Ligand-induced conformational change in the alpha7 nicotinic receptor ligand binding domain

Molecular dynamics simulations of a homology model of the ligand binding domain of the alpha7 nicotinic receptor are conducted with a range of bound ligands to induce different conformational states. Four simulations of 15 ns each are run with no ligand, antagonist d-tubocurarine (dTC), agonist acetylcholine (ACh), and agonist ACh with potentiator Ca(2+), to give insight into the conformations of the active and inactive states of the receptor and suggest the mechanism for conformational change. The main structural factor distinguishing the active and inactive states is that a more open, symmetric arrangement of the five subunits arises for the two agonist simulations, whereas a more closed and asymmetric arrangement results for the apo and dTC cases. Most of the difference arises in the lower portion of the ligand binding domain near its connection to the adjacent transmembrane domain. The transfer of the more open state to the transmembrane domain could then promote ion flow through the channel. Variation in how subunits pack together with no ligand bound appears to give rise to asymmetry in the apo case. The presence of dTC expands the receptor but induces rotations in alternate directions in adjacent subunits that lead to an asymmetric arrangement as in the apo case. Ca(2+) appears to promote a slightly greater expansion in the subunits than ACh alone by stabilizing the C-loop and ACh positions. Although the simulations are unlikely to be long enough to view the full conformational changes between open and closed states, a collection of different motions at a range of length scales are observed that are likely to participate in the conformational change.     

Hormone-induced conformational changes in the hepatic insulin receptor

The insulin receptor can exist in either a lower or a higher affinity state. Hormone binding alters the equilibrium between the two states of the insulin receptor, favoring the formation of that of higher affinity (Corin, R.E., and Donner, D.B. (1982), J. Biol. Chem. 257, 104-110). After brief or extended incubations with hormone, during which the fraction of higher affinity receptors increased, 125I-insulin was covalently coupled to the alpha subunits of its receptor using disuccinimidyl suberate. Some 125I-insulin remained bound to higher affinity receptors after dissociation of hormone from lower affinity sites. This hormone could also be covalently coupled to the alpha subunit of the receptor. During extended incubations between 125I-insulin and liver plasma membranes, components of the receptor were cleaved to yield degradation products of 120,000 and 23,000 Da. The significance of this process remains undetermined. Unoccupied insulin receptors were cleaved by trypsin to produce fragments of 94,000 and 37,000 Da which remained membrane-bound and could be covalently coupled to 125I-insulin. Trypsin treatment after binding yielded an additional receptor fragment of 64,000 Da. As the incubation time between 125I-insulin and membranes was lengthened, components of the receptor became progressively less sensitive to trypsin. Higher affinity binding sites isolated after release of rapid dissociating insulin were less sensitive to trypsin than were mixtures of higher and lower affinity receptors. These observations suggest that hormone binding produces two conformational changes (alterations of tryptic lability) in the hepatic insulin receptor. The first change is rapid and exposes parts of the receptor to tryptic degradation. The second, slower conformational change renders the receptor less sensitive to trypsin and occurs with the same time course as the increase of receptor affinity mediated by site occupancy.     

Epidermal growth factor binding induces a conformational change in the external domain of its receptor.

