Effects Of Ligand Binding Research Articles

Large-scale allosteric conformational transitions of adenylate kinase appear to involve a population-shift mechanism

Large-scale conformational changes in proteins are often associated with the binding of a substrate. Because conformational changes may be related to the function of an enzyme, understanding the kinetics and energetics of these motions is very important. We have delineated the atomically detailed conformational transition pathway of the phosphotransferase enzyme adenylate kinase (AdK) in the absence and presence of an inhibitor. The computed free energy profiles associated with conformational transitions offer detailed mechanistic insights into, as well as kinetic information on, the ligand binding mechanism. Specifically, potential of mean force calculations reveal that in the ligand-free state, there is no significant barrier separating the open and closed conformations of AdK. The enzyme samples near closed conformations, even in the absence of its substrate. The ligand binding event occurs late, toward the closed state, and transforms the free energy landscape. In the ligand-bound state, the closed conformation is energetically most favored with a large barrier to opening. These results emphasize the underlying dynamic nature of the enzyme and indicate that the conformational transitions in AdK are more intricate than a mere two-state jump between the crystal-bound and -unbound states. Based on the existence of the multiple conformations of the enzyme in the open and closed states, a different viewpoint of ligand binding is presented. Our estimated activation energy barrier for the conformational transition is also in reasonable accord with the experimental findings.

Transporter-to-Trap Conversion: a Disulfide Bond Formation in Cellular Retinoic Acid Binding Protein I Mutant Triggered by Retinoic Acid Binding Irreversibly Locks the Ligand Inside the Protein

Transport proteins must bind their ligands reversibly to enable release at the point of delivery, while irreversible binding is usually associated with the extreme cases of ligand sequestration. Protein conformational dynamics is an important modulator of binding kinetics, as increased flexibility in the regions adjacent to the binding site may facilitate both association and dissociation processes. Ligand entry to, and exit from, the internal binding site of the cellular retinoic acid binding protein I (CRABP I) occurs via a flexible portal region, which functions as a dynamic aperture. We designed and expressed a CRABP I mutant (A35C/T57C), in which a small-scale conformational switch caused by the ligand binding event triggers formation of a disulfide bond in the portal region, thereby arresting structural fluctuations and effectively locking the ligand inside the binding cavity. At the same time, no formation of the disulfide bond is observed in the apo form of the mutant, and most characteristics of the mutant, including protein stability, are very similar to those of the wild-type protein in the absence of retinoic acid. The mutation does not alter the kinetics of retinoic acid binding to the protein, although the disulfide formation makes the binding effectively irreversible, as suggested by the absence of retinoic acid transfer from the holo form of the mutant to lipid vesicles in the absence of a reducing agent. Taken together, these data suggest that the disulfide bond formation in the portal region arrests large-scale structural fluctuations, which are required for retinoic acid release from the protein. The unique properties of the CRABP I mutant described in this work can be used to inspire and guide a design of nanodevices for multiple tasks ranging from sequestering small-molecule toxins in both tissue and circulation to nutrient deprivation of pathogens.

The A, B, Cs of G-protein-coupled receptor pharmacology in assay development for HTS

G-protein-coupled receptors represent one of the most important areas of research in the pharmaceutical industry, being one of the largest druggable gene families. Recognising this fact, manufacturers have developed a huge variety of homogeneous assay technologies that facilitate the quantification of receptor ligand binding events and their downstream signalling cascades. However, while early emphasis was placed on the most sensitive, high-throughput and cost-effective screening technologies to enable identification of the most lead matter for further development, in recent years emphasis has shifted to a focus on maximising the identification of compounds that are new and developing assays that are more biologically/pharmacologically relevant. Therefore, this review provides an overview of the binding and functional techniques available for high-throughput screening, with particular attention on how assay application and configuration can be maximised to ensure their successful identification of relevant chemical matter and thereby optimising project success.

Zinc pyrithione-mediated activation of voltage-gated KCNQ potassium channels rescues epileptogenic mutants

KCNQ potassium channels are activated by changes in transmembrane voltage and play an important role in controlling electrical excitability. Human mutations of KCNQ2 and KCNQ3 potassium channel genes result in reduction or loss of channel activity and cause benign familial neonatal convulsions (BFNCs). Thus, small molecules capable of augmenting KCNQ currents are essential both for understanding the mechanism of channel activity and for developing therapeutics. We performed a high-throughput screen in search for agonistic compounds potentiating KCNQ potassium channels. Here we report identification of a new opener, zinc pyrithione (1), which activates both recombinant and native KCNQ M currents. Interactions with the channel protein cause an increase of single-channel open probability that could fully account for the overall conductance increase. Separate point mutations have been identified that either shift the concentration dependence or affect potentiation efficacy, thereby providing evidence for residues influencing ligand binding and downstream events. Furthermore, zinc pyrithione is capable of rescuing the mutant channels causal to BFNCs.

