Entropic and enthalpic contributions to annexin V-membrane binding: a comprehensive quantitative model

Annexin V binds to membranes with very high affinity, but the factors responsible remain to be quantitatively elucidated. Analysis by isothermal microcalorimetry and calcium titration under conditions of low membrane occupancy showed that there was a strongly positive entropy change upon binding. For vesicles containing 25% phosphatidylserine at 0.15 m ionic strength, the free energy of binding was -53 kcal/mol protein, whereas the enthalpy of binding was -38 kcal/mol. Addition of 4 m urea decreased the free energy of binding by about 30% without denaturing the protein, suggesting that hydrophobic forces make a significant contribution to binding affinity. This was confirmed by mutagenesis studies that showed that binding affinity was modulated by the hydrophobicity of surface residues that are likely to enter the interfacial region upon protein-membrane binding. The change in free energy was quantitatively consistent with predictions from the Wimley-White scale of interfacial hydrophobicity. In contrast, binding affinity was not increased by making the protein surface more positively charged, nor decreased by making it more negatively charged, ruling out general ionic interactions as major contributors to binding affinity. The affinity of annexin V was the same regardless of the head group present on the anionic phospholipids tested (phosphatidylserine, phosphatidylglycerol, phosphatidylmethanol, and cardiolipin), ruling out specific interactions between the protein and non-phosphate moieties of the head group as a significant contributor to binding affinity. Analysis by fluorescence resonance energy transfer showed that multimers did not form on phosphatidylserine membranes at low occupancy, indicating that annexin-annexin interactions did not contribute to binding affinity. In summary, binding of annexin V to membranes is driven by both enthalpic and entropic forces. Dehydration of hydrophobic regions of the protein surface as they enter the interfacial region makes an important contribution to overall binding affinity, supplementing the role of protein-calcium-phosphate chelates.     

The specificity of carboxypeptidase Y may be altered by changing the hydrophobicity of the S'1 binding pocket.

The S'1 binding pocket of carboxypeptidase Y is hydrophobic, spacious, and open to solvent, and the enzyme exhibits a preference for hydrophobic P'1 amino acid residues. Leu272 and Ser297, situated at the rim of the pocket, and Leu267, slightly further away, have been substituted by site-directed mutagenesis. The mutant enzymes have been characterized kinetically with respect to their P'1 substrate preferences using the substrate series FA-Ala-Xaa-OH (Xaa = Leu, Glu, Lys, or Arg) and FA-Phe-Xaa-OH (Xaa = Ala, Val, or Leu). The results reveal that hydrophobic P'1 residues bind in the vicinity of residue 272 while positively charged P'1 residues interact with Ser297. Introduction of Asp or Glu at position 267 greatly reduced the activity toward hydrophobic P'1 residues (Leu) and increased the activity two- to three-fold for the hydrolysis of substrates with Lys or Arg in P'1. Negatively charged substituents at position 272 reduced the activity toward hydrophobic P'1 residues even more, but without increasing the activity toward positively charged P'1 residues. The mutant enzyme L267D + L272D was found to have a preference for substrates with C-terminal basic amino acid residues. The opposite situation, where the positively charged Lys or Arg were introduced at one of the positions 267, 272, or 297, did not increase the rather low activity toward substrates with Glu in the P'1 position but greatly reduced the activity toward substrates with C-terminal Lys or Arg due to electrostatic repulsion. The characterized mutant enzymes exhibit various specificities, which may be useful in C-terminal amino acid sequence determinations.     

