Ab initio computational modeling of long loops in G-protein coupled receptors

A newly developed approach for predicting the structure of segments that connect known elements of secondary structure in proteins has been applied to some of the longer loops in the G-protein coupled receptors (GPCRs) rhodopsin and the dopamine receptor D2R. The algorithm uses Monte Carlo (MC) simulation in a temperature annealing protocol combined with a scaled collective variables (SCV) technique to search conformation space for loop structures that could belong to the native ensemble. Except for rhodopsin, structural information is only available for the transmembrane helices (TMHs), and therefore the usual approach of finding a single conformation of lowest energy has to be abandoned. Instead the MC search aims to find the ensemble located at the absolute minimum free energy, i.e., the native ensemble. It is assumed that structures in the native ensemble can be found by an MC search starting from any conformation in the native funnel. The hypothesis is that native structures are trapped in this part of conformational space because of the high-energy barriers that surround the native funnel. In this work it is shown that the crystal structure of the second extracellular loop (e2) of rhodopsin is a member of this loop’s native ensemble. In contrast, the crystal structure of the third intracellular loop is quite different in the different crystal structures that have been reported. Our calculations indicate, that of three crystal structures examined, two show features characteristic of native ensembles while the other one does not. Finally the protocol is used to calculate the structure of the e2 loop in D2R. Here, the crystal structure is not known, but it is shown that several side chains that are involved in interaction with a class of substituted benzamides assume conformations that point into the active site. Thus, they are poised to interact with the incoming ligand.     

Synthesis and spectroscopic characterization of photo-affinity peptide ligands to study rhodopsin-G protein interaction

G protein-coupled receptors (GPCRs) are involved in the control of virtually all aspects of our behavior and physiology. Activated receptors catalyze nucleotide exchange in heterotrimeric G proteins (composed of alpha.GDP, beta and gamma subunits) on the inner surface of the cell membrane. The GPCR rhodopsin and the G protein transducin (G(t)) are key proteins in the early steps of the visual cascade. The main receptor interaction sites on G(t) are the C-terminal tail of the G(t)alpha-subunit and the farnesylated C-terminal tail of the G(t)gamma-subunit. Synthetic peptides derived from these C-termini specifically bind and stabilize the active rhodopsin conformation (R*). Here we report the synthesis of R*-interacting peptides containing photo-reactive groups with a specific isotope pattern, which can facilitate detection of cross-linked products by mass spectrometry. In a preliminary set of experiments, we characterized such peptides derived from the farnesylated G(t)gamma C-terminus (G(t)gamma(60-71)far) in terms of their capability to bind R*. Here, we describe novel peptides with photo-affinity labels that bind R* with affinities similar to that of the native G(t)gamma(60-71)far peptide. Such peptides will enable an improved experimental strategy to probe rhodopsin-G(t) interaction and to map so far unknown interaction sites between both proteins.     

Enzyme‐Mediated,Site‐Specific Protein Coupling Strategies for Surface‐Based Binding Assays

Covalent surface immobilization of proteins for binding assays is typically performed non‐specifically via lysine residues. However, receptors that either have lysines near their binding pockets, or whose presence at the sensor surface is electrostatically disfavoured, can be hard to probe. To overcome these limitations and to improve the homogeneity of surface functionalization, we adapted and optimized three different enzymatic coupling strategies (4′‐phosphopantetheinyl transferase, sortase A, and asparaginyl endopeptidase) for biolayer interferometry surface modification. All of these enzymes can be used to site‐specifically and covalently ligate proteins of interest via short recognition sequences. The enzymes function under mild conditions and thus immobilization does not affect the receptors’ functionality. We successfully employed this enzymatic surface functionalization approach to study the binding kinetics of two different receptor–ligand pairs.     

Enzyme‐Mediated,Site‐Specific Protein Coupling Strategies for Surface‐Based Binding Assays

Covalent surface immobilization of proteins for binding assays is typically performed non‐specifically via lysine residues. However, receptors that either have lysines near their binding pockets, or whose presence at the sensor surface is electrostatically disfavoured, can be hard to probe. To overcome these limitations and to improve the homogeneity of surface functionalization, we adapted and optimized three different enzymatic coupling strategies (4′‐phosphopantetheinyl transferase, sortase A, and asparaginyl endopeptidase) for biolayer interferometry surface modification. All of these enzymes can be used to site‐specifically and covalently ligate proteins of interest via short recognition sequences. The enzymes function under mild conditions and thus immobilization does not affect the receptors’ functionality. We successfully employed this enzymatic surface functionalization approach to study the binding kinetics of two different receptor–ligand pairs.     