The solution structure of REF2-I reveals interdomain interactions and regions involved in binding mRNA export factors and RNA

The RNA binding and export factor (REF) family of mRNA export adaptors are found in several nuclear protein complexes including the spliceosome, TREX, and exon junction complexes. They bind RNA, interact with the helicase UAP56/DDX39, and are thought to bridge the interaction between the export factor TAP/NXF1 and mRNA. REF2-I consists of three domains, with the RNA recognition motif (RRM) domain positioned in the middle. Here we dissect the interdomain interactions of REF2-I and present the solution structure of a functionally competent double domain (NM; residues 1-155). The N-terminal domain comprises a transient helix (N-helix) linked to the RRM by a flexible arm that includes an Arg-rich region. The N-helix, which is required for REF2-I function in vivo, overlaps the highly conserved REF-N motif and, together with the adjacent Arg-rich region, interacts transiently with the RRM. RNA interacts with REF2-I through arginine-rich regions in its N- and C-terminal domains, but we show that it also interacts weakly with the RRM. The mode of interaction is unusual for an RRM since it involves loops L1 and L5. NMR signal mapping and biochemical analysis with NM indicate that DDX39 and TAP interact with both the N and RRM domains of REF2-I and show that binding of these proteins and RNA will favor an open conformation for the two domains. The proximity of the RNA, TAP, and DDX39 binding sites on REF2-I suggests their binding may be mutually exclusive, which would lead to successive ligand binding events in the course of mRNA export.

Identification of cognate ligands for the Escherichia coli phnD protein product and engineering of a reagentless fluorescent biosensor for phosphonates

The Escherichia coli phnD gene is hypothesized to code for the periplasmic binding component of a phosphonate uptake system. Here we report the characterization of the phosphonate-binding properties of the phnD protein product. We find that PhnD exhibits high affinity for 2-aminoethylphosphonate (5 nM), the most commonly occurring natural phosphonate produced by lower eukaryotes, but also binds several other phosphonates with micromolar affinities. A significant number of man-made phosphonates, such as insecticides and chemical warfare agents, are chemical threats and environmental pollutants. Consequently, there is an interest in developing methods for the detection and bioremediation of phosphonates. Bacterial periplasmic-binding proteins have been utilized for developing reagentless biosensors that report analytes by coupling ligand-binding events to changes in the emission properties of a covalently conjugated environmentally-sensitive fluorophore. Several PhnD conjugates described here show large changes in fluorescence upon binding to methylphosphonate (MP), with two conjugates exhibiting up to 50% decrease in emission intensity. Since MP is the final degradation product of many nerve agents, these PhnD conjugates can function as components in a biosensor system for chemical warfare agents.

Physical and chemical characterization of enolase immobilized polydiacetylene Langmuir–Blodgett film

Hexa histidine tagged recombinant Plasmodium falciparum enolase (His6-Pfen) was covalently immobilized on a Langmuir–Blodgett film of a self assembled mixture of 10,12-pentacosadiynoic acid and its N-succinimidyl ester derivative (PDA LB-film). The film was polymerized with a UV-lamp at 254nm to obtain a blue coloured, protein hooked polydiacetylene film. Atomic force microscopy (AFM) was used to characterize the surface morphology of the protein-immobilized film. The colorimetric response (CR) of the His6-Pfen hooked PDA LB-film to 2-phosphoglyceric acid (2-PGA), the substrate of enolase, in the presence of magnesium ions was studied spectrophotometrically. The CR of glutathione S-transferase-Pfen (GST-Pfen) immobilized film prepared by using similar procedure was also examined. The results suggest that binding of Mg (II) to the enzyme facilitates the interaction of the enzyme with 2-phosphoglycerate. This ligand-binding event could be detected by an observed increase in colorimetric response of the film by ∼10%. Thus the incorporation of enolase on a PDA film resulted in the formation of a novel material, which can serve as a biosensor.