Specific interaction to PIP2 increases the kinetic rate of membrane binding of VILIPs,a subfamily of Neuronal Calcium Sensors (NCS) proteins

VIsinin-LIke Proteins (VILIPs) are a subfamily of the Neuronal Calcium Sensor (NCS) proteins, which possess both N-myristoylation and EF-hand motifs allowing for a putative ‘calcium–myristoyl switch’ regulation mechanism. It has previously been established that myristoyl conjugation increases the affinity of proteins for membranes, but, in many cases, a second feature such as a cluster of positively-charged residues is needed for stable membrane binding. The interaction of two members of this family, VILIP-1 and VILIP-3, with Langmuir monolayers as membrane models has been investigated in order to study the effects of both myristoylation and the highly basic region containing conserved poly-lysine residues on membrane association kinetics and binding properties. Results show that in the presence of calcium, N-myristoylation significantly increases the kinetic rate of VILIP adsorption to the membrane. Additionally, the proteins bind to negatively charged phospholipids independently of the conjugated myristate moiety. Besides the regulatory effect of calcium on the rate of binding presumably due to exposure of the myristoyl moiety ascribed to their putative ‘calcium–myristoyl switch’, VILIP-1 and -3 also engage specific interactions with biomimetic membranes containing phosphatidylinositol 4,5-bisphosphate (PIP₂). The presence of PIP₂ increases the membrane association rates of both VILIPs. Taken together, these results show the major kinetic role of N-myristoylation for membrane binding, and highlight the critical role of specific phosphoinositide interactions for membrane association of members of the VILIP family.     

Identification of amino acids essential for calmodulin binding and activation of smooth muscle myosin light chain kinase.

Smooth muscle myosin light chain kinase (smMLCK) is a Ca(2+)-calmodulin (CaM)-dependent enzyme that phosphorylates the 20-kDa light chains of myosin. In a previous study (Bagchi, I.C., Kemp, B.E., and Means, A.R. (1989) J. Biol. Chem. 264, 15843-15849), we expressed in bacteria a 40-kDa fragment of smMLCK that displayed Ca(2+)-CaM-regulated catalytic activity. Initial mutagenesis experiments indicated that Gly811 and Arg812 were important for CaM-dependent activation of this 40-kDa enzyme. We have now carried out site-directed mutagenesis within the CaM-binding domain (Ser787 to Leu813) of this enzyme to identify amino acids that are critical for CaM binding and activation. Our studies reveal that the individual mutation of several hydrophobic amino acid residues such as Leu813, Ile810, and Trp800 and the glycine residue Gly804 also resulted in a severe decrease in or complete loss of CaM binding and activation of smMLCK. The hydrophobic residue (Trp800) and the basic residue (Arg812), both of which are mandatory for CaM binding to smMLCK, occur in analogous positions within the CaM-binding domain of a number of CaM-regulated enzymes. We conclude from these results that CaM binding by smMLCK is determined by an interplay of specific hydrophobic and electrostatic interactions which appear to be conserved among various target enzymes of CaM.     

Binding features of chloroplast fructose-1,6-bisphosphatase-thioredoxin interaction

It has been proposed that a hydrophobic groove surrounded by positively charged amino acids on thioredoxin (Trx) serves as the recognition and docking site for the interaction of Trx with target proteins. This model for Trx-protein interactions fits well with the Trx-mediated fructose-1,6-bisphosphatase (FBPase) activation, where a protruding negatively charged loop of FBPase would bind to this Trx groove, in a process involving both electrostatic and hydrophobic interactions. This model facilitates the prediction of Trx amino acid residues likely to be involved in enzyme binding. Site-directed mutagenesis of some of these amino acids, in conjunction with measurements of the FBPase activation capacity of the wild type and mutated Trxs, was used to check the model and provided evidence that lysine-70 and arginine-74 of pea Trx m play an essential role in FBPase binding. The binding parameters for the interaction between chloroplast FBPase and the wild type pea Trxs f and m, as well as mutated pea Trx m, determined by equilibrium dialysis in accordance with the Koshland-Nemethy-Filmer model of saturation kinetics, provided additional support for the role of these basic Trx residues in the interaction with FBPase. These data, in conjunction with the midpoint redox potential (E(m)) determinations of Trxs, support the hydrophobic groove model for the interaction between chloroplast FBPase and Trx. This model predicts that differences in the FBPase activation capacity of Trxs arise from their different binding abilities.     

