RESUMO
AIM: Glucagon-like peptide-1 (GLP-1) receptor agonists for the treatment of type 2 diabetes are administered by daily injection because of short plasma half-lives, which result partly from the biochemical instability of these peptides. There is a medical need for GLP-1 analogues that can be administered less frequently for patient convenience. METHODS: We synthesized a series of human GLP-1 (hGLP-1(7-36)NH(2) ) derivatives containing α-aminoisobutyric acid (Aib) substitutions, analysed their enzymatic stabilities and evaluated their secondary structures using circular dichroism (CD) and nuclear magnetic resonance (NMR). RESULTS: Plasma stability experiments showed that only the analogue containing Aib substitutions in both the N-terminus (position 8) and the C-terminus (position 35), [Aib8(,)³5]hGLP-1(7-36)NH2 (BIM-51077), was fully resistant to enzymatic cleavage. Incubation with human plasma kallikrein or plasmin confirmed that the Aib substitution at position 35 prevented protease cleavage around this residue, which contributes to the significantly enhanced plasma stability and increased plasma half-life. CD revealed increased C-terminal α-helicity in Aib³5-substituted analogues compared with both hGLP-1(7-36)NH2 and analogues containing only Aib8 substitutions. Based on NMR studies, the secondary structure of BIM-51077 is similar to hGLP-1(7-36)NH2 with a slight increase in α-helicity in the C-terminus. Compared with hGLP-1(7-36)NH2, BIM-51077 had similar binding affinity for the human GLP-1 receptor and activated this receptor with similar potency. CONCLUSIONS: We have discovered an Aib8(,)³5-substituted analogue of native hGLP-1(7-36)NH2 (BIM-51077) that retains the structure of the native peptide, and has similar activity and enhanced stability. A sustained-release formulation of this molecule (taspoglutide) is in phase-3 clinical development.
Assuntos
Diabetes Mellitus Tipo 2/tratamento farmacológico , Peptídeo 1 Semelhante ao Glucagon/farmacologia , Peptídeos/farmacologia , Descoberta de Drogas/métodos , Estabilidade de Medicamentos , Peptídeo 1 Semelhante ao Glucagon/administração & dosagem , Peptídeo 1 Semelhante ao Glucagon/análogos & derivados , Meia-Vida , Humanos , Peptídeos/administração & dosagemRESUMO
The structure of cardiotoxin CTX I from Naja naja atra has been investigated by NMR spectroscopy. Sequence specific resonance assignments have been obtained for all backbone protons as well as for most side-chain protons. Distance geometry calculations were carried out using a metric matrix DG program. A total of 715 NOE constraints, 27 phi angle constraints and a list of the hydrogen bond donors were used for the metric matrix DG calculations and refinement. The average pairwise r.m.s.d. of the resulting structures was 1.01 A for the backbone heavy atoms, and 1.69 A for all heavy atoms. The protein is rich in beta structure and consists of a large triple-stranded, antiparallel beta sheet as well as a short double-stranded, antiparallel beta sheet. Non-regular hydrogen bonding is found between side-chains of the carboxy-terminal end and the rest of the core region. The structure is discussed in terms of evolutionary aspects as well as recent investigations about the biological function and active site.
