Five residues in the HtrI transducer membrane-proximal domain close the cytoplasmic proton-conducting channel of sensory rhodopsin I

Transducer-free sensory rhodopsins carry out light-driven proton transport in Halobacterium salinarum membranes. Transducer binding converts the proton pumps to signal-relay devices in which the transport is inhibited. In sensory rhodopsin I (SRI) binding of its cognate transducer HtrI inhibits transport by closing a cytoplasmic proton-conducting channel necessary for proton uptake during the SRI photochemical reaction cycle. To investigate the channel closure, a series of HtrI mutants truncated in the membrane-proximal cytoplasmic portion of an SRI-HtrI fusion were constructed and expressed in H. salinarum membranes. We found that binding of the membrane-embedded portion of HtrI is insufficient for channel closure, whereas cytoplasmic extension of the second HtrI transmembrane helix by 13 residues blocks proton conduction through the channel as well as full-length HtrI. Specifically the closure activity is localized in this 13-residue membrane-proximal cytoplasmic domain to the 5 final residues, each of which incrementally contributes to reduction of proton conductivity. Moreover, these same residues in the dark incrementally and proportionally increase the pKa of the Asp-76 counterion to the protonated Schiff base chromophore in the membrane-embedded photoactive site. We conclude that this critical region of HtrI alters the dark conformation of SRI as well as light-induced channel opening. The 5 residues in HtrI correspond in position to 5 residues demonstrated on the homologous NpHtrII to interact with the E-F loop of its cognate receptor NpSRII in the accompanying article (Yang, C.-S., Sineshchekov, O., Spudich, E. N., and Spudich, J. L. (2004) J. Biol. Chem. 279, 42970-42976). These results strongly suggest that the membrane-proximal region of Htr proteins interact with their cognate sensory rhodopsin cytoplasmic domains as part of the signal-relay coupling between the proteins.     

Suppressor Mutation Analysis of the Sensory Rhodopsin I-Transducer Complex: Insights into the Color-Sensing Mechanism

The molecular complex containing the phototaxis receptor sensory rhodopsin I (SRI) and transducer protein HtrI (halobacterial transducer for SRI) mediates color-sensitive phototaxis responses in the archaeon Halobacterium salinarum. One-photon excitation of the complex by orange light elicits attractant responses, while two-photon excitation (orange followed by near-UV light) elicits repellent responses in swimming cells. Several mutations in SRI and HtrI cause an unusual mutant phenotype, called orange-light-inverted signaling, in which the cell produces a repellent response to normally attractant light. We applied a selection procedure for intragenic and extragenic suppressors of orange-light-inverted mutants and identified 15 distinct second-site mutations that restore the attractant response. Two of the 3 suppressor mutations in SRI are positioned at the cytoplasmic ends of helices F and G, and 12 suppressor mutations in HtrI cluster at the cytoplasmic end of the second HtrI transmembrane helix (TM2). Nearly all suppressors invert the normally repellent response to two-photon stimulation to an attractant response when they are expressed with their suppressible mutant alleles or in an otherwise wild-type strain. The results lead to a model for control of flagellar reversal by the SRI-HtrI complex. The model invokes an equilibrium between the A (reversal-inhibiting) and R (reversal-stimulating) conformers of the signaling complex. Attractant light and repellent light shift the equilibrium toward the A and R conformers, respectively, and mutations are proposed to cause intrinsic shifts in the equilibrium in the dark form of the complex. Differences in the strength of the two-photon signal inversion and in the allele specificity of suppression are correlated, and this correlation can be explained in terms of different values of the equilibrium constant (K_eq) for the conformational transition in different mutants and mutant-suppressor pairs.     