Inhibition of PhoE translocation across Escherichia coli inner-membrane vesicles by synthetic signal peptides suggests an important role of acidic phospholipids in protein translocation

To obtain insight into the mechanism of precursor protein translocation across membranes, the effect of synthetic signal peptides and other relevant (poly)peptides on in vitro PhoE translocation was studied. The PhoE signal peptide, associated with inner membrane vesicles, caused a concentration-dependent inhibition of PhoE translocation, as a result of a specific interaction with the membrane. Using a PhoE signal peptide analog and PhoE signal peptide fragments, it was demonstrated that the hydrophobic part of the peptide caused the inhibitory effect, while the basic amino terminus is most likely important for an optimal interaction with the membrane. A quantitative analysis of our data and the known preferential interaction of synthetic signal peptides with acidic phospholipids in model membranes strongly suggest the involvement of negatively charged phospholipids in the inhibitory interaction of the synthetic PhoE signal peptide with the inner membrane. The important role of acidic phospholipids in protein translocation was further confirmed by the observation that other (poly)peptides, known to have both a high affinity for acidic lipids and hydrophobic interactions with model membranes, also caused strong inhibition of PhoE translocation. The implication of these results with respect to the role of signal peptides in protein translocation is indicated.     

Design and characterization of anchoring amphiphilic peptides and their interactions with lipid vesicles.

In an effort to develop a polymer/peptide assembly for the immobilization of lipid vesicles, we have made and characterized four water-soluble amphiphilic peptides designed to associate spontaneously and strongly with lipid vesicles without causing significant leakage from anchored vesicles. These peptides have a primary amphiphilic structure with the following sequences: AAAAAAAAAAAAWKKKKKK, AALLLAAAAAAAAAAAAAAAAAAAWKKKKKK, and KKAALLLAAAAAAAAAAAAAAAAAAAWKKKKKK and its reversed homologue KKKKKKWAAAAA AAAAAAAAAAAAAALLLAAKK. Two of the four peptides have their hydrophobic segments capped at both termini with basic residues to stabilize the transmembrane orientation and to increase the affinity for negatively charged vesicles. We have studied the secondary structure and the membrane affinity of the peptides as well as the effect of the different peptides on the membrane permeability. The influence of the hydrophobic length and the role of lysine residues were clearly established. First, a hydrophobic segment of 24 amino acids, corresponding approximately to the thickness of a lipid bilayer, improves considerably the affinity to zwitterionic lipids compared to the shorter one of 12 amino acids. The shorter peptide has a low membrane affinity since it may not be long enough to adopt a stable conformation. Second, the presence of lysine residues is essential since the binding is dominated by electrostatic interactions, as illustrated by the enhanced binding with anionic lipids. The charges at both ends, however, prevent the peptide from inserting spontaneously in the bilayer since it would involve the translocation of a charged end through the apolar core of the bilayer. The direction of the amino acid sequence of the peptide has no significant influence on its behavior. None of these peptides perturbs membrane permeability even at an incubation lipid to peptide molar ratio of 0.5. Among the four peptides, AALLLAAAAAAAAAAAAAAAAAAAWKKKKKK is identified as the most suitable anchor for the immobilization of lipid vesicles.     

A single-residue deletion alters the lipid selectivity of a K+ channel-associated peptide in the beta-conformation: spin label electron spin resonance studies.