Assuntos
Proteínas Cardiotóxicas de Elapídeos/química , Estrutura Secundária de Proteína , Sequência de Aminoácidos , Animais , Evolução Biológica , Elapidae , Ligação de Hidrogênio , Espectroscopia de Ressonância Magnética , Dados de Sequência Molecular , Estrutura MolecularRESUMO
The three-dimensional structure of the two hepatotoxic microcystins LR and LY has been determined by two-dimensional nuclear magnetic resonance (2D NMR) spectroscopy and distance geometry calculations. For the microcystin LY a single family of highly convergent structures was obtained. This family is characterized by a relatively compact boat-like ring structure with the large side chain of the Adda residue protruding from the concave side, in close proximity to the Tyr side chain. Conversely, for the microcystin LR the calculations result in three conformational families characterized by an even more compact ring structure. The Adda and Arg side chains protrude from the ring distal from one another caused by the repulsion between the guanido function of Arg and the hydrophobic Adda. The lower toxicity of the LY microcystin could result from the restricted access of the Adda side chain, an essential residue for activity, which results from the close proximity of the aromatic Tyr residue. A significant enthalpic cost would be expected for disturbance of this hydrophobic collapse and correspondingly lower binding affinity to receptor molecules would be predicted. From the structures of the two related microcystins, and homology with other known toxins, we propose a working hypothesis of the Adda side chain interacting with a hydrophobic pore of the phosphatases while the rest of the microcystin acts as a scaffold to help stabilize the interdigitation of the Adda with additional intermolecular interactions.
Assuntos
Toxinas Bacterianas/química , Carcinógenos/química , Cianobactérias/química , Peptídeos Cíclicos/química , Sequência de Aminoácidos , Toxinas Marinhas , Microcistinas , Conformação Molecular , Dados de Sequência Molecular , Peptídeos Cíclicos/classificaçãoRESUMO
The parathyroid hormone (PTH) plays a vital role in the homeostasis of calcium within the blood stream. Given its unique ability to increase bone density, an understanding of the molecular mechanism by which the hormone is recognized and binds to its receptor should provide targets for the development of PTH-based, anabolic agents for the treatment of osteoporosis. Parathyroid hormone related protein (PTHrP), a genetically and structurally distinct hormone which displays similar binding and activation profiles as PTH, has greatly facilitated the effort to establish a structure-biological function relationship by allowing for direct comparisons. In an analogous manner, the presence of two receptors, PTH/PTHrP (PTH1) and PTH2, which differ in their ligand selectivity (PTH2 is activated by PTH, not PTHrP) has provided a unique vehicle for probing the structural motifs of the receptor required for ligand recognition and binding. Recent photo-affinity cross-linking studies of PTH and PTHrP binding to PTH1 have produced direct points of contact between the ligand and receptor. Here, we review each of the components involved in this important hormone system, with particular emphasis on the structural features of each: the ligands (PTH and PTHrP), the receptors (PTH1 and PTH2), and the interaction between ligand and receptor. Although the current understanding of the molecular mechanism of ligand binding and receptor activation does not allow for the rational design of drug candidates, and indeed contains much conjecture, significant strides have been made towards this end.
Assuntos
Osteoporose/tratamento farmacológico , Hormônio Paratireóideo/uso terapêutico , Proteínas/uso terapêutico , Sequência de Aminoácidos , Animais , Humanos , Dados de Sequência Molecular , Hormônio Paratireóideo/química , Proteína Relacionada ao Hormônio Paratireóideo , Conformação Proteica , Proteínas/química , Receptor Tipo 1 de Hormônio Paratireóideo , Receptores de Hormônios Paratireóideos/química , Receptores de Hormônios Paratireóideos/fisiologia , Relação Estrutura-AtividadeRESUMO
The natural peptide [Thr6]-bradykinin, Arg1-Pro2-Pro3-Gly4-Phe5-Thr6-Pro7-Phe8-Arg9, has been conformationally examined by molecular dynamics simulations using a two-phase box consisting of H2O and CCl4 to mimic the micellar environment utilized in the 1H-NMR studies. The different conformations generated from distance geometry calculations were refined with extensive molecular dynamics simulations. The resulting conformations provide additional structural insight into the differing biological activities of native bradykinin and [Thr6]-bradykinin, produced by the one conservative substitution Thr6 for Ser6. In addition, the simulations give some indication of the interaction of the peptide with the biphasic, hydrophilic/hydrophobic environment of the micelle. Such information is vital given the accumulating data indicating that the peptide first interacts with the membrane before the membrane-bound receptor. The structures of membrane-bound [Thr6]-bradykinin developed here provide experimental support for the interaction of residues 7 and 8 with the core of the membrane-bound receptor and the N-terminus and C-terminal arginine interacting with the extracellular portion of the receptor.
