N-terminal Fatty Acyl Research Articles

New variochelins from soil-isolated Variovorax sp. H002.

The soil bacterial genus Variovorax produce distinct photoreactive siderophores that may play a crucial role in the iron cycle within the rhizosphere. This study focused on exploring the natural products of the soil-isolated Variovorax sp. H002, leading to the isolation of variochelins A-E (1-5), a series of lipohexapeptide siderophores. NMR and MS/MS analyses revealed that these siderophores share a common core structure - a linear hexapeptide with β-hydroxyaspartate and hydroxamate functional groups, serving in iron-binding coordination. Three new variochelins C-E (3-5) were characterized by varied fatty acyl groups at their N-termini; notably, 4 and 5 represent the first variochelins with N-terminal unsaturated fatty acyl groups. Furthermore, the variochelin biosynthetic gene cluster was identified through draft genome sequencing and gene knockout experiments. Compounds 1-5 exhibited antimicrobial activities against Gram-negative bacteria, including several soil-isolated plant pathogens.

Studies of antibacterial activity (in vitro and in vivo) and mode of action for des-acyl tridecaptins (DATs)

Tridecaptins comprise a class of linear cationic lipopeptides with an N-terminal fatty acyl moiety. These 13-mer antimicrobial peptides consist of a combination of d- and l-amino acids, conferring increased proteolytic stability. Intriguingly, they are biosynthesized by non-ribosomal peptide synthetases in the same bacterial species that also produce the cyclic polymyxins displaying similar fatty acid tails. Previously, the des-acyl analog of TriA1 (termed H-TriA1) was found to possess very weak antibacterial activity, albeit it potentiated the effect of several antibiotics. In the present study, two series of des-acyl tridecaptins were explored with the aim of improving the direct antibacterial effect. At the same time, overall physico-chemical properties were modulated by amino acid substitution(s) to diminish the risk of undesired levels of hemolysis and to avoid an impairment of mammalian cell viability, since these properties are typically associated with highly hydrophobic cationic peptides. Microbiology and biophysics tools were used to determine bacterial uptake, while circular dichroism and isothermal calorimetry were used to probe the mode of action. Several analogs had improved antibacterial activity (as compared to that of H-TriA1) against Enterobacteriaceae. Optimization enabled identification of the lead compound 29 that showed a good ADMET profile as well as in vivo efficacy in a variety of mouse models of infection.

Nitric oxide synthases in GtoPdb v.2023.1

Nitric oxide synthases (NOS, E.C. 1.14.13.39) are a family of oxidoreductases that synthesize nitric oxide (NO.) via the NADPH and oxygen-dependent consumption of L-arginine with the resultant by-product, L-citrulline. There are 3 NOS isoforms and they are related by their capacity to produce NO, highly conserved organization of functional domains and significant homology at the amino acid level. NOS isoforms are functionally distinguished by the cell type where they are expressed, intracellular targeting and transcriptional and post-translation mechanisms regulating enzyme activity. The nomenclature suggested by NC-IUPHAR of NOS I, II and III [12] has not gained wide acceptance, and the 3 isoforms are more commonly referred to as neuronal NOS (nNOS), inducible NOS (iNOS) and endothelial NOS (eNOS) which reflect the location of expression (nNOS and eNOS) and inducible expression (iNOS). All are dimeric enzymes that shuttle electrons from NADPH, which binds to a C-terminal reductase domain, through the flavins FAD and FMN to the oxygenase domain of the other monomer to enable the BH4-dependent reduction of heme bound oxygen for insertion into the substrate, L-arginine. Electron flow from reductase to oxygenase domain is controlled by calmodulin binding to canonical calmodulin binding motif located between these domains. eNOS and nNOS isoforms are activated at concentrations of calcium greater than 100 nM, while iNOS shows higher affinity for Ca2+/calmodulin with great avidity and is essentially calcium-independent and constitutively active. Efficient stimulus-dependent coupling of nNOS and eNOS is achieved via subcellular targeting through respective N-terminal PDZ and fatty acid acylation domains whereas iNOS is largely cytosolic and function is independent of intracellular location. nNOS is primarily expressed in the brain and neuronal tissue, iNOS in immune cells such as macrophages and eNOS in the endothelial layer of the vasculature although exceptions in other cells have been documented. L-NAME and related modified arginine analogues are inhibitors of all three isoforms, with IC50 values in the micromolar range.

