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Hideto Miyoshi - One of the best experts on this subject based on the ideXlab platform.
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identification of the binding sites for ubiquinone and inhibitors in the na pumping nadh ubiquinone oxidoreductase from vibrio cholerae by Photoaffinity Labeling
Journal of Biological Chemistry, 2017Co-Authors: Takeshi Ito, Masatoshi Murai, Satoshi Ninokura, Yuki Kitazumi, Katherine G Mezic, Brady F Cress, Mattheos A G Koffas, Joel E Morgan, Blanca Barquera, Hideto MiyoshiAbstract:Abstract The Na+-pumping NADH-quinone oxidoreductase (Na+-NQR) is the first enzyme of the respiratory chain and the main ion transporter in many marine and pathogenic bacteria, including Vibrio cholerae. The V. cholerae Na+-NQR has been extensively studied, but its binding sites for ubiquinone and inhibitors remain controversial. Here, using a photoreactive ubiquinone PUQ-3 as well as two aurachin-type inhibitors [125I]PAD-1 and [125I]PAD-2 and Photoaffinity Labeling experiments on the isolated enzyme, we demonstrate that the ubiquinone ring binds to the NqrA subunit in the regions Leu-32–Met-39 and Phe-131–Lys-138, encompassing the rear wall of a predicted ubiquinone-binding cavity. The quinolone ring and alkyl side chain of aurachin bound to the NqrB subunit in the regions Arg-43–Lys-54 and Trp-23–Gly-89, respectively. These results indicate that the binding sites for ubiquinone and aurachin-type inhibitors are in close proximity but do not overlap one another. Unexpectedly, although the inhibitory effects of PAD-1 and PAD-2 were almost completely abolished by certain mutations in NqrB (i.e. G140A and E144C), the binding reactivities of [125I]PAD-1 and [125I]PAD-2 to the mutated enzymes were unchanged compared with those of the wild-type enzyme. We also found that Photoaffinity Labeling by [125I]PAD-1 and [125I]PAD-2, rather than being competitively suppressed in the presence of other inhibitors, is enhanced under some experimental conditions. To explain these apparently paradoxical results, we propose models for the catalytic reaction of Na+-NQR and its interactions with inhibitors on the basis of the biochemical and biophysical results reported here and in previous work.
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syntheses of photoreactive cardiolipins for a Photoaffinity Labeling study
Tetrahedron Letters, 2015Co-Authors: Masato Abe, Masaaki Nakano, Ayumi Kosaka, Hideto MiyoshiAbstract:The Photoaffinity Labeling technique using photoreactive cardiolipin (CL) is a powerful means for investigating the molecular mechanism of the formation of a specific cytochrome c–cardiolipin complex. Using phosphoramidite chemistry, we synthesized three photoreactive CLs, that possess an unstable diazirine ring at different positions; that is, the acyl chain in the sn-1 or sn-2 position and the central glycerol moiety.