Assuntos
Bradicinina/análogos & derivados , Bradicinina/química , Espectroscopia de Ressonância Magnética , Micelas , Modelos Moleculares , Conformação ProteicaRESUMO
High-resolution, three-dimensional structures of vancomycin and aglyco-vancomycin in DMSO were determined by nuclear magnetic resonance, metric matrix distance geometry, and molecular dynamics calculations. Conformational flexibility fast on the NMR time scale was examined by ensemble-based calculations which apply the experimentally derived restraints as an ensemble average. Two families of conformations of vancomycin, differing in the positioning of the vancosamine substituent, were observed. In contrast, the aglyco-vancomycin adopts only one conformation in solution. The conformations of vancomycin and the aglyco-vancomycin differ in the alignment of the amide protons which participate in the hydrogen-bonding network with the cell-wall precursor and orientation of the aromatic rings relative to the backbone. Therefore, the high-resolution structural characterization provides insight into a possible role of glycosylation on the activity of this important family of antibiotics.
Assuntos
Antibacterianos/química , Hexosaminas/química , Vancomicina/química , Dimetil Sulfóxido , Ressonância Magnética Nuclear Biomolecular , Conformação ProteicaRESUMO
The conformation of the natural peptide [Thr6]-bradykinin, Arg1-Pro2-Pro3-Gly4-Phe5-Thr6-Pro7-Phe8-Arg9, is investigated by NMR spectroscopy and computer simulations in an aqueous solution of sodium dodecyl sulfate micelles. The structural analysis of the peptide is of particular interest since it displays a different biological profile from bradykinin despite the high sequence homology (only one conservative substitution: Ser6/Thr6) and the fact that both peptides bind and activate common receptors. The SDS micelles provide a model system for the membrane-interface environment the peptide experiences when interacting with the membrane-embedded receptor and allow for the conformational examination of the peptide using high-resolution NMR techniques. The NMR spectra show that the micellar system induces a secondary structure in the otherwise inherently flexible peptide (as observed in benign aqueous solution). The distance geometry calculations indicate a beta-turn of type I about residues 7-8 as the preferred conformation. The results of ensemble calculations reveal conformational changes occurring rapidly on the NMR time scale and allow for the identification of three different families of conformations that average to reproduce the NMR observables. The three families differ in the type of conformation adopted at the C-terminus: type I beta-turn, type II beta-turn and a third conformation, intermediate between the two beta-turns. The structural results support the hypothesis of the determining role of the C-terminal conformation for biological activity and can provide an explanation of the different activities observed for bradykinin and [Thr6]-bradykinin.
Assuntos
Bradicinina/análogos & derivados , Bradicinina/química , Fenômenos Químicos , Físico-Química , Espectroscopia de Ressonância Magnética , Micelas , Modelos MolecularesRESUMO
Both the parathyroid hormone (PTH) and the functionally similar parathyroid hormone-related protein (PTHrP) have served as templates for the development of novel bone anabolic agents for the treatment of osteoporosis. The PTHrP analog RS-66271 (Vickery, B. H.; Avnur, Z.; Cheng Y.; Chiou, S.-S.; Leaffer, D.; Caulfield, J. P.; Kimmel, D. B.; Ho, T.; Krstenansky, J. L. J. Bone Miner. Res. 1996, 11, 1943-1951), in which the amino acids 22-31 have been substituted by the sequence E22-L-L-E-K-L-L-E-K-L31 (a model amphiphilic peptide), is a potent bone anabolic agent in vivo. Therefore, RS-66271 is a good candidate for structural analysis with the aim of developing a structure-activity relationship. The structural characterization described here was carried out in aqueous solution employing circular dichroism and nuclear magnetic resonance spectroscopy. We find that the incorporated amphiphilic decapeptide is indeed helical. In addition, it induces the adjacent residues, up to residue 16, to adopt the helical conformation. The helical domain, including residues 16-32, incorporates most of the previously identified principal receptor binding domain PTHrP(25-34). We discuss the relevance of the distinct and extensive helicity in light of the reduced in vitro receptor affinity/ activity and the enhanced in vivo bone anabolic efficacy of RS-66271.
