The Dynamics of the Neuropeptide Y Receptor Type 1 Investigated by Solid-State NMR and Molecular Dynamics Simulation (original) (raw)
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Structural studies of fragments of G-protein coupled receptors and their ligands by NMR
2009
In the course of my doctoral studies I characterized the structure and dynamics of G-protein coupled receptor (GPCRs) fragments and their ligands by high-resolution NMR. The receptors of the GPCR family are transmembrane proteins of prime biological importance. All members of this family possess similar architecture of seven membrane-spanning-helices and are involved in various signal transduction processes. First part of my work is devoted to the investigation of the structural determinants of the GPCR ligand peptide YY and monitoring the folding process of this peptide in solution. PYY is a 36-residue C-terminally amidated polypeptide that belongs to the neuropeptide Y family of peptide hormones. These molecules are involved in the regulation of a variety of physiological processes, such as for example food uptake. In the second part of my thesis I directed my efforts towards elucidation of the structure and probing the dynamic properties of the transmembrane fragments of the GPCRs in nativelike environments. The subject of my studies was the-factor G-protein coupled Ste2p receptor, which is involved in sensing pheromones in yeast. Two large polypeptide fragments including the first and the second (peptide TM1TM2) and the seventh (peptide TM7) transmembrane domains of the Ste2p receptor were structurally characterized in micellar solution. The obtained results provide important insights into the GPCR architecture in a membrane bilayer. In the first part of my work I focused on the structural determinants and the folding process of the peptide YY (PYY) in solution. Some of the peptides from neuropeptide Y family adopt a well-defined hairpin structure in water that was first shown for avian pancreatic peptide (aPP) using X-ray crystallography. This helical hairpin is commonly referred to as PP-fold and is characterized by a N-terminal polyproline helix, which is back-folded via a-turn onto a C-terminal-helix. The solution structure of the PYY displayed a highly similar helical hairpin, however in the highly homologous neuropeptide Y we were surprised by the absence of the tertiary structure. To investigate the significance of the tertiary contacts, Tyr and Pro residues at the hydrophobic interface of the hairpin-type structure of PYY were replaced by Ala residues, and the conformational and dynamical properties of the resulting peptides were analyzed by high-resolution NMR spectroscopy. Previously we established the 15N1H-NOE as a convenient method to quantify the extent of back-folding. A comparison of the data from different Ala mutant peptides to those of native PYY nicely reflected the differences in backbone rigidity of the N-terminus. Most of the Pro->Ala or the Tyr->Ala mutants possessed increased backbone dynamics, and the differences in N-terminal mobility among them reflected various degrees to which they sample conformations close to the PP-fold. By varying temperature or the methanol content of the aqueous solvent and monitoring chemical shifts we followed the residue-specific formation of tertiary contacts while changing the physical or chemical environment. The PYY peptide in methanol solution was characterized both by determining its solution structure as well as by its internal backbone dynamics as derived from 15N relaxation data. The latter is characterized by a complete loss of tertiary structure. Chemical shifts of C in the heat-denaturation experiments displayed sigmoidal curves with very similar points of inflection indicating that both secondary, as well as tertiary structure in the heat denaturation, was lost synchronously. The results suggest that helical hairpin formation in PYY peptide is both reversible and cooperative and that specific N-and C-terminal tertiary hydrophobic contacts between the polyproline and the-helix promote the folding process. In addition, structural analysis of substitutions in the turn region indicates that the loop does not constrain the hairpin structure. The results may also annimmt, die dem 'PP-fold' ähneln. Durch Variation der Temperatur oder des Methanolgehalts des wässrigen Lösungsmittels