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PubMed1997

Selective interaction of hsp90 with an estrogen receptor ligand-binding domain containing a point mutation.

The 90-kDa heat shock protein (hsp90) has been implicated in modulating steroid receptor function in vitro and in vivo. Previous studies have suggested that hsp90 interacts with large portions of the estrogen receptor (ER) ligand-binding domain and sequences of the receptor required for stable DNA binding. To characterize the interaction of the ER ligand-binding domain with hsp90, we have compared the properties of chimeras created by coupling the ligand-binding domain to the constitutive transactivator VP16-GAL. Two types of chimeras were created: VP16-GAL-ERG, containing the wild-type ligand-binding domain derived from the cDNA HEG0, and VP16-GAL-ERV, containing the substitution G400V derived from the ligand-binding domain of the original ER cDNA isolate, HE0. The G400V mutation alters the physical properties of VP16-GAL-ERV by rendering it hormone-dependent for DNA binding and more strongly dependent on estradiol for transactivation compared with VP16-GAL-ERG. Glycerol gradient analyses and chemical cross-linking/coimmunoprecipitation showed that, unlike VP16-GAL-ERG, VP16-GAL-ERV formed stable complexes with hsp90 in vitro. These data show that hsp90 selectively recognizes the altered ER ligand-binding domain containing the G400V substitution and indicate that the wild-type ER ligand-binding domain of VP16-GAL-ERG does not interact with hsp90 in vitro. Hormone binding studies showed that the ligand-binding domain of VP16-GAL-ERV was destabilized by incubation in the presence of high concentrations of salt or in the absence of sodium molybdate, conditions that disrupt its interaction with hsp90. The ligand-binding domain of the Val-400 ER thus behaves similarly to that of the wild-type glucocorticoid receptor, which has previously been shown to interact with hsp90 in vitro. These results provide evidence for the action of hsp90 as a molecular chaperone by selectively recognizing destabilized proteins.

Animals↗
PubMed2004

Tyr702 is an important determinant of agonist binding and domain closure of the ligand-binding core of GluR2.

Ionotropic glutamate receptors mediate most rapid excitatory synaptic transmission in the mammalian central nervous system, and their involvement in neurological diseases has stimulated widespread interest in their structure and function. Despite a large number of agonists developed so far, few display selectivity among (S)-2-amino-3-(3-hydroxy-5-methylisoxazol-4-yl) propionic acid (AMPA)-receptor subtypes. The present study provides X-ray structures of the glutamate receptor 2 (GluR2)-selective partial agonist (S)-2-amino-3-(1,3,5,6,7-pentahydro-2,4-dioxocyclopenta[e] pyrimidin-1-yl) propanoic acid [(S)-CPW399] in complex with the ligand-binding core of GluR2 (GluR2-S1S2J) and with a (Y702F)GluR2-S1S2J mutant. In addition, the structure of the nonselective partial agonist kainate in complex with (Y702F)GluR2-S1S2J was determined. The results show that the selectivity of (S)-CPW399 toward full-length GluR2 relative to GluR3 is reflected in the binding data on the two soluble constructs, allowing the use of (Y702F)GluR2-S1S2J as a model system for studying GluR2/GluR3 selectivity. Structural comparisons suggest that selectivity arises from disruption of a water-mediated network between ligand and receptor. A D1-D2 domain closure occurs upon agonist binding. (S)-CPW399 and kainate induce greater domain closure in the Y702F mutant, indicating that these partial agonists here act in a manner more reminiscent of full agonists. Both kainate and (S)-CPW399 exhibited higher efficacy at (Y702F)GluR2(Q)i than at wild-type GluR2(Q)i. Whereas an excellent correlation exists between domain closure and efficacy of a range of agonists at full-length GluR2 determined by electrophysiology in Xenopus laevis oocytes, a direct correlation between agonist induced domain closure of (Y702F)GluR2-S1S2J and efficacy at the GluR3 receptor is not observed. Although it clearly controls selectivity, mutation of this residue alone is insufficient to explain agonist-induced conformational rearrangements occurring in this variant.

Amino Acid Sequence↗
PubMed2001

Mutations in the Escherichia coli receptor FepA reveal residues involved in ligand binding and transport.