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Identification of the Binding Site of the Quinone-Head Group in Mitochondrial Coq10 by Photoaffinity Labeling
2014Co-Authors: Masatoshi Murai, Kohei Matsunobu, Sawako Kudo, Kentaro Ifuku, Makoto Kawamukai, Hideto MiyoshiAbstract:Mitochondrial Coq10 is a ubiquinone (UQ)-binding protein that is a member of the steroidogenic acute regulatory protein (StAR)-related lipid transfer (START) domain superfamily. Deletion of the COQ10 gene was previously shown to cause a marked respiratory defect in Saccharomyces cerevisiae and Schizosaccharomyces pombe, which indicated that Coq10 may support efficient electron transfer between the respiratory complexes; however, its physiological role remains elusive. To elucidate the role of Coq10, we attempted to identify the binding site of UQ in recombinant S. pombe Coq10 expressed in an Escherichia coli cell membrane through Photoaffinity Labeling with the photoreactive UQ probe, UQ-1, in combination with biotinylation of the labeled peptide by means of the so-called click chemistry. Comprehensive proteomic analyses revealed that the quinone-head ring of UQ-1 specifically binds to the N-terminal region of Phe39–Lys45 of Coq10, which corresponds to the ligand-binding pocket of many proteins containing the START domain. The Labeling was completely suppressed in the presence of an excess amount of artificial short-chain UQ analogues, such as UQ2. In the Phe39Ala and Pro41Ala mutants, the extents of Labeling were ∼40 and ∼60%, respectively, of that of wild-type Coq10. While Coq10 has been thought to bind UQ, our work first provides the direct evidence of Coq10 accommodating the quinone-head ring of UQ in its START domain. On the basis of these results, the physiological role of Coq10 has been discussed
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exploring interactions between the 49 kda and nd1 subunits in mitochondrial nadh ubiquinone oxidoreductase complex i by Photoaffinity Labeling
Biochemistry, 2011Co-Authors: Masatoshi Murai, Yuko Mashimo, Judy Hirst, Hideto MiyoshiAbstract:Quinazolines are strong inhibitors of NADH-ubiquinone oxidoreductase (complex I) from bovine heart mitochondria. Using a photoreactive quinazoline, [125I]AzQ, and bovine heart submitochondrial particles (SMPs), we demonstrated previously that [125I]AzQ binds at the interface of the 49 kDa and ND1 subunits in complex I; it labeled a site in the N-terminal (Asp41–Arg63) region of the 49 kDa subunit, suggesting that this region contacts the ND1 subunit [Murai, M., et al. (2009) Biochemistry 48, 688–698]. The labeled region of ND1 could not be identified because it is highly hydrophobic, and the SMPs did not yield sufficient amounts of labeled protein. Here, we describe how Photoaffinity Labeling of isolated complex I by [125I]AzQ yielded sufficient material for identification of the labeled region of the ND1 subunit. The inhibition of the isolated enzyme by AzQ is comparable to that of SMPs. Our results reveal that the labeled site in ND1 is between Asp199 and Lys262, mostly likely in the third matrix loop t...
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characterization of the inhibitor binding site in mitochondrial nadh ubiquinone oxidoreductase by Photoaffinity Labeling using a quinazoline type inhibitor
Biochemistry, 2009Co-Authors: Masatoshi Murai, Koji Sekiguchi, Takaaki Nishioka, Hideto MiyoshiAbstract:The diverse inhibitors of bovine heart mitochondrial complex I (NADH−ubiquinone oxidoreductase) are believed to share a common large binding domain with partially overlapping sites, though it remains unclear how these binding sites relate to each other. To obtain new insight into the inhibitor binding domain in complex I, we synthesized a photoreactive azidoquinazoline {[125I]-6-azido-4-(4-iodophenethylamino)quinazoline, [125I]AzQ}, in which a photolabile azido group was introduced into the toxophoric quinazoline ring to allow specific cross-linking, and carried out a Photoaffinity Labeling study using bovine heart submitochondrial particles. Analysis of the photo-cross-linked proteins by peptide mass fingerprinting and immunoblotting revealed that [125I]AzQ specifically binds to the 49 kDa and ND1 subunits with a frequency of ∼4:1. The cross-linking was completely blocked by excess amounts of other inhibitors such as acetogenin and fenpyroximate. Considerable cross-linking was also detected in the ADP/AT...
Masatoshi Murai - One of the best experts on this subject based on the ideXlab platform.