Lipid-peptide interactions with the 27-residue peptide of sequence KLEALYILMVLGFFGFFTLGIMLSYIR reconstituted as beta-sheet assemblies in dimyristoylphosphatidylcholine bilayers have been studied by electron spin resonance (ESR) spectroscopy with spin-labeled lipids. The peptide corresponds to residues 42-68 of the IsK voltage-gated K+ channel protein and contains the single putative transmembrane span of this protein. Lipid-peptide interactions give rise to a second component in the ESR spectra of lipids spin-labeled on the 14C atom of the chain that corresponds to restriction of the lipid mobility by direct interaction with the peptide assemblies. From the dependence on the lipid/peptide ratio, the stoichiometry of lipid interaction is found to be about two phospholipids/peptide monomer. The sequence of selectivity for lipid association with the peptide assemblies is in the order phosphatidic acid > stearic acid = phosphatidylserine > phosphatidylglycerol = phosphatidylcholine. Comparison with previous data for a corresponding 26-residue mutant peptide with a single deletion of the apolar residue Leu2 (Horvath et al., 1995. Biochemistry 34:3893-3898), indicates a very similar mode of membrane incorporation for native and mutant peptides, but a strongly modified pattern and degree of specificity for the interaction with negatively charged lipids. The latter is interpreted in terms of the relative orientations of the charged amino acid side chains in the beta-sheet assemblies of the native and deletion-mutant peptides.     

Phospholipase A(2)s and lipid peroxidation

Lipid peroxidation of membrane phospholipids can proceed both enzymatically via the mammalian 15-lipoxygenase-1 or the NADPH-cytochrome P-450 reductase system and non-enzymatically. In some cells, such as reticulocytes, this process is biologically programmed, whereas in the majority of biological systems lipid peroxidation is a deleterious process that has to be repaired via a deacylation-reacylation cycle of phospholipid metabolism. Several reports in the literature pinpoint a stimulation by lipid peroxidation of the activity of secretory phospholipase A(2)s (mainly pancreatic and snake venom enzymes) which was originally interpreted as a repair function. However, recent experiments from our laboratory have demonstrated that in mixtures of lipoxygenated and native phospholipids the former are not preferably cleaved by either secretory or cytosolic phospholipase A(2)s. We propose that the platelet activating factor (PAF) acetylhydrolases of type II, which cleave preferentially peroxidised or lipoxygenated phospholipids, are competent for the phospholipid repair, irrespective of their role in PAF metabolism. A corresponding role of Ca(2+)-independent phospholipase A(2), which has been proposed to be involved in phospholipid remodelling in biomembranes, has not been addressed so far. Direct and indirect 15-lipoxygenation of phospholipids in biomembranes modulates cell signalling by several ways. The stimulation of phospholipase A(2)-mediated arachidonic acid release may constitute an alternative route of the arachidonic acid cascade. Thus, 15-lipoxygenase-mediated oxygenation of membrane phospholipids and its interaction with phospholipase A(2)s may play a crucial role in the pathogenesis of diseases, such as bronchial asthma and atherosclerosis.     

Specific binding of mitochondrial protein precursors to liposomes containing cardiolipin

In vitro synthesized precursors of several mitochondrial proteins, including P-450(SCC), adrenodoxin, and malate dehydrogenase, bound to liposomes prepared from mitochondrial phospholipids, but not to those from microsomal phospholipids. When liposomes were prepared from various pure phospholipids, adrenodoxin precursor was bound only to the liposomes that contained cardiolipin. The liposomes containing other phospholipids did not show the binding affinity for the precursor. The binding was observed only with the precursor peptides of adrenodoxin and malate dehydrogenase, and their mature forms were not bound to the liposomes. The binding of the precursors was dependent on the concentration of cardiolipin in the liposomes. Liposomes containing various cardiolipin derivatives with modified polar head groups showed very different binding affinity for adrenodoxin precursor, suggesting the importance of the structure of the polar head of the cardiolipin molecule. Two or three positively charged amino acid residues in the extension peptide of P-450(SCC) precursor were replaced by neutral amino acid residues by site-directed mutagenesis. The mutated P-450(SCC) precursors did not bind to the liposomes containing cardiolipin. The results indicated that mitochondrial protein precursors have specific affinity for cardiolipin, and the affinity was due to the interaction between the extension peptides of the precursors and the polar head of the cardiolipin molecule.     