FepA is the Escherichia coli outer membrane receptor for ferric enterobactin, colicin D and colicin B. The transport processes through FepA are energy-dependent, relying on the periplasmic protein TonB to interact with FepA. Through this interaction, TonB tranduces energy derived from the cytoplasmic membrane across the periplasmic space to FepA. In this study, random mutagenesis strategies were used to define residues of FepA important for its function. Both polymerase chain reaction (PCR)-generated random mutations in the N-terminal 180 amino acids of FepA and spontaneous chromosomal fepA mutations were selected by resistance to colicin B. The PCR mutagenesis strategy targeted the N-terminus because it forms a plug inside the FepA barrel that is expected to be involved in ligand binding, ligand transport, and interaction with TonB. We report the characterization of 15 fepA missense mutations that were localized to three regions of the FepA receptor. The first region was a stretch of eight amino acids referred to as the TonB box. The second region included extracellular loops of both the barrel and the plug. A third region formed a cluster near the barrel wall around positions 75 and 126 of the plug. These mutations provide initial insight into the mechanisms of ligand binding and transport through the FepA receptor.

Amino Acid Motifs↗
PubMed2002

Receptor-ligand interaction between vitellogenin receptor (VtgR) and vitellogenin (Vtg), implications on low density lipoprotein receptor and apolipoprotein B/E. The first three ligand-binding repeats of VtgR interact with the amino-terminal region of Vtg.

The vitellogenin receptor (VtgR) belongs to the low density lipoprotein receptor (LDLR) gene family. It mediates the uptake of vitellogenin (Vtg) in oocyte development of oviparous animals. In this study, we cloned and characterized two forms of Oreochromis aureus VtgR. Northern analysis showed that VtgR was specifically expressed in ovarian tissues. However, reverse transcription-PCR indicates that either there are trace levels of expression of VtgR or a homolog of LDLR exists in nonovarian tissues. The VtgR is highly homologous to the very low density lipoprotein receptor. To better understand the mechanism by which similar structural modules in the ligand-binding domain bind different ligands, we used the yeast two-hybrid system to screen for the minimal interaction motifs in Vtg and VtgR. The amino-terminal region of the lipovitellin I domain of Vtg interacts with the ligand-binding domain of VtgR. The first three ligand-binding repeats of the receptor were found to be essential for ligand binding. Computational analysis of the binding sequence indicates that Vtg has a similar receptor-binding region to apolipoprotein (apo) E and apoB. Site-directed mutagenesis of this region indicates electrostatic interaction between Vtg and its receptor. Sequence analysis suggests the coevolution of receptor-ligand pairs for the LDLR/apo superfamily and suggests that the mode of binding of LDLR/very low density lipoprotein receptor to apoB and apoE is inherited from the electrostatic attraction of VtgR and Vtg.

Amino Acid Sequence↗
PubMed2001

Allosteric interaction of minor groove binding ligands with UL9-DNA complexes.

The herpes simplex virus type 1 origin binding protein (UL9) is a sequence-specific DNA binding protein. Several studies have demonstrated that UL9 binds to the 11-base pair sequence 5'-CGTTCGCACTT-3' primarily, or solely, through interaction with the major groove. Minor groove binding ligands, such as distamycin, netropsin, and GLX, an indole-linked dimer of netropsin, can effectively disrupt the UL9-DNA complex only when their DNA binding sites are coincident with the right side of the DNA binding site of the protein and overlap with the protein binding site by two (TT) base pairs. These results suggest that the right side of the UL9-DNA complex has a unique structure that is sensitive to minor groove ligand binding. In addition, a biphasic displacement curve was observed with GLX, which suggests two modes of ligand binding which have different effects on UL9-DNA complexes. Using a fluorescence-based hybridization stabilization assay, we determined that GLX can bind to its binding site as an overlapping dimer (i.e., 2:1 stoichiometry). Footprinting of UL9-DNA complexes with the minor groove directed chemical nuclease 1,10-phenanthroline copper confirms that the DNA conformation at the position of the right-side ligand binding site of GLX is altered and has a widened minor groove. In contrast, it is well established that at 1:1 stoichiometries, AT sequence specific ligands, such as netropsin, distamycin, and GLX, prefer uniform, narrow minor grooves. The opposing conformational requirements of UL9 and lower concentrations of GLX at the ligand binding A-tract overlapping the right side of the protein binding site indicate that allosteric inhibition, rather than direct steric competition, contributes to ligand-induced protein displacement. At higher GLX concentrations, giving 2:1 binding in a widened minor groove, co-binding with UL9 is allowed. A model is presented that is consistent with these observations, and implications for targeted regulation of gene transcription are discussed.

Allosteric Regulation↗
PubMed1999

A cyanobacterial hemoglobin with unusual ligand binding kinetics and stability properties.