Demonstration of 2:2 stoichiometry in the functional SRI-HtrI signaling complex in Halobacterium membranes by gene fusion analysis

A fusion protein in which the C-terminus of Halobacterium salinarum sensory rhodopsin I (SRI) is connected by a flexible linker to the N-terminus of its transducer (HtrI) was constructed and expressed in H. salinarum. The fusion protein mediated attractant responses to orange light and repellent responses to UV/violet light that were comparable to those produced by the wild-type SRI-HtrI complex. Immunoblot analysis of H. salinarum membrane proteins demonstrated intact fusion protein and no detectable proteolytic cleavage products. Rapid oxidative cross-linking of a monocysteine mutant in the HtrI domain confirmed that the fusion protein exists as a homodimer in the membrane. HtrI-free SRI and HtrI-complexed SRI have been shown previously to exhibit large differences in the pH dependence of their photocycle kinetics and in the pK(a) of Asp76 that controls a pH-dependent spectral transition in SRI. These differences were used to assess whether only one or both SRI domains in the fusion protein were complexed properly to the HtrI homodimer. Measurement of the photochemical activity, the photocycle kinetics, and the absorption spectra at various pH values established that both SRI domains are complexed to HtrI in the fusion protein, and therefore the stoichiometry is 2:2. Closer examination of the HtrI effect on SRI revealed that Asp76 titration in HtrI-free SRI fits two pK(a) values, with 98% and 2% of the molecules titrating with pK(a)'s of 7 and 9, respectively. The same two pK(a)'s of Asp76 are evident in HtrI-complexed SRI, but with 13% with pK(a) of 7 and 87% with pK(a) of 9 and a similar bias toward the pK(a) of 9 in the fusion protein. Titration of the fusion protein with Ala substitution at Arg73, a residue in the photoactive site, in the SRI domain indicates that a basic residue at Arg73 is necessary for the lower pK(a) to be observed. A model in which Arg73 plays a role in the HtrI effect on SRI is discussed.     

Deletion mapping of the sites on the HtrI transducer for sensory rhodopsin I interaction.

The phototaxis receptor sensory rhodopsin I (SRI) transmits signals through a membrane-bound transducer protein, HtrI. The genes for the receptor and transducer, sopI and htrI, respectively, are normally cotranscribed; however, previous work has established that fully functional interacting proteins are produced when htrI is expressed from the chromosome and sopI is expressed from a different promoter on a plasmid. In this report we show that in the membrane, concentrations of SRI from plasmid expression of wild-type sopI are negligible in the absence of HtrI protein in the cell. This requirement for HtrI is eliminated when sopI is extended at the 5'-end with 63 nucleotides of the bop gene, which encodes the N-terminal signal sequence of the bacteriorhodopsin protein. The signal is cleaved from the chimeric protein, and processed SRI is stable in the HtrI-free membrane. These results suggest a chaperone-like function for HtrI that facilitates membrane insertion or proper folding of the SRI protein. Six deletion constructs of HtrI were examined to localize the interaction sites for its putative chaperone function and for HtrI control of the SRI photocycle, a phenomenon described previously. The smallest HtrI fragment identified, which contained interaction sites for both SRI stability and photocycle control, consisted of the N-terminal 147 residues of the 536-residue HtrI protein. The active fragment is predicted to contain two transmembrane helices and the first approximately 20% of the cytoplasmic portion of the protein.     

Different dark conformations function in color-sensitive photosignaling by the sensory rhodopsin I-HtrI complex

The haloarchaeal phototaxis receptor sensory rhodopsin I (SRI) in complex with its transducer HtrI delivers an attractant signal from excitation with an orange photon and a repellent signal from a second near-UV photon excitation. Using a proteoliposome system with purified SRI in complex with its transducer HtrI, we identified by site-directed fluorescence labeling a site (Ser(155)) on SRI that is conformationally active in signal relay to HtrI. Using site-directed spin labeling of Ser(155)Cys with a nitroxide side chain, we detected a change in conformation following one-photon excitation such that the spin probe exhibits a splitting of the outer hyperfine extrema (2A'(zz)) significantly smaller than that of the electron paramagnetic resonance spectrum in the dark state. The dark conformations of five mutant complexes that do not discriminate between orange and near-UV excitation show shifts to lower or higher 2A'(zz) values correlated with the alterations in their motility behavior to one- and two-photon stimuli. These data are interpreted in terms of a model in which the dark complex is populated by two conformers in the wild type, one that inhibits the CheA kinase (A) and the other that activates it (R), shifted in the dark by mutations and shifted in the wild-type SRI-HtrI complex in opposite directions by one-photon and two-photon reactions.     