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identification of the binding sites for ubiquinone and inhibitors in the na pumping nadh ubiquinone oxidoreductase from vibrio cholerae by Photoaffinity Labeling
Journal of Biological Chemistry, 2017Co-Authors: Takeshi Ito, Masatoshi Murai, Satoshi Ninokura, Yuki Kitazumi, Katherine G Mezic, Brady F Cress, Mattheos A G Koffas, Joel E Morgan, Blanca Barquera, Hideto MiyoshiAbstract:Abstract The Na+-pumping NADH-quinone oxidoreductase (Na+-NQR) is the first enzyme of the respiratory chain and the main ion transporter in many marine and pathogenic bacteria, including Vibrio cholerae. The V. cholerae Na+-NQR has been extensively studied, but its binding sites for ubiquinone and inhibitors remain controversial. Here, using a photoreactive ubiquinone PUQ-3 as well as two aurachin-type inhibitors [125I]PAD-1 and [125I]PAD-2 and Photoaffinity Labeling experiments on the isolated enzyme, we demonstrate that the ubiquinone ring binds to the NqrA subunit in the regions Leu-32–Met-39 and Phe-131–Lys-138, encompassing the rear wall of a predicted ubiquinone-binding cavity. The quinolone ring and alkyl side chain of aurachin bound to the NqrB subunit in the regions Arg-43–Lys-54 and Trp-23–Gly-89, respectively. These results indicate that the binding sites for ubiquinone and aurachin-type inhibitors are in close proximity but do not overlap one another. Unexpectedly, although the inhibitory effects of PAD-1 and PAD-2 were almost completely abolished by certain mutations in NqrB (i.e. G140A and E144C), the binding reactivities of [125I]PAD-1 and [125I]PAD-2 to the mutated enzymes were unchanged compared with those of the wild-type enzyme. We also found that Photoaffinity Labeling by [125I]PAD-1 and [125I]PAD-2, rather than being competitively suppressed in the presence of other inhibitors, is enhanced under some experimental conditions. To explain these apparently paradoxical results, we propose models for the catalytic reaction of Na+-NQR and its interactions with inhibitors on the basis of the biochemical and biophysical results reported here and in previous work.
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Identification of the Binding Site of the Quinone-Head Group in Mitochondrial Coq10 by Photoaffinity Labeling
2014Co-Authors: Masatoshi Murai, Kohei Matsunobu, Sawako Kudo, Kentaro Ifuku, Makoto Kawamukai, Hideto MiyoshiAbstract:Mitochondrial Coq10 is a ubiquinone (UQ)-binding protein that is a member of the steroidogenic acute regulatory protein (StAR)-related lipid transfer (START) domain superfamily. Deletion of the COQ10 gene was previously shown to cause a marked respiratory defect in Saccharomyces cerevisiae and Schizosaccharomyces pombe, which indicated that Coq10 may support efficient electron transfer between the respiratory complexes; however, its physiological role remains elusive. To elucidate the role of Coq10, we attempted to identify the binding site of UQ in recombinant S. pombe Coq10 expressed in an Escherichia coli cell membrane through Photoaffinity Labeling with the photoreactive UQ probe, UQ-1, in combination with biotinylation of the labeled peptide by means of the so-called click chemistry. Comprehensive proteomic analyses revealed that the quinone-head ring of UQ-1 specifically binds to the N-terminal region of Phe39–Lys45 of Coq10, which corresponds to the ligand-binding pocket of many proteins containing the START domain. The Labeling was completely suppressed in the presence of an excess amount of artificial short-chain UQ analogues, such as UQ2. In the Phe39Ala and Pro41Ala mutants, the extents of Labeling were ∼40 and ∼60%, respectively, of that of wild-type Coq10. While Coq10 has been thought to bind UQ, our work first provides the direct evidence of Coq10 accommodating the quinone-head ring of UQ in its START domain. On the basis of these results, the physiological role of Coq10 has been discussed
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exploring interactions between the 49 kda and nd1 subunits in mitochondrial nadh ubiquinone oxidoreductase complex i by Photoaffinity Labeling
Biochemistry, 2011Co-Authors: Masatoshi Murai, Yuko Mashimo, Judy Hirst, Hideto MiyoshiAbstract:Quinazolines are strong inhibitors of NADH-ubiquinone oxidoreductase (complex I) from bovine heart mitochondria. Using a photoreactive quinazoline, [125I]AzQ, and bovine heart submitochondrial particles (SMPs), we demonstrated previously that [125I]AzQ binds at the interface of the 49 kDa and ND1 subunits in complex I; it labeled a site in the N-terminal (Asp41–Arg63) region of the 49 kDa subunit, suggesting that this region contacts the ND1 subunit [Murai, M., et al. (2009) Biochemistry 48, 688–698]. The labeled region of ND1 could not be identified because it is highly hydrophobic, and the SMPs did not yield sufficient amounts of labeled protein. Here, we describe how Photoaffinity Labeling of isolated complex I by [125I]AzQ yielded sufficient material for identification of the labeled region of the ND1 subunit. The inhibition of the isolated enzyme by AzQ is comparable to that of SMPs. Our results reveal that the labeled site in ND1 is between Asp199 and Lys262, mostly likely in the third matrix loop t...