The glbN gene of the cyanobacterium Nostoc commune UTEX 584 encodes a hemoprotein, named cyanoglobin, that has high oxygen affinity. The basis for the high oxygen affinity of cyanoglobin was investigated through kinetic studies that utilized stopped-flow spectrophotometry and flash photolysis. Association and dissociation rate constants were measured at 20 degrees C for oxygen, carbon monoxide, nitric oxide, and methyl and ethyl isocyanides. The association rate constants for the binding of these five ligands to cyanoglobin are the highest reported for any naturally occurring hemoglobin, suggesting an unhindered and apolar ligand binding pocket. Cyanoglobin also shows high rates of autoxidation and hemin loss, indicating that the prosthetic group is readily accessible to solvent. The ligand binding behavior of cyanoglobin was more similar to that of leghemoglobin a than to that of sperm whale myoglobin. Collectively, the data support the model of cyanoglobin function described by Hill et al. [(1996) J. Bacteriol. 178, 6587-6598], in which cyanoglobin sequesters oxygen, and presents it to, or is a part of, a terminal cytochrome oxidase complex in Nostoc commune UTEX 584 under microaerobic conditions, when nitrogen fixation, and thus ATP demand, is maximal.

Bacterial Proteins↗
PubMed1981

Measurement of ligand binding with nuclear magnetic resonance spectroscopy.

A method is described for measuring with nuclear magnetic resonance (NMR) spectroscopy the binding of ligands to particulate components of cells. This method is sensitive to intermediate-strength binding, which is difficult to measure with standard radiotracer binding assays, and is insensitive to 'tight' binding. Nonspecific binding and entrapment can be distinguished from specific binding even when a pharmacological agent is not available for competitive binding assays. Thus, the experimental approach presented provides a means of studying the possible physiological roles in neural tissue of metabolites that have not been implicated by neuropharmacological and neurophysiological experiments. The utility of this technique is discussed in terms of the information it has provided about the binding of enkephalin, epinephrine, acetylcholine and carnosine. This technique, although it uses technology new to most neuroscience laboratories, should be relatively easy to apply to existing research topics. The NMR spectrometer required for the binding assay is the type already found on most academic campuses, and the ligand need not have any special isotopic label. The particulate fraction can be prepared in the same way as for the radiotracer binding assays, which means that both tight and intermediate-strength ligand binding can be measured with the same preparation.

Binding Sites↗
PubMed1998

Solution structure of the human Hck SH3 domain and identification of its ligand binding site.

SH3 domains are protein binding domains that occur widely among signal transduction proteins. Here, we present the NMR-determined solution structure of the SH3 domain from the cytoplasmic protein tyrosine kinase, Hck. Hck is involved in a number of cell signal transduction pathways, frequently in pathways associated with immune response. SH3 domains bind proteins via a left-handed polyproline type II helix on the target protein. We have assessed the structural impact of binding to a ligand through addition of a peptide corresponding to a proline-rich region of a Hck target, the GTPase activating protein of the Ras pathway. Ligand binding effects small structural changes and stabilizes the SH3 domain structure. Also, we have compared the solution structure of the Hck SH3 domain to the crystal structure of Hck, in which the SH3 domain exhibits an intramolecular binding to an interdomain linker region. These structures are interpreted as the apo- and holo- forms of the Hck SH3 domain.

Amino Acid Sequence↗
PubMed1999

Orientation of heparin-binding sites in native vitronectin. Analyses of ligand binding to the primary glycosaminoglycan-binding site indicate that putative secondary sites are not functional.

A primary heparin-binding site in vitronectin has been localized to a cluster of cationic residues near the C terminus of the protein. More recently, secondary binding sites have been proposed. In order to investigate whether the binding site originally identified on vitronectin functions as an exclusive and independent heparin-binding domain, solution binding methods have been used in combination with NMR and recombinant approaches to evaluate ligand binding to the primary site. Evaluation of the ionic strength dependence of heparin binding to vitronectin according to classical linkage theory indicates that a single ionic bond is prominent. It had been previously shown that chemical modification of vitronectin using an arginine-reactive probe results in a significant reduction in heparin binding (Gibson, A., Baburaj, K., Day, D. E., Verhamme, I. , Shore, J. D., and Peterson, C. B. (1997) J. Biol. Chem. 272, 5112-5121). The label has now been localized to arginine residues within the cyanogen bromide fragment-(341-380) that contains the primary heparin-binding site on vitronectin. One- and two-dimensional NMR on model peptides based on this primary heparin-binding site indicate that an arginine residue participates in the ionic interaction and that other nonionic interactions may be involved in forming a complex with heparin. A recombinant polypeptide corresponding to the C-terminal 129 amino acids of vitronectin exhibits heparin-binding affinity that is comparable to that of full-length vitronectin and is equally effective at neutralizing heparin anticoagulant activity. Results from this broad experimental approach argue that the behavior of the primary site is sufficient to account for the heparin binding activity of vitronectin and support an exposed orientation for the site in the structure of the native protein.