Multicolored protein conformation states in the photocycle of transducer-free sensory rhodopsin-I

Sensory rhodopsin-I (SRI), a phototaxis receptor of archaebacteria, is a retinal-binding protein that exists in the cell membrane intimately associated with a signal-transducing protein (HtrI) homologous to eubacterial chemotaxis receptors. Transducer-free sensory rhodopsin-I (fSRI), from cells devoid of HtrI, undergoes a photochemical cycle kinetically different from that of native SRI. We report here on the measurement and analysis of the photochemical kinetics of fSRI reactions in the 350-750-nm spectral range and in a 10(-7) s to 1 s time window. The lack of specific intermolecular interactions between SRI and HtrI results in early return of the ground form via distinct branching reactions in fSRI, not evident in the photocycle of native SRI. The chromophore transitions are loosely coupled to protein structural transitions. The coexistence of multiple spectral forms within kinetic intermediates is interpreted within the concept of multicolored protein conformational states.     

Structural changes of sensory rhodopsin I and its transducer protein are dependent on the protonated state of Asp76

Sensory rhodopsin I (SRI) functions in both positive and negative phototaxis in complex with halobacterial transducer protein I (HtrI). Orange light activation of SRI results in deprotonation of the retinylidene chromophore of SRI to produce the S 373 photocycle intermediate, the signaling state for positive phototaxis. In this study, we observed pH dependence on structural coupling between the two molecules upon the formation of the S 373 intermediate by means of Fourier transform infrared spectroscopy. At alkaline pH, where Asp76 (one of the counterions of the protonated retinylidene Schiff base) is deprotonated, HtrI-dependent alteration of the light-induced difference spectra is limited to reduction of amide I bands at 1661 (+)/ 1647 (-) cm (-1), and perturbation of one of the protonated carboxylic acid bands occurs at 1734 (-) cm (-1) (which appears to become ionized only when complexed with HtrI). However, at acidic pH, HtrI-complexed SRI exhibits not only light-induced reduction of the amide I changes but a wider range of spectral alterations including the appearance of several new amide I bands, perturbation of the chromophore-related vibrational modes, and other additional changes characteristic of tyrosine, glutamate, and aspartate residues. Since such pH dependence of structural changes was not observed in the complex of the D76N mutant of SRI, which behaves much like HtrI-complexed SRI in acidic conditions, we conclude that extensive orange light-induced conformational coupling between SRI and HtrI occurs only when Asp76 is neutralized.     

Phototaxis of Halobacterium salinarium requires a signalling complex of sensory rhodopsin I and its methyl-accepting transducer HtrI.

Sensory rhodopsin I (SRI) is a photoreceptor that mediates phototaxis in the archaeon Halobacterium salinarium. Receptor excitation is relayed to the motility system of the cell by the methyl-accepting transducer protein HtrI. In membranes prepared from cells that lack HtrI the absorbance difference maximum of SRI was shifted from 587 to 565 nm. The thermal decay of the metastable photocycle intermediate SRI373 was measured as time-dependent recovery of the absorbance at 590 nm. In the absence of HtrI the decay was slowed down by two orders of magnitude. When SRI was overproduced in cells that contained normal levels of HtrI, the decay of SRI373 was biexponential indicating two kinetically distinct species. Spectroscopic measurements on intact cells revealed the same effect of HtrI on SRI photocycling as found in isolated membranes. By transient exposure of membranes from wild-type cells to low ionic strength, the decay of SR373 was slowed to the same value found for untreated membranes in the absence of HtrI. In parallel, the absorbance difference maximum was shifted to 565 nm indicating that a physical interaction of HtrI and SRI had been irreversibly destroyed. Overproduction of SRI in the presence of wild-type amounts of HtrI did not increase the light sensitivity of the cells to orange light step down stimulation. It is concluded that SRI and HtrI form a stable complex in the cell membrane that signals to the flagellar motor and defines absorbance maximum, photocycling rate and photochemical efficiency of SRI.     

Transducer-binding and transducer-mutations modulate photoactive-site-deprotonation in sensory rhodopsin I.