NMR Analyses of the Activation of the Arp2/3 Complex by Neuronal Wiskott−Aldrich Syndrome Protein

The VCA domain of the neuronal Wiskott-Aldrich syndrome protein (N-WASP) is a potent activator of the Arp2/3 complex, a 240 kDa heteroheptameric actin-nucleating assembly. We used site-directed spin labeling of N-WASP peptides in conjunction with methyl-TROSY spectra of the intact, selectively labeled Arp2/3 complex to identify regions of the VCA that are proximal to the ARPC3 subunit of the assembly. We also cross-linked CA peptides to the Arp3, Arp2, ARPC1, and ARPC3 subunits. The combined data suggest that the extreme C-terminus of the A region and the C-terminus of the C region of N-WASP are proximal to ARPC3. These results have implications for the mechanism of Arp2/3 complex activation by VCA peptides. This study also demonstrates the utility of NMR spectroscopy for studying ligand binding events in large, asymmetric, macromolecular assemblies.

Receptor Flexibility in de Novo Ligand Design and Docking

One of the major problems in computational drug design is incorporation of the intrinsic flexibility of protein binding sites. This is particularly crucial in ligand binding events, when induced fit can lead to protein structure rearrangements. As a consequence of the huge conformational space available to protein structures, receptor flexibility is rarely considered in ligand design procedures. In this work, we present an algorithm for integrating protein binding-site flexibility into de novo ligand design and docking processes. The approach allows dynamic rearrangement of amino acid side chains during the docking and design simulations. The impact of protein conformational flexibility is investigated in the docking of highly active inhibitors in the binding sites of acetylcholinesterase and human collagenase (matrix metalloproteinase-1) and in the design of ligands in the S1' pocket of MMP-1. The results of corresponding simulations for both rigid and flexible binding sites are compared in order to gauge the influence of receptor flexibility in drug discovery protocols.

Teaching the Basics of Nuclear Receptor Action: A Simple Laboratory Exercise Using the Yeast Two-Hybrid Method

Objectives. Nuclear receptors (NRs) are ligand-activated transcription factors that regulate development and cellular differentiation, endocrine functions, and metabolism and transport of nutrients and drugs. Understanding of NR action is therefore essential for students of pharmacy and biology. Currently, there are no convenient laboratory exercises that clearly demonstrate the components and mechanisms of NR action. Design. Employing the yeast two-hybrid method, we developed a simple hands-on exercise that demonstrated ligand binding, dose responses, and consequences of ligand binding that are critical for NR function. Assessment. The exercise uses inexpensive, non-radioactive reagents and standard laboratory equipment. The results are directly visible, easy to interpret, and reproducible. The whole exercise can be conducted in a single day. In a real life test, a majority of the students who participated reported that their understanding of NR action had increased. Conclusions. This simple exercise can...

Kinetics of the Initial Steps of G Protein‐Coupled Receptor‐Mediated Cellular Signaling Revealed by Single‐Molecule Imaging

We report on an in vivo single-molecule study of the signaling kinetics of G protein-coupled receptors (GPCR) performed using the neurokinin 1 receptor (NK1R) as a representative member. The NK1R signaling cascade is triggered by the specific binding of a fluorescently labeled agonist, substance P (SP). The diffusion of single receptor-ligand complexes in plasma membrane of living HEK 293 cells is imaged using fast single-molecule wide-field fluorescence microscopy at 100 ms time resolution. Diffusion trajectories are obtained which show intra- and intertrace heterogeneity in the diffusion mode. To investigate universal patterns in the diffusion trajectories we take the ligand-binding event as the common starting point. This synchronization allows us to observe changes in the character of the ligand-receptor-complex diffusion. Specifically, we find that the diffusion of ligand-receptor complexes is slowed down significantly and becomes more constrained as a function of time during the first 1000 ms. The decelerated and more constrained diffusion is attributed to an increasing interaction of the GPCR with cellular structures after the ligand-receptor complex is formed.

