Conotoxins and structural biology: a prospective paradigm for drug discovery.

Understanding the interactions between activating or antagonizing ligands and their cognate receptors at a molecular level offers promise for the development of pharmacological therapeutics for CNS disorders. The discovery of novel molecules that are capable of discriminating between the varied molecular subunits or isoforms of ion channels should provide a more detailed understanding of the pathophysiology of many CNS disorders. Abundant natural sources of pharmacologically active agents that demonstrate this refined selectivity and specificity are found in the animal toxins of venomous species including: snakes, spiders and the marine snail of the genus Conus. The uniquely fascinating combinatorial ability of the marine snail, genus Conus to modify the pharmacological properties of these neurotoxins or conopeptides within its venom is depicted throughout this review. The myriad of posttranslational modifications and disulfide bonded architectures that have been identified in the conopeptides, are described with an emphasis on the unique pharmacological properties and receptor target specificities that have been ascribed to each of these modifications. The ability of NMR spectroscopy to provide three-dimensional structural information within the interaction interface for both the ligand and target protein following complex formation and its application to conopeptide drug discovery are discussed. Similarly, the strength of merging NMR spectroscopy data with ab initio "restrained soft-docking" for rational pharmacophore design and the identification of lead compounds from in silico library screens will also be discussed. The initial phases of this stratagem are illustrated using two toxin antagonists and the recently determined structure of the KcsA potassium channel. These data exemplify the utility of this approach in elucidating important molecular interfaces of specific toxin-receptor/ion channel complexes, which can be further exploited in drug discovery initiatives.

A novel approach for assessing macromolecular complexes combining soft-docking calculations with NMR data.

We present a novel and efficient approach for assessing protein-protein complex formation, which combines ab initio docking calculations performed with the protein docking algorithm BiGGER and chemical shift perturbation data collected with heteronuclear single quantum coherence (HSQC) or TROSY nuclear magnetic resonance (NMR) spectroscopy. This method, termed "restrained soft-docking," is validated for several known protein complexes. These data demonstrate that restrained soft-docking extends the size limitations of NMR spectroscopy and provides an alternative method for investigating macromolecular protein complexes that requires less experimental time, effort, and resources. The potential utility of this novel NMR and simulated docking approach in current structural genomic initiatives is discussed.

The omega-loop region of the human prothrombin gamma-carboxyglutamic acid domain penetrates anionic phospholipid membranes.

The hydrophobic omega-loop within the prothrombin gamma-carboxyglutamic acid-rich (Gla) domain is important in membrane binding. The role of this region in membrane binding was investigated using a synthetic peptide, PT-(1-46)F4W, which includes the N-terminal 46 residues of human prothrombin with Phe-4 replaced by Trp providing a fluorescent probe. PT-(1-46)F4W and PT-(1-46) bind calcium ions and phospholipid membranes, and inhibit the prothrombinase complex. PT-(1-46)F4W, but not PT-(1-46), exhibits a blue shift (5 nm) and red-edge excitation shift (28 nm) in the presence of phosphatidylserine (PS)-containing vesicles, suggesting Trp-4 is located within the motionally restricted membrane interfacial region. PS-containing vesicles protect PT-(1-46)F4W, but not PT-(1-46), fluorescence from potassium iodide-induced quenching. Stern-Volmer analysis of the quenching of PT-(1-46)F4W in the presence and absence of 80% phosphatidylcholine/20% PS vesicles suggested that Trp-4 is positioned within the membrane and protected from aqueous quenching agents whereas Trp-41 remains solvent-accessible in the presence of PS-containing vesicles. Fluorescence quenching of membrane-bound PT-(1-46)F4W is optimal with 7- and 10-doxyl-labeled lipids, indicating that Trp-4 is inserted 5 to 7 A into the bilayer. This report demonstrates that the omega-loop region of prothrombin specifically interacts with PS-containing membranes within the interfacial membrane region.

Amphipathic helices support function of blood coagulation factor IXa.

