Liposome Binding Assays Research Articles

Interplay of Membrane Lipids Differentially Affects Lipid Binding of Phosphatidic Acid Effectors

The interaction of phosphatidic acid (PA) with membrane proteins is responsible for a host of cellular functions. To date no PA specific binding domain has been identified. Instead, electrostatic and hydrophobic interactions are likely to work in tandem to regulate PA effector-PA binding. Electrostatic interactions with the PA headgroup are explained by the electrostatic-hydrogen bond switch model, whereas hydrophobic interactions are explained by the negative curvature of (unsaturated) PA. In order to shed light on PA-protein binding we study the interaction of PA with PA effectors in complex lipid mixture, not just phosphatidylcholine (PC) bilayers, using liposome binding assays. Previously we showed that PE differentially affects the binding of PA effectors. We extended this work to now show that the opposite effect is observed in the presence of lyso-phosphocholine (LPC). We also show that under the right conditions diolyeoyl glycerol (DOG) stimulates binding to PA for a well-known and extensively characterized PA-binding protein. PA-effector-PA binding is thus significantly affected by the presence of other membrane lipids. These studies show the need to incorporate other membrane lipids when investigating the interaction of putative PA binding proteins with PA, thereby further our understanding of PA mediated signaling.

Deletion of the Phytophthora sojae Avirulence Gene Avr1d Causes Gain of Virulence on Rps1d

Phytophthora sojae is an oomycete and a pathogen of soybean that causes root rot. During infection P. sojae delivers effector proteins into host cells to foster disease. However, effector-triggered immunity (ETI) results when pathogen factors are recognized by host resistance (R) proteins. We have now identified the P. sojae Avr1d gene, which encodes a predicted effector protein with the amino acid motif Arg-X-Leu-Arg (RXLR). Genetic mapping of 16 different P. sojae isolates and of a segregating F2 population of 40 individuals shows that the predicted RXLR effector gene Avh6 precisely cosegregates with the Avr1d phenotype. Transient expression assays confirm that Avr1d triggers cell death specifically in Rps1d soybean plants. The Avr1d gene is present in P. sojae strains that are avirulent on Rps1d, whereas the gene is deleted from the genome of virulent strains. Two sequence variants of the Avr1d gene encoding different protein products occur in P. sojae strains, but both are recognized by Rps1d and cause ETI. Liposome binding assays show that Avr1d has affinity for phosphatidylinositol 4-phosphate and that binding can be disrupted by mutation of lysine residues in the carboxy-terminal effector domain of the protein. The identification of Avr1d aids pathogen diagnostics and soybean cultivar development.

Large Scale Structural Rearrangement of a Serine Hydrolase from Francisella tularensis Facilitates Catalysis

Tularemia is a deadly, febrile disease caused by infection by the gram-negative bacterium, Francisella tularensis. Members of the ubiquitous serine hydrolase protein family are among current targets to treat diverse bacterial infections. Herein we present a structural and functional study of a novel bacterial carboxylesterase (FTT258) from F. tularensis, a homologue of human acyl protein thioesterase (hAPT1). The structure of FTT258 has been determined in multiple forms, and unexpectedly large conformational changes of a peripheral flexible loop occur in the presence of a mechanistic cyclobutanone ligand. The concomitant changes in this hydrophobic loop and the newly exposed hydrophobic substrate binding pocket suggest that the observed structural changes are essential to the biological function and catalytic activity of FTT258. Using diverse substrate libraries, site-directed mutagenesis, and liposome binding assays, we determined the importance of these structural changes to the catalytic activity and membrane binding activity of FTT258. Residues within the newly exposed hydrophobic binding pocket and within the peripheral flexible loop proved essential to the hydrolytic activity of FTT258, indicating that structural rearrangement is required for catalytic activity. Both FTT258 and hAPT1 also showed significant association with liposomes designed to mimic bacterial or human membranes, respectively, even though similar structural rearrangements for hAPT1 have not been reported. The necessity for acyl protein thioesterases to have maximal catalytic activity near the membrane surface suggests that these conformational changes in the protein may dually regulate catalytic activity and membrane association in bacterial and human homologues.