Mechanism of group IVA cytosolic phospholipase A(2) activation by phosphorylation

Group IVA cytosolic phospholipase A2 (cPLA2) has been shown to play a critical role in the agonist-induced release of arachidonic acid. To understand the mechanism by which phosphorylation of Ser505 and Ser727 activates cPLA2, we systematically analyzed the effects of S505A, S505E, S727A, S727E, S505A/S727A, S505A/S727E, and S505E/S727E mutations on its enzyme activity and membrane affinity. In vitro membrane binding measurements showed that S505A has lower affinity than the wild type or S505E for phosphatidylcholine membranes, which is exclusively due to faster desorption of the membrane-bound S505A. In contrast, neither S727A nor S727E mutation had a significant effect on the phosphatidylcholine vesicle binding affinity of cPLA2. The difference in in vitro membrane affinity between wild type (or S505E) and S505A increased with the decrease in Ca2+ concentration, reaching >60-fold at 2.5 microm Ca2+. When HEK293 cells transfected with cPLA2 and mutants were stimulated with ionomycin, the wild type and S505E translocated to the perinuclear region and caused the arachidonic acid release at 0.4 microm Ca2+, whereas S505A showed no membrane translocation and little activity to release arachidonic acid. Further mutational analysis of hydrophobic residues in the active site rim (Ile399, Leu400, and Leu552) indicate that a main role of the Ser505 phosphorylation is to promote membrane penetration of these residues, presumably by inducing a conformational change of the protein. These enhanced hydrophobic interactions allow the sustained membrane interaction of cPLA2 in response to transient calcium increases. On the basis of these results, we propose a mechanism for cPLA2 activation by calcium and phosphorylation.     

Structural characterization of type II dockerin module from the cellulosome of Clostridium thermocellum: calcium-induced effects on conformation and target recognition

The assembly of a functional cellulose-degrading complex termed the cellulosome involves two specific calcium-dependent cohesin-dockerin interactions: type I and type II. Extensive structural and mutagenesis studies have been performed on the type I modules and their interaction in an attempt to identify the underlying molecular determinants responsible for this specificity. However, very little structural information exists for the type II interaction. We have performed a variety of biophysical studies on the type II dockerin-X-module modular pair (DocX), which comprises the C-terminal region of cellulosomal scaffoldin subunit from Clostridium thermocellum, to determine the effect of calcium on its structure and interaction with type II cohesin. Our results indicate that calcium binding to type II dockerin occurs with an apparent dissociation constant (K(d)) of 7 microM, induces stable secondary and tertiary structure, and leads to the exposure of a hydrophobic surface. Calcium binding also results in the homodimerization of DocX. Analytical ultracentrifugation experiments indicate that the DocX homodimer has an elongated shape and a K(d) of approximately 40 microM. However, addition of the SdbA type II cohesin binding partner led to the dissociation of the DocX homodimer and to the formation of a 1:1 heterodimer. We propose that the exposed hydrophobic surface forms, at least in part, the type II cohesin-binding site, which in the absence of cohesin results in the dimerization of DocX.     

Computational study of coagulation factor VIIa’s affinity for phospholipid membranes

The interaction between the γ-carboxyglutamic acid-rich domain of coagulation factor VIIa (FVIIa), a vitamin-K-dependent enzyme, and phospholipid membranes plays a major role in initiation of blood coagulation. However, despite a high sequence and structural similarity to the Gla domain of other vitamin-K-dependent enzymes with a high membrane affinity, its affinity for negatively charged phospholipids is poor. A few amino acid differences are responsible for this observation. Based on the X-ray structure of lysophosphatidylserine (lysoPS) bound to the Gla domain of bovine prothrombin (Prth), models of the Gla domain of wildtype FVIIa and mutated FVIIa Gla domains in complex with lysoPS were built. Molecular dynamics (MD) and steered molecular dynamics (SMD) simulations on the complexes were applied to investigate the significant difference in the binding affinity. The MD simulation approach provides a structural and dynamic support to the role of P10Q and K32E mutations in the improvement of the membrane contact. Hence, rotation of the Gly11 main chain generated during the MD simulation results in a hydrogen bond with Q10 side chain as well as the appearance of a hydrogen bond between E32 and Q10 forcing the loop harbouring Arg9 and Arg15 to shrink and thereby enhances the accessibility of the phospholipids to the calcium ions. Furthermore, the application of the SMD simulation method to dissociate C6-lysoPS from a series of Gla domain models exhibits a ranking of the rupture force that can be useful in the interpretation of the PS interaction with Gla domains. Finally, adiabatic mapping of Gla6 residue in FVIIa with or without insertion of Tyr4 confirms the critical role of the insertion on the conformation of the side chain Gla6 in FVIIa and the corresponding Gla7 in Prth.     