Sensory rhodopsin I (SRI) is a seven-transmembrane helix retinylidene protein that mediates color-sensitive phototaxis responses through its bound transducer HtrI in the archaeon Halobacterium salinarum. Deprotonation of the Schiff base attachment site of the chromophore accompanies formation of the SRI signaling state, S(373). We measured the rate of laser flash-induced S(373) formation in the presence and absence of HtrI, and the effects of mutations in SRI or HtrI on the kinetics of this process. In the absence of HtrI, deprotonation occurs rapidly (halftime 10 micros) if the proton acceptor Asp76 is ionized (pK(a) = approximately 7), and only very slowly (halftime > 10 ms) when Asp76 is protonated. Transducer-binding, although it increases the pK(a) of Asp76 so that it is protonated throughout the range of pH studied, results in a first order, pH-independent rate of S(373) formation of approximately 300 micros. Therefore, the complexation of HtrI facilitates the proton-transfer reaction, increasing the rate approximately 50-fold at pH6. Arrhenius analysis shows that HtrI-binding accelerates the reaction primarily by an entropic effect, suggesting HtrI constrains the SRI molecule in the complex. Function-perturbing mutations in SRI and HtrI also alter the rate of S(373) formation and the lambda(max) of the parent state as assessed by laser flash-induced kinetic difference spectroscopy, and shifts to longer wavelength are correlated with slower deprotonation. The data indicate that HtrI affects electrostatic interactions of the protonated Schiff base and not only receives the signal from SRI but also optimizes the photochemical reaction process for SRI signaling.     

Salinibacter sensory rhodopsin: sensory rhodopsin I-like protein from a eubacterium

Halobacterium salinarum sensory rhodopsin I (HsSRI), a dual receptor regulating both negative and positive phototaxis in haloarchaea, transmits light signals through changes in protein-protein interactions with its transducer, halobacterial transducer protein I (HtrI). Haloarchaea also have another sensor pigment, sensory rhodopsin II (SRII), which functions as a receptor regulating negative phototaxis. Compared with HsSRI, the signal relay mechanism of SRII is well characterized because SRII from Natronomonus pharaonis (NpSRII) is much more stable than HsSRI and HsSRII, especially in dilute salt solutions and is much more resistant to detergents. Two genes encoding SRI homologs were identified from the genome sequence of the eubacterium Salinibacter ruber. Those sequences are distantly related to HsSRI ( approximately 40% identity) and contain most of the amino acid residues identified as necessary for its function. To determine whether those genes encode functional protein(s), we cloned and expressed them in Escherichia coli. One of them (SrSRI) was expressed well as a recombinant protein having all-trans retinal as a chromophore. UV-Vis, low-temperature UV-Vis, pH-titration, and flash photolysis experiments revealed that the photochemical properties of SrSRI are similar to those of HsSRI. In addition to the expression system, the high stability of SrSRI makes it possible to prepare large amounts of protein and enables studies of mutant proteins that will allow new approaches to investigate the photosignaling process of SRI-HtrI.     

Structural characteristics around the β-ionone ring of the retinal chromophore in Salinibacter sensory rhodopsin I

Organisms sense and respond to environmental stimuli through membrane-embedded receptors and transducers. Sensory rhodopsin I (SRI) and sensory rhodopsin II (SRII) are the photoreceptors for the positive and negative phototaxis in microorganisms, respectively. They form signaling complexes in the membrane with their cognate transducer proteins, HtrI and HtrII, and these SRI-HtrI and SRII-HtrII complexes transmit a light signal through their cytoplasmic sensory signaling system, inducing opposite effects (i.e., the inactivation or activation of the kinase CheA). Here we found, by using Fourier transformed infrared spectroscopy, that a conserved residue, Asp102 in Salinibacter SRI (SrSRI), which is located close to the β-ionone ring of the retinal chromophore, is deprotonated upon formation of the active M-intermediate. Furthermore, the D102E mutant of SrSRI affects the structure and/or structural changes of Cys130. This mutant shows a large spectral shift and is comparably unstable, especially in the absence of Cl(-). These phenomena have not been observed in the wild-type, or the N105Q and N105D mutants of Natronomonas pharaonis SRII (NpSRII), indicating differences in the structure and structural changes between SrSRI and NpSRII around the β-ionone ring. These differences could also be supported by the measurements of the reactivity with the water-soluble reagent azide. On the basis of these results, we discuss the structure and structural changes around the retinal chromophore in SrSRI.     