Molecular Basis of the Interaction between Signaling Lipids and Proteins; the Special Case of Phosphatidic Acid

Intricate networks of signaling cascades regulate cellular development and response to environmental factors. within these cascades, the lipid second messenger phosphatidic acid (PA) is an important regulator in all eukaryotes. Rapid but transient increase of PA levels regulate growth, acclimatization and survival during development and stress response. Signaling lipids, including PA, function by selectively recruiting proteins to the membrane. This facilitates either the activation or inhibition of these target proteins. However, the exact details of phospholipid-mediated stress response mechanisms are still largely unclear, partially due to the lack of characterized target proteins of PA.Here we report on characterization of the interaction between various regulatory proteins and lipid second messengers by determining their lipid binding specificity and affinity for PA. Furthermore, to assess relative binding affinity in variable structural lipid environments, in vitro liposome-binding assays with liposomes covering a range of lipid compositions are performed. In our binding studies, phosphatidylethanolamine is utilized to study the role of membrane curvature and electrostatics in lipid-binding.Specific attention is given to the electrostatic/hydrogen bond switch model. Hydrogen bond formation between basic amino acids and the PA phosphomonoester headgroup increases the negative charge of PA and allows for docking of the protein binding domain. The elucidation of the mechanism behind the binding of proteins to lipid second messengers such as PA will further develop our understanding of their function in plant growth and stress responses. This in turn can help in understanding regulation and cell functioning in numerous organisms.

The Calmodulin-like Calcium Binding Protein EhCaBP3 of Entamoeba histolytica Regulates Phagocytosis and Is Involved in Actin Dynamics

Phagocytosis is required for proliferation and pathogenesis of Entamoeba histolytica and erythrophagocytosis is considered to be a marker of invasive amoebiasis. Ca2+ has been found to play a central role in the process of phagocytosis. However, the molecular mechanisms and the signalling mediated by Ca2+ still remain largely unknown. Here we show that Calmodulin-like calcium binding protein EhCaBP3 of E. histolytica is directly involved in disease pathomechanism by its capacity to participate in cytoskeleton dynamics and scission machinery during erythrophagocytosis. Using imaging techniques EhCaBP3 was found in phagocytic cups and newly formed phagosomes along with actin and myosin IB. In vitro studies confirmed that EhCaBP3 directly binds actin, and affected both its polymerization and bundling activity. Moreover, it also binds myosin 1B in the presence of Ca2+. In cells where EhCaBP3 expression was down regulated by antisense RNA, the level of RBC uptake was reduced, myosin IB was found to be absent at the site of pseudopod cup closure and the time taken for phagocytosis increased, suggesting that EhCaBP3 along with myosin 1B mediate the closure of phagocytic cups. Experiments with EhCaBP3 mutant defective in Ca2+ -binding showed that Ca2+ binding is required for phagosome formation. Liposome binding assay revealed that EhCaBP3 recruitment and enrichment to membrane is independent of any cellular protein as it binds directly to phosphatidylserine. Taken together, our results suggest a novel pathway mediating phagocytosis in E. histolytica, and an unusual mechanism of modulation of cytoskeleton dynamics by two calcium binding proteins, EhCaBP1 and EhCaBP3 with mostly non-overlapping functions.

Phosphatidic Acid Binds and Stimulates Arabidopsis Sphingosine Kinases

Phosphatidic acid (PA) and phytosphingosine-1-phosphate (phyto-S1P) have both been identified as lipid messengers mediating plant response to abscisic acid (ABA). To determine the relationship of these messengers, we investigated the direct interaction of PA with Arabidopsis sphingosine kinases (SPHKs) that phosphorylate phytosphingosine to generate phyto-S1P. Two unique SPHK cDNAs were cloned from the annotated At4g21540 locus of Arabidopsis, and the two transcripts are differentially expressed in Arabidopsis tissues. Both SPHKs are catalytically active, phosphorylating various long-chain sphingoid bases (LCBs) and are associated with the tonoplast. They both interact with PA as demonstrated by lipid-filter binding, liposome binding, and surface plasmon resonance (SPR). SPHK1 and SPHK2 exhibited strong binding to 18:1/18:1, 16:0/18:1, and 16:0/18:2 PA, but poor binding to 16:0/16:0, 8:0/8:0, 18:0/18:0, and 18:2/18:2 PA. Surface dilution kinetics analysis indicates that PA stimulates SPHK activity by increasing the specificity constant through decreasing K(m)(B). The results show that the annotated At4g21540 locus is actually comprised of two separate SPHK genes. PA binds to both SPHKs, and the interaction promotes lipid substrate binding to the catalytic site of the enzyme. The PA-SPHK interaction depends on the PA molecular species. The data suggest that these two Arabidopsis SPHKs are molecular targets of PA, and the PA stimulation of SPHK is part of the signaling networks in Arabidopsis.