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characterization of the inhibitor binding site in mitochondrial nadh ubiquinone oxidoreductase by Photoaffinity Labeling using a quinazoline type inhibitor
Biochemistry, 2009Co-Authors: Masatoshi Murai, Koji Sekiguchi, Takaaki Nishioka, Hideto MiyoshiAbstract:The diverse inhibitors of bovine heart mitochondrial complex I (NADH−ubiquinone oxidoreductase) are believed to share a common large binding domain with partially overlapping sites, though it remains unclear how these binding sites relate to each other. To obtain new insight into the inhibitor binding domain in complex I, we synthesized a photoreactive azidoquinazoline {[125I]-6-azido-4-(4-iodophenethylamino)quinazoline, [125I]AzQ}, in which a photolabile azido group was introduced into the toxophoric quinazoline ring to allow specific cross-linking, and carried out a Photoaffinity Labeling study using bovine heart submitochondrial particles. Analysis of the photo-cross-linked proteins by peptide mass fingerprinting and immunoblotting revealed that [125I]AzQ specifically binds to the 49 kDa and ND1 subunits with a frequency of ∼4:1. The cross-linking was completely blocked by excess amounts of other inhibitors such as acetogenin and fenpyroximate. Considerable cross-linking was also detected in the ADP/AT...
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characterization of the inhibitor binding site in mitochondrial nadh ubiquinone oxidoreductase by Photoaffinity Labeling using a quinazoline type inhibitor
Biochemistry, 2009Co-Authors: Masatoshi Murai, Koji Sekiguchi, Takaaki Nishioka, Hideto MiyoshiAbstract:The diverse inhibitors of bovine heart mitochondrial complex I (NADH-ubiquinone oxidoreductase) are believed to share a common large binding domain with partially overlapping sites, though it remains unclear how these binding sites relate to each other. To obtain new insight into the inhibitor binding domain in complex I, we synthesized a photoreactive azidoquinazoline {[(125)I]-6-azido-4-(4-iodophenethylamino)quinazoline, [(125)I]AzQ}, in which a photolabile azido group was introduced into the toxophoric quinazoline ring to allow specific cross-linking, and carried out a Photoaffinity Labeling study using bovine heart submitochondrial particles. Analysis of the photo-cross-linked proteins by peptide mass fingerprinting and immunoblotting revealed that [(125)I]AzQ specifically binds to the 49 kDa and ND1 subunits with a frequency of approximately 4:1. The cross-linking was completely blocked by excess amounts of other inhibitors such as acetogenin and fenpyroximate. Considerable cross-linking was also detected in the ADP/ATP carrier and 3-hydroxybutyrate dehydrogenase, though it was not associated with dysfunction of the two proteins. The partial proteolysis of the [(125)I]AzQ-labeled 49 kDa subunit by V8-protease and N-terminal sequencing of the resulting peptides revealed that the amino acid residue cross-linked by [(125)I]AzQ is within the sequence region Thr25-Glu143 (118 amino acids). Furthermore, examination of fragment patterns generated by exhaustive digestion of the [(125)I]AzQ-labeled 49 kDa subunit by V8-protease, lysylendopeptidase, or trypsin strongly suggested that the cross-linked residue is located within the region Asp41-Arg63 (23 amino acids). The present study has revealed, for the first time, the inhibitor binding site in complex I at the sub-subunit level.
Christina M Woo - One of the best experts on this subject based on the ideXlab platform.