Molecular modeling of neurokinin B and tachykinin NK₃ receptor complex

The tachykinin receptor NK? is a member of the rhodopsin family of G-protein coupled receptors. The NK? receptor has been regarded as an important drug target due to diverse physiological functions and its possible role in the pathophysiology of psychiatric disorders, including schizophrenia. The NK3 receptor is primarily activated by the tachykinin peptide hormone neurokinin B (NKB) which is the most potent natural agonist for the NK? receptor. NKB has been reported to play a vital role in the normal human reproduction pathway and in potentially life threatening diseases such as pre-eclampsia and as a neuroprotective agent in the case of neurodegenerative diseases. Agonist binding to the receptor is a critical event in initiating signaling, and therefore a characterization of the structural features of the agonists can reveal the molecular basis of receptor activation and help in rational design of novel therapeutics. In this study a molecular model for the interaction of the primary ligand NKB with its G-protein coupled receptor NK? has been developed. A three-dimensional model for the NK? receptor has been generated by homology modeling using rhodopsin as a template. A knowledge based docking of the NMR derived bioactive conformation of NKB to the receptor has been performed utilizing limited ligand binding data obtained from photoaffinity labeling and site-directed mutagenesis studies. A molecular model for the NKB-NK? receptor complex obtained sheds light on the topographical features of the binding pocket of the receptor and provides insight into the biochemical data currently available for the receptor.     

一种糖生物传感芯片制备的新方法

建立了一种用于SPR生物传感器分析的糖生物传感芯片制备新方法。在已二胺大量过量的情况下，海洋硫酸多糖911还原末端的半缩醛基与已二胺的一个氨基进行还原胺化反应，引入一个伯氨基，该伯氨基进一步与sulfo-NHS-biotin反应，使911的还原末端生物素化，通过预偶联到GM5芯片上的链酶抗生素抗体，以俘获法将生物素化的911固定到芯片表面。该方法避免已有固定方法对糖内部结构的破坏，减少了非特异性耦联，能更好地反映糖类与其它分子的相互作用特性，且操作简单，适应于多数具有还原末端的糖类。911经该方法固定后，利用SPR生物传感器，研究了其与HIV gp120蛋白V3区的相互作用动力学，初步证明两者之间存在强烈的相互作用，KA与KD分别为：2.25e5与4.44e-6。     

MODELING RHODOPSIN, A MEMBER OF G-PROTEIN COUPLED RECEPTORS, BY COMPUTER GRAPHICS. INTERPRETATION OF CHEMICAL SHIFTS OF FLUORINATED RHODOPSINS

An attempt has been made to construct a 3-D model of rhodopsin, a member of G-protein coupled receptors. Sequence homology of rhodopsin with the latter was a factor considered in the modeling procedure. The constructed model has been used to compare currently available specific protein/substrate interaction information, the shape of the binding cavity derived from shape of binding retinal isomers and analogs and challenged to explain recently available results from a series of fluorinated rhodopsins.     

Molecular dynamics simulation of the human adenosine A3 receptor: agonist induced conformational changes of Trp243

The adenosine A(3) receptor together with rhodopsin belongs to Class A of the G-protein coupled receptors. As the crystal structure of bovine rhodopsin represents the dark (inactive) state of the receptor, the details of GPCR activation are still unknown. In this molecular dynamics study we investigate how the homology model of the human adenosine A(3) receptor responds to ligand exposure. To this end we placed the homology model in a POPC membrane model. After equilibrating for 13 ns an agonist (Cl-IB-MECA) and an inverse agonist (PSB-10) were placed inside the putative binding pocket. In the following 10 ns molecular dynamics simulation we observed a different behaviour of the side-chain torsions of Trp243(6.48), depending on the presence or absence of the agonist or inverse agonist. This conformational change of Trp243 correlates with the assumed influence of ligands on receptor activation. Other predicted conformational changes of the receptor could not be observed yet. So Trp243 may represent the first switch in receptor activation.     

Structure-activity relationship studies: methods and ligand design for G-protein coupled peptide receptors

The exchange of information between cells represents an important regulatory mechanism for cellular activities. Such regulation processes mainly occur by hydrophilic compounds, unable to penetrate the cell membrane. Accordingly such signals have to be transmitted into the cell that is performed by transmembrane receptors. The widespread group of G-protein coupled receptors plays a decisive role in extracellular signal recognition and transition into cellular response. The importance of this interaction is evidently shown by the severe diseases that correlate with dysfunction of the interaction between ligand and G-protein coupled receptor. The development of drugs against these diseases needs the comprehension of signal recognition and transition as well as the understanding of intracellular signal pathways. In this review, we describe concepts and methods to identify the structure-activity relationships of G-protein coupled peptide receptors and their successful application. Furthermore we provide an insight into peptide based drug design. Examples are taken from the field of CGRP, orexin and growth hormone secretagogue receptor ligands.     