An Amphipathic Alpha‐Helical Domain Identified in Nucleoporins Plays a Role at The Nuclear Pore Complex of Saccharomyces Cerevisiae

All macromolecular transport between the nucleus and cytoplasm occurs via nuclear pore complexes (NPCs) embedded in the nuclear envelope. NPCs are composed of multiple copies of proteins, termed nucleoporin (Nups) which assemble sequentially into nascent nuclear pores generated following fusion of the inner and outer nuclear membranes. While much is known about NPC structure, the mechanisms governing NPC biogenesis remain largely undefined. Recently, an amphipathic α‐helix (ALPS for ArfGAP1 lipid packing sensor) motif, which mediates protein association with highly curved liposomes, has been defined. This ALPS motif was identified in protein components of two early associating S. Cerevisiae (sc) NPC subcomplexes; the scNup84 subcomplex (specifically Nup85 and Nup120) and the scNup170 subcomplex (specifically Nup170 and Nup188) and may help proteins associate with newly developing NPCs. Utilizing microscopy techniques and yeast genetics we have identified an important role for the ALPS domain in vivo and our evidence suggests that ALPS domains of different NUPs have redundant functions. Utilizing in vitro liposome binding assays we find that the ALPS domain is sufficient for liposome binding and that this binding is dependent on liposome size. Together, this data provides the first evidence for an in vivo function of the ALPS motif with an impact in the broad membrane biology field, and concomitantly expands our understanding of NPC assembly.Grant Funding Source: IRACDA & RO1

Phosphoinositide 3-kinase γ has Multiple Phospholipid Binding Sites

Phosphoinositide 3-kinase gamma is a multifunctional enzyme with lipid and protein kinase activities that also acts as a scaffold protein in many diverse signalling processes. The enzyme contains five different domains, but their individual contributions to membrane binding are not fully understood. Here, using in vitro liposome binding assays of individual domains and deletion constructs of human phosphoinositide 3-kinase gamma, we show that each domain is capable of binding anionic phospholipids to varying degrees, depending on the charge of the anionic substrate. Moreover, with the exception of the C2-domain, deletion of any single protein domain results in a complete loss of kinase activity toward both lipids and proteins.

Binding of GAPR-1 to negatively charged phospholipid membranes: Unusual binding characteristics to phosphatidylinositol

Golgi-Associated Plant Pathogenesis-Related protein 1 (GAPR-1) is a mammalian protein that belongs to the superfamily of plant pathogenesis-related proteins group 1 (PR-1). GAPR-1 strongly associates with lipid rafts at the cytosolic leaflet of the Golgi membrane. The myristoyl moiety at the N-terminus of GAPR-1 contributes to membrane binding but is not sufficient for stable membrane anchorage. GAPR-1 is positively charged at physiological pH, which allows for additional membrane interactions with proteins or lipids. To determine the potential contribution of lipids to membrane binding of GAPR-1, we used a liposome binding assay. Here we report that non-myristoylated GAPR-1 stably binds liposomes that contain the negatively charged lipids phosphatidylserine, phosphatidylglycerol, phosphatidylinositol, or phosphatidic acid. GAPR-1 displays the highest preference for phosphatidic acid-containing liposomes. In contrast, lysozyme, which contains a similar surface charge, did not bind to these liposomes, except for a weak membrane association with PA-containing liposomes. Interestingly, GAPR-1 binds to phosphatidylinositol with unusual characteristics. Denaturation or organic extraction of GAPR-1 does not result in dissociation of phosphatidylinositol from GAPR-1. The association of phosphatidylinositol with GAPR-1 results in a diffuse gel-shift in SDS-PAGE. Mass spectrometric analysis of gel-shifted GAPR-1 showed the association of up to 3 molecules of phosphatidylinositol with GAPR-1. These results suggest that the lipid composition contributes to the GAPR-1 binding to biological membranes.