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a binding site hotspot map of the fkbp12 rapamycin frb ternary complex by Photoaffinity Labeling and mass spectrometry based proteomics
Journal of the American Chemical Society, 2019Co-Authors: Hope A Flaxman, Chiafu Chang, Carter H Nakamoto, Christina M WooAbstract:Structural characterization of small molecule binding site hotspots within the global proteome is uniquely enabled by Photoaffinity Labeling (PAL) coupled with chemical enrichment and unbiased analysis by mass spectrometry (MS). MS-based binding site maps provide structural resolution of interaction sites in conjunction with identification of target proteins. However, binding site hotspot mapping has been confined to relatively simple small molecules to date; extension to more complex compounds would enable the structural definition of new binding modes in the proteome. Here, we extend PAL and MS methods to derive a binding site hotspot map for the immunosuppressant rapamycin, a complex macrocyclic natural product that forms a ternary complex with the proteins FKBP12 and FRB. Photo-rapamycin was developed as a diazirine-based PAL probe for rapamycin, and the FKBP12-photo-rapamycin-FRB ternary complex formed readily in vitro. Photoirradiation, digestion, and MS analysis of the ternary complex revealed a McLafferty rearrangement product of photo-rapamycin conjugated to specific surfaces on FKBP12 and FRB. Molecular modeling based on the binding site map revealed two distinct conformations of complex-bound photo-rapamycin, providing a 5.0 A distance constraint between the conjugated residues and the diazirine carbon and a 9.0 A Labeling radius for the diazirine upon photoactivation. These measurements may be broadly useful in the interpretation of binding site measurements from PAL. Thus, in characterizing the ternary complex of photo-rapamycin by MS, we applied binding site hotspot mapping to a macrocyclic natural product and extracted precise structural measurements for interpretation of PAL products that may enable the discovery of new binding sites in the "undruggable" proteome.
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small molecule interactome mapping by Photoaffinity Labeling reveals binding site hotspots for the nsaids
Journal of the American Chemical Society, 2018Co-Authors: Jinxu Gao, Adelphe Mfuh, Yuka Amako, Christina M WooAbstract:Many therapeutics elicit cell-type specific polypharmacology that is executed by a network of molecular recognition events between a small molecule and the whole proteome. However, measurement of the structures that underpin the molecular associations between the proteome and even common therapeutics, such as the nonsteroidal anti-inflammatory drugs (NSAIDs), is limited by the inability to map the small molecule interactome. To address this gap, we developed a platform termed small molecule interactome mapping by Photoaffinity Labeling (SIM-PAL) and applied it to the in cellulo direct characterization of specific NSAID binding sites. SIM-PAL uses (1) photochemical conjugation of NSAID derivatives in the whole proteome and (2) enrichment and isotope-recoding of the conjugated peptides for (3) targeted mass spectrometry-based assignment. Using SIM-PAL, we identified the NSAID interactome consisting of over 1000 significantly enriched proteins and directly characterized nearly 200 conjugated peptides representing direct binding sites of the photo-NSAIDs with proteins from Jurkat and K562 cells. The enriched proteins were often identified as parts of complexes, including known targets of NSAID activity (e.g., NF-κB) and novel interactions (e.g., AP-2, proteasome). The conjugated peptides revealed direct NSAID binding sites from the cell surface to the nucleus and a specific binding site hotspot for the three photo-NSAIDs on histones H2A and H2B. NSAID binding stabilized COX-2 and histone H2A by cellular thermal shift assay. Since small molecule stabilization of protein complexes is a gain of function regulatory mechanism, it is conceivable that NSAIDs affect biological processes through these broader proteomic interactions. SIM-PAL enabled characterization of NSAID binding site hotspots and is amenable to map global binding sites for virtually any molecule of interest.