To study the properties of the extracellular epidermal growth factor (EGF) binding domain of the human EGF receptor, we have infected insect cells with a suitably engineered baculovirus vector containing the cDNA encoding the entire ectodomain of the parent molecule. This resulted in a correctly folded, stable, 110 kd protein which possessed an EGF binding affinity of 200 nM. The protein was routinely purified in milligram amounts from 1 litre insect cell cultures using a series of three standard chromatographic steps. The properties of the ectodomain were studied before and after the addition of different EGF ligands, using both circular dichroism and fluorescence spectroscopic techniques. A secondary structural analysis of the far UV CD spectrum of the ectodomain indicated significant proportions of alpha-helix and beta-sheet in agreement with a published model of the EGF receptor. The ligand additions to the receptor showed differences in both the near- and far-UV CD spectra, and were similar for each ligand used, suggesting similar conformational differences between uncomplexed and complexed receptor. Steady-state fluorescence measurements indicated that the tryptophan residues present in the ectodomain are buried and that the solvent-accessible tryptophans in the ligands become buried on binding the receptor. The rotational correlation times measured by fluorescence anisotropy decay for the receptor-ligand complexes were decreased from 6 to 2.5 ns in each case. This may indicate a perturbation of the tryptophan environment of the receptor on ligand binding. Ultracentrifugation studies showed that no aggregation occurred on ligand addition, so this could not explain the observed differences from CD or fluorescence.(ABSTRACT TRUNCATED AT 250 WORDS)     

Intronic nature of the rat luteinizing hormone receptor gene defines a soluble receptor subspecies with hormone binding activity

A rat testicular luteinizing hormone (LH) receptor cDNA containing a 266-base pair deletion resulting in the omission of the 1st transmembrane region and truncation of the open reading frame was isolated using a rat ovarian LH receptor cDNA probe. Comparison of this clone with a restriction fragment from the LH receptor genomic DNA revealed potential alternative splice sites following the consensus sequence TTXCAG that is present at an intron acceptor splice site and also within the next exon, accounting for the specific deletion mutation observed in this cDNA. Expression of the testicular cDNA in COS1 cells resulted in synthesis and secretion of a soluble binding protein with high affinity and specificity for LH and human chorionic gonadotropin. These studies have demonstrated that the LH receptor gene contains intron(s) within the region coding for the extracellular domain of the molecule, which determine the nature and generation of LH receptor isoforms. Expression of the soluble form of the LH receptor has indicated that the amino-terminal extracellular region plays a major role in gonadotropin binding. These features of the LH receptor are distinct from those of most other G protein-coupled receptors, which are intronless and contain their binding sites within the transmembrane region rather than the extracellular domain.     

Triple-resonance multidimensional NMR study of calmodulin complexed with the binding domain of skeletal muscle myosin light-chain kinase: indication of a conformational change in the central helix

Heteronuclear 3D and 4D NMR experiments have been used to obtain 1H, 13C, and 15N backbone chemical shift assignments in Ca(2+)-loaded calmodulin complexed with a 26-residue synthetic peptide (M13) corresponding to the calmodulin-binding domain (residues 577-602) of rabbit skeletal muscle myosin light-chain kinase. Comparison of the chemical shift values with those observed in peptide-free calmodulin [Ikura, M., Kay, L. E., & Bax, A. (1990) Biochemistry 29, 4659-4667] shows that binding of M13 peptide induces substantial chemical shift changes that are not localized in one particular region of the protein. The largest changes are found in the first helix of the Ca(2+)-binding site I (E11-E14), the N-terminal portion of the central helix (M72-D78), and the second helix of the Ca(2+)-binding site IV (F141-M145). Analysis of backbone NOE connectivities indicates a change from alpha-helical to an extended conformation for residues 75-77 upon complexation with M13. This conformational change is supported by upfield changes in the C alpha and carbonyl chemical shifts of these residues relative to M13-free calmodulin and by hydrogen-exchange experiments that indicate that the amide protons of residues 75-82 are in fast exchange (kexch greater than 10 s-1 at pH 7, 35 degrees C) with the solvent. No changes in secondary structure are observed for the first helix of site I or the C-terminal helix of site IV. Upon complexation with M13, a significant decrease in the amide exchange rate is observed for residues T110, L112, G113, and E114 at the end of the second helix of site III.     

Characterization of a soluble follitropin receptor from porcine testis.