und Verfolgung des 'chemical shift' konnten wir die aminosäure-spezifische Bildung der Tertiärkontakte während der Änderung der physikalischen oder chemischen Umgebung verfolgen. Das PYY Peptid in Methanollösung wurde charakterisiert sowohl durch die Bestimmung seiner Lösungsstruktur als auch durch ihre interne 'backbone'-Dynamik mittels 15N-relaxation-Daten. Die 'backbone'-Dynamik zeichnet sich durch einen vollständigen Verlust der tertiären Struktur aus. Die 'Chemical shifts' der C in den Hitze-Denaturierungs-Experimenten zeigten sigmoidale Kurven mit sehr ähnliche Wendepunkten, was darauf hinweist, dass sowohl Sekundär-als auch Tertiärstruktur in der Hitzedenaturierung synchron verloren werden. Die Ergebnisse deuten darauf hin, dass die Bildung des helikalen 'hairpin' im PYY Peptid reversibel und kooperativ ist und dass spezifische N-und C-terminale hydrophobe Tertiärkontakte zwischen der Polyprolinhelix und der-Helix den Faltungsprozess fördern. Darüber hinaus deutet die Strukturanalyse von Substitutionen in der 'turn'-Region darauf hin, dass der 'loop' die 'hairpin'-Struktur nicht hemmt. Die Ergebnisse können auch Auswirkungen für unser Verständnis der Bindung dieser Peptide auf ihren Rezeptoren haben. Im zweiten Teil der Dissertation wurde die Struktur und Dynamik von zwei großen Fragmenten von Ste2p, dem G-Protein-gekoppelten-Faktor-Rezeptor von Hefe untersucht. Beide GPCR-Fragmente wurden exprimiert und aufgereinigt von unseren Kollegen aus der Arbeitsgruppe von Prof. Fred Naider (College of Staten Island, NY). Zuerst untersuchte ich das 73-aminosäure-Peptid TM7 (Ste2p (267-339)) bestehend aus dem dritten extrazellulären 'loop', der siebten Transmembran-Helix und 40 Aminosäuren aus der zytosolische C-terminalen Domäne in Dodecylphosphocholin-Micellen mittels NMR-Spektroskopie. Die Struktur offenbarte die Anwesenheit einer-Helix im Segment von Aminosäurerest 10 bis 30, die um das interne Pro24 gestört wird. 15N-relaxation und RDC-Daten unterstützten einen recht stabilen 'fold' für den Transmembran-Anteil des TM7, hingegen die ausgesetzten Segmente waren flexibler. Die Spin-Label-Daten weisten darauf hin, dass die TM7-Helix in die Dodecylphosphocholin-Micellen integriert wurde, aber zeigten Flexibilität rund um das interne Pro24, da die Aminosäuren 22 bis 26 in die Lösung zeigen, desweiteren zeigten sie einen zweiten Interaktionsort mit der Micelle innerhalb der Region von Aminosäurerest 43 bis 58, die einen Teil einer weniger gut definierten im Entstehen begriffenen Helix bildet. Im weiteren verlängerte ich meine Arbeit an einem einfachen Transmembran-Fragment TM7 zu einem längeren 80-Aminosäure-Doppel-Transmembran-Peptid TM1TM2 (Ste2p (31-110)), bestehend vom 19 Aminosäuren aus der N-terminalen Domäne, die erste Transmembran-Helix, der erste zytoplasmatische 'loop', die zweite Transmembran-Helix und 7 Aminosäuren aus dem ersten extrazellulären 'loop' des Ste2p-Rezeptors. Aufgrund der größeren Komplexität des doppelten Transmembran-Fragments wurden verschiedene Isotopen-Labeling-Muster genutzt: [15N], [15N, 13C], [15N, 13C, 2H]-markiert und selektiv [15N]-markiert an bestimmten Aminosäuren oder protoniert nur an ausgewählten Methyl-Gruppen-Peptiden. Die Struktur des TM 1 TM 2-Peptids in LYSO-palmitoylphosphatidylglycerol-Micellen zeigte das Vorhandensein von drei-Helices, von Aminosäure 39-47, 49-72 und 80-103, mit einer größeren Flexibilität rund um das interne Arg58 der ersten Transmembran-Domäne. Mehrere 'long range-interhelical NOE' Verbindungen unterstützen die Faltung von TM1TM2 in eine Tertiärstruktur, die eine gekreuzte Helix bildet, die sich ausdehnt in Richtung der extrazellulären Regionen und die erhebliche Flexibilität in der G56VRSG60 Region enthält. 15N-relaxation-und Wasserstoff-Deuterium-Austausch-Daten unterstützten einen stabilen 'fold' für die Transmembran-Teile von TM1TM2, während die lösungsmittel-exponierten Segmente flexibler waren. Interessanterweise ist die NMR-Struktur im Einklang mit den Ergebnissen der biochemischen Experimente, die die Ligandenbindungsort in dieser Region des Rezeptors identifizierten. Die erzielten Ergebnisse während meiner Promotionsstudien zeigen wichtige Aspekte der GPCR-Peptid-Liganden PYY-Struktur und seiner Faltung in der Lösung, sowie geben sie Aufschluss über die Struktur der großen Fragmente des Hefe-Pheromon-Rezeptor Ste2p in nativer Micellenumgebung.