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Small Molecule Interactome Mapping by Photoaffinity Labeling Reveals Binding Site Hotspots for the NSAIDs
2018Co-Authors: Jinxu Gao, Adelphe Mfuh, Yuka Amako, Christina M WooAbstract:Many therapeutics elicit cell-type specific polypharmacology that is executed by a network of molecular recognition events between a small molecule and the whole proteome. However, measurement of the structures that underpin the molecular associations between the proteome and even common therapeutics, such as the nonsteroidal anti-inflammatory drugs (NSAIDs), is limited by the inability to map the small molecule interactome. To address this gap, we developed a platform termed small molecule interactome mapping by Photoaffinity Labeling (SIM-PAL) and applied it to the in cellulo direct characterization of specific NSAID binding sites. SIM-PAL uses (1) photochemical conjugation of NSAID derivatives in the whole proteome and (2) enrichment and isotope-recoding of the conjugated peptides for (3) targeted mass spectrometry-based assignment. Using SIM-PAL, we identified the NSAID interactome consisting of over 1000 significantly enriched proteins and directly characterized nearly 200 conjugated peptides representing direct binding sites of the photo-NSAIDs with proteins from Jurkat and K562 cells. The enriched proteins were often identified as parts of complexes, including known targets of NSAID activity (e.g., NF-κB) and novel interactions (e.g., AP-2, proteasome). The conjugated peptides revealed direct NSAID binding sites from the cell surface to the nucleus and a specific binding site hotspot for the three photo-NSAIDs on histones H2A and H2B. NSAID binding stabilized COX-2 and histone H2A by cellular thermal shift assay. Since small molecule stabilization of protein complexes is a gain of function regulatory mechanism, it is conceivable that NSAIDs affect biological processes through these broader proteomic interactions. SIM-PAL enabled characterization of NSAID binding site hotspots and is amenable to map global binding sites for virtually any molecule of interest
Julio Martin - One of the best experts on this subject based on the ideXlab platform.
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identification of a putative sordarin binding site incandida albicans elongation factor 2 by Photoaffinity Labeling
Journal of Biological Chemistry, 2001Co-Authors: Juan Manuel Dominguez, Julio MartinAbstract:Candida albicans EF-2 binds sordarin to a single class of binding sites with K(d) = 1.26 microm. Equimolar mixtures of EF-2 and ribosomes, in the presence of a non-hydrolyzable GTP analog, reveal two classes of high affinity sordarin binding sites with K(d) = 0.7 and 41.5 nm, probably due to the existence of two ribosome populations. Photoaffinity Labeling of C. albicans EF-2 in the absence of ribosomes has been performed with [(14)C]GM258383, a photoactivatable sordarin derivative. Labeling is saturable and can be considered specific, because it can be prevented with another sordarin analog. The fragment Gln(224)-Lys(232) has been identified as the modified peptide within the EF-2 sequence, Lys(228) being the residue to which the photoprobe was linked. This fragment is included within the G"-subdomain of EF-2. These results are discussed in the light of the high sordarin specificity toward fungal systems.
Laurence J Miller - One of the best experts on this subject based on the ideXlab platform.
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Secretin occupies a single protomer of the homodimeric secretin receptor complex: insights from Photoaffinity Labeling studies using dual sites of covalent attachment.
The Journal of biological chemistry, 2010Co-Authors: Maoqing Dong, Delia I. Pinon, Polo C.-h. Lam, Andrew Orry, Ruben Abagyan, Laurence J MillerAbstract:The secretin receptor, a prototypic family B G protein-coupled receptor, forms a constitutive homodimeric complex that is stable even in the presence of hormone. Recently, a model of this agonist-bound receptor was built based on high resolution structures reported for amino-terminal domains of other family members. Although this model provided the best solution for all extant data, including 10 Photoaffinity Labeling constraints, a new such constraint now obtained with a position 16 photolabile probe was inconsistent with this model. As the secretin receptor forms constitutive homodimers, we explored whether secretin might dock across both protomers of the complex, an observation that could also contribute to the negative cooperativity observed. To directly explore this, we prepared six secretin analogue probes that simultaneously incorporated two photolabile benzoylphenylalanines as sites of covalent attachment, in positions known to label distinct receptor subdomains. Each bifunctional probe was a full agonist that labeled the receptor specifically and saturably, with electrophoretic migration consistent with Labeling a single protomer of the homodimeric secretin receptor. No band representing radiolabeled receptor dimer was observed with any bifunctional probe. The labeled monomeric receptor bands were cleaved with cyanogen bromide to demonstrate that both of the photolabile benzoylphenylalanines within a single probe had established covalent adducts with a single receptor in the complex. These data are consistent with a model of secretin occupying a single secretin receptor protomer within the homodimeric receptor complex. A new molecular model accommodating all constraints is now proposed.