Dynamic models of G-protein coupled receptor dimers: indications of asymmetry in the rhodopsin dimer from molecular dynamics simulations in a POPC bilayer

Based on the growing evidence that G-protein coupled receptors (GPCRs) form homo- and hetero-oligomers, models of GPCR signaling are now considering macromolecular assemblies rather than monomers, with the homo-dimer regarded as the minimal oligomeric arrangement required for functional coupling to the G-protein. The dynamic mechanisms of such signaling assemblies are unknown. To gain some insight into properties of GPCR dimers that may be relevant to functional mechanisms, we study their current structural prototype, rhodopsin. We have carried out nanosecond time-scale molecular dynamics (MD) simulations of a rhodopsin dimer and compared the results to the monomer simulated in the same type of bilayer membrane model composed of an equilibrated unit cell of hydrated palmitoyl-oleoyl-phosphatidyl choline (POPC). The dynamic representation of the homo-dimer reveals the location of structural changes in several regions of the monomeric subunits. These changes appear to be more pronounced at the dimerization interface that had been shown to be involved in the activation process [Proc Natl Acad Sci USA 102:17495, 2005]. The results are consistent with a model of GPCR activation that involves allosteric modulation through a single GPCR subunit per dimer.     

alpha(2A)-adrenergic receptor derived peptide adsorbates: a G-protein interaction study

The affinity of alpha(2A)-adrenergic receptor (alpha(2A)-AR) derived peptide adsorbates for the functional bovine brain G-protein is studied in the search for the minimum sequence recognition. Three short peptides (GPR-i2c, GPR-i3n, and GPR-i3c) are designed to mimic the second and third intracellular loops of the receptor. X-ray photoelectron spectroscopy is used to study the chemical composition of the peptides and the binding strength to the surfaces. Chemisorption of the peptides to the gold substrates is observed. Infrared spectroscopy is used to study the characteristic absorption bands of the peptides. The presence of peptides on the surfaces is verified by prominent amide I and amide II bands. The interaction between the peptides and the G-protein is studied with surface plasmon resonance. It is shown that GPR-i3n has the highest affinity for the G-protein. Equilibrium analysis of the binding shows that the G-protein keeps its native conformation when interacting with GPR-i3c, but during the interaction with GPR-i2c and GPR-i3n the conformation of G-protein is changed, leading to the formation of aggregates and/or multilayers.     

Analysis of the activation mechanism of the guinea-pig Histamine H<Subscript>1</Subscript>-receptor

The Histamine H(1)-receptor (H1R), belonging to the amine receptor-class of family A of the G-protein coupled receptors (GPCRs) gets activated by agonists. The consequence is a conformational change of the receptor, which may involve the binding-pocket. So, for a good prediction of the binding-mode of an agonist, it is necessary to have knowledge about these conformational changes. Meanwhile some experimental data about the structural changes of GPCRs during activation exist. Based on homology modeling of the guinea-pig H1R (gpH1R), using the crystal structure of bovine rhodopsin as template, we performed several MD simulations with distance restraints in order to get an inactive and an active structure of the gpH1R. The calculations led to a Phe6.44/Trp6.48/Phe6.52-switch and linearization of the proline kinked transmembrane helix VI during receptor activation. Our calculations showed that the Trp6.48/Phe6.52-switch induces a conformational change in Phe6.44, which slides between transmembrane helices III and VI. Additionally we observed a hydrogen bond interaction of Ser3.39 with Asn7.45 in the inactive gpH1R, but because of a counterclockwise rotation of transmembrane helix III Ser3.39 establishes a water-mediated hydrogen bond to Asp2.50 in the active gpH1R. Additionally we simulated a possible mechanism for receptor activation with a modified LigPath-algorithm.     