Assuntos
Osteogênese/efeitos dos fármacos , Conformação Proteica , Teriparatida/análogos & derivados , Sequência de Aminoácidos , Dicroísmo Circular , Humanos , Espectroscopia de Ressonância Magnética , Hormônio Paratireóideo/química , Proteína Relacionada ao Hormônio Paratireóideo , Proteínas/química , Receptor Tipo 1 de Hormônio Paratireóideo , Receptores de Hormônios Paratireóideos/metabolismo , Relação Estrutura-Atividade , Teriparatida/química , Teriparatida/metabolismo , Teriparatida/farmacologiaRESUMO
The conformational features of H-Lys-Arg-Ado-Ser-Pro-Phe-OH (Ado = 12-aminododecanoic acid), a des-Arg(9) analogue of Lys-bradykinin, have been determined by high-resolution NMR in the presence of a zwitterionic lipid environment. The analogue is the most active member of a series of analogues designed to probe the topological arrangement of the N- and C-termini required for agonistic activity at the B1 kinin receptor. A novel computational procedure for the utilization of NOE constraints from cis and trans configurational isomers is illustrated. Only with this computational methodology could the structural features of the N-terminus of the peptide be determined. Using radical-induced relaxation of the (1)H NMR signals, we measured the topological orientation of the peptide with respect to the zwitterionic lipid interface. The results indicate that the long, alkyl chain of the Ado amino acid imbeds into the lipid surface. The structural features of the C-terminus of the B1-selective analogue consist of a well-defined turn. Although removed from a standard beta-turn, required for activity at the B2 kinin receptor, the topological orientation of the side chains of the des-Arg(9) compound are surprisingly similar to those previously observed for beta-turn-containing bradykinin analogues. Therefore, we attribute the high B1 receptor selectivity, observed upon removal of Arg(9) from bradykinin, solely to the loss of a charged amino acid and not to altered structural features.
Assuntos
Calidina/análogos & derivados , Calidina/química , Oligopeptídeos/química , Receptores da Bradicinina/agonistas , Calidina/síntese química , Lipídeos/química , Espectroscopia de Ressonância Magnética , Membranas Artificiais , Micelas , Modelos Moleculares , Oligopeptídeos/síntese química , Conformação Proteica , Receptor B1 da BradicininaRESUMO
The conformations of two backbone-cyclized substance P analogs as derived from 1H NMR and molecular dynamics simulations carried out in DMSO and water are described. The method of floating chiralities is used in the simulations to facilitate the diastereotopic assignment of methylene protons. One of the analogs, cyclo-[-(CH2)3-NH-CO-(CH2)4-Arg-Phe-Phe-N-]-CH2-CO-Leu-Met-NH2, is a highly active, selective agonist for the NK-receptor, while the other, cyclo[-(CH2)2-NH-CO-(CH2)2-Gly-Arg-Phe-Phe-N-]-CH2-CO-Leu-Met-NH2, is inactive. Both analogs contain cyclic ring systems of the same size, varying in only the number of amide linkages. From the conformational analysis, the lack of activity can be attributed to the introduction of too much constraint into the ring system. This has an effect on the topological array of the important residues Arg-Phe-Phe. The results presented here are compared with biologically active analogs previously examined. The differences between conformations of active and inactive compounds are used to develop insight into the conformational requirements for biological activity.