Transmembrane domains 4 and 7 of the M(1) muscarinic acetylcholine receptor are critical for ligand binding and the receptor activation switch.

Activation of the muscarinic acetylcholine receptors requires agonist binding followed by a conformational change, but the ligand binding and conformation-switching residues have not been completely identified. Systematic alanine-scanning mutagenesis has been used to assess residues 142-164 in transmembrane helix 4 and 402-421 in transmembrane helix 7 of the M(1) muscarinic acetylcholine receptor. Several inward-facing amino acid side chains in the exofacial parts of transmembrane helices 4 and 7 contribute to acetylcholine binding. Alanine substitution of the aromatic residues in this group reduced signaling efficacy, suggesting that they may form part of a charge-stabilized aromatic cage, which triggers rotation and movement of the transmembrane helices. The mutation of adjacent residues modulated receptor activation, either reducing signaling or causing constitutive activation. In the buried endofacial section of transmembrane helix 7, alanine substitution mutants of the conserved NSXXNPXXY motif displayed strongly reduced signaling efficacy, despite having increased or unchanged acetylcholine affinity. These residues may have dual functions, forming intramolecular contacts that stabilize the receptor in the inactive ground state, but that are broken, allowing them to form new intramolecular bonds in the activated state. This conformational rearrangement is critical to produce a G protein binding site and may represent a key mechanism of receptor activation.     

Removal of the transducer protein from sensory rhodopsin I exposes sites of proton release and uptake during the receptor photocycle.

The phototaxis receptor sensory rhodopsin-I (SR-I) was genetically truncated in the COOH terminus which leads to overexpression in Halobacterium salinarium and was expressed in the presence and absence of its transducer, HtrI. Pyranine (8-hydroxyl-1,3,6-pyrene-trisulfonate) was used as a pH probe to show that proton release to the bulk phase results from the SR-I587 to S373 photoconversion, but only in the absence of transducer. The stoichiometry is 1 proton/S373 molecule formed. When SR-I is overexpressed in the presence of HtrI, the kinetics of the thermal return of S373 to SR-I587 is biphasic. A kinetic dissection indicates that overexpressed SR-I is present in two pools: one pool which generates an SR-I molecule possessing a normal (i.e., transducer-interacting) pH-independent rate of S373 decay, and a second pool which shows the pH-dependent kinetics of transducer-free S373 decay. The truncated SR-I receptor functions normally based on the following criteria: (i) Truncated SR-I restores phototaxis (attractant and repellent responses) when expressed in a strain lacking native SR-I, but containing HtrI. (ii) The absorption spectrum and the flash-induced absorption difference spectrum are indistinguishable from those of native SR-I. (iii) The rate of decay of S373 is pH-dependent in the absence of HtrI but not in the presence of HtrI. The data presented here indicate that a proton-conducting path exists between the protonated Schiff base nitrogen and the extramembranous environment in the transducer-free receptor, and transducer binding blocks this path.     

Identification of methylation sites and effects of phototaxis stimuli on transducer methylation in Halobacterium salinarum.