Properties of the N -terminal domains from Y receptors probed by NMR spectroscopy
Journal of Peptide Science, 2009
Binding of neurohormones from the NPY family to their receptors, the so-called Y receptors, that belong to the superfamily 1b of G-protein coupled receptors might include transient binding to the N-terminal domains of the receptors. Accordingly, we have studied structural features of the N-terminal domains from the Y1, Y2, Y4, and Y5 receptor subtypes (N-Y1, N-Y2, N-Y4, N-Y5). We developed efficient strategies for their recombinant expression. N-Y4 and N-Y1 were expressed as insoluble fusions to enforce accumulation into inclusion bodies, whereas N-Y2 and N-Y5 were expressed as soluble fusion proteins. All N-terminal domains are fully flexible in aqueous buffer. In the presence of phospholipid micelles some stretches within the polypeptides adopt helical conformations, but these are too unstable to be characterized in detail. Using chemical shift mapping techniques, interactions of NPY, peptide YY (PYY), and pancreatic polypeptide (PP), the three members of the neurohormone family that are the Y receptors' natural ligands, with N-Y1, N-Y2, and N-Y5 revealed chemical shift changes in all cases, with the largest values being encountered for PP interacting with N-Y1 or N-Y5 both in the presence and in the absence of phospholipid micelles. The strength of the interactions, however, is generally weak, and the data also point to nonspecific contacts. Previously, in case of the interaction of N-Y4 with PP, the contacts were shown to be electrostatic in nature. This work indicates that association of the peptides with the N-terminal domains may generally be part of their binding trajectory.
Journal of Peptide Science, 1996
Neuropeptide Y (NFV), a peptide amide comprising 36 residue has been shown to act as a potent vasoconstrictor. In order to shed light on the structural requirements for the biological activities with respect to the different prerequisites for afflnty to the NPY receptor subtypes Y1 and Yz. in the present study the syntheses and conformational analyses of two C-terminal segments, NPY(18-36) and NFT(13-36), are described.
Membrane interaction of neuropeptide Y detected by EPR and NMR spectroscopy
Biochimica et Biophysica Acta (BBA) - Biomembranes, 2005
Neuropeptide Y (NPY) is one of the most abundant peptides in the central nervous system of mammals. It belongs to the best-conserved peptides in nature, i.e., the amino acid sequences of even evolutionary widely separated species are very similar to each other. Using porcine NPY, which differs from human NPY only at position 17 (a leucine residue exchanged for a methionine), labeled with a TOAC spin probe at the 2nd, 32nd, or 34th positions of the peptide backbone, the membrane binding and penetration of NPY was determined using EPR and NMR spectroscopy. The vesicular membranes were composed of phosphatidylcholine and phosphatidylserine at varying mixing ratios. From the analysis of the EPR line shapes, the spectral contributions of free, dimerized, and membrane bound NPY could be separated. This analysis was further supported by quenching experiments, which selected the contributions of the bound NPY fraction. The results of this study give rise to a model where the a-helical part of NPY (amino acids 13 -36) penetrates the membrane interface. The unstructured N-terminal part (amino acids 1 -12) extends into the aqueous phase with occasional contacts with the lipid headgroup region. Besides the mixing ratio of zwitterionic and negatively charged phospholipid species, the electrostatic peptide membrane interactions are influenced by the pH value, which determines the net charge of the peptide resulting in a modified membrane binding affinity. The results of these variations indicate that NPY binding to phospholipid membranes depends strongly on the electrostatic interactions. An estimation of the transfer energy of the peptide from aqueous solution to the membrane interface DG supports the preferential interaction of NPY with negatively charged membranes. D
Journal of Peptide Research, 2009
The solution structure of the Y1 receptor agonist, porcine [Leu31, Pro34]NPY, has been investigated by two-dimensional NMR and molecular modeling. A complete assignment of the NMR resonances was achieved and 201 inter-residue nuclear Overhauser enhancement spectroscopy (NOESY) connectivities could be identified, comprising several connectivities between the N- and C-terminal segments. A molecular model was calculated by distance geometry, simulated annealing and conjugate gradients energy minimization using the NOE constraints. The results indicate that, like NPY and other peptides of the family, [Leu31, Pro34]NPY adopts a folded hairpin structure with the terminal segments in close proximity. Analysis of the secondary chemical shifts for the CHα's and of the temperature dependence of the NH chemical shifts combined with the NOE constraints indicates a tendency toward helix structure for the segment 18-30 and the presence of turn structure for the C-terminal segment (residues 31-36). Native NPY and [Leu31, Pro34]NPY have most of their structures in common but differ slightly in their C-terminal portion. Based on the structures of NPY and of its specific agonists, [Leu31, Pro34]NPY and NPY 13-36, conclusions can be drawn about the structural requirements for binding to the Y1 and Y2 receptor subtypes.
