Photoaffinity labeling approach to map the Taxol-binding site on the microtubule.
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Animals↗
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Characterization of the binding site for inhibitors of the HPV11 E1-E2 protein interaction on the E2 transactivation domain by photoaffinity labeling and mass spectrometry.
An indandione-containing class of inhibitors abrogates DNA replication of human papillomavirus (HPV) types 6 and 11 by binding reversibly to the transactivation domain (TAD) of the viral E2 protein and inhibiting its interaction with the viral E1 helicase. To locate the binding site of this class of protein-protein interaction inhibitors, a benzophenone derivative was used to generate an irreversibly labeled E2-TAD polypeptide. The single site of covalent modification of the E2-TAD was identified by proteolytic digestions using trypsin, LysC, and V8 proteases and characterization of the resulting peptides by LC-MS procedures. Through this methodology, the benzophenone attachment point was located at the terminal methyl of residue Met101. Evidence further pinpointed the site of photoaffinity attachment to the terminal carbon atom, which is significant in providing a definitive example of the ability to locate photoinduced cross-linking to a polypeptide with atomic resolution using solely mass spectrometric detection. The location of the inhibitor binding site vis-à-vis the Glu39 and Glu100 residues sensitive to mutation for HPV 11 E2-TAD is discussed in relation to the crystal structure of the E2-TAD from the related HPV type 16.
Antiviral Agents↗
Design, synthesis, and characterization of a novel, 4-[2-(diphenylmethoxy)ethyl]-1-benzyl piperidine-based, dopamine transporter photoaffinity label.
The dopamine transporter (DAT) has been implicated strongly in cocaine's reinforcing effects. Many derivatives of piperidine analogs of GBR 12909 have been developed and were found to be quite potent and selective for the DAT. In this regard, most of these derivatives were found to be much more selective for the DAT than conventional GBR compounds e.g. GBR 12909 when their selectivity was compared with the serotonin transporter (SERT). A brief structure-activity relationship (SAR) study has been carried out in the development of a novel photoaffinity ligand which illustrated the effect of the presence of a sterically bulky iodine atom next to the azido group in activity and selectivity for the DAT. This SAR study also led to the development of the compound 4 which is one of the most potent and selective blockers for the DAT known today. The photoaffinity ligand [125I]AD-96-129 was incorporated into the DAT molecule as was demonstrated by immunoprecipitation with serum 16 which is specific for DAT. This photolabeling was antagonized by DAT-specific blockers and was unaffected by specific SERT and norepinephrine transporter (NET) blockers indicating interaction of this novel ligand with the DAT.
Animals↗
Photoaffinity labeling of the brevetoxin receptor on sodium channels in rat brain synaptosomes.
Brevetoxin, a neurotoxin isolated from the marine dinoflagellate Ptychodiscus brevis, has been derivatized into a photoaffinity probe by carbodiimide linkage to p-azidobenzoic acid. Rosenthal analysis of a tritiated p-azidobenzoate brevetoxin derivative indicates that specific binding of the toxin occurs at two distinct and separate sites, with Kd and Bmax values of 0.21 nM and 2.12 pmol/mg of protein for the high affinity site and 50.7 nM and 91.5 pmol/mg of protein for the low affinity site, respectively. Binding of tritiated photoaffinity probe to the high affinity/low capacity site can be displaced in a competitive manner by native brevetoxin (Kd = 1.9 nM), demonstrating a specific competitive interaction with the receptor site. Rat brain synaptosomes, covalently labeled with the brevetoxin photoaffinity probe, were subjected to detergent solubilization. The covalently labeled membrane protein was estimated to have a Stokes radius of 55 +/- 3 A. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed specific labeling of a 260-kDa protein. Treatment with 2-mercaptoethanol and neuraminidase resulted in retention of brevetoxin binding to this high molecular weight protein. The affinity-purified membrane protein-brevetoxin photoaffinity probe complex was specifically recognized by a sodium channel antibody directed against the intracellular side of transmembrane segment IS6. The sodium channel alpha subunit is implicated as the specific site of brevetoxin interaction.
