Ligand-induced rearrangement of the dimeric metabotropic glutamate receptor 1alpha

The extracellular domain of the metabotropic glutamate receptor 1alpha (mGluR1alpha) forms a dimer and the ligand, glutamate, induces a structural rearrangement in this domain. However, the conformational change in the cytoplasmic domain, which is critical for mGluR1alpha's interaction with G proteins, remains unclear. Here we investigated the ligand-induced conformational changes in the cytoplasmic domain by fluorescence resonance energy transfer (FRET) analysis of mGluR1alpha labeled with fluorescent protein(s) under total internal reflection field microscopy. Upon ligand binding, the intersubunit FRET efficiency between the second loops increased, whereas that between first loops decreased. In contrast, the intrasubunit FRET did not change clearly. These results show that ligand binding does not change the structure of each subunit, but does change the dimeric allocation of the cytoplasmic regions, which may underlie downstream signaling.     

Binding and fluorescence studies of the relationship between neurophysin-peptide interaction and neurophysin self-association: an allosteric system exhibiting minimal cooperativity

The mechanism of peptide-enhanced neurophysin self-association was investigated to address questions raised by the crystal structure of a neurophysin-dipeptide complex. The dependence on protein concentration of the binding of a broad range of peptides to the principal hormone-binding site confirmed that occupancy of this site alone, and not a site that bridges the monomer-monomer interface, is the trigger for enhanced dimerization. For the binding of most peptides to the principal hormone-binding site on bovine neurophysin I, the affinity of each dimer site was at least 10 times that of monomer under the conditions used. No interactions between the two sites of the dimer were evident. Fluorescence polarization studies of pressure-induced dimer dissociation indicated that the volume change for this reaction was almost 4 times greater in the liganded than in the unliganded state, pointing to a significant alteration of the monomer-monomer interface upon peptide binding. Novel conformational changes in the vicinity of the single neurophysin tyrosine, Tyr-49, induced by pressures lower than required for subunit dissociation, were also observed. The bovine neurophysin I dimer therefore appears to represent an allosteric system in which there is thermodynamic and functional communication between each binding site and the monomer-monomer interface, but no communication across the interface to the binding site of the other subunit. A model for the peptide-enhanced dimerization is proposed in which intersubunit contacts between monomers reduce the large unfavorable free energy associated with binding-induced intrasubunit conformational change. Structural origins of the lack of communication across the interface are suggested on the basis of the low volume change associated with dimerization in the unliganded state and monomer-monomer contacts in the crystal structure. Potential roles for the peptide alpha-amino group and position 2 phenyl ring in triggering conformational change are discussed.     

Comparative fluorescence resonance energy transfer analysis of metabotropic glutamate receptors: implications about the dimeric arrangement and rearrangement upon ligand bindings

Dimerization of G protein-coupled receptors has received much attention as a regulatory system of physiological function. Metabotropic glutamate receptors (mGluRs) are suitable models for studying the physiological significance of G protein-coupled receptor dimers because they form constitutive homodimers and function through dimeric rearrangement of their extracellular ligand binding domains. However, the molecular architecture of the transmembrane domains (TMDs) and their rearrangement upon agonist binding are still largely unknown. Here we show that the two helix Vs are arranged as the closest part in the dimeric TMDs and change their positions through synergistic control by the binding of two glutamates. The possibility that helix V is involved in an inter-protomer communication was first suggested by the finding that constitutively active mutation sites were identified on both sides of helix V. Then, comprehensive fluorescence resonance energy transfer (FRET) analysis using mGluRs whose cytoplasmic loops were labeled with donor and acceptor fluorescent proteins revealed that the third intracellular loop connecting helices V and VI of one protomer was in close proximity to the second and third intracellular loops of the other protomer and that all the intracellular loops became closer during the activation. Furthermore, FRET analysis of heterodimers in which only one protomer had ligand binding ability revealed the synergistic effect of the binding of two glutamates on the dimeric rearrangements of the TMD. Thus, the glutamate-dependent synergistic relocation of the helix Vs in the dimer is important for the signal flow from the extracellular ligand binding domain to the cytoplasmic surface of the mGluR.     