Amino Acid Sequence↗
PubMed2006

The change of protein intradomain mobility on ligand binding: is it a commonly observed phenomenon?

Analysis of changes in the dynamics of protein domains on ligand binding is important in several aspects: for the understanding of the hierarchical nature of protein folding and dynamics at equilibrium; for analysis of signal transduction mechanisms triggered by ligand binding, including allostery; for drug design; and for construction of biosensors reporting on the presence of target ligand in studied media. In this work we use the recently developed HCCP computational technique for the analysis of stabilities of dynamic domains in proteins, their intrinsic motions and of their changes on ligand binding. The work is based on comparative studies of 157 ligand binding proteins, for which several crystal structures (in ligand-free and ligand-bound forms) are available. We demonstrate that the domains of the proteins presented in the Protein DataBank are far more robust than it was thought before: in the majority of the studied proteins (152 out of 157), the ligand binding does not lead to significant change of domain stability. The exceptions from this rule are only four bacterial periplasmic transport proteins and calmodulin. Thus, as a rule, the pattern of correlated motions in dynamic domains, which determines their stability, is insensitive to ligand binding. This rule may be the general feature for a vast majority of proteins.

Biosensing Techniques↗
PubMed2006

Minimum sequence requirements for selective RNA-ligand binding: a molecular mechanics algorithm using molecular dynamics and free-energy techniques.

In vitro evolution techniques allow RNA molecules with unique functions to be developed. However, these techniques do not necessarily identify the simplest RNA structures for performing their functions. Determining the simplest RNA that binds to a particular ligand is currently limited to experimental protocols. Here, we introduce a molecular-mechanics based algorithm employing molecular dynamics simulations and free-energy methods to predict the minimum sequence requirements for selective ligand binding to RNA. The algorithm involves iteratively deleting nucleotides from an experimentally determined structure of an RNA-ligand complex, performing energy minimizations and molecular dynamics on each truncated structure, and assessing which truncations do not prohibit RNA binding to the ligand. The algorithm allows prediction of the effects of sequence modifications on RNA structural stability and ligand-binding energy. We have implemented the algorithm in the AMBER suite of programs, but it could be implemented in any molecular mechanics force field parameterized for nucleic acids. Test cases are presented to show the utility and accuracy of the methodology.

Algorithms↗
PubMed2003

Homology modeling of rat and human cytochrome P450 2D (CYP2D) isoforms and computational rationalization of experimental ligand-binding specificities.

The ligand-binding characteristics of rat and human CYP2D isoforms, i.e., rat CYP2D1-4 and human CYP2D6, were investigated by measuring IC(50) values of 11 known CYP2D6 ligands using 7-methoxy-4-(aminomethyl)coumarin (MAMC) as substrate. Like CYP2D6, all rat CYP2D isozymes catalyzed the O-demethylation of MAMC with K(m) and V(max) values ranging between 78 and 145 microM and 0.048 and 1.122 min(-1), respectively. To rationalize observed differences in the experimentally determined IC(50) values, homology models of the CYP2D isoforms were constructed. A homology model of CYP2D6 was generated on the basis of crystallized rabbit CYP2C5 and was validated on its ability to reproduce binding orientations corresponding to metabolic profiles of the substrates and to remain stable during unrestrained molecular dynamics simulations at 300 K. Twenty-two active site residues, sharing up to 59% sequence identity, were identified in the CYP2D binding pockets and included CYP2D6 residues Phe120, Glu216, and Asp301. Electrostatic potential calculations displayed large differences in the negative charge of the CYP2D active sites, which was consistent with observed differences in absolute IC(50) values. MD studies on the binding mode of sparteine, quinidine, and quinine in CYP2D2 and CYP2D6 furthermore concurred well with experimentally determined IC(50) values and metabolic profiles. The current study thus provides new insights into differences in the active site topology of the investigated CYP2D isoforms.