Assuntos
Substância P/química , Sequência de Aminoácidos , Simulação por Computador , Dimetil Sulfóxido , Espectroscopia de Ressonância Magnética , Conformação Molecular , Dados de Sequência Molecular , Relação Estrutura-Atividade , Substância P/análogos & derivados , Substância P/farmacologiaRESUMO
A series of analogues of desArg(9)-Lys-bradykinin (BK), Lys-Arg-X-Ac(n)c-X-Ser-Pro-Phe, in which the spacer X-Ac(n)c-X replaces the central tetrapeptide Pro-Pro-Gly-Phe of BK, have been synthesized and functionally characterized at the B1 receptor. The 1-aminocycloalkane-1-carboxylic acids (Ac(6)c, Ac(7)c, Ac(8)c, Ac(9)c, Ac(12)c) were incorporated to impart conformational constraint and probe the importance of the hydrophobicity of the residue in the central position. The linker is varied in length (X = Gly, betaAla, gammaAbu) to examine the optimal distance between the biologically important residues at the N- and C-termini. The biological assays indicate that the optimal length is obtained with X = Gly, with reduced activities for the longer linkers. Although the size of the central cyclic amino acid does not significantly alter the biological activity, the hydrophobic residue Ac(n)c which may tether the peptide in the membrane environment is required (Lys-Arg-Gly-Gly-Gly-Ser-Pro-Phe is inactive). Two of the analogues, Lys-Arg-Gly-Ac(7)c-Gly-Ser-Pro-Phe and Lys-Arg-gammaAbu-Ac(7)c-gammaAbu-Ser-Pro-Phe, have been structurally characterized in the presence of a zwitterionic lipid environment by high-resolution NMR. Both compounds have similar structural features, differing greatest in the distance between the termini (9 and 15 A for the Gly- and gammaAbu-containing analogues, respectively). The correlation of the smaller distance with activity at the B1 receptor is in complete accord with the results from our previous examination of Lys-Arg-NH-(CH(2))(11)-CO-Ser-Pro-Phe. With the results from this series of compounds we are beginning to define some of the molecular descriptors important for activity at the B1 BK receptor.
Assuntos
Bradicinina/análogos & derivados , Bradicinina/síntese química , Oligopeptídeos/química , Fragmentos de Peptídeos/química , Receptores da Bradicinina/agonistas , Animais , Bradicinina/química , Bradicinina/farmacologia , Íleo/efeitos dos fármacos , Íleo/fisiologia , Técnicas In Vitro , Espectroscopia de Ressonância Magnética , Espectrometria de Massas , Músculo Liso/efeitos dos fármacos , Músculo Liso/fisiologia , Ratos , Receptor B1 da Bradicinina , Relação Estrutura-AtividadeRESUMO
The molecular mechanism of constitutive activity of the G protein-coupled receptor for human parathyroid hormone (PTH1) has been examined by molecular dynamics (MD) simulations. The single point mutations H223R, T410P, and I458R, of the PTH1 receptor result in ligand-independent receptor activation. Extensive MD simulations indicate that each of the mutations, through different mechanisms, lead to very similar conformational changes of the third intracellular loop. The structural changes, centered on K405 in the C-terminus of the third intracellular loop, can be traced back to the single-point mutations by calculation of the forces and torques responsible for the collective motions of the receptor. This analysis indicates a direct correlation between the conformational preferences of the cytoplasmic loop and the mutations in different locations of the receptor: TM2 (H223R), TM6 (T410P), and TM7 (1458R). Given the pivotal role of the third intracellular loop of PTH1 in coupling to the G proteins, the structural changes induced by these single-point mutations may be responsible for the ligand-free activation of the receptor. These results coupled with the high-resolution structure of the third cytoplasmic loop of PTH1, previously determined in our laboratory, provide unique insight into the mechanism of ligand free activation of the PTH1 receptor.
