinositide-related cellular processes; however, no interaction between HA and the major plasma membrane phosphoinositide PIP2 (phosphatidylinositol-(4,5)-biphosphate) has yet been shown. Super-resolution imaging in fixed cells reveals that HA and PIP2 clusters are colocalized with similar radial distribution functions. Furthermore, single particle tracking experiments in living cells indicate that PIP2 molecules have reduced mobility and increased confinement in the presence of HA clusters. This evidence suggests an underlying interaction between HA and PIP2 and merits further investigation. Understanding cellular interactions with influenza viral proteins may lead to new anti-viral therapies that are strain independent.

Influenza Hemagglutinin Modulates Phosphatidylinositol 4,5-Bisphosphate Membrane Clustering

The lipid phosphatidylinositol 4,5-bisphosphate (PIP2) forms nanoscopic clusters in cell plasma membranes; however, the processes determining PIP2 mobility and thus its spatial patterns are not fully understood. Using super-resolution imaging of living cells, we find that PIP2 is tightly colocalized with and modulated by overexpression of the influenza viral protein hemagglutinin (HA). Within and near clusters, HA and PIP2 follow a similar spatial dependence, which can be described by an HA-dependent potential gradient; PIP2 molecules move as if they are attracted to the center of clusters by a radial force of 0.079 ± 0.002 pN in HAb2 cells. The measured clustering and dynamics of PIP2 are inconsistent with the unmodified forms of the raft, tether, and fence models. Rather, we found that the spatial PIP2 distributions and how they change in time are explained via a novel, to our knowledge, dynamic mechanism: a radial gradient of PIP2 binding sites that are themselves mobile. This model may be useful for understanding other biological membrane domains whose distributions display gradients in density while maintaining their mobility.

Interaction of the human erythrocyte Band 3 anion exchanger 1 (AE1, SLC4A1) with lipids and glycophorin A: Molecular organization of the Wright (Wr) blood group antigen.

The Band 3 (AE1, SLC4A1) membrane protein is found in red blood cells and in kidney where it functions as an electro-neutral chloride/bicarbonate exchanger. In this study, we have used molecular dynamics simulations to provide the first realistic model of the dimeric membrane domain of human Band 3 in an asymmetric lipid bilayer containing a full complement of phospholipids, including phosphatidylinositol 4,5–bisphosphate (PIP2) and cholesterol, and its partner membrane protein Glycophorin A (GPA). The simulations show that the annular layer in the inner leaflet surrounding Band 3 was enriched in phosphatidylserine and PIP2 molecules. Cholesterol was also enriched around Band 3 but also at the dimer interface. The interaction of these lipids with specific sites on Band 3 may play a role in the folding and function of this anion transport membrane protein. GPA associates with Band 3 to form the Wright (Wr) blood group antigen, an interaction that involves an ionic bond between Glu658 in Band 3 and Arg61 in GPA. We were able to recreate this complex by performing simulations to allow the dimeric transmembrane portion of GPA to interact with Band 3 in a model membrane. Large-scale simulations showed that the GPA dimer can bridge Band 3 dimers resulting in the dynamic formation of long strands of alternating Band 3 and GPA dimers.

PtdIns(4,5)P2 stabilizes active states of GPCRs and enhances selectivity of G-protein coupling.

G protein-coupled receptors (GPCRs) are involved in many physiological processes and are therefore key drug targets 1. Despite detailed structural information however the effects of lipids on the receptors themselves, or on their downstream coupling to G proteins, are ill defined. Here we use native mass spectrometry to identify endogenous lipids bound to three class A GPCRs. We demonstrate preferential binding of phosphatidylinositol 4,5 bisphosphate (PIP2) over related lipids and confirm hotspots for PIP2 binding on the intracellular face of the receptors. Endogenous lipids were also observed bound directly to the entire adenosine A2A receptor (A2AR) trimeric Gαsβγ protein complex in the gas phase. Recruiting engineered Gα subunits (mini-Gs, i, 12) 2 we found that the β1AR: mini-Gαs complex is stabilised by the binding of two PIP2 molecules. By contrast PIP2 did not stabilise coupling between β1AR and other Gα subunits (mini- Gαi, 12) or a high affinity nanobody. Other endogenous lipids that bound to receptors showed no effect on coupling, highlighting the specificity of PIP2. Potential of mean force calculations and increased GTP turnover of the activated neurotensin receptor: trimeric Gαiβγ complex in the presence of PIP2 further support the specific effect of PIP2 on coupling. Together our results identify key residues on cognate Gα subunits that mediate simultaneous PIP2 bridging interactions between basic residues on class A receptors, which do not correspond with those on class B GPCRs. By uncovering the effects of lipid modulation of receptors we highlight consequences for understanding function, G protein selectivity and drug targeting of class A GPCRs.

Molecular details of spontaneous insertion and interaction of HCV non-structure 3 protease protein domain with PIP2-containing membrane.