Blood coagulation factor IXa gains proteolytic efficiency upon binding to a phospholipid membrane. We have found that an amphipathic, membrane-binding peptide from the C2 domain of factor VIII, fVIII(2303)(-23), enhances proteolytic efficiency of factor IXa in the absence of phospholipid membranes. This enhancement is the result of a reduction in the K(M) for the substrate, factor X, with little effect on the k(cat). Enhanced function requires interaction of the gamma-carboxyglutamic acid (Gla) domains of factor IXa and factor X since (i) a synthetic peptide comprising the Gla domain of factor IXa and antibodies directed to the Gla domain of factor IXa inhibit this acceleration, (ii) the acceleration is Ca(II) dependent, and (iii) conversion of Gla-domainless factor X is not affected by the presence of fVIII(2303)(-23). The effect of fVIII(2303)(-23) on factor IXa parallels the enhanced function produced by phosphatidylserine-containing bilayers, and fVIII(2303)(-23) does not further enhance function of factor IXa when phospholipid vesicles are present. The critical feature of fVIII(2303)(-23) is apparently its amphipathic helix-forming structure [Gilbert, G. E., and Baleja, J. D. (1995) Biochemistry 34, 3022-3031] because other alpha-helical peptides such as a homologous peptide from the C2 domain of factor V and melittin have similar effects. Diastereomeric analogues of fVIII(2303)(-23) and melittin, which have reduced helical content, do not support factor IXa activity. A truncated peptide of fVIII(2303)(-23) with three C-terminal residues deleted retains alpha-helical content but loses capacity to enhance factor X cleavage, suggesting that a minimum length of alpha-helix is required. Although these results probably do not illuminate the physiologic function of the factor VIII peptide corresponding to fVIII(2303)(-23), they demonstrate a novel, membrane-mimetic role of amphipathic helical peptides in supporting function of factor IXa.

The Gla domain of human prothrombin has a binding site for factor Va.

The role of the Gla domain of human prothrombin in interaction with the prothrombinase complex was studied using a peptide with the sequence of the first 46 residues of human prothrombin, PT-(1-46). Intrinsic fluorescence measurements showed that PT-(1-46) undergoes a conformational alteration upon binding calcium; this conclusion is supported by one-dimensional (1)H NMR spectroscopy, which identifies a change in the chemical environment of tryptophan 41. PT-(1-46) binds phospholipid membranes in a calcium-dependent manner with a K(d) of 0.5 microm and inhibits thrombin generation by the prothrombinase complex with a K(i) of 0.8 microm. In the absence of phospholipid membranes, PT-(1-46) inhibits thrombin generation by factor Xa in the presence but not absence of factor Va, suggesting that PT-(1-46) inhibits prothrombin-factor Va binding. The addition of factor Va to PT-(1-46) labeled with the fluorophore sulfosuccinimidyl-7-amino-4-methylcoumarin-3-acetic acid (PT-(1-46)AMCA) caused a concentration-dependent quenching of AMCA fluorescence, providing direct evidence of a PT-(1-46)-factor Va interaction. The K(d) for this interaction was 1.3 microm. These results indicate that the N-terminal Gla domain of human prothrombin is a functional unit that has a binding site for factor Va. The prothrombin Gla domain is important for interaction of the substrate with the prothrombinase complex.

A conotoxin from Conus textile with unusual posttranslational modifications reduces presynaptic Ca2+ influx.

Cone snails are gastropod mollusks of the genus Conus that live in tropical marine habitats. They are predators that paralyze their prey by injection of venom containing a plethora of small, conformationally constrained peptides (conotoxins). We report the identification, characterization, and structure of a gamma-carboxyglutamic acid-containing peptide, conotoxin epsilon-TxIX, isolated from the venom of the molluscivorous cone snail, Conus textile. The disulfide bonding pattern of the four cysteine residues, an unparalleled degree of posttranslational processing including bromination, hydroxylation, and glycosylation define a family of conotoxins that may target presynaptic Ca2+ channels or act on G protein-coupled presynaptic receptors via another mechanism. This conotoxin selectively reduces neurotransmitter release at an Aplysia cholinergic synapse by reducing the presynaptic influx of Ca2+ in a slow and reversible fashion. The three-dimensional structure, determined by two-dimensional 1H NMR spectroscopy, identifies an electronegative patch created by the side chains of two gamma-carboxyglutamic acid residues that extend outward from a cavernous cleft. The glycosylated threonine and hydroxylated proline enclose a localized hydrophobic region centered on the brominated tryptophan residue within the constrained intercysteine region.