The two transducers in the phototaxis system of the archaeon Halobacterium salinarum, HtrI and HtrII, are methyl-accepting proteins homologous to the chemotaxis transducers in eubacteria. Consensus sequences predict three glutamate pairs containing potential methylation sites in HtrI and one in HtrII. Mutagenic substitution of an alanine pair for one of these, Glu265-Glu266, in HtrI and for the homologous Glu513-Glu514 in HtrII eliminated methylation of these two transducers, as demonstrated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis autofluorography. Photostimulation of the repellent receptor sensory rhodopsin II (SRII) induced reversible demethylation of HtrII, while no detectable change in the extent of methylation of HtrI was observed in response to stimulation of its cognate sensory rhodopsin, the attractant receptor SRI. Cells containing HtrI or HtrII with all consensus sites replaced by alanine still exhibited phototaxis responses and behavioral adaptation, and methanol release assays showed that methyl group turnover was still induced in response to photostimulation of SRI or SRII. By pulse-chase experiments with in vivo L-[methyl-(3)H]methionine-labeled cells, we found that repetitive photostimulation of SRI complexed with wild-type (or nonmethylatable) HtrI induced methyl group turnover in transducers other than HtrI to the same extent as in wild-type HtrI. Both attractant and repellent stimuli cause a transient increase in the turnover rate of methyl groups in wild-type H. salinarum cells. This result is unlike that obtained with Escherichia coli, in which attractant stimuli decrease and repellent stimuli increase turnover rate, and is similar to that obtained with Bacillus subtilis, which also shows turnover rate increases regardless of the nature of the stimulus. We found that a CheY deletion mutant of H. salinarum exhibited the E. coli-like asymmetric pattern, as has recently also been observed in B. subtilis. Further, we demonstrate that the CheY-dependent feedback effect does not require the stimulated transducer to be methylatable and operates globally on other transducers present in the cell.     

Retroviral entry mediated by receptor priming and low pH triggering of an envelope glycoprotein

Avian leukosis virus (ALV) has been used as a model system to understand the mechanism of pH-independent viral entry involving receptor-induced conformational changes in the viral envelope (Env) glycoprotein that lead to membrane fusion. Here, we report the unexpected finding that ALV entry depends on a critical low pH step that was overlooked when this virus was directly compared to the classical pH-dependent influenza A virus. In contrast to influenza A virus, receptor interaction plays an essential role in priming ALV Env for subsequent low pH triggering. Our results reveal a novel principle in viral entry, namely that receptor interaction can convert a pH-insensitive viral glycoprotein to a form that is responsive to low pH.     

The cytoplasmic end of transmembrane domain 3 regulates the activity of the Saccharomyces cerevisiae G-protein-coupled alpha-factor receptor

The binding of alpha-factor to its receptor (Ste2p) activates a G-protein-signaling pathway leading to conjugation of MATa cells of the budding yeast S. cerevisiae. We conducted a genetic screen to identify constitutively activating mutations in the N-terminal region of the alpha-factor receptor that includes transmembrane domains 1-5. This approach identified 12 unique constitutively activating mutations, the strongest of which affected polar residues at the cytoplasmic ends of transmembrane domains 2 and 3 (Asn84 and Gln149, respectively) that are conserved in the alpha-factor receptors of divergent yeast species. Targeted mutagenesis, in combination with molecular modeling studies, suggested that Gln149 is oriented toward the core of the transmembrane helix bundle where it may be involved in mediating an interaction with Asn84. These residues appear to play specific roles in maintaining the inactive conformation of the protein since a variety of mutations at either position cause constitutive receptor signaling. Interestingly, the activity of many mammalian G-protein-coupled receptors is also regulated by conserved polar residues (the E/DRY motif) at the cytoplasmic end of transmembrane domain 3. Altogether, the results of this study suggest a conserved role for the cytoplasmic end of transmembrane domain 3 in regulating the activity of divergent G-protein-coupled receptors.     

Different modes of proton translocation by sensory rhodopsin I.

The membrane-bound complex between sensory rhodopsin I (SRI) and its transducer HtrI forms the functional photoreceptor unit that allows transmission of light signals to the flagellar motor. Although being a photosensor, SRI, the mutant SRI-D76N and the HtrI-SRI complex can transport protons, as we demonstrate by using the sensitive and ion-specific black lipid membrane technique. SRI sustains an orange light-driven (one-photon-driven) outward proton transport which is enhanced by additional blue light (two-photon-driven). The vectoriality of the two-photon-driven transport could be reversed at neutral pH from the outward to the inward direction by switching the cut-off wavelength of the long wavelength light from 550 to 630 nm. The cut-off wavelength determining the reversal point decreases with decreasing pH. The currents could be enhanced by azide. A two-photon-driven inward proton transport by SRI-D76N (catalyzed by azide) and by the complex HtrI-SRI is demonstrated. The influence of pH and azide concentration on the rise and decay kinetics of the SRI380 intermediate is analyzed. The different modes of proton translocation of the SRI species are discussed on the basis of a general model of proton translocation of retinal proteins and in the context of signal transduction.     