Distinct Roles of N-Terminal Fatty Acid Acylation of the Salinity-Sensor Protein SOS3.

The Salt-Overly-Sensitive (SOS) pathway controls the net uptake of sodium by roots and the xylematic transfer to shoots in vascular plants. SOS3/CBL4 is a core component of the SOS pathway that senses calcium signaling of salinity stress to activate and recruit the protein kinase SOS2/CIPK24 to the plasma membrane to trigger sodium efflux by the Na/H exchanger SOS1/NHX7. However, despite the well-established function of SOS3 at the plasma membrane, SOS3 displays a nucleo-cytoplasmic distribution whose physiological meaning is not understood. Here, we show that the N-terminal part of SOS3 encodes structural information for dual acylation with myristic and palmitic fatty acids, each of which commands a different location and function of SOS3. N-myristoylation at glycine-2 is essential for plasma membrane association and recruiting SOS2 to activate SOS1, whereas S-acylation at cysteine-3 redirects SOS3 toward the nucleus. Moreover, a poly-lysine track in positions 7–11 that is unique to SOS3 among other Arabidopsis CBLs appears to be essential for the correct positioning of the SOS2-SOS3 complex at the plasma membrane for the activation of SOS1. The nuclear-localized SOS3 protein had limited bearing on the salt tolerance of Arabidopsis. These results are evidence of a novel S-acylation dependent nuclear trafficking mechanism that contrasts with alternative subcellular targeting of other CBLs by S-acylation.

Simulations of octapeptin–outer membrane interactions reveal conformational flexibility is linked to antimicrobial potency

The octapeptins are lipopeptide antibiotics that are structurally similar to polymyxins yet retain activity against polymyxin-resistant Gram-negative pathogens, suggesting they might be used to treat recalcitrant infections. However, the basis of their unique activity is unclear because of the difficulty in generating high-resolution experimental data of the interaction of antimicrobial peptides with lipid membranes. To elucidate these structure-activity relationships, we employed all-atom molecular dynamics simulations with umbrella sampling to investigate the conformational and energetic landscape of octapeptins interacting with bacterial outer membrane (OM). Specifically, we examined the interaction of octapeptin C4 and FADDI-115, lacking a single hydroxyl group compared with octapeptin C4, with the lipid A-phosphoethanolamine modified OM of Acinetobacter baumannii Octapeptin C4 and FADDI-115 both penetrated into the OM hydrophobic center but experienced different conformational transitions from an unfolded to a folded state that was highly dependent on the structural flexibility of their respective N-terminal fatty acyl groups. The additional hydroxyl group present in the fatty acyl group of octapeptin C4 resulted in the molecule becoming trapped in a semifolded state, leading to a higher free energy barrier for OM penetration. The free energy barrier for the translocation through the OM hydrophobic layer was ∼72 kcal/mol for octapeptin C4 and 62 kcal/mol for FADDI-115. Our results help to explain the lower antimicrobial activity previously observed for octapeptin C4 compared with FADDI-115 and more broadly improve our understanding of the structure-function relationships of octapeptins. These findings may facilitate the discovery of next-generation octapeptins against polymyxin-resistant Gram-negative 'superbugs.'

Water-Alcohol Bigels from Fatty Acylated Dipeptides.

Peptide-based gels are emerging as an interesting class of biocompatible soft materials. 9-Fluorenylmethoxycarbonyl-protected amino acids and short peptides have gained considerable attention as promising gelators. Peptide amphiphiles, wherein an alkyl chain is appended to a polar peptidic moiety, are another important class of peptide-based gelators. Here, we report the alcohol/water bigels formed by the rather simple fatty acylated dipeptides wherein the peptidic moiety is made up of hydrophobic amino acids, viz., Val, Ile, and Leu. Lauroyl, myristoyl, and palmitoyl were investigated as the N-terminal fatty acyl groups. None of the lauroylated peptides caused gelation of methanol/water and ethanol/water mixtures up to 2 wt % peptide concentration. Eight out of the 27 peptides resulted in distinct bigels. The gels are composed of fibrous aggregates as characterized by electron microscopy. Infrared spectroscopy suggests the β-sheet conformation of the peptidic region in the gels. Using the Ma-IV ethanol/water bigel as the representative gel, entrapment and steady release of the anticancer drug docetaxel are demonstrated. Such bigels from rather simple amphipathic peptides that are easily synthesized and purified through solvent extraction could be attractive gelator candidates with potential application in drug delivery.