Assuntos
Receptores de Hormônios Paratireóideos/química , Proteínas de Ligação ao GTP/metabolismo , Humanos , Ligantes , Modelos Moleculares , Mutação Puntual , Conformação Proteica , Receptores de Hormônios Paratireóideos/genética , Receptores de Hormônios Paratireóideos/metabolismoRESUMO
Over the years, the association of peptide ligands to Family B GPCRs (G-protein coupled receptors) has been characterized by a number of experimental and theoretical techniques. For the PTH (parathyroid hormone) ligand-receptor system, important insight has been provided by photoaffinity labelling experiments and the elucidation of direct contact points between ligand and receptor. Our research has focused on the structural elucidation of the receptor domains shown to be involved in the binding of PTH. Employing a combination of carefully designed receptor domains, solution-state NMR carried out in the presence of membrane mimetics and extensive computer simulations, we have obtained a well-resolved model of the ligand-receptor complex for PTH. Here, we review the development of this model and highlight some inherent limitations of the methods employed and their consequences on interpretation of the ligand-receptor model.
Assuntos
Proteína Relacionada ao Hormônio Paratireóideo/metabolismo , Hormônio Paratireóideo/metabolismo , Receptor Tipo 1 de Hormônio Paratireóideo/química , Receptor Tipo 1 de Hormônio Paratireóideo/metabolismo , Animais , Humanos , Ligantes , Hormônio Paratireóideo/química , Ligação Proteica , Estrutura Terciária de ProteínaRESUMO
The Na(+)/H(+) exchanger regulatory factor-1 (NHERF1) is a molecular scaffold important for the signaling of the G-protein coupled receptor for the parathyroid hormone (PTH1R). The two PDZ (PSD-95, Discs-large, ZO1) domains of NHERF1 through association with the C-termini of PTH1R and phospholipase C enhance the signaling pathway associated with PTH. To examine these interactions, we have produced the individual PDZ1 and PDZ2 domains as well as the tandem PDZ1-PDZ2 domains (PDZ12) of NHERF1 and have characterized the binding affinities of the C-terminal motifs of PTH1R and PLCbeta using fluorescence anisotropy. Circular dichroism indicates that the PDZ1 and PDZ2 are properly folded. Based on fluorescence anisotropy we find that the C-terminus of PTH1R, containing ETVM, has similar affinities (approximately 10 microm) for both PDZ1 and PDZ2. The PTH1R displayed reduced binding affinity for the tandem PDZ12 (16 microm) compared with the individual domains or a solution of equal molar concentrations of PDZ1 and PDZ2 (5.8 microm), suggesting negative cooperativity between the PDZ domains or intervening region. The C-termini of PLCbeta (both beta1 and beta2 isozymes were examined, containing DTPL and ESRL, respectively) displayed a diminished affinity for PDZ2 (approximately 30 microm) over that of PDZ1 (approximately 8 microm). Finally, we demonstrate trans PDZ1-PDZ2 association that is enhanced in the presence of the C-terminus of PTH1R or PLCbeta, suggesting oligomerization of NHERF as a mode for enhancing the signaling associated with PTH.