Linking agonist binding to histamine H1 receptor activation

G protein-coupled receptors (GPCRs) constitute a large and functionally diverse family of transmembrane proteins. They are fundamental in the transfer of extracellular stimuli to intracellular signaling pathways and are among the most targeted proteins in drug discovery. The detailed molecular mechanism for agonist-induced activation of rhodopsin-like GPCRs has not yet been described. Using a combination of site-directed mutagenesis and molecular modeling, we characterized important steps in the activation of the human histamine H1 receptor. Both Ser3.36 and Asn7.45 are important links between histamine binding and previously proposed conformational changes in helices 6 and 7. Ser3.36 acts as a rotamer toggle switch that, upon agonist binding, initiates the activation of the receptor through Asn7.45. The proposed transduction involves specific residues that are conserved among rhodopsin-like GPCRs.     

Molecular dynamics simulations reveal insights into key structural elements of adenosine receptors

The crystal structure of the human A(2A) adenosine receptor, a member of the G protein-coupled receptor (GPCR) family, is used as a starting point for the structural characterization of the conformational equilibrium around the inactive conformation of the human A(2) (A(2A) and A(2B)) adenosine receptors (ARs). A homology model of the closely related A(2B)AR is reported, and the two receptors were simulated in their apo form through all-atom molecular dynamics (MD) simulations. Different conditions were additionally explored in the A(2A)AR, including the protonation state of crucial histidines or the presence of the cocrystallized ligand. Our simulations reveal the role of several conserved residues in the ARs in the conformational equilibrium of the receptors. The "ionic lock" absent in the crystal structure of the inactive A(2A)AR is rapidly formed in the two simulated receptors, and a complex network of interacting residues is presented that further stabilizes this structural element. Notably, the observed rotameric transition of Trp6.48 ("toggle switch"), which is thought to initiate the activation process in GPCRs, is accompanied by a concerted rotation of the conserved residue of the A(2)ARs, His6.52. This new conformation is further stabilized in the two receptors under study by a novel interaction network involving residues in transmembrane (TM) helices TM5 (Asn5.42) and TM3 (Gln3.37), which resemble the conformational changes recently observed in the agonist-bound structure of β-adrenoreceptors. Finally, the interaction between Glu1.39 and His7.43, a pair of conserved residues in the family of ARs, is found to be weaker than previously thought, and the role of this interaction in the structure and dynamics of the receptor is thoroughly examined. All these findings suggest that, despite the commonalities with other GPCRs, the conformational equilibrium of ARs is also modulated by specific residues of the family.     

Angiotensin IV is a potent agonist for constitutive active human AT1 receptors. Distinct roles of the N-and C-terminal residues of angiotensin II during AT1 receptor activation

The octapeptide hormone, angiotensin II (Ang II), exerts its major physiological effects by activating AT(1) receptors. In vivo Ang II is degraded to bioactive peptides, including Ang III (angiotensin-(2-8)) and Ang IV (angiotensin-(3-8)). These peptides stimulate inositol phosphate generation in human AT(1) receptor expressing CHO-K1 cells, but the potency of Ang IV is very low. Substitution of Asn(111) with glycine, which is known to cause constitutive receptor activation by disrupting its interaction with the seventh transmembrane helix (TM VII), selectively increased the potency of Ang IV (900-fold) and angiotensin-(4-8), and leads to partial agonism of angiotensin-(5-8). Consistent with the need for the interaction between Arg(2) of Ang II and Ang III with Asp(281), substitution of this residue with alanine (D281A) decreased the peptide's potency without affecting that of Ang IV. All effects of the D281A mutation were superseded by the N111G mutation. The increased affinity of Ang IV to the N111G mutant was also demonstrated by binding studies. A model is proposed in which the Arg(2)-Asp(281) interaction causes a conformational change in TM VII of the receptor, which, similar to the N111G mutation, eliminates the constraining intramolecular interaction between Asn(111) and TM VII. The receptor adopts a more relaxed conformation, allowing the binding of the C-terminal five residues of Ang II that switches this "preactivated" receptor into the fully active conformation.