Library Design for Fragment Based Screening

According to Hann's model of molecular complexity an increased probability of detection binding to a target protein can be expected when small, low complex molecular fragments are screened with high sensitivity instead of full-sized ligands with lower sensitivity. Analysis of the HTS summary data of Novartis and comparison with NMR screening results obtained on generic fragment libraries indicate this expectation to be true with hitrates of 0.001% - 0.151% observed in the identification of ligands with an IC(50) threshold in the micromolar range in an HTS setup and hitrates above or equal to 3% observed in NMR screening of fragments with an affinity threshold in the millimolar range. It is however necessary to keep in mind that the sets of target studied were not identical for both method and the experience in NMR screening is too limited for a final conclusion. The term hitrate as used here reflects only the success rate in the observation of ligand binding event. It must not be confused with the overall success rate of fragment and high throughput screening in the lead finding process, which can be entirely different, since the steps required to follow-up a ligand binding event to a lead are different for both methods. A survey of fragment-based lead discovery case studies given in the literature shows that in approximately half of the cases the initial hit fragment was discovered by screening a generic library, whereas in the other cases some knowledge about an initial ligands or the protein binding site has been used, whereas systematic virtual screening of fragment databases has been only rarely reported. As comparatively high hitrates were obtained, further consideration to optimize the generic fragment screening library were directed to the chemical tractability of the fragment. As several functional groups preferred by chemists for modification and linking of the fragments are also preferentially involved in interactions between the fragments and the target protein, a set of screening fragments was derived from chemical building blocks by masking its linker group by a chemical transformation which can be later on used in the chemical follow-up of the fragment hit. For example primary amines can be masked as acetamides. If the screening fragment is active the related building block can then be used for synthesis of a follow-up library.

Kinetic Stabilization of an Oligomeric Protein by a Single Ligand Binding Event

Protein native state stabilization imposed by small molecule binding is an attractive strategy to prevent the misfolding and misassembly processes associated with amyloid diseases. Transthyretin (TTR) amyloidogenesis requires rate-limiting tetramer dissociation before misassembly of a partially denatured monomer ensues. Selective stabilization of the native TTR tetramer over the dissociative transition state by small molecule binding to both thyroxine binding sites raises the kinetic barrier of tetramer dissociation, preventing amyloidogenesis. Assessing the amyloidogenicity of a TTR tetramer having only one amyloidogenesis inhibitor (I) bound is challenging because the two small molecule binding constants are generally not distinct enough to allow for the exclusive formation of TTR.I in solution to the exclusion of TTR.I(2) and unliganded TTR. Herein, we report a method to tether one fibril formation inhibitor to TTR by disulfide bond formation. Occupancy of only one of the two thyroxine binding sites is sufficient to inhibit tetramer dissociation in 6.0 M urea and amyloidogenesis under acidic conditions by imposing kinetic stabilization on the entire tetramer. The sufficiency of single occupancy for stabilizing the native state of TTR provides the incentive to search for compounds displaying striking negative binding cooperativity (e.g., K(d1) in nanomolar range and K(d2) in the micromolar to millimolar range), enabling lower doses of inhibitor to be employed in the clinic, mitigating potential side effects.

Insights into heme-based O 2 sensing from structure–function relationships in the FixL proteins

FixL proteins are bacterial heme-containing signal transduction proteins responsible for sensing the O 2 concentration in the organism’s environment. In Sinorhizobium meliloti FixL is a protein histidine kinase that, together with its response regulator FixJ, constitute an oxygen-sensitive switch for regulation of the organism’s nitrogen fixation and microaerobic respiration genes. The O 2 sensitivity of the switch is such that it transitions during the process of symbiosis in alfalfa roots. Bradyrhizobium japonicum FixL similarly regulates microaerobic and anaerobic respiration genes during symbiosis in soybean roots. FixLs responds to low oxygen concentrations with increased autophosphorylation activity of their kinase domains. The phosphorylated FixL provides a phosphoryl group to FixJ within a FixLJ complex. The phosphorylated FixJs are transcriptionally active toward their target genes. The FixL kinase domain is inhibited when the heme in FixL is oxygenated. Kinetic and thermodynamic studies of ligand binding to both ferrous and ferric FixLs have shown a generally low affinity for ligands relative to myoglobins. These relatively low ligand affinities are attributable almost completely to diminished rates of ligand binding. The heme and its environment in liganded and unliganded FixLs have been characterized by UV–visible spectroscopy, resonance Raman spectroscopy, EXAFS, and X-ray crystallography. These studies have revealed that in the purified proteins, the heme is converted from a six-coordinate low spin state to a five-coordinate high spin state upon O 2 release. Comparisons of spectroscopic and structural characteristics of deoxyFixL with oxyFixL, met-FixL–CN, FixL–CO, and FixL–NO complexes indicate that distal affects in the heme pocket are, at least in part, responsible for communicating the ligation state of the heme to the kinase domain. The mechanisms by which ligand binding events are communicated from the heme to the kinase domain involves propagation and/or amplification of the ligation-coupled conformational transitions of the heme and its immediate protein environment. More recently, time-resolved experiments examining the nonequilibrium, ligand-coupled dynamics initiated by O 2, CO, and NO photolysis from the corresponding FixL complexes have begun to shed light on the landscape of the switching coordinate. Current thinking and understanding of the mechanism for signal transduction in the FixLJ systems are discussed in the context of these physical investigations.