Alcohol Oxidoreductases↗
PubMed1989

Cloning of murine adipocyte lipid binding protein in Escherichia coli. Its purification, ligand binding properties, and phosphorylation by the adipocyte insulin receptor.

The murine adipocyte lipid binding protein (ALBP) has been cloned into Escherichia coli, purified from expressing cultures, and its ligand binding and phosphorylation properties studied. In the cloning strategy, the recombinant, pT7-5 rALBP, was transformed into E. coli strain K38 harboring plasmid pGP1-2 which directs the synthesis of T7 RNA polymerase. Upon shifting the temperature from 30 to 42 degrees C to induce T7 RNA polymerase expression, the 14.6-kDa recombinant ALBP (rALBP) was expressed for approximately 2 h and accumulated to about 1% of total E. coli protein. The recombinant ALBP was soluble in E. coli extracts and resistant to bacterial proteolysis. A procedure for purifying rALBP was developed utilizing immuno-chemical detection based upon reactivity with anti-murine ALBP antiserum. A combination of acidic ammonium sulfate fractionation, gel permeation chromatography, and carboxymethyl ion-exchange high performance liquid chromatography separation was used to prepare homogeneous rALBP. Sequence analysis of rALBP indicated that the initiating methionine residue had been removed and the amino-terminal cysteine residue was not blocked. Purified rALBP exhibited stoichiometric, saturable binding of oleic acid (n = 1.0, K0.5 approximately 100 microM) and retinoic acid (n = 1.0, K0.5 approximately 170 microM). Incubation of rALBP with wheat germ agglutinin-purified insulin receptor, ATP, and 100 nM insulin resulted in a 5-fold stimulation of rALBP phosphorylation above the basal state. Kinetic analysis of rALBP phosphorylation by the 3T3-L1 insulin receptor kinase yielded a Michaelis constant (Km) of 50 microM and a maximal velocity of 1 mol of rALBP phosphorylated/min/mol insulin binding sites. Phosphoamino acid analysis indicated that phosphorylation occurred upon tyrosine. These results indicate that murine ALBP has been cloned and expressed in E. coli, purified to homogeneity, and is a substrate for the insulin receptor tyrosyl kinase in vitro.

Adipose Tissue↗
PubMed2004

Critical residues for ligand binding in blade 2 of the propeller domain of the integrin alphaIIb subunit.

Ligand binding to integrin alphaIIbbeta3 is a key event of thrombus formation. The propeller domain of the alphaIIb subunit has been implicated in ligand binding. Recently, the ligand binding site of the alphaV propeller was determined by crystal structure analysis. However, the structural basis of ligand recognition by the alphaIIb propeller remains to be determined. In this study, we conducted site-directed mutagenesis of all residues located in the loops extending above blades 2 and 4 of the alphaIIb propeller, which are spatially close to, but distinct from, the loops that contain the binding site for an RGD ligand in the crystal structure of the alphaV propeller. Replacement by alanine of Q111, H112 or N114 in the loop within the blade 2 (the W2:2-3 loop in the propeller model) abolished binding of a ligand-mimetic antibody and fibrinogen to alphaIIbbeta3 induced by different types of integrin activation including activation of alphaIIbbeta3 by beta3 cytoplasmic mutation. CHO cells stably expressing recombinant alphaIibbeta3 bearing Q111A, H112A or N114A mutation did not exhibit alphaIibbeta3-mediated adhesion to fibrinogen. According to the crystal structure of alphaVbeta3, the alphaV residue corresponding to alphaIIbN114 is exposed on the integrin surface and close to the RGD binding site. These results suggest that the Q111, H112 and N114 residues in the loop within blade 2 of the alphaIIb propeller are critical for ligand binding, possibly because of direct interaction with ligands or modulation of the RGD binding pocket.

Alanine↗
PubMed1998

The Ah receptor can bind ligand in the absence of receptor-associated heat-shock protein 90.