Solution and solid state conformation of the human EGF receptor transmembrane region.

The epidermal growth factor receptor (EGFR) is a member of the tyrosine kinase family of signalling cell surface molecules. Signalling by this protein is mediated through binding of epidermal growth factor to its extracellular region ultimately leading to phosphorylation of several residues on the intracellular portion of the receptor. The only means of communication between the intracellular and extracellular domains is via the transmembrane region of the protein. In this work we describe the first structural studies of a 34-residue synthetic peptide (hEGFRp), representative of the human EGFR transmembrane region, using two-dimensional and 2H wideline NMR and CD spectroscopies. In water the peptide demonstrated a lack of regular secondary structure and existed as oligomers. Addition of the lipomimetic solvent, trifluoroethanol (TFE), led to the production of monomeric structured species. Analysis of NMR spectra of the hEGFRp indicated that an alpha-helix was present between residues M626 and R647. This observation was reinforced by solid state 2H NMR studies in lipid bilayers which showed typical 'Pake' spectra indicating axially symmetric motion. The helical region in hEGFRp commences four residues later than predicted via hydrophobicity profiles, and extends to include several charged arginine residues which would lie on the cytosolic side of the membrane. These observations provide the first evidence that the transmembrane alpha-helical region in EGFR may not only traverse the membrane but may continue to the cytosolic region near T654, an important phosphorylation site.

Perceived sources of occupational stress in general dental practitioners.

AIM: To evaluate levels of occupational stress in general dental practitioners (GDPs) and compare them with those reported by Cooper et al. (1987). DESIGN/SETTING: Postal questionnaire in England and Wales. SUBJECTS: A sample of 1007 GDPs was chosen from the Dental Register. MAIN OUTCOME MEASURES: 30 separate stressors were scored on a 5-point scale. Summated scores within time-, job-, income-, staff- and patient-related categories were submitted to analysis of variance by sex, type of practice, years since qualification and geographic location. RESULTS: 823 questionnaires were returned (82%) of which 667 were from practising GDPs (63% male, 26% female, 1% undetermined) working full- (87%) or part- (13%) time in NHS (61%), private (8%), mixed (26%), or community (5%) practice. In general, problems associated with time management were the most stressful. The highest ranked individual stressors were 'running behind schedule' (percentage responding with scores 4 or 5 = 61.9, 95% confidence intervals = 58.2-65.6), followed by 'coping with difficult or uncooperative patients' (58.2, 54.5-61.9) and the 'working constraints set by the NHS' (57.5, 53.8-61.2). Differences in levels of reported stress (P < 0.05) were observed for at least one group category of stressor between practitioners with different types of practice, between males and females, by geographical area (north against south) and by the length of time since qualification. CONCLUSIONS: The results indicate that GDPs now rank factors related to time management as major job stressors, with an apparently dramatic elevation of 'NHS working constraints' to a top-ranking stressor since 1986.

Oligomerization of the EGF receptor transmembrane domain: a 2H NMR study in lipid bilayers.

During the course of a previous study by wideline 2H NMR, we noted spectral features suggesting the possibility of monitoring homodimer/oligomer interactions between transmembrane domains of the EGF receptor in lipid bilayers [Rigby, A. R., Shaw, G. S., Barber, K. R., & Grant, C. W. M. (1996) Biochemistry 35, 12591-12601]. In the present work this possibility was explored using the 34-residue peptide EGFRtm. The peptide sequence included the 23 amino acid hydrophobic stretch thought to span the membrane (Ile622-Met644 of the EGF receptor), plus the first 10 amino acids of the receptor's cytoplasmic domain (Arg645-Thr654). Selective deuteration was carried out at sites corresponding to Ala623, Met644, and Val650. Samples were studied from 12 to 65 degrees C by 2H NMR in fluid membranes having low peptide concentration (1 mol %) or high peptide concentration (6 mol %). Methyl groups proved to be technically particularly attractive probe locations. Reversible homodimer/oligomer interactions were detected in membranes of the common natural phospholipid 1-palmitoyl-2-oleoylphosphatidylcholine (POPC), without cholesterol. Effects on the EGF receptor transmembrane domain included alterations in peptide backbone motional order and/or conformation at the site of Ala623 within the membrane, and alterations in motional properties of the Val650 side chain in the cytoplasmic domain. There was little spectral evidence of stable oligomer formation except at the lowest temperature studied. Addition of 33% cholesterol to these membranes was accompanied by spectral changes consistent with the formation of more stable peptide oligomers, and by evidence that peptide-peptide interactions were sensed at all three probe locations. Peptide-peptide interactions remained easily reversible, particularly at higher temperatures. Freeze-fracture electron microscopy of the NMR samples demonstrated peptide-related intramembranous particles traversing the membranes. To our knowledge, this is the first electron microscopy description of receptor tyrosine kinases or their fragments in model membranes. In the presence of cholesterol, the peptide-related particles were generally larger, more sharply demarcated, and showed a tendency to cluster. These observations relate to models of receptor lateral association as an aspect of signal transduction, and to forces that may determine protein sorting and organization in cell membranes. We suggest that the cholesterol effects reflect a general phenomenon rather than one specific to the EGF receptor.