Site-specific mutations in the COOH-terminus of placental alkaline phosphatase: a single amino acid change converts a phosphatidylinositol- glycan-anchored protein to a secreted protein

Placental alkaline phosphatase (PLAP) is anchored in the plasma membrane by a phosphatidylinositol-glycan moiety (PI-glycan). PI-glycan is added posttranslationally to the nascent peptide chain after the removal of 29 amino acids from the COOH-terminus. The contribution of selected COOH-terminal amino acids to the signal for PI-glycan addition was tested by creating a fusion protein with the COOH-terminus of PLAP and a secreted protein and by mutagenesis of specific PLAP COOH-terminal amino acids. The cDNA encoding the COOH-terminus of PLAP was fused in frame to the cDNA for human clotting Factor X and expressed in transfected COS-1 cells. Fusion proteins containing 32 amino acids of the PLAP COOH-terminus were modified by PI-glycan addition. Thus, the signal for PI-glycan modification must reside in these amino acids. Next, the region between the hydrophobic domain and the cleavage site was examined for additional determinants. Mutations of the hydrophilic residues in the spacer region demonstrated that these amino acids do not contribute to the signal for PI-glycan addition. Deletion of amino acids in the spacer region prevented the addition of PI-glycan suggesting that the length of the spacer domain or the amino acids around the cleavage site are important determinants. Finally, we demonstrated that interruption of the hydrophobic domain by a charged residue prevents PI-glycan addition and results in a protein that is secreted into the medium. The finding that a single Leu to Arg substitution in the hydrophobic domain converts a PI-glycan anchored, membrane protein to a secreted protein suggests that an essential signal for the correct sorting of PI-glycan anchored proteins versus secreted proteins resides in the hydrophobic domain. Substitution of a charged amino acid for a hydrophobic amino acid may be a mechanism for producing membrane bound and secreted forms of the same protein.     

Analysis of antimicrobial peptide interactions with hybrid bilayer membrane systems using surface plasmon resonance

The lipid binding behaviour of the antimicrobial peptides magainin 1, melittin and the C-terminally truncated analogue of melittin (21Q) was studied with a hybrid bilayer membrane system using surface plasmon resonance. In particular, the hydrophobic association chip was used which is composed of long chain alkanethiol molecules upon which liposomes adsorb spontaneously to create a hybrid bilayer membrane surface. Multiple sets of sensorgrams with different peptide concentrations were generated. Linearisation analysis and curve fitting using numerical integration analysis were performed to derive estimates for the association (k(a)) and dissociation (k(d)) rate constants. The results demonstrated that magainin 1 preferentially interacted with negatively charged dimyristoyl-L-alpha-phosphatidyl-DL-glycerol (DMPG), while melittin interacted with both zwitterionic dimyristoyl-L-alpha-phosphatidylcholine and anionic DMPG. In contrast, the C-terminally truncated melittin analogue, 21Q, exhibited lower binding affinity for both lipids, showing that the positively charged C-terminus of melittin greatly influences its membrane binding properties. Furthermore the results also demonstrated that these antimicrobial peptides bind to the lipids initially via electrostatic interactions which then enhances the subsequent hydrophobic binding. The biosensor results were correlated with the conformation of the peptides determined by circular dichroism analysis, which indicated that high alpha-helicity was associated with high binding affinity. Overall, the results demonstrated that biosensor technology provides a new experimental approach to the study of peptide-membrane interactions through the rapid determination of the binding affinity of bioactive peptides for phospholipids.     