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molecular approximation between residue 10 of secretin and its receptor demonstrated by Photoaffinity Labeling
Annals of the New York Academy of Sciences, 2006Co-Authors: Maoqing Dong, Laurence J MillerAbstract:Using Photoaffinity Labeling, we have previously explored the molecular approximations between multiple positions of secretin and its receptor. Interestingly, the amino-terminal secretin probe incorporating a photolabile residue in position 1 labels the top of the sixth transmembrane domain of the receptor, whereas other probes with photolabile residue in positions 6, 12, 13, 14, 18, 22, and 26 all label the long amino-terminal domain of the secretin receptor. Recently, we have developed a secretin probe that incorporated a radioiodinatable photolabile p-(4-hydroxybenzoyl)phenylalanine in position 10 and demonstrated that it efficiently labeled the secretin receptor in a saturable and specific manner. In this work, we attempted to further map its domain of Labeling by cyanogen bromide (CNBr) cleavage of the wild-type and mutant receptors. Surprisingly, this position 10 probe labeled the top of the sixth transmembrane domain of the receptor, a domain labeled by the position 1 probe. These data provide an important constraint for modeling the agonist-bound G protein-coupled secretin receptor and should add substantially to our current understanding of the molecular basis of ligand binding of this important receptor.
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differential spatial approximation between secretin and its receptor residues in active and inactive conformations demonstrated by Photoaffinity Labeling
Molecular Endocrinology, 2006Co-Authors: Maoqing Dong, Delia I. Pinon, Keiko Hosohata, Natesa Muthukumaraswamy, Laurence J MillerAbstract:Understanding of the conformational changes in G protein-coupled receptors associated with activation and inactivation is of great interest. We previously used Photoaffinity Labeling to elucidate spatial approximations between photolabile residues situated throughout the pharmacophore of secretin agonist probes and this receptor. The aim of the current work was to develop analogous photolabile secretin antagonist probes and to explore their spatial approximations. The most potent secretin antagonist reported is a pseudopeptide ([ψ4, 5]secretin) in which the peptide bond between residues 4 and 5 was replaced by a ψ(CH2-NH) peptide bond isostere. We have developed a series of [ψ4, 5]secretin analogs incorporating photolabile benzoyl phenylalanine residues in positions 6, 22, and 26. Each bound to the secretin receptor saturably and specifically, with affinity similar to their parental peptide. At concentrations with no measurable agonist activity, each probe covalently labeled the secretin receptor. Peptide...
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identification of peptide ligand binding domains within the human motilin receptor using Photoaffinity Labeling
Journal of Biological Chemistry, 2001Co-Authors: Bernard Coulie, Maoqing Dong, Delia I. Pinon, Bunzo Matsuura, Elizabeth M Hadac, Andrew D Howard, Laurence J MillerAbstract:Abstract The cDNA encoding the human motilin receptor was recently cloned and found to represent a G protein-coupled receptor that is structurally related to the growth hormone secretagogue receptors. Together, these represent a new Class I receptor family. Our aim in the present work is to gain insight into the molecular basis of binding of motilin to its receptor using Photoaffinity Labeling. To achieve this, we developed a Chinese hamster ovary cell line that overexpressed functional motilin receptor (CHO-MtlR; 175,000 sites per cell, with K i = 2.3 ± 0.4 nmmotilin and EC50 = 0.3 ± 0.1 nm motilin) and a radioiodinatable peptide analogue of human motilin that incorporated a photolabilep-benzoyl-l-phenylalanine (Bpa) residue into its pharmacophoric domain. This probe, [Bpa1,Ile13]motilin, was a full agonist at the motilin receptor that increased intracellular calcium in a concentration-dependent manner (EC50 = 1.5 ± 0.4 nm). This photolabile ligand bound specifically and with high affinity to the motilin receptor (K i = 12.4 ± 1.0 nm), and covalently labeled that molecule within its M r = 45,000 deglycosylated core. Cyanogen bromide cleavage demonstrated its covalent attachment to fragments of the receptor having apparent M r = 6,000 and M r = 31,000. These were demonstrated to represent fragments that included both the first and the large second extracellular loop domains, with the latter representing a unique structural feature of this receptor. The spatial approximation of the pharmacophoric domain of motilin with these receptor domains support their functional importance as well.