Porcine testis receptors for follitropin (FSH) were solubilized by treatment with the non-ionic detergent Nonidet P-40 and receptor-bound and free ¹²⁵I-porcine FSH were separated by ammonium sulfate precipitation. The soluble receptor retained both its high affinity and specificity for FSH. The soluble hormone-receptor complex exhibited an equilibrium association constant of 4.7 × 10¹⁰ M^?1 at 4°C. Its hydrodynamic properties were consistent with those obtained for other solubilized peptide hormone receptors, and its molecular weight estimated to 244,000.     

Role of the transmembrane domain 4/extracellular loop 2 junction of the human gonadotropin-releasing hormone receptor in ligand binding and receptor conformational selection

Recent crystal structures of G protein-coupled receptors (GPCRs) show the remarkable structural diversity of extracellular loop 2 (ECL2), implying its potential role in ligand binding and ligand-induced receptor conformational selectivity. Here we have applied molecular modeling and mutagenesis studies to the TM4/ECL2 junction (residues Pro(174(4.59))-Met(180(4.66))) of the human gonadotropin-releasing hormone (GnRH) receptor, which uniquely has one functional type of receptor but two endogenous ligands in humans. We suggest that the above residues assume an α-helical extension of TM4 in which the side chains of Gln(174(4.60)) and Phe(178(4.64)) face toward the central ligand binding pocket to make H-bond and aromatic contacts with pGlu(1) and Trp(3) of both GnRH I and GnRH II, respectively. The interaction between the side chains of Phe(178(4.64)) of the receptor and Trp(3) of the GnRHs was supported by reciprocal mutations of the interacting residues. Interestingly, alanine mutations of Leu(175(4.61)), Ile(177(4.63)), and Met(180(4.66)) decreased mutant receptor affinity for GnRH I but, in contrast, increased affinity for GnRH II. This suggests that these residues make intramolecular or intermolecular contacts with residues of transmembrane (TM) domain 3, TM5, or the phospholipid bilayer, which couple the ligand structure to specific receptor conformational switches. The marked decrease in signaling efficacy of I177A and F178A also indicates that IIe(177(4.63)) and Phe(178(4.64)) are important in stabilizing receptor-active conformations. These findings suggest that the TM4/ECL2 junction is crucial for peptide ligand binding and, consequently, for ligand-induced receptor conformational selection.     

Properties of the naturally occurring soluble surface glycoprotein of ecotropic murine leukemia virus: binding specificity and possible conformational change after binding to receptor

Ecotropic murine leukemia virus (MuLV) infection is initiated by the interaction between the surface glycoprotein (SU) of the virus and its cell-surface receptor mCAT-1. We investigated the SU-receptor interaction by using a naturally occurring soluble SU which was encoded by the envelope (env) gene of a defective endogenous MuLV, Fv-4(r). Binding of the SU to mCAT-1-positive mouse cells was completed by 1 min at 37 degrees C. The SU could not bind to mouse cells that were persistently infected by ecotropic MuLVs (but not amphotropic or dualtropic MuLVs) or transfected with wild-type ecotropic env genes or a mutant env gene which can express only precursor Env protein that is restricted to retention in the endoplasmic reticulum. These cells were also resistant to superinfection by ecotropic MuLVs. Thus, superinfection resistance correlated with the lack of SU-binding capacity. After binding to the cells, the SU appeared to undergo some conformational changes within 1 min in a temperature-dependent manner. This was suggested by the different properties of two monoclonal antibodies (MAbs) reactive with the same C-terminal half of the Fv-4(r) SU domain, including a proline-rich motif which was shown to be important for conformation of the SU and interaction between the SU and the transmembrane protein. One MAb reacting with the soluble SU bound to cells was dissociated by a temperature shift from 4 to 37 degrees C. Such dissociation was not observed in cells synthesizing the SU or when another MAb was used, indicating that the dissociation was not due to a temperature-dependent release of the MAb but to possible conformational changes in the SU.     