Molecular dynamics study of 4-OH-phenylacetyl- D-Y(Me)FQNRPR-NH 2 selectivity to V1a receptor
2003
G protein-coupled receptors relay diverse extracellular signals into cells via a common mechanism, involving activation of cytosol G proteins. The mechanism underlies the actions of~50% of all drugs. In this work, we focus on simulating three protein-ligand complexes of the neurohypophyseal hormone analog 4-OH-phenylacetyl-D-Y(Me)FQNRPR-NH 2 (I) with the human V1a, V2 and oxytocin receptors. The peptide I is a potent selective V1a receptor antagonist. To obtain relaxed models of the complexes, the following techniques were used: docking of I into the vasopressin V1a, V2 and oxytocin receptor models, optimization of the geometry of the resulting complexes and molecular dynamics in a fully hydrated 1-palmitoyl-2-oleoyl-snglycero-3-phosphatidylcholine lipid bilayer. The results of the simulations allow us to draw some conclusions about the ligand selectivity to V1aR.
Biochimica et Biophysica Acta (BBA) - Biomembranes, 2011
Fragments of integral membrane proteins have been used to study the physical chemical properties of regions of transporters and receptors. Ste2p(G31-T110) is an 80-residue polypeptide which contains a portion of the N-terminal domain, transmembrane domain 1 (TM1), intracellular loop 1, TM2 and part of extracellular loop 1 of the α-factor receptor (Ste2p) from Saccharomyces cerevisiae. The structure of this peptide was previously determined to form a helical hairpin in lyso-palmitoylphosphatidyl-glycerol micelles (LPPG) [1]. Herein, we perform a systematic comparison of the structure of this protein fragment in micelles and trifluoroethanol (TFE):water in order to understand whether spectra recorded in organic:aqueous medium can facilitate the structure determination in a micellar environment. Using uniformly labeled peptide and peptide selectively protonated on Ile, Val and Leu methyl groups in a perdeuterated background and a broad set of 3D NMR experiments we assigned 89% of the observable atoms. NOEs and chemical shift analysis were used to define the helical regions of the fragment. Together with constraints from paramagnetic spin labeling, NOEs were used to calculate a transiently folded helical hairpin structure for this peptide in TFE:water. Correlation of chemical shifts was insufficient to transfer assignments from TFE:water to LPPG spectra in the absence of further information.
Relationships between Structural Dynamics and Functional Kinetics in Oligomeric Membrane Receptors
Biophysical Journal, 2010
Recent efforts to broaden understanding of the molecular mechanisms of membrane receptors in signal transduction make use of rate-equilibrium free-energy relationships (REFERs), previously applied to chemical reactions, enzyme kinetics, and protein folding. For oligomeric membrane receptors, we distinguish between a), the Leffler parameter a L , to characterize the global transition state for the interconversion between conformations; and b), the Fersht parameter, f F , to assign the degree of progression of individual residue positions at the transition state. For both a L and f F , insights are achieved by using harmonic energy profiles to reflect the dynamic nature of proteins, as illustrated with single-channel results reported for normal and mutant nicotinic receptors. We also describe new applications of a L based on published results. For large-conductance calciumactivated potassium channels, data are satisfactorily fit with an a L value of 0.65, in accord with REFERs. In contrast, results reported for the flip conformational state of glycine and nicotinic receptors are in disaccord with REFERs, since they yield a L values outside the usual limits of 0-1. Concerning published f F values underlying the conformational wave hypothesis for nicotinic receptors, we note that interpretations may be complicated by variations in the width of harmonic energy profiles.
Archives of Biochemistry and Biophysics, 2008
The three-dimensional structure of full-length structure of the M 1 muscarinic receptor was obtained through the fragmental homology modeling procedure. A 10-ns molecular dynamics (MD) simulation of the protein imbedded in a lipid slab and surrounded by water molecules was further used to relax the model. It was found that the homology model corresponded to the conformation in the ground state, since no significant motions of the backbone of transmembrane domains were observed. Furthermore, the reliability of the model was validated by analyzing key inter-helical contacts, sidechain-sidechain interactions, the formation of stable aromatic microdomains (clusters) and the docking of acetylcholine to its binding site. Moreover, a few conserved interactions observed in the X-ray structure of rhodopsin, such as inter-helical sidechain-sidechain hydrogen bonds were accurately reproduced in the MD simulation. The coupling of ACh to its binding site was found to be dominated by p-cation and salt bridge interactions, while its conformational space was restrained through van der Waals and hydrogen bond interactions. In general, such features were in very good agreement with the available experimental as well as with theoretical data. Considering the above, the structural information obtained in this study can be used a starting point to investigate the activation mechanism of the receptor and the ability to develop selective agonists and allosteric modulators which could be used for the treatment of Alzheimer's disease.