G protein activation by the leukotriene B4 receptor dimer. Evidence for an absence of trans-activation

There is compelling evidence that G protein-coupled receptors exist as homo- and heterodimers, but the way these assemblies function at the molecular level remains unclear. We used here the purified leukotriene B(4) receptor BLT1 stabilized in its dimeric state to analyze how a receptor dimer activates G proteins. For this, we produced heterodimers between the wild-type BLT1 and a BLT1/ALXR chimera. The latter is no longer activated by leukotriene B(4) but is still activated by ALXR agonists. In this heterodimer, agonist binding to either one of the two protomers induced asymmetric conformational changes within the receptor dimer. Of importance, no G protein activation was observed when using a dimer where the ligand-loaded protomer was not able to trigger GDP/GTP exchange due to specific mutations in its third intracellular loop, establishing that the conformation of the agonist-free protomer is not competent for G protein activation. Taken together, these data indicate that although ligand binding to one protomer in the heterodimer is associated with cross-conformational changes, a trans-activation mechanism where the ligand-free subunit would trigger GDP/GTP exchange cannot be considered in this case for G protein activation. This observation sheds light into the way GPCR dimers, in particular heterodimers, could activate their cognate G proteins.     

Ligand-induced asymmetry as observed through fluorophore rotations and free energy couplings: application to neurophysin

Changes that occur in subunit neurophysin structure upon ligand binding were explored by two methods. First, the thermal coefficient of the viscosity around the subunit tyrosine was monitored, which yields information on the environmental flexibility and free rotational space of the fluorophore. Initially, it was determined that the environmental flexibility and the free space around each subunit tyrosine are unperturbed upon dimerization. Binding of the tripeptide analogue of oxytocin causes the once homologous environments of the subunit tyrosines to become drastically different such that one moves onto a closely packed environment whereas the other moves into a region of larger free space. Even though the subunits as seen by each tyrosine are very different, the specific binding sites as seen by the ligands are similar. It was also found that ligand binding is stabilized by ring stacking and that energy transfer occurs between the tyrosine of the ligand and the neurophysin subunit tyrosine. Second, changes in subunit structure upon ligation were also followed by the determination of the order of free energy coupling between ligand binding and oligomerization, which tells how each ligand affects the subunit affinity. Since the binding of ligand is cooperative and induces dimer formation, there is second-order coupling between ligand binding and dimerization and the binding of the second ligand is responsible for the increase in subunit affinity.     

Observation of signal transduction in three-dimensional domain swapping

p13suc1 (suc1) has two native states, a monomer and a domain-swapped dimer. The structure of each subunit in the dimer is identical to that of the monomer, except for the hinge loop that connects the exchanging domains. Here we find that single point mutations at sites throughout the protein and ligand binding both shift the position of the equilibrium between monomer and dimer. The hinge loop was shown previously to act as a loaded molecular spring that releases tension present in the monomer by adopting an alternative conformation in the dimer. The results here indicate that the release of strain propagates throughout the entire protein and alters the energetics of regions remote from the hinge. Our data illustrate how the signal conferred by the conformational change of a protein loop, elicited by domain swapping, ligand binding or mutation, can be sensed by a distant active site. This work highlights the potential role of strained loops in proteins: the energy they store can be used for both signal transduction and allostery, and they could steer the evolution of protein function. Finally, a structural mechanism for the role of suc1 as an adapter molecule is proposed.     

Dynamically driven protein allostery

Allosteric interactions are typically considered to proceed through a series of discrete changes in bonding interactions that alter the protein conformation. Here we show that allostery can be mediated exclusively by transmitted changes in protein motions. We have characterized the negatively cooperative binding of cAMP to the dimeric catabolite activator protein (CAP) at discrete conformational states. Binding of the first cAMP to one subunit of a CAP dimer has no effect on the conformation of the other subunit. The dynamics of the system, however, are modulated in a distinct way by the sequential ligand binding process, with the first cAMP partially enhancing and the second cAMP completely quenching protein motions. As a result, the second cAMP binding incurs a pronounced conformational entropic penalty that is entirely responsible for the observed cooperativity. The results provide strong support for the existence of purely dynamics-driven allostery.     