Molecular structure of the rat vitamin D receptor ligand binding domain complexed with 2-carbon-substituted vitamin D3 hormone analogues and a LXXLL-containing coactivator peptide

We have determined the crystal structures of the ligand binding domain (LBD) of the rat vitamin D receptor in ternary complexes with a synthetic LXXLL-containing peptide and the following four ligands: 1alpha,25-dihydroxyvitamin D(3); 2-methylene-19-nor-(20S)-1alpha,25-dihydroxyvitamin D(3) (2MD); 1alpha-hydroxy-2-methylene-19-nor-(20S)-bishomopregnacalciferol (2MbisP), and 2alpha-methyl-19-nor-1alpha,25-dihydroxyvitamin D(3) (2AM20R). The conformation of the LBD is identical in each complex. Binding of the 2-carbon-modified analogues does not change the positions of the amino acids in the ligand binding site and has no effect on the interactions in the coactivator binding pocket. The CD ring of the superpotent analogue, 2MD, is tilted within the binding site relative to the other ligands in this study and to (20S)-1alpha,25-dihydroxyvitamin D(3) [Tocchini-Valentini et al. (2001) Proc. Natl. Acad. Sci. U.S.A. 98, 5491-5496]. The aliphatic side chain of 2MD follows a different path within the binding site; nevertheless, the 25-hydroxyl group at the end of the chain occupies the same position as that of the natural ligand, and the hydrogen bonds with histidines 301 and 393 are maintained. 2MbisP binds to the receptor despite the absence of the 25-hydroxyl group. A water molecule is observed between His 301 and His 393 in this structure, and it preserves the orientation of the histidines in the binding site. Although the alpha-chair conformer is highly favored in solution for the A ring of 2AM20R, the crystal structures demonstrate that this ring assumes the beta-chair conformation in all cases, and the 1alpha-hydroxyl group is equatorial. The peptide folds as a helix and is anchored through hydrogen bonds to a surface groove formed by helices 3, 4, and 12. Electrostatic and hydrophobic interactions between the peptide and the LBD stabilize the active receptor conformation. This stablization appears necessary for crystal growth.     

Thermodynamics of follitropin binding to solubilized calf testis receptor

Thermodynamic parameters of follitropin binding to solubilized testicular receptors were measured in order to assess the forces involved in the binding reaction. Reversibility of follitropin binding to solubilized receptor decreased only 20% over the temperature range 4-24 degrees C, whereas earlier studies indicated reversibility of binding to membrane-bound receptor decreased by more than 40% over the same range [Anderson, T. T., Curatolo, L. M., & Reichert, L. E., Jr. (1983) Mol. Cell. Endocrinol. 33, 37-52]. Thermodynamic analysis of follitropin binding to solubilized receptors showed that the hydrophobic effect was important in the binding reaction. The mean values, at 25 degrees C, for delta H and delta S were -31.8 kcal/mol and -66.0 cal mol-1 K-1, respectively, and delta Cp was -3.0 kcal mol-1 K-1. This is an unusually large heat capacity for protein-protein association reactions, indicating an enhanced role for the hydrophobic effect with the solubilized (compared to membrane-bound) receptor. Since glycerol was necessary to stabilize the solubilized receptor, we determined whether glycerol affected the thermodynamic parameters measured for the binding reaction. Control experiments, performed with membrane-bound receptor in the presence or absence of glycerol, indicated that delta Cp actually decreased upon addition of glycerol (-0.8 kcal mol-1 K-1 in the presence of glycerol compared to -2.3 kcal mol-1 K-1 in the absence of glycerol). Thus, the large negative delta Cp observed for the soluble receptor was a result of its removal from the membrane and was not due to the presence of glycerol.(ABSTRACT TRUNCATED AT 250 WORDS)     