Affinity Labels↗
Differential docking of high-affinity peptide ligands to type A and B cholecystokinin receptors demonstrated by photoaffinity labeling.
Type A and B cholecystokinin (CCK) receptors are highly homologous members of the class-I family of G protein-coupled receptors that bind CCK with high affinity. However, they have divergent structural specificities, with the type A receptor requiring seven carboxyl-terminal residues including a sulfated tyrosine and the type B receptor requiring only the carboxyl-terminal tetrapeptide. The aim of this work was to utilize affinity labeling to determine spatial approximations with photolabile p-benzoyl-l-phenylalanine (Bpa) residues sited at each end of CCK as docked at the type B CCK receptor, contrasting this with analogous work using similar probes docked at the type A receptor. Both probes were fully efficacious, potent agonists that stimulated intracellular calcium in receptor-bearing CHO-CCKBR cells (EC(50) values: Bpa(24) probe, 41 +/- 9 pM; Bpa(33) probe, 15 +/- 3.3 pM). They bound specifically, with high affinity (K(i) values: Bpa(24) probe, 0.60 +/- 0.17 nM; Bpa(33) probe, 0.58 +/- 0.11 nM). Cyanogen bromide cleavage of the covalently labeled receptor suggested the first extracellular loop as the region of labeling by each probe, distinct from the type A CCK receptor regions labeled using the same probes (third loop and amino-terminal tail, respectively). This was confirmed by subsequent enzymatic and chemical cleavage of labeled wild-type and mutant receptors. Sequential cycles of Edman degradation of labeled receptor fragments identified the specific residues within loop one labeled by each probe (Bpa(24) probe labeled Phe(122); Bpa(33) probe labeled Thr(119)). This provides a direct demonstration of distinct modes of docking the same high-affinity ligand to highly homologous receptors.
Amino Acid Sequence↗
Stereoselective synthesis of (E)- and (Z)-beta-bromostyrene containing trifluoromethyldiazirine for photoaffinity labeling.
Summary A convenient procedure for the synthesis of (E)- and (Z)-beta-bromostyrene containing trifluoromethyldiazirine is described, which involves the stereoselective reduction of the corresponding trifluoromethyldiazirinyl beta,beta-dibromostyrene without damaging the photophor. The synthetic route easily introduced deuterium, which can be utilized for the detection of a photolabeled component by MS spectrometry, after construction of a trifluoromethyldiazirinyl skeleton.
Diazomethane↗
Ecdysteroid 7,9(11)-dien-6-ones as potential photoaffinity labels for ecdysteroid binding proteins.
Three ecdysteroid 7,9(11)-dien-7-ones (dacryhainansterone, 25-hydroxydacryhainansterone and kaladasterone) were prepared by dehydration of the corresponding 11a-hydroxy ecdysteroids (ajugasterone C, turkesterone and muristerone A, respectively). The biological activities of the dienones in the Drosophila melanogaster B(II) cell bioassay, which reflect the affinity for the ecdysteroid receptor complex, showed that the dienones retain high biological activity. Irradiation at 350 nm of the ecdysteroid dienones (100 nM) with bacterially-expressed dipteran and lepidopteran ecdysteroid receptor proteins (DmEcR/DmUSP or CfEcR/CfUSP), followed by loading with [(3)H]ponasterone A revealed that irradiation of dacryhainansterone or kaladasterone resulted in blocking of >70% of the specific binding sites. Thus, ecdysteroid dienones show considerable potential as photoaffinity analogues for ecdysteroid binding proteins.
Animals↗
Photoaffinity labeling of the Klenow fragment with 8-azido-dATP.