Sulfatides Partition Disabled-2 in Response to Platelet Activation

BackgroundPlatelets contact each other at the site of vascular injury to stop bleeding. One negative regulator of platelet aggregation is Disabled-2 (Dab2), which is released to the extracellular surface upon platelet activation. Dab2 inhibits platelet aggregation through its phosphotyrosine-binding (PTB) domain by competing with fibrinogen for αIIbβ3 integrin receptor binding by an unknown mechanism.Methodology/Principal FindingsUsing protein-lipid overlay and liposome-binding assays, we identified that the N-terminal region of Dab2, including its PTB domain (N-PTB), specifically interacts with sulfatides. Moreover, we determined that such interaction is mediated by two conserved basic motifs with a dissociation constant (Kd) of 0.6 µM as estimated by surface plasmon resonance (SPR) analysis. In addition, liposome-binding assays combined with mass spectroscopy studies revealed that thrombin, a strong platelet agonist, cleaved N-PTB at a site located between the basic motifs, a region that becomes protected from thrombin cleavage when bound to sulfatides. Sulfatides on the platelet surface interact with coagulation proteins, playing a major role in haemostasis. Our results show that sulfatides recruit N-PTB to the platelet surface, sequestering it from integrin receptor binding during platelet activation. This is a transient recruitment that follows N-PTB internalization by an actin-dependent process.Conclusions/SignificanceOur experimental data support a model where two pools of Dab2 co-exist at the platelet surface, in both sulfatide- and integrin receptor-bound states, and their balance controls the extent of the clotting response.

Regulation of the Substrate Preference of p190RhoGAP by Protein Kinase C-Mediated Phosphorylation of a Phospholipid Binding Site

The Rho family GTPases are stringently regulated through the action of a large family of GTPase activating proteins (GAPs) that stimulate their relatively weak intrinsic GTP hydrolyzing activity. The p190RhoGAPs, which include the p190A and p190B proteins, are potent and widely expressed GAPs acting on both Rho and Rac GTPases. We have observed that several acidic phospholipids inhibit the RhoGAP activity and promote the RacGAP activity of p190 proteins. In liposome binding assays we have demonstrated that binding of p190A to phospholipids is controlled by electrostatic interactions. Using mapping techniques, we determined that a small polybasic peptide stretch within p190A is a common site for both the phospholipid binding and PKC phosphorylation. Moreover, PKC-mediated phosphorylation of two amino acids (serine-1221 and threonine-1226) within this region of p190A prevents the binding and substrate specificity regulation by phospholipids. Transfection of COS-7 cells with mutant forms of p190A either unable to bind to phospholipids or unable to become phosphorylated induced distinct morphological changes. Together, these findings reveal a novel GAP regulatory mechanism in which phosphorylation indirectly alters GTPase substrate preference by affecting the interaction with acidic phospholipids. Our observations provide a potential mechanism of Rac/Rho antagonism described in several cellular functions.

Two-Step Conformational Changes in a Coronavirus Envelope Glycoprotein Mediated by Receptor Binding and Proteolysis

The coronaviruses mouse hepatitis virus type 2 (MHV-2) and severe acute respiratory syndrome coronavirus (SARS-CoV) utilize proteases to enter host cells. Upon receptor binding, the spike (S) proteins of both viruses are activated for membrane fusion by proteases, such as trypsin, present in the environment, facilitating virus entry from the cell surface. In contrast, in the absence of extracellular proteases, these viruses can enter cells via an endosomal pathway and utilize endosomal cathepsins for S protein activation. We demonstrate that the MHV-2 S protein uses multistep conformational changes for membrane fusion. After interaction with a soluble form of the MHV receptor (CEACAM1a), the metastable form of S protein is converted to a stable trimer, as revealed by mildly denaturing sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Liposome-binding assays indicate that the receptor-bound virions are associated with the target membrane through hydrophobic interactions. The exposure of receptor-bound S protein to trypsin or cathepsin L (CPL) induces the formation of six-helix bundles (6HB), the final conformation. This trypsin- or CPL-mediated conversion to 6HB can be blocked by a heptad repeat peptide known to block the formation of 6HB. Although trypsin treatment enabled receptor-bound MHV-2 to enter from the cell surface, CPL failed to do so. Interestingly, consecutive treatment with CPL and then chlorpromazine enabled a portion of the virus to enter from cell surface. These results suggest that trypsin suffices for the induction of membrane fusion of receptor-primed S protein, but an additional unidentified cellular factor is required to trigger membrane fusion by CPL.