Homology modeling of rabbit prolactin hormone complexed with its receptor

The pituitary hormone prolactin (prl) is implicated in a number of biological functions, especially lactation, which is mediated through specific lactogenic receptors (PrlR). Human growth hormone (hGH) is also a pituitary hormone responsible for linear growth. While the growth hormone receptor (hGHR) binds only hGH, hPrlR can interact with both hGH and hPrl. Using structural information from the human growth hormone (hGH)/receptor (hGHR) complex, we modeled by homology a complex between rabbit prolactin hormone (rbPrl) and its receptor (rbPrlR). While the somatogenic hormone/somatogenic receptor (hGH/hGHR) and somatogenic hormone/lactogenic receptor (hGH/hPrlR) interactions are now known and well studied, here we propose a model for the interaction of the lactogenic hormone with its receptor (rbPrl/rbPrlR), and compare these three kinds of ligand/receptor interaction. We identified residues contributing to the active site and tested the potential dimerization of the receptor. Biochemical studies and information deduced from the modeled complex do not exclude a homodimeric form but point to a functional heterodimeric complex. Proteins 27: 459–468, 1997. © 1997 Wiley-Liss, Inc.     

Crystal structure of guanosine-free ribonuclease T1, complexed with vanadate (V), suggests conformational change upon substrate binding

Ribonuclease T1 was crystallized in the presence of vanadate(V). The crystal structure was solved by molecular replacement and refined by least-squares methods using stereochemical restraints. The refinement was based on data between 10 and 1.8 A and converged at a crystallographic R factor of 0.137. Except for the substrate-recognition site the three-dimensional structure of ribonuclease T1 closely resembles the structure of the enzyme complexed with guanosine 2'-phosphate and its derivatives. A tetrahedral anion was found at the catalytic site and identified as H2VO4-. This is the first crystal structure of ribonuclease T1 determined in the absence of bound substrate analogue. Distinct structural differences between guanosine-free and complexed ribonuclease T1 are observed at the base-recognition site: The side chains of Tyr45 and Glu46 and the region around Asn98 changed their conformations, and the peptide bond between Asn43 and Asn44 has turned around by 140 degrees. We suggest that the structural differences seen in the crystal structures of free and complexed ribonuclease T1 are related to conformational adjustments associated with the substrate binding process.     

Comparative structural analysis of the binding domain of follicle stimulating hormone receptor

Proteins with leucine-rich repeats (LRRs) specialize in mediating protein-protein interactions. The hormone binding portion of the receptor for follicle stimulating hormone (FSH) is an LRR protein by sequence, and the crystal structure of this domain from human FSH receptor in a complex with FSH shows that it does indeed have an LRR structure. It differs from other LRR domains, however, in being an all-beta protein composed of highly irregular repeats and having only slight overall curvature. Despite these distinctions and a superficial resemblance to beta-helical proteins, the binding domain of FSH receptor clearly is an LRR protein. The structure does consist of two parts with distinctively different curvatures. Comparison with the structures of other LRR-containing proteins shows a correlation between curvature and main-chain hydrogen bonding pattern of the parallel beta-sheet. The hormone-binding site is located at the concave surface of the receptor structure, a feature common to proteins with LRR motifs. Analysis of the ligand-binding site of LRR-containing proteins reveals that they generally utilize extensive interface area and a large number of charged residues to facilitate high-affinity protein-protein interactions.     

Residues in the stalk domain of the hendra virus g glycoprotein modulate conformational changes associated with receptor binding