Negative Cooperativity of Glutamate Binding in the Dimeric Metabotropic Glutamate Receptor Subtype 1

Metabotropic glutamate receptor (mGluR) subtype 1 is a Class III G-protein-coupled receptor that is mainly expressed on the post-synaptic membrane of neuronal cells. The receptor has a large N-terminal extracellular ligand binding domain that forms a homodimer, however, the intersubunit communication of ligand binding in the dimer remains unknown. Here, using the intrinsic tryptophan fluorescence change as a probe for ligand binding events, we examined whether allosteric properties exist in the dimeric ligand binding domain of the receptor. The indole ring of the tryptophan 110, which resides on the upper surface of the ligand binding pocket, sensed the ligand binding events. From saturation binding curves, we have determined the apparent dissociation constants (K(0.5)) of representative agonists and antagonists for this receptor (3.8, 0.46, 40, and 0.89 microm for glutamate, quisqualate, (S)-alpha-methyl-4-carboxyphenylglycine ((S)-MCPG), and (+)-2-methyl-4-carboxyphenylglycine (LY367385), respectively). Calcium ions functioned as a positive modulator for agonist but not for antagonist binding (K(0.5) values were 1.3, 0.21, 59, and 1.2 microm for glutamate, quisqualate, (S)-MCPG, and LY367385, respectively, in the presence of 2.0 mm calcium ion). Moreover, a Hill analysis of the saturation binding curves revealed the strong negative cooperativity of glutamate binding between each subunit in the dimeric ligand binding domain. As far as we know, this is the first direct evidence that the dimeric ligand binding domain of mGluR exhibits intersubunit cooperativity of ligand binding.

Regulation of Hyperpolarization-Activated HCN Channels by cAMP through a Gating Switch in Binding Domain Symmetry

Recent X-ray structures show that the binding domains of tetrameric ligand-gated channels form either a 4-fold symmetric gating ring or a 2-fold symmetric dimer of dimers. To determine how such structures function to coordinate the binding of multiple ligands during channel activation, we examined the action of cAMP to enhance the opening of the hyperpolarization-activated HCN2 channels, whose cytoplasmic C terminus forms a gating ring in the presence of cAMP. Using tandem dimers and tetramers in which cAMP binding to selected HCN2 subunits was prevented by a point mutation or deletion, we provide the first direct determination of the energetic effects on gating of each of four ligand binding events and demonstrate the importance of the gating ring for cAMP regulation. We suggest that cAMP binding enhances channel opening by promoting assembly of the gating ring from an unliganded state in which the four subunits interact as a 2-fold symmetric dimer of dimers.

Applications of laser-polarized 129Xe to biomolecular assays.

The chemical shift sensitivity and significant signal enhancement afforded by laser-polarized 129Xe have motivated the application of 129Xe NMR to biological imaging and spectroscopy. Recent research done by our group has used laser-polarized 129Xe in biomolecular assays that detect ligand-binding events and distinguish protein conformations. The successful application of unfunctionalized and functionalized 129Xe NMR to in vitro biomolecular assays suggests the potential future use of a functionalized xenon biosensor for in vivo imaging.

Solution Studies and Structural Model of the Extracellular Domain of the Human Amyloid Precursor Protein

The amyloid precursor protein (APP) is the precursor of the β-amyloid peptide (A β), which is centrally related to the genesis of Alzheimer's disease (AD). In addition, APP has been suggested to mediate and/or participate in events that lead to neuronal degeneration in AD. Despite the fact that various aspects of the cell biology of APP have been investigated, little information on the structure of this protein is available. In this work, the solution structure of the soluble extracellular domain of APP (sAPP, composing 89% of the amino acid residues of the whole protein) has been investigated through a combination of size-exclusion chromatography, circular dichroism, and synchrotron radiation small-angle x-ray scattering (SAXS) studies. sAPP is monomeric in solution (65 kDa obtained from SAXS measurements) and exhibits an anisometric molecular shape, with a Stokes radius of 39 or 51 Å calculated from SAXS or chromatographic data, respectively. The radius of gyration and the maximum molecular length obtained by SAXS were 38 Å and 130 Å, respectively. Analysis of SAXS data further allowed building a structural model for sAPP in solution. Circular dichroism data and secondary structure predictions based on the amino acid sequence of APP suggested that a significant fraction of APP (30% of the amino acid residues) is not involved in standard secondary structure elements, which may explain the elongated shape of the molecule recovered in our structural model. Possible implications of the structure of APP in ligand binding and molecular recognition events involved in the biological functions of this protein are discussed.