A hydrophobic sequence at position 313-316 (Leu-Ala-Phe-Trp) in the fifth domain of apolipoprotein H (beta2-glycoprotein I) is crucial for cardiolipin binding.

Apolipoprotein H (apoH, protein; APOH, gene) binds to negatively charged phospholipids, which triggers the production of a subset of autoantibodies against phospholipid in patients with autoimmune diseases. We have demonstrated that two naturally occurring missense mutations in the fifth domain of apoH, Trp316Ser and Cys306Gly, disrupt the binding of native apoH to phosphatidylserine [Sanghera, D. K., Wagenknecht, D. R., McIntyre, J. A. & Kamboh, M. I. (1997) Hum. Mol. Genet. 6, 311-316]. To confirm whether these are functional mutations, we mutagenized APOH cDNAs and transiently expressed them in COS-1 cells. The cardiolipin ELISA of wild-type and mutant recombinant apoH confirmed that the Gly306 and Ser316 mutations are responsible for abolishing the binding of recombinant apoH to cardiolipin. These mutations, however, had no effect on the levels of expression or secretion of recombinant apoH in transfected COS-1 cells. While the Cys306Gly mutation disrupts a disulfide bond between Cys306 and Cys281, which appears to be critical for clustering positively charged amino acids, the Trp316Ser mutation affects the integrity of an evolutionarily conserved hydrophobic sequence at position 313-316 (Leu-Ala-Phe-Trp), which is hypothesized to interact with anionic phospholipid. To test this hypothesis, we exchanged the remaining three hydrophobic amino acids with neutral amino acids by site-directed mutagenesis (Leu313Gly, Ala314Ser and Phe315Ser). Binding of the Leu313Gly and Phe315Ser mutants to cardiolipin was significantly reduced to 25% and 13%, respectively, of that of the wild-type. On the other hand, the Ala314Ser mutation showed normal cardiolipin binding. Taken together with our previous findings, these results strongly suggest that the configuration of the fifth domain of apoH, as well as the integrity of the highly conserved hydrophobic amino acids at positions 313-316, is essential for the binding of apoH to anionic phospholipid.     

A structure-function study of the C2 domain of cytosolic phospholipase A2. Identification of essential calcium ligands and hydrophobic membrane binding residues

The C2 domain of cytosolic phospholipase A2 (cPLA2) is involved in the Ca2+-dependent membrane binding of this protein. To identify protein residues in the C2 domain of cPLA2 essential for its Ca2+ and membrane binding, we selectively mutated Ca2+ ligands and putative membrane-binding residues of cPLA2 and measured the effects of mutations on its enzyme activity, membrane binding affinity, and monolayer penetration. The mutations of five Ca2+ ligands (D40N, D43N, N65A, D93N, N95A) show differential effects on the membrane binding and activation of cPLA2, indicating that two calcium ions bound to the C2 domain have differential roles. The mutations of hydrophobic residues (F35A, M38A, L39A, Y96A, Y97A, M98A) in the calcium binding loops show that the membrane binding of cPLA2 is largely driven by hydrophobic interactions resulting from the penetration of these residues into the hydrophobic core of the membrane. Leu39 and Val97 are fully inserted into the membrane, whereas Phe35 and Tyr96 are partially inserted. Finally, the mutations of four cationic residues in a beta-strand (R57E/K58E/R59E/R61E) have modest and negligible effects on the binding of cPLA2 to zwitterionic and anionic membranes, respectively, indicating that they are not directly involved in membrane binding. In conjunction with our previous study on the C2 domain of protein kinase C-alpha (Medkova, M., and Cho, W. (1998) J. Biol. Chem. 273, 17544-17552), these results demonstrate that C2 domains are not only a membrane docking unit but also a module that triggers membrane penetration of protein and that individual Ca2+ ions bound to the calcium binding loops play differential roles in the membrane binding and activation of their parent proteins.     