The Toca-1-N-WASP Complex Links Filopodial Formation to Endocytosis

The transducer of Cdc42-dependent actin assembly (Toca-1)-N-WASP complex was isolated as an essential cofactor for Cdc42-driven actin polymerization in vitro. Toca-1 consists of an N-terminal F-BAR domain, followed by a Cdc42 binding site (HR1 domain) and an SH3 domain, (the N-WASP interacting site). N-WASP is an activator of actin nucleation through the Arp2/3 complex. The aim of the present study was to investigate the cellular function of the Toca-1-N-WASP complex. We report that Toca-1 induces filopodia and neurites as does N-WASP in N1E115 neuroblastoma cells. Toca-1 requires the F-BAR domain, Cdc42 binding site, and SH3 domain to induce filopodia. Toca-1 and N-WASP both require each other to induce filopodia. The expression of Toca-1 and N-WASP affects the distribution, size, and number of Rab5 positive membranes. Toca-1 interacts directly with N-WASP in filopodia and Rab5 membrane as seen by Forster resonance energy transfer. Thus the Toca-1-N-WASP complex localizes to and induces the formation of filopodia and endocytic vesicles. Last, three inhibitors of endocytosis, Dynamin-K44A, Eps15Delta95/295, and clathrin heavy chain RNA interference, block Toca-1-induced filopodial formation. Taken together, these data suggest that the Toca-1-N-WASP complex can link filopodial formation to endocytosis.

Microplate-Based Analysis of Protein−Membrane Binding Interactions via Immobilization of Whole Liposomes Containing a Biotinylated Anchor

Cellular membranes play key roles in the regulation of a range of important biological processes. However, the characterization of membrane involvement in these events is difficult to achieve due to the complexity of the membrane bilayer and the challenges associated with handling and analyzing these systems. As such, rapid and reliable approaches for characterizing membrane-based processes are necessary. To address this issue, we have first developed an azide-tagged modular lipid anchor scaffold (2) that can be conveniently derivatized via click chemistry to functionalize the membrane surface. This was used to access biotin- and fluorescein-lipid conjugates 1a and 1b, respectively. These compounds were then employed to perform and characterize the immobilization of liposomes containing biotin-anchor 1a onto streptavidin-coated microplates. Results from these studies indicated clean, biotin-dependent surface deposition. This strategy for liposome attachment was then applied to a microplate-based platform to detect the binding of receptor proteins to immobilized liposomes, specifically for the membrane binding of protein kinase Calpha (PKCalpha). The resulting data indicated direct detection of binding to the membrane-functionalized surface. The reported approaches provide efficient methods for the derivatization of the membrane surface, which is applicable to the study of membrane-based processes. Finally, the described microplate-based liposome binding assay allows for high-throughput analysis of important protein-membrane binding events.

Functional assessment of perforin C2 domain mutations illustrates the critical role for calcium-dependent lipid binding in perforin cytotoxic function

AbstractPerforin-mediated lymphocyte cytotoxicity is critical for pathogen elimination and immune homeostasis. Perforin disruption of target cell membranes is hypothesized to require binding of a calcium-dependent, lipid-inserting, C2 domain. In a family affected by hemophagocytic lymphohistiocytosis, a severe inflammatory disorder caused by perforin deficiency, we identified 2 amino acid substitutions in the perforin C2 domain: T435M, a previously identified mutant with disputed pathogenicity, and Y438C, a novel substitution. Using biophysical modeling, we predicted that the T435M substitution, but not Y438C, would interfere with calcium binding and thus cytotoxic function. The capacity for cytotoxic function was tested after expression of the variant perforins in rat basophilic leukemia cells and murine cytotoxic T lymphocytes. As predicted, cells transduced with perforin-T435M lacked cytotoxicity, but those expressing perforin-Y438C displayed intact cytotoxic function. Using novel antibody-capture and liposome-binding assays, we found that both mutant perforins were secreted; however, only nonmutated and Y438C-substituted perforins were capable of calcium-dependent lipid binding. In addition, we found that perforin-Y438C was capable of mediating cytotoxicity without apparent proteolytic maturation. This study clearly demonstrates the pathogenicity of the T435M mutation and illustrates, for the first time, the critical role of the human perforin C2 domain for calcium-dependent, cytotoxic function.

A high‐resolution solution structure of a trypanosomatid FYVE domain

FYVE domain proteins play key roles in regulating membrane traffic in eukaryotic cells. The FYVE domain displays a remarkable specificity for the head group of the target lipid, phosphatidylinositol 3-phosphate (PtdIns[3]P). We have identified five putative FYVE domain proteins in the genome of the protozoan parasite Leishmania major, three of which are predicted to contain a functional PtdIns(3)P-binding site. The FYVE domain of one of these proteins, LmFYVE-1, bound PtdIns(3)P in liposome-binding assays and targeted GFP to acidified late endosomes/lysosomes in mammalian cells. The high-resolution solution structure of its N-terminal FYVE domain (LmFYVE-1[1-79]) was solved by nuclear magnetic resonance. Functionally significant clusters of residues of the LmFYVE-1 domain involved in PtdIns(3)P binding and dependence on low pH for tight binding were identified. This structure is the first trypanosomatid membrane trafficking protein to be determined and has been refined to high precision and accuracy using residual dipolar couplings.