Dimeric Crystal Structure of Rabbit l-Gulonate 3-Dehydrogenase/λ-Crystallin: Insights into the Catalytic Mechanism

l-Gulonate 3-dehydrogenase (GDH) is a bifunctional dimeric protein that functions not only as an NAD⁺-dependent enzyme in the uronate cycle but also as a taxon-specific λ-crystallin in rabbit lens. Here we report the first crystal structure of GDH in both apo form and NADH-bound holo form. The GDH protomer consists of two structural domains: the N-terminal domain with a Rossmann fold and the C-terminal domain with a novel helical fold. In the N-terminal domain of the NADH-bound structure, we identified 11 coenzyme-binding residues and found 2 distinct side-chain conformers of Ser124, which is a putative coenzyme/substrate-binding residue. A structural comparison between apo form and holo form and a mutagenesis study with E97Q mutant suggest an induced-fit mechanism upon coenzyme binding; coenzyme binding induces a conformational change in the coenzyme-binding residues Glu97 and Ser124 to switch their activation state from resting to active, which is required for the subsequent substrate recruitment. Subunit dimerization is mediated by numerous intersubunit interactions, including 22 hydrogen bonds and 104 residue pairs of van der Waals interactions, of which those between two cognate C-terminal domains are predominant. From a structure/sequence comparison within GDH homologues, a much greater degree of interprotomer interactions (both polar and hydrophobic) in the rabbit GDH would contribute to its higher thermostability, which may be relevant to the other function of this enzyme as λ-crystallin, a constitutive structural protein in rabbit lens. The present crystal structures and amino acid mutagenesis studies assigned the role of active-site residues: catalytic base for His145 and substrate binding for Ser124, Cys125, Asn196, and Arg231. Notably, Arg231 participates in substrate binding from the other subunit of the GDH dimer, indicating the functional significance of the dimeric state. Proper orientation of the substrate-binding residues for catalysis is likely to be maintained by an interprotomer hydrogen-bonding network of residues Asn196, Gln199, and Arg231, suggesting a network-based substrate recognition of GDH.     

GPCR homo-oligomerization

Cartoon depictions of (a) Dynamic interchange between receptor monomers, (homo, orange GPCR1, and hetero, orange GPCR1 and green GPCR2) dimers and oligomers which may be ligand regulated (orange sphere). (b) Signalling cross-talk between protomers of a dimer. Left: ligand (orange sphere) binds to one protomer and the conformational change transmitted to the second protomer results in signalling through activation of a Gα subunit (blue). Right: ligand (green sphere) binding to the second protomer may modulate signalling (large or small yellow flash).

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A tweezers-like motion of the ATP-binding cassette dimer in an ABC transport cycle

The ATPase components of ATP binding cassette (ABC) transporters power the transporters by binding and hydrolyzing ATP. Major conformational changes of an ATPase are revealed by crystal structures of MalK, the ATPase subunit of the maltose transporter from Escherichia coli, in three different dimeric configurations. While other nucleotide binding domains or subunits display low affinity for each other in the absence of the transmembrane segments, the MalK dimer is stabilized through interactions of the additional C-terminal domains. In the two nucleotide-free structures, the N-terminal nucleotide binding domains are separated to differing degrees, and the dimer is maintained through contacts of the C-terminal regulatory domains. In the ATP-bound form, the nucleotide binding domains make contact and two ATPs lie buried along the dimer interface. The two nucleotide binding domains of the dimer open and close like a pair of tweezers, suggesting a regulatory mechanism for ATPase activity that may be tightly coupled to translocation.     