N-terminal and C-terminal domains of arrestin both contribute in binding to rhodopsin

Visual arrestin terminates the signal amplification cascade in photoreceptor cells by blocking the interaction of light activated phosphorylated rhodopsin with the G-protein transducin. Although crystal structures of arrestin and rhodopsin are available, it is still unknown how the complex of the two proteins is formed. To investigate the interaction sites of arrestin with rhodopsin various surface regions of recombinant arrestin were sterically blocked by different numbers of fluorophores (Alexa 633). The binding was recorded by time-resolved light scattering. To accomplish site-specific shielding of protein regions, in a first step all three wild-type cysteines were replaced by alanines. Nevertheless, regarding the magnitude and specificity of rhodopsin binding, the protein is still fully active. In a second step, new cysteines were introduced at selected sites to allow covalent binding of fluorophores. Upon attachment of Alexa 633 to the recombinant cysteines we observed that these bulky labels residing in the concave area of either the N- or the C-terminal domain do not perturb the activity of arrestin. By simultaneously modifying both domains with one Alexa 633 the binding capacity was reduced. The presence of two Alexa 633 molecules in each domain prevented binding of rhodopsin to arrestin. This observation indicates that both concave sites participate in binding.     

Rhodopsin exhibits a preference for solvation by polyunsaturated docosohexaenoic acid

An all-atom molecular dynamics simulation of rhodopsin in a membrane environment has been carried out with lipid composition similar to that of the retinal membrane. The initial conformation of the protein was taken from the X-ray crystallographic structure (1F88), while those of the lipids came from a previous molecular dynamics simulation. During the course of the 12.5 ns simulation, the initially randomly placed lipids adopt an anisotropic solvation structure around the protein. The lipids, having one saturated stearic acid chain and one polyunsaturated docosohexaenoic acid chain with a zwitterionic phosphatidylcholine headgroup, arrange themselves to maximize contact between the polyunsaturated chain and the protein surface. This organization is driven by energetically favorable interactions between the transmembrance helices and the docosohexaenoyl chains that are largely of the van der Waals type. These observations are consistent with various experimental studies on rhodopsin and other G-protein coupled receptors and with the picture of extreme flexibility in polyunsaturated fatty acid chains that has arisen from recent NMR and computational work.     

Engineering a rhodopsin protein mimic

Due to the difficulties in handling and manipulating membrane-bound proteins, such as rhodopsin, and the lack of crystallographic information on the cone opsins, we have opted to engineer a protein mimic of the transmembrane G-protein coupled receptor. Human cellular retinoic acid binding protein (CRABPII), a well studied and characterized protein, has been reengineered into a protein that now will bind retinal as a protonated Schiff base with high binding affinity (Kd = 2 nM) mimicking that of rhodopsin.     

Determining the folding and unfolding rate constants of nucleic acids by biosensor. Application to telomere G-quadruplex

Nucleic acid molecules may fold into secondary structures, and the formation of such structures is involved in many biological processes and technical applications. The folding and unfolding rate constants define the kinetics of conformation interconversion and the stability of these structures and is important in realizing their functions. We developed a method to determine these kinetic parameters using an optical biosensor based on surface plasmon resonance. The folding and unfolding of a nucleic acid is coupled with a hybridization reaction by immobilization of the target nucleic acid on a sensor chip surface and injection of a complementary probe nucleic acid over the sensor chip surface. By monitoring the time course of duplex formation, both the folding and unfolding rate constants for the target nucleic acid and the association and dissociation rate constants for the target-probe duplex can all be derived from the same measurement. We applied this method to determine the folding and unfolding rate constants of the G-quadruplex of human telomere sequence (TTAGGG)(4) and its association and dissociation rate constants with the complementary strand (CCCTAA)(4). The results show that both the folding and unfolding occur on the time scale of minutes at physiological concentration of K(+). We speculate that this property might be important for telomere elongation. A complete set of the kinetic parameters for both of the structures allows us to study the competition between the formation of the quadruplex and the duplex. Calculations indicate that the formation of both the quadruplex and the duplex is strand concentration-dependent, and the quadruplex can be efficiently formed at low strand concentration. This property may provide the basis for the formation of the quadruplex in vivo in the presence of a complementary strand.     

Structural determinants of the supramolecular organization of G protein-coupled receptors in bilayers

The G protein-coupled receptor (GPCR) rhodopsin self-assembles into supramolecular structures in native bilayers, but the structural determinants of receptor oligomerization are not known. We carried out multiple self-assembly coarse-grained molecular dynamics (CGMD) simulations of model membranes containing up to 64 molecules of the visual receptor rhodopsin over time scales reaching 100 μs. The simulations show strong preferential interaction modes between receptors. Two primary modes of receptor-receptor interactions are consistent with umbrella sampling/potential of mean force (PMF) calculations as a function of the distance between a pair of receptors. The preferential interfaces, involving helices (H) 1/8, 4/5 and 5, present no energy barrier to forming a very stable receptor dimer. Most notably, the PMFs show that the preferred rhodopsin dimer exists in a tail-to-tail conformation, with the interface comprising transmembrane H1/H2 and amphipathic H8 at the extracellular and cytoplasmic surfaces, respectively. This dimer orientation is in line with earlier electron microscopy, X-ray, and cross-linking experiments of rhodopsin and other GPCRs. Less stable interfaces, involving H4 and H6, have a free energy barrier for desolvation (delipidation) of the interfaces and appear to be designed to stabilize "lubricated" (i.e., lipid-coated) dimers. The overall CGMD strategy used here is general and can be applied to study the homo- and heterodimerization of GPCRs and other transmembrane proteins. Systematic extension of the work will deepen our understanding of the forces involved in the membrane organization of integral membrane proteins.     