Hendra virus (HeV) is a member of the broadly tropic and highly pathogenic paramyxovirus genus Henipavirus. HeV is enveloped and infects cells by using membrane-anchored attachment (G) and fusion (F) glycoproteins. G possesses an N-terminal cytoplasmic tail, an external membrane-proximal stalk domain, and a C-terminal globular head that binds the recently identified receptors ephrinB2 and ephrinB3. Receptor binding is presumed to induce conformational changes in G that subsequently trigger F-mediated fusion. The stalk domains of other attachment glycoproteins appear important for oligomerization and F interaction and specificity. However, this region of G has not been functionally characterized. Here we performed a mutagenesis analysis of the HeV G stalk, targeting a series of isoleucine residues within a hydrophobic α-helical domain that is well conserved across several attachment glycoproteins. Nine of 12 individual HeV G alanine substitution mutants possessed a complete defect in fusion-promotion activity yet were cell surface expressed and recognized by a panel of conformation-dependent monoclonal antibodies (MAbs) and maintained their oligomeric structure. Interestingly, these G mutations also resulted in the appearance of an additional electrophoretic species corresponding to a slightly altered glycosylated form. Analysis revealed that these G mutants appeared to adopt a receptor-bound conformation in the absence of receptor, as measured with a panel of MAbs that preferentially recognize G in a receptor-bound state. Further, this phenotype also correlated with an inability to associate with F and in triggering fusion even after receptor engagement. Together, these data suggest the stalk domain of G plays an important role in the conformational stability and receptor binding-triggered changes leading to productive fusion, such as the dissociation of G and F.     

Expression of a soluble glycine binding domain of the NMDA receptor in Escherichia coli

Glycine is an essential co-agonist of the excitatory N-methyl-D-aspartate (NMDA) receptor. The glycine binding site of this subtype of ionotropic glutamate receptors is formed by the S1 and S2 regions of the NR1 subunit. Here, different S1S2 fusion proteins were expressed and purified from Escherichia coli cultures, and refolding protocols were established allowing the production of 30 mg of soluble S1S2 fusion protein from 1 liter bacterial culture. After affinity purification and renaturation, two of the fusion proteins (S1S2 and S1S2-V1) bound the competitive glycine site antagonist [3H]MDL105,519 with K(d) values of 9.35 and 3.9 nM, respectively. In contrast, with three other constructs (S1S2M, S1S2-V2, and -V3) saturable ligand binding could not be obtained. These results redefine the S1S2 domains required for high-affinity glycine binding. Furthermore, our high-affinity binding proteins may be used for the large-scale production of the glycine binding core region for future structural studies.     

LRET investigations of conformational changes in the ligand binding domain of a functional AMPA receptor

The structural investigations using the soluble ligand binding domain of the AMPA subtype of the glutamate receptor have provided invaluable insight into the mechanistic pathway by which agonist binding to this extracellular domain mediates the formation of cation-selective channels in this protein. These structures, however, are in the absence of the transmembrane segments, the primary functional component of the protein. Here, we have used a modified luminescence resonance energy transfer based method to obtain distance changes due to agonist binding in the ligand binding domain in the presence of the transmembrane segments. These distance changes show that the cleft closure conformational change observed in the isolated ligand binding domain upon binding agonist is conserved in the receptor with the channel segments, thus establishing that the isolated ligand binding domain is a good model of the domain in the receptor containing the transmembrane segments.     

An agonist-induced conformational change in the growth hormone receptor determines the choice of signalling pathway

The growth and metabolic actions of growth hormone (GH) are believed to be mediated through the GH receptor (GHR) by JAK2 activation. The GHR exists as a constitutive homodimer, with signal transduction by ligand-induced realignment of receptor subunits. Based on the crystal structures, we identify a conformational change in the F'G' loop of the lower cytokine module, which results from binding of hGH but not G120R hGH antagonist. Mutations disabling this conformational change cause impairment of ERK but not JAK2 and STAT5 activation by the GHR in FDC-P1 cells. This results from the use of two associated tyrosine kinases by the GHR, with JAK2 activating STAT5, and Lyn activating ERK1/2. We provide evidence that Lyn signals through phospholipase C gamma, leading to activation of Ras. Accordingly, mice with mutations in the JAK2 association motif respond to GH with activation of hepatic Src and ERK1/2, but not JAK2/STAT5. We suggest that F'G' loop movement alters the signalling choice between JAK2 and a Src family kinase by regulating TMD realignment. Our findings could explain debilitated ERK but not STAT5 signalling in some GH-resistant dwarfs and suggest pathway-specific cytokine agonists.