Contribution of the C1A and C1B domains to the membrane interaction of protein kinase C

The hallmark for protein kinase C activation is its "translocation" to membranes following generation of lipid second messengers. This translocation is mediated by the C1 and C2 domains, two membrane-targeting modules, whose engagement on membranes provides the energy for an activating conformational change in which an autoinhibitory pseudosubstrate sequence is released from the active site. Novel and conventional protein kinase C isozymes contain a tandem repeat of C1 domains, the C1A and C1B, which each contain a binding pocket for phorbol esters/diacylglycerol. This study addresses the contribution of the C1A and C1B domains in the regulation of protein kinase C's membrane interaction using bisfunctional (dimeric) phorbol myristate acetate (PMA) molecules. We show that dimeric bisphorbols are an order of magnitude more effective at recruiting full-length PKC betaII to membranes compared with monomeric PMA and that the effectiveness of the interaction depends on the nature and length of the cross-link between the PMA moieties. Most effective were dimeric phorbol 12-acetate 13-esters linked at the 13 position with a 14 carbon spacer. The increased potency of dimeric phorbol esters is reduced if either the C1A or C1B domains are mutated so that they are unable to bind PMA, if one moiety of the dimer contains a nonfunctional phorbol, or if the binding to the isolated C1B domain is measured. Thus, the increased potency of the dimeric phorbol esters results primarily from their ability to engage, to a limited extent, both C1 modules on the same molecule. Although dimeric phorbols were more potent than monomeric phorbol esters in recruiting protein kinase C to membranes, the magnitude of the increase was still several orders of magnitude lower than what would be predicted on the basis of the reduction in dimensionality that occurs when the first C1 domain is engaged on the membrane. Thus, engaging both domains can be forced but is highly unfavored. In summary, our data reveal that both C1 domains are oriented for potential membrane interaction but only one C1 domain binds ligand in a physiological context.     

Quaternary structure of SecA in solution and bound to SecYEG probed at the single molecule level

Dual-color fluorescence-burst analysis (DCFBA) was applied to measure the quaternary structure and high-affinity binding of the bacterial motor protein SecA to the protein-conducting channel SecYEG reconstituted into lipid vesicles. DCFBA is an equilibrium technique that enables the direct observation and quantification of protein-protein interactions at the single molecule level. SecA binds to SecYEG as a dimer with a nucleotide- and preprotein-dependent dissociation constant. One of the SecA protomers binds SecYEG in a salt-resistant manner, whereas binding of the second protomer is salt sensitive. Because protein translocation is salt sensitive, we conclude that the dimeric state of SecA is required for protein translocation. A structural model for the dimeric assembly of SecA while bound to SecYEG is proposed based on the crystal structures of the Thermotoga maritima SecA-SecYEG and the Escherichia coli SecA dimer.     

Structure of the topoisomerase VI-B subunit: implications for type II topoisomerase mechanism and evolution

Type IIA and type IIB topoisomerases each possess the ability to pass one DNA duplex through another in an ATP-dependent manner. The role of ATP in the strand passage reaction is poorly understood, particularly for the type IIB (topoisomerase VI) family. We have solved the structure of the ATP-binding subunit of topoisomerase VI (topoVI-B) in two states: an unliganded monomer and a nucleotide-bound dimer. We find that topoVI-B is highly structurally homologous to the entire 40-43 kDa ATPase region of type IIA topoisomerases and MutL proteins. Nucleotide binding to topoVI-B leads to dimerization of the protein and causes dramatic conformational changes within each protomer. Our data demonstrate that type IIA and type IIB topoisomerases have descended from a common ancestor and reveal how ATP turnover generates structural signals in the reactions of both type II topoisomerase families. When combined with the structure of the A subunit to create a picture of the intact topoisomerase VI holoenzyme, the ATP-driven motions of topoVI-B reveal a simple mechanism for strand passage by the type IIB topoisomerases.     

Structure-function relationship of islet-activating protein, pertussis toxin: biological activities of hybrid toxins reconstituted from native and methylated subunits