Escherichia coli Signal Recognition Particle Receptor FtsY Contains an Essential and Autonomous Membrane-binding Amphipathic Helix

Escherichia coli membrane protein biogenesis is mediated by a signal recognition particle and its membrane-associated receptor (FtsY). Although crucial for its function, it is still not clear how FtsY interacts with the membrane. Analysis of the structure/function differences between severely truncated active (NG+1) and inactive (NG) mutants of FtsY enabled us to identify an essential membrane-interacting determinant. Comparison of the three-dimensional structures of the mutants, combined with site-directed mutagenesis, modeling, and liposome-binding assays, revealed that FtsY contains a conserved autonomous lipid-binding amphipathic alpha-helix at the N-terminal end of the N domain. Deletion experiments showed that this helix is essential for FtsY function in vivo, thus offering, for the first time, clear evidence for the functionally important, physiologically relevant interaction of FtsY with lipids.

Clustering and synaptic targeting of PICK1 requires direct interaction between the PDZ domain and lipid membranes

Protein interacting with c kinase 1 (PICK1) regulates the trafficking of receptors and ion-channels such as AMPA receptors. Traditionally, the PICK1 PDZ domain is regarded as an adaptor capable of binding to receptors trafficked by PICK1, and the lipid-binding BAR domain functions to tether PICK1 directly to membranes. Here, we show that the PICK1 PDZ domain can directly interact with lipid membranes. The PDZ domain and lipid membrane interaction is mediated by both a polybasic amino-acid cluster and a conserved 'Cys-Pro-Cys' motif located away from the peptide ligand-binding groove. Disruption of the PDZ and lipid membrane interaction totally abolished synaptic targeting of PICK1. Although mutation of the CPC motif did not affect the interaction between PICK1 and AMPA receptors, the mutant PICK1 was unable to cluster the GluR2 subunit of the receptor. In neurons, PICK1 containing the same mutation displayed dramatically compromised capacity in the trafficking of AMPA receptors. Taken together, our findings not only uncovered the novel lipid membrane-binding property of the PICK1 PDZ domain, but also provided direct evidence supporting the functional relevance of the PDZ-lipid interaction.

An Electrostatic/Hydrogen Bond Switch as the Basis for the Specific Interaction of Phosphatidic Acid with Proteins

Phosphatidic acid (PA) is a minor but important phospholipid that, through specific interactions with proteins, plays a central role in several key cellular processes. The simple yet unique structure of PA, carrying just a phosphomonoester head group, suggests an important role for interactions with the positively charged essential residues in these proteins. We analyzed by solid-state magic angle spinning 31P NMR and molecular dynamics simulations the interaction of low concentrations of PA in model membranes with positively charged side chains of membrane-interacting peptides. Surprisingly, lysine and arginine residues increase the charge of PA, predominantly by forming hydrogen bonds with the phosphate of PA, thereby stabilizing the protein-lipid interaction. Our results demonstrate that this electrostatic/hydrogen bond switch turns the phosphate of PA into an effective and preferred docking site for lysine and arginine residues. In combination with the special packing properties of PA, PA may well be nature's preferred membrane lipid for interfacial insertion of positively charged membrane protein domains.

The C2A-C2B Linker Defines the High Affinity Ca2+ Binding Mode of Rabphilin-3A

The Ca(2+) binding properties of C2 domains are essential for the function of their host proteins. We present here the first crystal structures showing an unexpected Ca(2+) binding mode of the C2B domain of rabphilin-3A in atomic detail. Acidic residues from the linker region between the C2A and C2B domains of rabphilin-3A interact with the Ca(2+)-binding region of the C2B domain. Because of these interactions, the coordination sphere of the two bound Ca(2+) ions is almost complete. Mutation of these acidic residues to alanine resulted in a 10-fold decrease in the intrinsic Ca(2+) binding affinity of the C2B domain. Using NMR spectroscopy, we show that this interaction occurred only in the Ca(2+)-bound state of the C2B domain. In addition, this Ca(2+) binding mode was maintained in the C2 domain tandem fragment. In NMR-based liposome binding assays, the linker was not released upon phospholipid binding. Therefore, this unprecedented Ca(2+) binding mode not only shows how a C2 domain increases its intrinsic Ca(2+) affinity, but also provides the structural base for an atypical protein-Ca(2+)-phospholipid binding mode of rabphilin-3A.