Assuntos
Fosfoproteínas/metabolismo , Receptores de Hormônios Paratireóideos/metabolismo , Fosfolipases Tipo C/metabolismo , Animais , Dicroísmo Circular , Clonagem Molecular , Polarização de Fluorescência , Camundongos , Fosfoproteínas/química , Ligação Proteica , Estrutura Terciária de Proteína , Proteínas Recombinantes de Fusão/metabolismo , Trocadores de Sódio-HidrogênioRESUMO
The Antennapedia homeodomain structure consists of four helices. The helices II and III are connected by a tripeptide that forms a turn, and constitute the well-known helix-turn-helix motif. The recognition helix penetrates the DNA major groove, gives specific protein-DNA contacts and forms direct, or water-mediated, intermolecular hydrogen bonds. It was suggested that helix III (and perhaps also helix IV) might represent the recognition helix of Antennapedia homeodomain, which makes contact with the surface of the major groove of the DNA. In an attempt to clarify the helix III capabilities of assuming an helical conformation when separated from the rest of the protein, we carried out the structural determination of the recognition helix III in different solvent media. The conformational study of fragments 42-53, where residues W48 and F49, not involved in the protein-DNA interaction, were substituted by two alanines, was conducted in sodium dodecyl sulfate (SDS), trifluoroethanol (TFE) and TFE/water, using circular dichroism, nuclear magnetic resonance (NMR) and distance geometry (DG) techniques. The fragment assumes a well-defined secondary structure in TFE and in TFE/water (90/10, v/v) with an alpha-helix encompassing residues 4-9, while in TFE/water (70/30, v/v) a less regular structure was found. The DG results in the micellar system evidence the presence of a distorted alpha-helical conformation involving residues 4-8. Our results reveal that the isolated Antennapedia recognition helix III tend to preserve in solution the alpha-helical conformation even if separated from the rest of the molecule.
Assuntos
Proteínas de Homeodomínio/química , Proteínas Nucleares/química , Peptídeos/química , Fatores de Transcrição/química , Animais , Proteína do Homeodomínio de Antennapedia , Espectroscopia de Ressonância Magnética , Estrutura Secundária de Proteína , Solventes/química , Trifluoretanol/químicaRESUMO
In this article a few methods or modifications proven to be useful in the conformational examination of peptides and related molecules by molecular dynamics are illustrated. The first is the explicit use of organic solvents in the simulations. For many cases such solvents are appropriate since the nmr measurements (or other experimental observations) were carried out in the same solvent. Here, the use of dimethylsulfoxide and chloroform in molecular dynamics is described, with some advantages of the use of these solvents high-lighted. A constant allowing for the scaling of the nonbonded interactions of the force field, an idea previously employed in distance geometry and simulated annealing, has been implemented. The usefulness of this method is that when the nonbonded term is turned to zero, atoms can pass through each other, while the connectivity of the molecule is maintained. It will be shown that such simulations, if a sufficient driving force is present (i.e., nuclear Overhauser effects restraints), can produce the correct stereoconfiguration (i.e., chiral center) as well as configurational isomer (i.e., cis/trans isomers). Lastly, a penalty term for coupling constants directly related to the Karplus curve has been implemented into the potential energy force field. The advantages of this method over the commonly used dihedral angle restraining are discussed. In particular, it is shown that with more than one coupling constant about a dihedral angle a great reduction of the allowed conformational space is obtained.
Assuntos
Simulação por Computador , Modelos Moleculares , Peptídeos/química , Conformação Proteica , Algoritmos , Sequência de Aminoácidos , Clorofórmio , Dimetil Sulfóxido , Dados de Sequência Molecular , Peptídeos Cíclicos/química , Solventes , EstereoisomerismoRESUMO
The interaction of the C-terminal octapeptide of cholecystokinin, CCK-8, with the third extracellular loop of human cholecystokinin-A receptor, CCK(A)-R(329-357), has been probed by high-resolution NMR and extensive computer simulations. The structure of CCK(A)-R(329-357) in the presence of dodecylphosphocholine micelles consists of three alpha-helices, with the first and third corresponding to the extracellular ends of transmembrane (TM) helices 6 and 7. The central helix, residues W335-R345, is found to lie on the zwitterionic surface. Titration with CCK-8 produces a stable complex with a number of intermolecular NOEs between the C-terminus of the ligand (Trp(30), Met(31), Asp(32)) and the interface of TM6 and the third extracellular loop (N333, A334, Y338) of the receptor fragment. The mode of ligand binding based on these intermolecular NOEs is in agreement with a number of published findings from receptor mutagenesis and photoaffinity cross-linking. Utilizing these ligand/receptor points of interaction, the structural features of CCK(A)-R(329-357), and also the structures of CCK-8 and CCK(A)-R(1-47) previously determined, extensive molecular dynamics simulations of the CCK-8/CCK(A)-R complex were carried out. The results provide unique insight into the molecular interactions and forces important for the binding of CCK-8 to CCK(A)-R.