Nitric oxide synthases (version 2019.4) in the IUPHAR/BPS Guide to Pharmacology Database

Nitric oxide synthases (NOS, E.C. 1.14.13.39) are a family of oxidoreductases that synthesize nitric oxide (NO.) via the NADPH and oxygen-dependent consumption of L-arginine with the resultant by-product, L-citrulline. There are 3 NOS isoforms and they are related by their capacity to produce NO, highly conserved organization of functional domains and significant homology at the amino acid level. NOS isoforms are functionally distinguished by the cell type where they are expressed, intracellular targeting and transcriptional and post-translation mechanisms regulating enzyme activity. The nomenclature suggested by NC-IUPHAR of NOS I, II and III [11] has not gained wide acceptance, and the 3 isoforms are more commonly referred to as neuronal NOS (nNOS), inducible NOS (iNOS) and endothelial NOS (eNOS) which reflect the location of expression (nNOS and eNOS) and inducible expression (iNOS). All are dimeric enzymes that shuttle electrons from NADPH, which binds to a C-terminal reductase domain, through the flavins FAD and FMN to the oxygenase domain of the other monomer to enable the BH4-dependent reduction of heme bound oxygen for insertion into the substrate, L-arginine. Electron flow from reductase to oxygenase domain is controlled by calmodulin binding to canonical calmodulin binding motif located between these domains. eNOS and nNOS isoforms are activated at concentrations of calcium greater than 100 nM, while iNOS shows higher affinity for Ca2+/calmodulin with great avidity and is essentially calcium-independent and constitutively active. Efficient stimulus-dependent coupling of nNOS and eNOS is achieved via subcellular targeting through respective N-terminal PDZ and fatty acid acylation domains whereas iNOS is largely cytosolic and function is independent of intracellular location. nNOS is primarily expressed in the brain and neuronal tissue, iNOS in immune cells such as macrophages and eNOS in the endothelial layer of the vasculature although exceptions in other cells have been documented. L-NAME and related modified arginine analogues are inhibitors of all three isoforms, with IC50 values in the micromolar range.

Protein Lipidation: Occurrence, Mechanisms, Biological Functions, and Enabling Technologies.

Protein lipidation, including cysteine prenylation, N-terminal glycine myristoylation, cysteine palmitoylation, and serine and lysine fatty acylation, occurs in many proteins in eukaryotic cells and regulates numerous biological pathways, such as membrane trafficking, protein secretion, signal transduction, and apoptosis. We provide a comprehensive review of protein lipidation, including descriptions of proteins known to be modified and the functions of the modifications, the enzymes that control them, and the tools and technologies developed to study them. We also highlight key questions about protein lipidation that remain to be answered, the challenges associated with answering such questions, and possible solutions to overcome these challenges.

Plasma Protein Binding Structure-Activity Relationships Related to the N-Terminus of Daptomycin.

Daptomycin is a lipopeptide antibiotic that is highly bound to plasma proteins. To date, the plasma components and structure-activity relationships responsible for the plasma protein binding profile of daptomycin remain uncharacterized. In the present study we have employed a surface plasmon resonance assay together with molecular docking techniques to investigate the plasma protein binding structure-activity relationships related to the N-terminal fatty acyl of daptomycin. Three compounds were investigated: (1) native daptomycin, which displays an N-terminal n-decanoyl fatty acid side chain, and two analogues with modifications to the N-terminal fatty acyl chain; (2) des-acyl daptomycin; and (3) acetyl-daptomycin. The surface plasmon resonance (SPR) data showed that the binding profile of native daptomycin was in the rank order human serum albumin (HSA) ≫ α-1-antitrypsin > low-density lipoprotein ≥ hemoglobin > sex hormone binding globulin > α-1-acid-glycoprotein (AGP) > hemopexin > fibrinogen > α-2-macroglobulin > β2-microglobulin > high-density lipoprotein > fibronectin > haptoglobulin > transferrin > immunoglobulin G. Notably, binding to fatty acid free HSA was greater than binding to nondelipidated HSA. SPR and ultrafiltration studies also indicated that physiological concentrations of calcium increase binding of daptomycin and acetyl-daptomycin to HSA and AGP. A molecular model of the daptomycin-human serum albumin A complex is presented that illustrates the pivotal role of the N-terminal fatty acyl chain of daptomycin for binding to drug site 1 of HSA. In proof-of-concept, the capacity of physiological cocktails of the identified plasma proteins to inhibit the antibacterial activity of daptomycin was assessed with in vitro microbiological assays. We show that HSA, α-1-antitrypsin, low-density lipoprotein, sex hormone binding globulin, α-1-acid-glycoprotein, and hemopexin are responsible for the majority of the sequestering activity in human plasma. The findings are relevant to medicinal chemistry programs focused on the development of next-generation daptomycin lipopeptides. Tailored modifications to the N-terminal fatty acyl domain of the daptomycin molecule should yield novel daptomycin lipopeptides with more ideal plasma protein binding profiles to increase the levels of active (free) drug in plasma and improved in vivo activity.