Three-dimensional structure of a gamma-carboxyglutamic acid-containing conotoxin, conantokin G, from the marine snail Conus geographus: the metal-free conformer.

Conantokin G is a gamma-carboxyglutamic acid-containing conotoxin from the venom of the marine cone snail Conus geographus. The 17-residue peptide, which contains five gamma-carboxyglutamic acid (Gla) residues and an amidated C-terminal asparagine amide, was synthesized chemically in a form identical to the natural conantokin G. To gain insight into the role of gamma-carboxyglutamic acid in the structure of this peptide, we determined the three-dimensional structure of conantokin G by 1H NMR and compared its structure to other conotoxins and to the gamma-carboxyglutamic acid-containing regions of the vitamin K-dependent blood-clotting proteins. Complete resonance assignments were made by two-dimensional 1H NMR spectroscopy in the absence of metal ions. NOE cross-peaks d(alphaN), d(NN), and d(betaN) provided interproton distance information, and vicinal spin-spin coupling constants 3J(HN alpha) were used to calculate phi torsion angles. Distance geometry and simulated annealing methods were used to derive 20 convergent structures from a set of 227 interproton distance restraints and 13 torsion angle measurements. The backbone rmsd to the geometric average for 20 final structures is 0.8 +/- 0.1 A. Conantokin G consists of a structured region commencing at Gla 3 and extending through arginine 13. This structure includes a partial loop centered around Gla 3 and Gla 4, a distorted type I turn between glutamine 6 and glutamine 9, and two type I turns involving Gla 10, leucine 11, and isoleucine 12 and arginine 13. Together, these two turns define approximately 1.6 turns of a distorted 3(10) helix. The observed structure possesses structural elements similar to those seen in the disulfide-linked conotoxins.

Role of gamma-carboxyglutamic acid in the calcium-induced structural transition of conantokin G, a conotoxin from the marine snail Conus geographus.

Conantokin G is a gamma-carboxyglutamic acid- (Gla-) containing conotoxin isolated from the venom of the marine cone snail Conus geographus. This 17-residue polypeptide, which contains five gamma-carboxyglutamic acid residues, is a N-methyl-d-aspartate- (NMDA-) type glutamate receptor antagonist. To investigate the role of gamma-carboxyglutamic acid in the calcium-induced structural transition of conantokin G, we determined the three-dimensional structure of the conantokin G/Ca2+ complex by two-dimensional 1H NMR spectroscopy and compared it to the high-resolution structure of conantokin G in the absence of metal ions [Rigby et al. (1997) Biochemistry 36, 6906]. Complete resonance assignments were made by two dimensional 1H NMR spectroscopy at pH 5.6 in the presence of saturating amounts of Ca2+. Distance geometry and simulated annealing methods were used to derive 23 convergent structures from a set of 302 interproton distance restraints and two torsion angle measurements. A high-resolution structure, with the backbone root mean square deviation to the geometric average of the 23 structures of 0.6 +/- 0.1 A, contains a linear alpha-helix from Gla 3 to Lys 15. Gla residues 3, 7, 10, and 14 are aligned in a linear array on one face of the helix. A genetic algorithm was applied to determine the calcium positions in conantokin G, and the conantokin G/Ca2+ complex refined by molecular simulation. Upon binding of Ca2+ to gamma-carboxyglutamic acid, conantokin G undergoes a conformational transition from a distorted curvilinear 310 helix to a linear alpha-helix. Occupancy of the metal binding sites, defined by gamma-carboxyglutamic acids, results in formation of a calcium-carboxylate network that linearizes the helix and exposes the hydrophobic amino acids on the opposite face of the helix.