Assuntos
Fragmentos de Peptídeos/química , Receptores da Colecistocinina/química , Sincalida/química , Sequência de Aminoácidos , Simulação por Computador , Cristalografia por Raios X , Espaço Extracelular/química , Espaço Extracelular/metabolismo , Humanos , Substâncias Macromoleculares , Modelos Moleculares , Dados de Sequência Molecular , Ressonância Magnética Nuclear Biomolecular , Fragmentos de Peptídeos/metabolismo , Estrutura Secundária de Proteína , Receptor de Colecistocinina A , Receptores da Colecistocinina/metabolismo , Sincalida/metabolismo , TermodinâmicaRESUMO
A penalty function for scalar coupling constants has been applied in molecular dynamics simulations as an experimental constraint. The function is based on the difference between the coupling constant calculated from the dihedral angle and the experimentally measured coupling constant. The method is illustrated on a model cyclic pentapeptide for which 3JHN-H alpha and 3JHN-C beta, both about the phi backbone dihedral angle, have been measured. The function is efficient in producing structures consistent with the scalar couplings, but removed from the conformation observed in solution. This arises from the lack of J restraints for the psi dihedral angle. Simulations with both nuclear Overhauser effect (NOE) and J-coupling restraints illustrates small but significant differences from simulations using only NOEs.
Assuntos
Peptídeos/química , Sequência de Aminoácidos , Modelos Químicos , Dados de Sequência Molecular , Oligopeptídeos/química , Peptídeos Cíclicos/química , Conformação Proteica , TermodinâmicaRESUMO
G-protein-coupled receptors (GPCRs) allow cells to respond to calcium, hormones, and neurotransmitters. Not surprisingly, they currently make up the largest family of validated drug targets. Rational drug design for molecular regulators targeting GPCRs has been limited to theoretical-based computational approaches. X-ray crystallography of intact GPCRs has provided the topological orientation of the seven transmembrane helices, but limited structural information of the extracellular and intracellular loops and protein termini. In this review we detail an NMR-based approach which provides the high-resolution structural features on the extracellular domains of GPCRs and the ligand/receptor complexes formed upon titration of the peptide hormone. The results provide important contact points and a high-resolution description of the ligand/receptor interactions, which may be useful for the rational design of therapeutic agents targeting GPCRs. Recent results from our investigation of the cholecystokinin peptide hormone system are used to highlight this approach.
Assuntos
Proteínas de Ligação ao GTP/química , Receptores de Superfície Celular/química , Sequência de Aminoácidos , Animais , Humanos , Espectroscopia de Ressonância Magnética , Modelos Moleculares , Dados de Sequência Molecular , Receptores da Colecistocinina/químicaRESUMO
The conformation of a dimer of the peptide antibiotic vancomycin is developed from computer simulations based on experimental distance constraints derived from high-resolution NMR measurements. The conformation and topological array of the dimer are determined by a distance geometry based method using the molecules cast into four spatial dimensions. This method was imperative for the refinement of vancomycin given the entwining of the monomers (including hydrogen bonding and interactions between the aromatic ring systems) within the dimer. The development of the high-resolution structure of the monomer and then simple molecular modeling to create the dimer which fulfills all of the experimental observations was not possible. In contrast, the refinement protocol using the dimer cast into four spatial dimensions was able to quickly locate conformations in which both the intra- and intermolecular nuclear Overhauser effects were satisfied. These structures, once converted back to three dimensions, were further refined using standard molecular mechanics energy minimization. The structural characteristics of the dimer with respect to binding to the cell-wall precursor are described.