Hydrophobic residues in small ankyrin 1 participate in binding to obscurin

Abstract Small ankyrin-1 is a splice variant of the ANK1 gene that binds to obscurin A. Previous studies have identified electrostatic interactions that contribute to this interaction. In addition, molecular dynamics (MD) simulations predict four hydrophobic residues in a 'hot spot' on the surface of the ankyrin-like repeats of sAnk1, near the charged residues involved in binding. We used site-directed mutagenesis, blot overlays and surface plasmon resonance assays to study the contribution of the hydrophobic residues, V70, F71, I102 and I103, to two different 30-mers of obscurin that bind sAnk1, Obsc????????? and Obsc?????????. Alanine mutations of each of the hydrophobic residues disrupted binding to the high affinity binding site, Obsc?????????. In contrast, V70A and I102A mutations had no effect on binding to the lower affinity site, Obsc?????????. Alanine mutagenesis of the five hydrophobic residues present in Obsc????????? showed that V6328, I6332, and V6334 were critical to sAnk1 binding. Individual alanine mutants of the six hydrophobic residues of Obsc????????? had no effect on binding to sAnk1, although a triple alanine mutant of residues V6233/I6234/I6235 decreased binding. We also examined a model of the Obsc?????????-sAnk1 complex in MD simulations and found I102 of sAnk1 to be within 2.2? of V6334 of Obsc?????????. In contrast to the I102A mutation, mutating I102 of sAnk1 to other hydrophobic amino acids such as phenylalanine or leucine did not disrupt binding to obscurin. Our results suggest that hydrophobic interactions contribute to the higher affinity of Obsc????????? for sAnk1 and to the dominant role exhibited by this sequence in binding.     

10N-nonyl acridine orange interacts with cardiolipin and allows the quantification of this phospholipid in isolated mitochondria.

The acridine orange derivative, 10N-nonyl acridine orange, is an appropriate marker of the inner mitochondrial membrane in whole cells. We use membrane model systems to demonstrate that 10N-nonyl acridine orange binds to negatively charged phospholipids (cardiolipin, phosphatidylinositol and phosphatidylserine). The stoichiometry has been found to be 2 mol 10N-nonyl acridine orange/mol cardiolipin and 1 mol dye/mol phosphatidylserine or phosphatidylinositol, while, with zwitterionic phospholipids, significant binding could not be detected. The affinity constants were 2 x 10(6) M-1 for cardiolipin-10N-nonyl-acridine-orange association and only 7 x 10(4) M-1 for that of phosphatidylserine and phosphatidylinositol association. The high affinity of the dye for cardiolipin may be explained by two essential interactions; firstly an electrostatic interaction between the quaternary ammonium of nonyl acridine orange and the ionized phosphate residues of cardiolipin and secondly, hydrophobic interactions between adjacent chromophores. A linear relationship was demonstrated between the cardiolipin content of model membranes and the incorporated dye. Consequently, a convenient and rapid method for cardiolipin quantification in membranes was established and applied to the cardiolipin-containing organelle, the mitochondrion.     

Anionic phospholipids are determinants of membrane protein topology.

The orientation of many membrane proteins is determined by the asymmetric distribution of positively charged amino acid residues in cytoplasmic and translocated loops. The positive-inside rule states that loops with large amounts of these residues tend to have cytoplasmic locations. Orientations of constructs derived from the inner membrane protein leader peptidase from Escherichia coli were found to depend on the anionic phospholipid content of the membrane. Lowering the contents of anionic phospholipids facilitated membrane passage of positively charged loops. On the other hand, elevated contents of acidic phospholipids in the membrane rendered translocation more sensitive to positively charged residues. The results demonstrate that anionic lipids are determinants of membrane protein topology and suggest that interactions between negatively charged phospholipids and positively charged amino acid residues contribute to the orientation of membrane proteins.