Insights into peptide-membrane interactions of newly synthesized, nitroxide-containing analogs of the peptaibiotic trichogin GA IV using EPR.

Trichogin GA IV is a short-length (10-amino acid long), mostly hydrophobic, peptaibiotic with an N-terminal fatty acyl chain and a C-terminal 1,2-amino alcohol. A cardinal role of the terminal moieties in the cytotoxic activity of trichogin has been recently found. Previously, peptide orientation and dynamics of trichogin analogs in the membrane were studied using methyl ester derivatives. Therefore, in the present work we synthesized several trichogin analogs with naturally occurring terminal groups to verify whether these moieties have any effect on peptide-membrane interaction. These trichogin analogs, both neutral and carrying a positively charged Lys residue, bear the nitroxide-containing α-amino acid TOAC to study them using EPR spectroscopy. Vesicles were used to investigate orientation and penetration depth of the peptide at room temperature. Bicelles were employed to evaluate the order, dynamics, and orientation of the peptide at a near physiological temperature. In addition, the position of the N-terminal 1-octanoyl chain in the membrane was studied by labeling it with a nitroxide. The secondary structure of the peptides in vesicles was studied by CD spectroscopy showing that they adopt a mostly α-helical structure. In vesicles, the analogs insert below the lipid headgroups with the helix axis oriented parallel to the membrane surface at a peptide-to-lipid (P:L) ratio of 1:100. The presence of the single, positively charged Lys residue does not alter the orientation adopted by the peptides. In bicelles at P:L ratios 1:100 and 1:60, the peptide adopts a transmembrane orientation characterized by a very low orientational order, whereas at a 1:15 P:L ratio it severely disrupts the membrane. Our data shows that overall orientation and insertion in model membranes of the native trichogin GA IV are strictly comparable to those of its methyl ester analogs previously examined.

A Novel Chemical Biology Approach for Mapping of Polymyxin Lipopeptide Antibody Binding Epitopes.

Polymyxins B and E (i.e., colistin) are a family of naturally occurring lipopeptide antibiotics that are our last line of defense against multidrug resistant (MDR) Gram-negative pathogens. Unfortunately, nephrotoxicity is a dose-limiting factor for polymyxins that limits their clinical utility. Our recent studies demonstrate that polymyxin-induced nephrotoxicity is a result of their extensive accumulation in renal tubular cells. The design and development of safer, novel polymyxin lipopeptides is hampered by our limited understanding of their complex structure-nephrotoxicity relationships. This is the first study to employ a novel targeted chemical biology approach to map the polymyxin recognition epitope of a commercially available polymyxin mAb and demonstrate its utility for mapping the kidney distribution of a novel, less nephrotoxic polymyxin lipopeptide. Eighteen novel polymyxin lipopeptide analogues were synthesized with modifications in the polymyxin core domains, namely, the N-terminal fatty acyl region, tripeptide linear segment, and cyclic heptapeptide. Surface plasmon resonance epitope mapping revealed that the monoclonal antibody (mAb) recognition epitope consisted of the hydrophobic domain (N-terminal fatty acyl and position 6/7) and diaminobutyric acid (Dab) residues at positions 3, 5, 8, and 9 of the polymyxin molecule. Structural diversity within the hydrophobic domains and Dab 3 position are tolerated. Enlightened with an understating of the structure-binding relationships between the polymyxin mAb and the core polymyxin scaffold, we can now rationally employ the mAb to probe the kidney distribution of novel polymyxin lipopeptides. This information will be vital in the design of novel, safer polymyxins through chemical tailoring of the core scaffold and exploration of the elusive/complex polymyxin structure-nephrotoxicity relationships.