The Ah receptor (AhR) is a soluble ligand-dependent DNA regulatory protein that mediates many of the biological responses to 2,3,7, 8-tetrachlorodibenzo-p-dioxin (TCDD, dioxin) and related chemicals. In the absence of ligand, the cytosolic form of the AhR is found complexed with at least two molecules of hsp90, a heat shock protein of 90 kDa. In addition to its role in AhR protein folding and ability to repress the inherent nuclear localization, dimerization, and DNA binding activity of the AhR, it has been reported that hsp90 is absolutely required for maintaining the AhR in its high-affinity ligand binding conformation. The ability of high salt conditions (0. 4 M KCl) to dissociate the multimeric AhR complex into its monomeric form provides us with an avenue to examine the role of hsp90 in AhR ligand binding activity. In contrast to previous reports, we demonstrate that salt-dissociated "hsp90-free" AhR from several species still retains the ability to specifically bind ligand ([3H]TCDD). Although partial inactivation of ligand binding of salt-dissociated rat hepatic AhR was observed (to a maximum of 50% of total AhR binding), the presence of bound ligand protected against this inactivation. Little or no inactivation of the ligand binding ability of salt-dissociated guinea pig or rabbit AhR occurred. Our results not only indicate a significant species-difference in AhR ligand binding stability and/or activity, but also demonstrate that AhR ligand binding activity does not absolutely require the presence of receptor-bound hsp90.

Animals↗
PubMed2006

Conformational flexibility, hydration and state parameter fluctuations of fibroblast growth factor-10: effects of ligand binding.

Differential effects of ligand binding on local and global fibroblast growth factor-10 (FGF-10) flexibility and stability have been investigated utilizing a variety of experimental and computational techniques. Normal mode analysis was used to predict the low frequency motions and regional flexibility of FGF-10. Similarly, regional variations in local folding/unfolding equilibria were characterized with the COREX/BEST algorithm. Experimental adiabatic and isothermal compressibilities of FGF-10 alone and in the presence of polyanions are compared. Furthermore, the effect of polyanions on the coefficient of thermal expansion is compared. Measurements of density, heat capacity, compressibility, and expansibility were combined to calculate experimentally determined volume and enthalpy fluctuations. Global effects of polyanions on FGF-10 flexibility, thermodynamic fluctuations, and hydration vary depending on the size and charge density of the polyanion. Local effects of polyanions were investigated utilizing time-resolved fluorescence spectroscopy and red edge excitation spectroscopy (REES). Increased rigidity of the protein matrix or an increased solvent response surrounding the Trp residues is observed in the presence of polyanions. Similarly, time-resolved spectroscopy reveals increased ground state heterogeneity and increased dipole relaxation on the time scale of fluorescence for FGF-10 in the presence of polyanions. These polyanions increase heterogeneity, global flexibility, and fluctuations while increasing the melting temperature (Tm) of FGF-10.

Crystallography, X-Ray↗
PubMed1990

The ligand binding site and transduction mechanism in the inositol-1,4,5-triphosphate receptor.

The inositol-1,4,5-triphosphate (InsP3) receptor consists of a homotetramer of highly conserved 313 kd subunits that contain multiple transmembrane regions in the C-terminal part of the protein. The receptor was expressed in COS cells and its domain structure was studied by mutagenesis. Deletion of the transmembrane regions from the receptor results in the synthesis of a soluble receptor protein that efficiently binds InsP3 but which instead of associating into homotetramers remains monomeric. This result suggests a role for the transmembrane regions in the association of the receptor subunits into tetramers but not in ligand binding. To localize the ligand binding site, further cDNAs encoding truncated receptor proteins were constructed. Assays of InsP3 binding to these truncated InsP3 receptors revealed that sequences in the N-terminal fourth of the InsP3 receptor are sufficient for ligand binding. Accordingly, each subunit of the InsP3 receptor homotetramer contains an independent ligand binding site that is located on the N-terminal ends of each subunit and is separated from the putative channel-forming transmembrane regions by greater than 1400 amino acids. Gel filtration experiments demonstrate a large conformational change of the receptor as a function of ligand binding, suggesting a mechanism by which ligand binding might cause channel opening.

Amino Acid Sequence↗
PubMed2004

Regulation of AMPA receptor gating by ligand binding core dimers.

Ionotropic glutamate receptors are tetramers, the isolated ligand binding cores of which assemble as dimers. Previous work on nondesensitizing AMPA receptor mutants, which combined crystallography, ultracentrifugation, and patch-clamp recording, showed that dimer formation by the ligand binding cores is required for activation of ion channel gating by agonists. To define the mechanisms responsible for stabilization of dimer assembly in native AMPA receptors, contacts between the adjacent ligand binding cores were individually targeted by amino acid substitutions, using the GluR2 crystal structure as a guide to design mutants. We show that disruption of a salt bridge, hydrogen bond network, and intermolecular van der Waals contacts between helices D and J in adjacent ligand binding cores greatly accelerates desensitization. Conservation of these contacts in AMPA and kainate receptors indicates that they are important determinants of dimer stability and that the dimer interface is a key structural element in the gating mechanism of these glutamate receptor families.

Binding Sites↗