Surface Charge Changes upon Formation of the Signaling State in Visual Rhodopsin†

The cytoplasmic surface of G protein‐coupled receptors plays a central role for activation and deactivation of the receptor. To understand the molecular mechanisms which underlie these processes, we determined the surface charge density and its changes upon activation directly at the cytoplasmic surface of bovine rhodopsin and correlated these changes with key events in receptor activation. The surface charge density was calculated from the ionic strength dependence of the apparent pK_a of the surface‐bound pH‐indicator dye fluorescein according to the Gouy‐Chapman theory. The surface charge density at pH 6.5 changes by 0.8 ± 0.2 elementary charge/1000 Ų in rod outer segment disk membranes and by 0.4 ± 0.2 elementary charge/1000 Ų in rhodopsin/dodecylmaltoside micelles upon formation of the active metarhodopsin‐II state. By comparison of these surface charge density values determined with and without the native lipid environment, we calculated the charge change to about 1 elementary charge/cytoplasmic rhodopsin surface. The more positive surface charge density in metarhodopsin‐II decreases back to the dark state level of σ = ?2.0 ± 0.2 elementary charges/1000 Ų in the opsin state, providing further evidence that the cytoplasmic surface properties after metarhodopsin‐II decay resemble almost those of the dark state.     

Label-free detection of C-reactive protein using reflectometric interference spectroscopy-based sensing system

Reflectometric interference spectroscopy (RIfS) is a label-free, time-resolved technique, and suitable for detecting antibody–antigen interaction. This work describes a continuous flow biosensor for C-reactive protein (CRP), involving an effective immobilization method of a monoclonal antibody against CRP (anti-CRP) to achieve highly sensitive RIfS-based detection of CRP. The silicon nitride-coated silicon chip (SiN chip) for the RIfS sensing was first treated with trimethylsilylchloride (TMS), followed by UV-light irradiation to in situ generation of homogeneous silanols on the surface. Following amination by 3-aminopropyltriethoxysilane, carboxymethyldextran (CMD) was grafted, and subsequently, protein A was immobilized to create the oriented anti-CRP surface. The immobilization process of protein A and anti-CRP was monitored with the RIfS system by consecutive injections of an amine coupling reagent, protein A and anti-CRP, respectively, to confirm the progress of each step in real time. The sensitivity was enhanced when all of the processes were adopted, suggesting that the oriented immobilization of anti-CRP via protein A that was coupled with the grafted CMD on the aminated surface of TMS-treated SiN chip. The feasibility of the present sensing system was demonstrated on the detection of CRP, where the silicon-based inexpensive chips and the simple optical setup were employed. It can be applied to other target molecules in various fields of life science as a substitute of surface plasmon resonance-based expensive sensors.     

Rhodopsin Activation Switches in a Native Membrane Environment†

The elucidation of structure–function relationships of membrane proteins still poses a considerable challenge due to the sometimes profound influence of the lipid bilayer on the functional properties of the protein. The visual pigment rhodopsin is a prototype of the family of G protein‐coupled transmembrane receptors and a considerable part of our knowledge on its activation mechanisms has been derived from studies on detergent‐solubilized proteins. This includes in particular the events associated with the conformational transitions of the receptor from the still inactive Meta I to the Meta II photoproduct states, which are involved in signaling. These events involve disruption of an internal salt bridge of the retinal protonated Schiff base, movement of helices and proton uptake from the solvent by the conserved cytoplasmic E(D)RY network around Glu134. As the equilibria associated with these events are considerably altered by the detergent environment, we set out to investigate these equilibria in the native membrane environment and to develop a coherent thermodynamic model of these activating steps using UV–visible and Fourier‐transform infrared spectroscopy as complementary techniques. Particular emphasis is put on the role of protonation of Glu134 from the solvent, which is a thermodynamic prerequisite for full receptor activation in membranes, but not in detergent. In view of the conservation of this carboxylate group in family A G protein‐coupled receptors, it may also play a similar role in the activation of other family members.