Antimicrobial activity of human α-defensin 5 and its linear analogs: N-terminal fatty acylation results in enhanced antimicrobial activity of the linear analogs

Human α-defensin 5 (HD5) exhibits broad spectrum antimicrobial activity and plays an important role in mucosal immunity of the small intestine. Although there have been several studies, the structural requirements for activity and mechanism of bacterial killing is yet to be established unequivocally. In this study, we have investigated the antimicrobial activity of HD5 and linear analogs. Cysteine deletions attenuated the antibacterial activity considerably. Candidacidal activity was affected to a lesser extent. Fatty acid conjugated linear analogs showed antimicrobial activity comparable activity to HD5. Effective surface charge neutralization of bacteria was observed for HD5 as compared to the non-fatty acylated linear analogs. Our results show that HD5 and non-fatty acylated linear analogs enter the bacterial cytoplasm without causing damage to the bacterial inner membrane. Although fatty acylated peptides exhibited antimicrobial activity comparable to HD5, their mechanism of action involved permeabilization of the Escherichia coli inner membrane. HD5 and analogs had the ability to bind plasmid DNA. HD5 had greater binding affinity to plasmid DNA as compared to the analogs. The three dimensional structure of HD5 favors greater interaction with the bacterial cell surface and also with DNA. Antibacterial activity of HD5 involves entry into bacterial cytoplasm and binding to DNA which would result in shut down of the bacterial metabolism leading to cell death. We show how a moderately active linear peptide derived from the α-defensin HD5 can be engineered to enhance antimicrobial activity almost comparable to the native peptide.

N-terminal fatty acylation of peptides spanning the cationic C-terminal segment of bovine β-defensin-2 results in salt-resistant antibacterial activity

Peptides spanning the C-terminal segment of bovine-β-defensin-2 (BNBD-2) rich in cationic amino acids, show antimicrobial activity. However, they exhibit considerably reduced activity at physiological concentration of NaCl. In the present study, we have investigated whether N-terminal acylation (acetylation and palmitoylation) of these peptides would result in improved antimicrobial activity. N-terminal palmitoylation though increased hydrophobicity of the peptides, did not enhance antimicrobial potency. However, antibacterial activity of these peptides was not attenuated by NaCl. Biophysical studies on the palmitoylated peptides have indicated that antibacterial activity in the presence of NaCl arises due to the ability of the peptides to interact with membranes more effectively. These peptides showed hemolytic activity which was attenuated considerably in the presence of serum and lipid vesicles. In defensin related peptides, fatty acylation would be a convenient way to generate analogs that are active in the presence of salt.

Biosynthesis of the new broad-spectrum lipopeptide antibiotic paenibacterin in Paenibacillus thiaminolyticus OSY-SE

Paenibacterin is a novel lipopeptide antibiotic with potent activity against Gram-negative and Gram-positive human pathogens. The antibiotic consists of a cyclic 13-residue peptide and an N-terminal C15 fatty acyl chain. To elucidate the biosynthesis of paenibacterin, we determined the whole genome sequence of the producer strain Paenibacillus thiaminolyticus OSY-SE, and the function of the peptide synthetase was confirmed experimentally. The gene cluster of paenibacterin was identified within a 52-kb DNA region, encoding thee non-ribosomal peptide synthetases, PbtA, PbtB and PbtC, and two ABC-transporters, PbtD and PbtE. Both PbtA and PbtB consist of five modules, whereas PbtC comprises three modules. Each of these 13 modules consists of three essential domains (condensation–adenylation–thiolation) and assembles an amino acid into the paenibacterin peptide. Selected adenylation domains in the NRPS were cloned and expressed in Escherichia coli; the substrate specificity of each recombinant A-domain was studied in vitro by protein function analysis. The presence of four epimerization domains in paenibacterin peptide synthetases suggests that Orn1, Orn4, Lys7 and Ser8 in the paenibacterin molecule have d-configuration; the absolute configuration of two ornithine residues in paenibacterin was confirmed by chiral amino acid analysis using Marfey's reagents. Taken together, the findings enabled us to propose the biosynthetic pathway of paenibacterin.