Sphingolipid-derived signalling modulators: interaction with phosphatidylserine.

We previously described the synthesis of two deuterium-labelled sphingoid bases, which made it possible to perform NMR spectroscopy on this family of signalling modulators in membranes (Rigby, A.C, Barber, K.R and Grant, C.W.M. (1995) Biochim. Biophys. Acta 1240, 75-82). In the present work we sought to test the concept that such mediators may display altered physical behaviour through association with anionic lipids - as a possible mechanism of involvement in signal transduction. Lyso-dihydrogalactosylceramide with deuterium nuclei at C4 and C5 of the sphingosine backbone and at C'3 and C'4 of the galactose ring ([2H4]lyso-GalCer), and N,N-dimethylsphingosine with deuterated amino-methyl groups ([2H6]dimethylsphingosine), were assembled as minor components into unsonicated fluid bilayer membranes of 1-palmitoyl-2-oleoylphosphatidylcholine/cholesterol. The effect of (anionic) phosphatidylserine was considered in this zwitterionic host matrix. The results present a picture of rapidly reversible interaction. The (-) charged phosphatidylserine exerted readily-measurable control over the orientation of the carbohydrate residue of [2H4]lyso-GalCer. In contrast, surrounding (-) charges exerted little spectral influence at the level of C4 and C5 of the lyso-GalCer, membrane-inserted, backbone; or at the level of the amino group of dimethylsphingosine. It would appear that packing alterations induced by the phosphatidylserine/sphingoid base association can translate into sizeable spatial constraints in the neighbouring aqueous domain.

Transmembrane region of the epidermal growth factor receptor: behavior and interactions via 2H NMR.

The first wide-line 2H NMR investigation of a receptor tyrosine kinase is reported. Selectively deuterated peptides from the membrane-associated portion of the human epidermal growth factor (EGF) receptor were synthesized for examination in lipid bilayers mimicking certain natural membrane features. The peptide sequence included the 23-amino acid hydrophobic stretch thought to span the membrane (Ile622-Met644 of the EGF receptor), plus the first 10 amino acids of the receptor's cytoplasmic domain (Arg645-Thr654). Dispersion of the peptide with lipid in the lipomimetic solvent, trifluoroethanol (TFE), was found to be a very useful initial step for sample preparation. TFE readily dissolved all components and was then easily removed in vacuo to yield thin films which could be subsequently hydrated to produce bilayers incorporating homogeneously dispersed peptide. Samples extensively studied consisted of 6 mol % peptide in multilamellar liposomes of 1-palmitoyl-2-oleoylphosphatidylcholine and similar liposomes containing cholesterol. 2H NMR spectra of the resulting unsonicated model membranes indicated the existence of peptide monomers undergoing rapid axially symmetric diffusion. It was possible to examine structural and behavioral effects of events often suggested as pivotal in signaling mechanisms and to consider by wide-line NMR for the first time the effect of cholesterol on hydrophobic peptides. When it was incorporated into bilayers by an alternative method involving dialysis of aqueous solutions prepared using a cationic detergent, spectra suggested that the peptide existed primarily as irreversibly aggregated oligomers which were relatively immobile on a time scale of 10(-3)-10(-4) s. For liposomes prepared by hydration of thin films, deuterated methyl groups on the peptide at locations corresponding to Ala623, Met644, and Val650 of the human EGF receptor were individually distinguishable. In highly fluid matrices, spectra suggested the presence of peptide monomers, diffusing symmetrically about axes perpendicular to the membrane. Studied as a function of temperature, 2H NMR spectra of such samples permitted independent consideration of membrane/peptide relationships at separate locations in the receptor tyrosine kinase. None of the locations probed demonstrated significant conformational sensitivity to temperature over a wide range. Effects seen at Ala623 and Met644, at opposite ends of the putative membrane-spanning domain, suggested slight increases in motional order with decreasing temperature. Addition of 33% cholesterol to the membrane caused little apparent conformational change at Val650 or Met644. However, in the presence of the sterol, Met644 and Ala623 exhibited nonaxially symmetric motion at low temperatures, perhaps as a result of peptide oligomerization. Moreover, the presence of cholesterol led to considerable change in spatial arrangement or order at Ala623. There was little evidence to support transmission of conformational changes along the peptide segment probed.