Hepatitis C virus (HCV), known as the leading cause of liver cirrhosis, viral hepatitis, and hepatocellular carcinoma, has been affecting more than 150 million people globally. The HCV non-structure 3 (NS3) protease protein domain plays a key role in HCV replication and pathogenesis; and is currently a primary target for HCV antiviral therapy. Through unbiased molecular dynamics simulations which take advantage of the novel highly mobile membrane mimetic model, we constructed the membrane-bound state of the protein domain at the atomic level. Our results indicated that protease domain of HCV NS3 protein can spontaneously bind and penetrate to an endoplasmic reticulum complex membrane containing phosphatidylinositol 4,5-bisphosphate (PIP2). An amphipathic helix α0 and loop S1 show their anchoring role to keep the protein on the membrane surface. Proper orientation of the protein domain at membrane surface was identified through measuring tilt angles of two specific vectors, wherein residue R161 plays a crucial role in its final orientation. Remarkably, PIP2 molecules were observed to bind to three main sites of the protease domain via specific electrostatic contacts and hydrogen bonds. PIP2-interaction determines the protein orientation at the membrane while both hydrophobic interplay and PIP2-interaction can stabilize the NS3 - membrane complex. Simulated results provide us with a detailed characterization of insertion, orientation and PIP2-interaction of NS3 protease domain at membrane environment, thus enhancing our understanding of structural functions and mechanism for the association of HCV non-structure 3 protein with respect to ER membranes.

How HIV-1 Takes Advantage of PI(4,5)P2 Clusters during Viral Assembly

Located at the inner leaflet of the plasma membrane (PM), phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2, here PIP2] has been proposed to exist in distinct pools or microdomains. The origin of such spatially separated pools and the contribution of this distribution to PIP2 function is unknown. PIP2 is critically involved not only in many cellular reactions, but also in HIV-1 particle assembly and release. For example, in vitro at a physiological level of 2 mol%, PIP2 significantly enhances membrane binding of the viral structural protein Gag. And the viral membrane is enriched in PIP2 compared with the PM. Here, we exploited model membranes with PM inner leaflet lipid compositions to characterize PIP2 clustering and to understand how HIV-1 takes advantage of these clusters. Using acyl-chain fluorescently labeled TopFluor-PIP2 in fluorometric assays, we demonstrated that PIP2 begins to form clusters at approximately 0.05 mol%, far below physiological levels. This clustering is dependent on multivalent metal ions. PIP2 clustering behavior is headgroup-specific and independent of acyl chain type. In cells PIP2 is likely to exist in two forms, either as individual PIP2 molecules or else clustered with multivalent metal ions and other PIP2 molecules. Manipulating PIP2 to either form clusters or stay as individual molecules in membranes enabled us to study whether proteins recognize PIP2 clusters, and whether protein binding reorganizes PIP2 distributions. We found that in microscopic-based giant unilamellar vesicle (GUVs) assays, naturally myristoylated HIV-1 MA, the membrane binding domain of Gag, strongly prefers binding to PIP2 in clusters compared with individual PIP2 molecules, whereas charge sensor proteins hardly sense any difference in between individual or clustered PIP2. We are currently examining whether the PIP2 lateral distribution is influenced upon MA binding. We hypothesize that HIV-1 exploits PIP2 clusters as sites of viral assembly.

Insight into Phosphatidylinositol-Dependent Membrane Localization of the Innate Immune Adaptor Protein Toll/Interleukin 1 Receptor Domain-Containing Adaptor Protein.

The toll/interleukin 1 receptor (TIR) domain-containing adaptor protein (TIRAP) plays an important role in the toll-like receptor (TLR) 2, TLR4, TLR7, and TLR9 signaling pathways. TIRAP anchors to phosphatidylinositol (PI) 4,5-bisphosphate (PIP2) on the plasma membrane and PI (3,4,5)-trisphosphate (PIP3) on the endosomal membrane and assists in recruitment of the myeloid differentiation primary response 88 protein to activated TLRs. To date, the structure and mechanism of TIRAP’s membrane association are only partially understood. Here, we modeled an all-residue TIRAP dimer using homology modeling, threading, and protein–protein docking strategies. Molecular dynamics simulations revealed that PIP2 creates a stable microdomain in a dipalmitoylphosphatidylcholine bilayer, providing TIRAP with its physiologically relevant orientation. Computed binding free energy values suggest that the affinity of PI-binding domain (PBD) for PIP2 is stronger than that of TIRAP as a whole for PIP2 and that the short PI-binding motif (PBM) contributes to the affinity between PBD and PIP2. Four PIP2 molecules can be accommodated by distinct lysine-rich surfaces on the dimeric PBM. Along with the known PI-binding residues (K15, K16, K31, and K32), additional positively charged residues (K34, K35, and R36) showed strong affinity toward PIP2. Lysine-to-alanine mutations at the PI-binding residues abolished TIRAP’s affinity for PIP2; however, K34, K35, and R36 consistently interacted with PIP2 headgroups through hydrogen bond (H-bond) and electrostatic interactions. TIRAP exhibited a PIP2-analogous intermolecular contact and binding affinity toward PIP3, aided by an H-bond network involving K34, K35, and R36. The present study extends our understanding of TIRAP’s membrane association, which could be helpful in designing peptide decoys to block TLR2-, TLR4-, TLR7-, and TLR9-mediated autoimmune diseases.