Structure–activity relationships for the binding of polymyxins with human α-1-acid glycoprotein

Here, for the first time, we have characterized binding properties of the polymyxin class of antibiotics for human α-1-acid glycoprotein (AGP) using a combination of biophysical techniques. The binding affinity of colistin, polymyxin B, polymyxin B3, colistin methansulfonate, and colistin nona-peptide was determined by isothermal titration calorimetry (ITC), surface plasma resonance (SPR) and fluorometric assay methods. All assay techniques indicated colistin, polymyxin B and polymyxin B3 display a moderate binding affinity for AGP. ITC and SPR showed there was no detectable binding affinity for colistin methansulfonate and colistin nona-peptide, suggesting both the positive charges of the diaminobutyric acid (Dab) side chains and the N-terminal fatty acyl chain of the polymyxin molecule are required to drive binding to AGP. In addition, the ITC and fluorometric data suggested that endogenous lipidic substances bound to AGP provide part of the polymyxin binding surface. A molecular model of the polymyxin B3–AGP F1*S complex was presented that illustrates the pivotal role of the N-terminal fatty acyl chain and the D-Phe6-L-Leu7 hydrophobic motif of polymyxin B3 for binding to the cleft-like ligand binding cavity of AGP F1*S variant. The model conforms with the entropy driven binding interaction characterized by ITC which suggests hydrophobic interactions coupled to desolvation events and conformational changes are the primary driving force for polymyxins binding to AGP. Collectively, the data are consistent with a role of this acute-phase reactant protein in the transport of polymyxins in plasma.

Interaction of GCAP1 with retinal guanylyl cyclase and calcium: sensitivity to fatty acylation

Guanylyl cyclase activating proteins (GCAPs) are calcium/magnesium binding proteins within neuronal calcium sensor proteins group (NCS) of the EF-hand proteins superfamily. GCAPs activate retinal guanylyl cyclase (RetGC) in vertebrate photoreceptors in response to light-dependent fall of the intracellular free Ca2+ concentrations. GCAPs consist of four EF-hand domains and contain N-terminal fatty acylated glycine, which in GCAP1 is required for the normal activation of RetGC. We analyzed the effects of a substitution prohibiting N-myristoylation (Gly2 → Ala) on the ability of the recombinant GCAP1 to co-localize with its target enzyme when heterologously expressed in HEK293 cells. We also compared Ca2+ binding and RetGC-activating properties of the purified non-acylated G2A mutant and C14:0 acylated GCAP1 in vitro. The G2A GCAP1 expressed with a C-terminal GFP tag was able to co-localize with the cyclase, albeit less efficiently than the wild type, but much less effectively stimulated cyclase activity in vitro. Ca2+ binding isotherm of the G2A GCAP1 was slightly shifted toward higher free Ca2+ concentrations and so was Ca2+ sensitivity of RetGC reconstituted with the G2A mutant. At the same time, myristoylation had little effect on the high-affinity Ca2+-binding in the EF-hand proximal to the myristoyl residue in three-dimensional GCAP1 structure. These data indicate that the N-terminal fatty acyl group may alter the activity of EF-hands in the distal portion of the GCAP1 molecule via presently unknown intramolecular mechanism.

Characterization of daptomycin oligomerization with perylene excimer fluorescence: Stoichiometric binding of phosphatidylglycerol triggers oligomer formation

Daptomycin is a lipopeptide antibiotic that binds to and depolarizes bacterial cell membranes. Its antibacterial activity requires calcium and correlates with the content of phosphatidylglycerol in the target membrane. Daptomycin has been shown to form oligomers on liposome membranes. We here use perylene excimer fluorescence to further characterize the membrane-associated oligomer. To this end, the N-terminal fatty acyl chain was replaced with perylene-butanoic acid. The perylene derivative retains one third of the antibacterial activity of native daptomycin. On liposomes containing phosphatidylcholine and phosphatidylglycerol, as well as on Bacillus subtilis cells, the perylene-labeled daptomycin forms excimers, which shows that the N-terminal acyl chains of neighboring oligomer subunits are in immediate contact with one another. In a lipid bicelle system, oligomer formation can be titrated with stoichiometric amounts of phosphatidylglycerol. Therefore, the interaction of daptomycin with a single molecule of phosphatidylglycerol is sufficient to trigger daptomycin oligomerization.