Receptor Mutant Missing Specific Active State

The precise mechanism by which heterotrimeric guanine nucleotide-binding protein (G protein)-coupled receptors translate a ligand-binding event that occurs in the extracellular part of the receptor in order to activate a G protein docked on the cytoplasmic portion of the receptor remains poorly understood. Lecat et al. used fluorescence resonance energy transfer (FRET) to monitor binding of neurokinin A tagged with the fluorophore Texas Red to the NK2 neurokinin receptors tagged with green fluorescent protein. They measured ligand binding and two biological effects of receptor activation--an increase in the concentration of intracellular free calcium and increased synthesis of cAMP (adenosine 3',5'-monophosphate). These measurements were made for both the wild-type receptor and a mutant receptor with two point mutations in the extracellular domain. These mutations were previously suggested to decrease the apparent affinity of neurokinin for the receptor, and it was thus suggested that the altered amino acids were critical for ligand binding. Lecat et al. report that although the wild-type receptor activates both calcium and cAMP signals, the mutant receptor can only enhance calcium signals. This result supports the hypothesis that the receptor has at least three states, an inactive one, an active one in which it stimulates calcium signals, and a second active state (which the mutant receptor cannot occupy) that enhances cAMP signals. The authors interpret their measurements and modeling to indicate that the mutations actually do not alter the affinity of the receptor for neurokinin, but rather inhibit the transitions of the receptor from the inactive to active states. The maximal FRET signal observed with the mutant receptor was less than that measured for the wild-type receptor, which may mean that the distance between the donor and acceptor fluorophores is greater when the receptor can no longer achieve the activated state in which it promotes synthesis of cAMP. S. Lecat, B. Bucher, Y. Mely, J.-L. Galzi, Mutations in the extracellular amino-terminal domain of the NK2 neurokinin receptor abolish cAMP signaling but preserve intracellular calcium responses. J. Biol. Chem. 277 , 42034-42048 (2002). [Abstract] [Full Text]

Receptor Mutant Missing Specific Active State

The precise mechanism by which heterotrimeric guanine nucleotide-binding protein (G protein)-coupled receptors translate a ligand-binding event that occurs in the extracellular part of the receptor in order to activate a G protein docked on the cytoplasmic portion of the receptor remains poorly understood. Lecat et al. used fluorescence resonance energy transfer (FRET) to monitor binding of neurokinin A tagged with the fluorophore Texas Red to the NK2 neurokinin receptors tagged with green fluorescent protein. They measured ligand binding and two biological effects of receptor activation--an increase in the concentration of intracellular free calcium and increased synthesis of cAMP (adenosine 3',5'-monophosphate). These measurements were made for both the wild-type receptor and a mutant receptor with two point mutations in the extracellular domain. These mutations were previously suggested to decrease the apparent affinity of neurokinin for the receptor, and it was thus suggested that the altered amino acids were critical for ligand binding. Lecat et al. report that although the wild-type receptor activates both calcium and cAMP signals, the mutant receptor can only enhance calcium signals. This result supports the hypothesis that the receptor has at least three states, an inactive one, an active one in which it stimulates calcium signals, and a second active state (which the mutant receptor cannot occupy) that enhances cAMP signals. The authors interpret their measurements and modeling to indicate that the mutations actually do not alter the affinity of the receptor for neurokinin, but rather inhibit the transitions of the receptor from the inactive to active states. The maximal FRET signal observed with the mutant receptor was less than that measured for the wild-type receptor, which may mean that the distance between the donor and acceptor fluorophores is greater when the receptor can no longer achieve the activated state in which it promotes synthesis of cAMP.S. Lecat, B. Bucher, Y. Mely, J.-L. Galzi, Mutations in the extracellular amino-terminal domain of the NK2 neurokinin receptor abolish cAMP signaling but preserve intracellular calcium responses. J. Biol. Chem. 277, 42034-42048 (2002). [Abstract] [Full Text]