Islet-activating protein (IAP), pertussis toxin, is a hexameric protein composed of an A protomer and a B oligomer, the residual pentamer having such a subunit assembly that two different dimers, dimer 1 and dimer 2, are connected with each other by means of the smallest C subunit. Incubation of IAP with formaldehyde and pyridine-borane produced the modified toxin in which most of the free amino groups were dimethylated. The methylated and nonmethylated (native) IAP were disintegrated into their respective constituent components, which were then cross combined to reconstitute hybrid toxins with the original hexameric structure. The binding of the B oligomer to the mammalian cell surface via dimer 2 was, but the binding via dimer 1 was not, seriously impaired by methylation of amino groups in the protein. The binding of the B oligomer allowed the A protomer to enter cells and to catalyze ADP-ribosylation of a membrane Mr 41 000 protein. The diverse biological activities of IAP occurring by this mechanism were mimicked by not only methylated IAP but also all hybrid toxins, indicating that the free amino groups in the protein were not essential for the enzyme activity of the A protomer and that the A protomer was able to enter cells if the B oligomer bound to cells "monovalently" via dimer 1. An additional effect of the B oligomer binding, i.e., the direct stimulation, without the transport of the A protomer, of cells leading to mitosis in lymphocytes in vitro or increases in circulating lymphocytes in vivo, was not mimicked by hybrid toxins containing methylated dimer 2.(ABSTRACT TRUNCATED AT 250 WORDS)     

NMR analysis of the monomeric form of a mutant unliganded bovine neurophysin: comparison with the crystal structure of a neurophysin dimer

Determination of the structure of the unliganded monomeric state of neurophysin is central to an understanding of the allosteric relationship between neurophysin peptide-binding and dimerization. We examined this state by NMR, using the weakly dimerizing H80E mutant of bovine neurophysin-I. The derived structure, to which more than one conformer appeared to contribute, was compared with the crystal structure of the unliganded des 1-6 bovine neurophysin-II dimer. Significant conformational differences between the two proteins were evident in the orientation of the 3,10 helix, in the 50-58 loop, in beta-turns, and in specific intrachain contacts between amino- and carboxyl domains. However, both had similar secondary structures, in independent confirmation of earlier circular dichroism studies. Previously suggested interactions between the amino terminus and the 50-58 loop in the monomer were also confirmed. Comparison of the observed differences between the two proteins with demonstrated effects of dimerization on the NMR spectrum of bovine neurophysin-I, and preliminary investigation of the effects of dimerization on H80E spectra, allowed tentative distinction between the contributions of sequence and self-association differences to the difference in conformation. Regions altered by dimerization encompass most binding site residues, providing a potential explanation of differences in binding affinity between the unliganded monomeric and dimeric states. Differences between monomer and dimer states in turns, interdomain contacts, and within the interdomain segment of the 50-58 loop suggest that the effects of dimerization on intrasubunit conformation reflect the need to adjust the relative positions of the interface segments of the two domains for optimal interaction with the adjacent subunit and/or reflect the dual role of some residues as participants both at the interface and in interdomain contacts.     

Amino acid mutagenesis of the ligand binding site and the dimer interface of the metabotropic glutamate receptor 1. Identification of crucial residues for setting the activated state

Previously, we determined the crystal structures of the dimeric ligand binding region of the metabotropic glutamate receptor subtype 1. Each protomer binds l-glutamate within the crevice between the LB1 and LB2 domains. We proposed that the two different conformations of the dimer interface between the two LB1 domains define the activated and resting states of the receptor protein. In this study, the residues in the ligand-binding site and the dimer interface were mutated, and the effects were analyzed in the full-length and truncated soluble receptor forms. The variations in the ligand binding activities of the purified truncated receptors are comparable with those of the full-length form. The mutated full-length receptors were also analyzed by inositol phosphate production and Ca(2+) response. The magnitude of the ligand binding capacities and the amplitude of the intracellular signaling were almost correlated. Alanine substitutions of four residues, Thr(188), Asp(208), Tyr(236), and Asp(318), which interact with the alpha-amino group of glutamate in the crystal, abolished their responses both to glutamate and quisqualate. The mutations of the Tyr(74), Arg(78), and Gly(293) residues, which interact with the gamma-carboxyl group of glutamate, lost their responsiveness to glutamate but not to quisqualate. Furthermore, a mutant receptor containing alanine instead of isoleucine at position 120 located within an alpha helix constituting the dimer interface showed no intracellular response to ligand stimulation. The results demonstrate the crucial role of the dimer interface in receptor activation.     

Base sequence-specific interactions of operator DNA fragments with the lambda-cro repressor coupled with changes in their conformations.