Conformational Changes in Guanylate Cyclase-Activating Protein 1 Induced by Ca2+ and N-Terminal Fatty Acid Acylation

Neuronal Ca(2+) sensors (NCS) are high-affinity Ca(2+)-binding proteins critical for regulating a vast range of physiological processes. Guanylate cyclase-activating proteins (GCAPs) are members of the NCS family responsible for activating retinal guanylate cyclases (GCs) at low Ca(2+) concentrations, triggering synthesis of cGMP and recovery of photoreceptor cells to the dark-adapted state. Here we use amide hydrogen-deuterium exchange and radiolytic labeling, and molecular dynamics simulations to study conformational changes induced by Ca(2+) and modulated by the N-terminal myristoyl group. Our data on the conformational dynamics of GCAP1 in solution suggest that Ca(2+) stabilizes the protein but induces relatively small changes in the domain structure; however, loss of Ca(+2) mediates a significant global relaxation and movement of N- and C-terminal domains. This model and the previously described "calcium-myristoyl switch" proposed for recoverin indicate significant diversity in conformational changes among these highly homologous NCS proteins with distinct functions.

N-Terminal Fatty Acylation of Transducin Profoundly Influences Its Localization and the Kinetics of Photoresponse in Rods

N-terminal acylation of the alpha-subunits of heterotrimeric G-proteins is believed to play a major role in regulating the cellular localization and signaling of G-proteins, but physiological evidence has been lacking. To examine the functional significance of N-acylation of a well understood G-protein alpha-subunit, transducin (G alpha(t)), we generated transgenic mice that expressed a mutant G alpha(t) lacking N-terminal acylation sequence (G alpha(t)G2A). Rods expressing G alpha(t)G2A showed a severe defect in transducin cellular localization. In contrast to native G alpha(t), which resides in the outer segments of dark-adapted rods, G alpha(t)G2A was found predominantly in the inner compartments of the photoreceptor cells. Remarkably, transgenic rods with the outer segments containing G alpha(t)G2A at 5-6% of the G alpha(t) levels in wild-type rods showed only a sixfold reduction in sensitivity and a threefold decrease in the amplification constant. The much smaller than predicted reduction may reflect an increase in the lateral diffusion of transducin and an increased activation rate by photoexcited rhodopsin or more efficient activation of cGMP phosphodiesterase 6 by G alpha(t)G2A; alternatively, nonlinear relationships between concentration and the activation rate of transducin also potentially contribute to the mismatch between the amplification constant and quantitative expression analysis of G alpha(t)G2A rods. Furthermore, the G2A mutation reduced the GTPase activity of transducin and resulted in two to three times slower than normal recovery of flash responses of transgenic rods, indicating the role of G alpha(t) membrane tethering for its efficient inactivation by the regulator of G-protein signaling 9 GTPase-activating protein complex. Thus, N-acylation is critical for correct compartmentalization of transducin and controls the rate of its deactivation.

Signal Transfer from GPCRs to G Proteins: ROLE OF THE Gα N-TERMINAL REGION IN RHODOPSIN-TRANSDUCIN COUPLING

Catalysis of nucleotide exchange in heterotrimeric G proteins (Galphabetagamma) is a key step in cellular signal transduction mediated by G protein-coupled receptors. The Galpha N terminus with its helical stretch is thought to be crucial for G protein/activated receptor (R(*)) interaction. The N-terminal fatty acylation of Galpha is important for membrane targeting of G proteins. By applying biophysical techniques to the rhodopsin/transducin model system, we studied the effect of N-terminal truncations in Galpha. In Galphabetagamma, lack of the fatty acid and Galpha truncations up to 33 amino acids had little effect on R(*) binding and R(*)-catalyzed nucleotide exchange, implying that this region is not mandatory for R(*)/Galphabetagamma interaction. However, when the other hydrophobic modification of Galphabetagamma, the Ggamma C-terminal farnesyl moiety, is lacking, R(*) interaction requires the fatty acylated Galpha N terminus. This suggests that the two hydrophobic extensions can replace each other in the interaction of Galphabetagamma with R(*). We propose that in native Galphabetagamma, these two terminal regions are functionally redundant and form a microdomain that serves both to anchor the G protein to the membrane and to establish an initial docking complex with R(*). Accordingly, we find that the native fatty acylated Galpha is competent to interact with R(*) even in the absence of Gbetagamma, whereas nonacylated Galpha requires Gbetagamma for interaction. Experiments with N-terminally truncated Galpha subunits suggest that in the second step of the catalytic process, the receptor binds to the alphaN/beta1-loop region of Galpha to reduce nucleotide affinity and to make the Galpha C terminus available for subsequent interaction with R(*).