2H-NMR study of two probe-labelled glycosphingolipid-derived signalling modulators in bilayer membranes.

We describe here the first report of sphingoid bases bearing non-perturbing 2H probe nuclei. These were produced, by two different routes of partial synthesis, to permit direct assessment of their arrangement and behaviour as minor components in membrane systems. Wideline 2H-NMR spectra of N,N-dimethylsphingosine with deuterated amino-methyl groups ([2H6]dimethylsphingosine), and of lyso-dihydrogalactosylceramide (lyso-GalCer) with deuterium nuclei at C4,C5 of the sphingosine backbone and at C3,C4 of the galactose ring ([2H4]lyso-GalCer), were recorded in unsonicated, cholesterol-containing fluid bilayer membranes. The sphingolipid metabolites behaved as single populations of lipid amphiphiles dispersed uniformly in the membrane and undergoing rapid symmetric motion about their long molecular axes. This was the case throughout the pH ranges examined, which included values generally considered for the cell cytoplasm. Spectra of [2H6]dimethyl sphingosine indicated that the methyl groups are equivalent on the NMR timescale, and that the molecule's orientation and behaviour are largely unaffected by pH over the range, 6 to 10.5. There was no spectral evidence of deprotonation of the tertiary amine function in this range. Similarly, variation of pH between 6.4 and 8.9 had virtually no effect on the average conformation and orientational order of lyso-GalCer at the level of C4,C5 in the sphingosine backbone. pH did, however, exert significant control over the orientation of the galactose residue--the effect being most marked in the region of the sphingoid base pKa. The lyso-glycolipid showed some evidence of being less motionally ordered than the corresponding parent species, presumably as a result of removal of constraints imposed by the fatty acid.

Behaviour of complex oligosaccharides at a bilayer membrane surface: probed by 2H-NMR.

Deuterium wideline NMR was used in an attempt to directly assess oligosaccharide arrangement and motional characteristics of complex glycosphingolipids dispersed as minor components in phospholipid membranes. A convenient, general synthetic approach was developed which involved replacement of the acetate group of amido sugars with deuteroacetate (-COCD3). This provided excellent signal-to-noise when applied to the terminal GalNAc residue of globoside, and the terminal NANA residue of GM1. Simultaneously, globoside and GM1 fatty acids were replaced with stearic acid deuterated at C-2- a probe location sensitive to glycolipid hydrophobic backbone orientation and rigid body motion. Deuterated GM1 and globoside were studied by 2H-NMR in bilayers of 1-palmitoyl-2-oleoyl phosphatidylcholine, in the presence and absence of physiological quantities of cholesterol. The monoglycosyl glycosphingolipid, glucosyl ceramide, which is the common skeleton of many complex glycosphingolipids including those studied here, was also deuterated at fatty acid C-2 for comparative study in the same matrices. Correlation with spectra of the complex glycolipids demonstrated that, for a given temperature and membrane composition, ceramide backbone conformation was very similar amongst the species studied. Spectral features of GM1 deuterated on terminal NANA and assembled at a membrane surface, were found to be highly consistent with the oligosaccharide conformation determined in studies of GM1 in solution. In contrast, globoside deuterated in the terminal GalNAc residue gave spectra very different from those predicted on the basis of the conformation considered to exist in solution. It seems likely that this result reflects a combination of greater oligosaccharide chain flexibility relative to GM1, and the presence of the membrane environment. Interestingly, although there was highly significant spatial geometry associated with the complex oligosaccharide chains, and although temperature and the presence of cholesterol exert measurable effects on the membrane-inserted portion, these factors had very little impact on the measured spectral parameters associated with the NANA residue of GM1 or the terminal GalNAc residue of globoside. This seems to indicate lack of sensitivity of the complex oligosaccharide chains to conformation and internal motions of the hydrophobic chain segments in these fluid and semi-fluid membranes; and has important implications for mechanisms of crypticity.