The mechanism of interaction of the operator DNA with the lambda-cro repressor protein was investigated using proton n.m.r. and photo CIDNP. Three kinds of DNA duplexes, the lambda-OR3 17-mer, phi80-OR2 19-mer and CRP binding site 22-mer, were prepared, and all of their imino proton resonances of the complexes with lambda-cro were assigned to individual base pairs. By monitoring the assigned signals of the DNA fragments and lambda-cro, it was found that in the complex of lambda-cro with lambda-OR3, two subunits of the cro dimer bind to the right and left halves of the OR3, respectively, and the bidentate binding induces a structural distortion in the middle of the 17-mer. lambda-cro itself also undergoes a conformational change including loosening of the dimeric form. In the complex of lambda-cro with phi 80-OR2, which has a 6-bp sequence common to that of lambda-OR3, one subunit of the cro dimer seems to bind specifically to the common part. However, there is only a slight conformational change in the cro dimer. In the mixture of the CRP binding site 22-mer and lambda-cro, soft contact without any conformational change was observed between them.     

Modulation of dimerization, binding, stability, and folding by mutation of the neurophysin subunit interface

Bovine neurophysins, which have typically served as the paradigm for neurophysin behavior, are metastable in their disulfide-paired folded state and require ligand stabilization for efficient folding from the reduced state. Studies of unliganded porcine neurophysin (oxytocin-associated class) demonstrated that its dimerization constant is more than 90-fold greater than that of the corresponding bovine protein at neutral pH and showed that the increased dimerization constant is accompanied by an increase in stability sufficient to allow efficient folding of the reduced protein in the absence of ligand peptide. Using site-specific mutagenesis of the bovine protein and expression in Escherichia coli, the functional differences between the bovine and porcine proteins were shown to be attributable solely to two subunit interface mutations in the porcine protein, His to Arg at position 80 and Glu to Phe at position 81. Mutation of His-80 alone to Arg had a relatively small impact on dimerization, while mutation to either Glu or Asp markedly reduced dimerization in the unliganded state, albeit with apparent retention of the positive linkage between dimerization and binding. Comparison of the peptide-binding constants of the different mutants additionally indicated that substitution of His-80 led to modifications in binding affinity and specificity that were independent of effects on dimerization. The results demonstrate the importance of the carboxyl domain segment of the subunit interface in modulating neurophysin properties and suggest a specific contribution of the energetics of ligand-induced conformational change in this region to the overall thermodynamics of binding. The potential utility to future studies of the self-folding and monomeric mutants generated by altering the interface is noted.     

The crystal structure of maleylacetate reductase from Rhizobium sp. strain MTP‐10005 provides insights into the reaction mechanism of enzymes in its original family

Maleylacetate reductase plays a crucial role in catabolism of resorcinol by catalyzing the NAD(P)H‐dependent reduction of maleylacetate, at a carbon–carbon double bond, to 3‐oxoadipate. The crystal structure of maleylacetate reductase from Rhizobium sp. strain MTP‐10005, GraC, has been elucidated by the X‐ray diffraction method at 1.5 Å resolution. GraC is a homodimer, and each subunit consists of two domains: an N‐terminal NADH‐binding domain adopting an α/β structure and a C‐terminal functional domain adopting an α‐helical structure. Such structural features show similarity to those of the two existing families of enzymes in dehydroquinate synthase‐like superfamily. However, GraC is distinct in dimer formation and activity expression mechanism from the families of enzymes. Two subunits in GraC have different structures from each other in the present crystal. One subunit has several ligands mimicking NADH and the substrate in the cleft and adopts a closed domain arrangement. In contrast, the other subunit does not contain any ligand causing structural changes and adopts an open domain arrangement. The structure of GraC reveals those of maleylacetate reductase both in the coenzyme, substrate‐binding state and in the ligand‐free state. The comparison of both subunit structures reveals a conformational change of the Tyr326 loop for interaction with His243 on ligand binding. Structures of related enzymes suggest that His243 is likely a catalytic residue of GraC. Mutational analyses of His243 and Tyr326 support the catalytic roles proposed from structural information. The crystal structure of GraC characterizes the maleylacetate reductase family as a third family in the dehydroquinate synthase‐like superfamily. Proteins 2016; 84:1029–1042. © 2016 Wiley Periodicals, Inc.