The photoaffinity compound 8-azido-dATP was used as a probe for the deoxyribonucleoside triphosphate-binding site of the large fragment of DNA polymerase I. Azido-dATP specifically modified a saturable binding site within the Klenow fragment, and each of the four natural deoxyribonucleoside triphosphate substrates competed with labeling at this site in proportion to its binding constant, as previously defined by equilibrium dialysis. Analysis of tryptic peptides after azido-dATP modification revealed five major cross-linking products, which apparently arose from five distinct photoadducts formed near Tyr-766.
Adenosine Triphosphate↗
Synthesis of photoreactive phosphatidic acid analogues displaying activatory properties on cyclic AMP-phosphodiesterases. Photoaffinity labeling of an isoform of phosphodiesterase.
We have previously shown that phosphatidic acid (PA) is a specific activator of some isoforms of type 4 cyclic nucleotide phosphodiesterases (PDE 4) and that accumulation of endogenous PA can, in this way, influence the cAMP signaling pathway in different cell types. Enzyme activation depends on direct binding of the effector to specific sites carried by the enzyme. To identify the binding domain, photoactivatable phosphatidic acid analogues 1-azidoPA (12) and 2-azidoPA (7 and 15), potentially suitable for covalent labeling of PDE4, have been synthesized. The ability of phospholipases A(2) and D to hydrolyze unnatural phospholipids has been considered in this paper. The effect of 1-azidoPA (12) and 2-azidoPA (7 and 15) on the activity of a recombinant PA-sensitive isoform PDE4D3 was evaluated. The three compounds were able to activate the enzyme with different efficiencies. A tritiated analogue of 15 was synthesized and used in PDE4D3 labeling experiments, which showed that this PA analogue was specifically and covalently linked to the enzyme after UV irradiation. Photoactivatable analogues thus appear as suitable tools for the characterization of PA binding sites.
3',5'-Cyclic-AMP Phosphodiesterases↗
Structural reorganization of the acetylcholine binding site of the torpedo nicotinic receptor as revealed by dynamic photoaffinity labeling.
We explored the structural changes that occur at the acetylcholine binding site of the Torpedo marmorata nicotinic receptor during activation by the tritiated photoactivatable agonist (diazocyclohexadienoylpropyl)trimethylammonium ([(3)H]DCTA). We quantified the incorporation of radioactivity into the receptor subunits as a function of the mixing time of [(3)H]DCTA with the receptor by using a rapid-mixing device adapted with a photochemical quenching system. A saturable increase of the specific photolabeling on the alpha and gamma subunits was observed with a half-time of about 2 minutes. We further analyzed this photoincorporation either after rapid mixing for 500 ms or after equilibration for 50 minutes. Under these conditions, [(3)H]DCTA explored transient state(s) and the stable desensitized state, respectively. Comparative analyses showed that at a probe concentration of 10 microM the relative variation of photoincorporation was more pronounced for the gamma subunit (three- to fourfold) than for the alpha subunit (about twofold). By contrast, the relative distribution of radioactivity among alpha-subunit labeled residues (alphaTyr190, alphaCys192, alphaCysC193, and alphaTyr198) did not change. Altogether, these results reveal that during the course of agonist-induced receptor desensitization, the site-lining peptide loops, which belong to adjacent alpha and gamma subunits, move closer to each other.
Acetylcholine↗
Structurally related peptide agonist, partial agonist, and antagonist occupy a similar binding pocket within the cholecystokinin receptor. Rapid analysis using fluorescent photoaffinity labeling probes and capillary electrophoresis.
The molecular basis of ligand binding to receptors provides important insights for drug development. Here, we explore domains of the cholecystokinin (CCK) receptor that are critical for ligand binding, using a novel series of fluorescent photolabile probes, receptor proteolysis, and rapid high resolution separation of peptide fragments by capillary electrophoresis. Each probe incorporated the same fluorophore and a photolabile p-benzoylphenylalanine at the amino terminus of the pharmacophoric domain (residue 24 of CCK-33) of CCK analogues representing full agonist, partial agonist, and antagonist of this receptor. Each was used to label the CCK receptor expressed on Chinese hamster ovary-CCKR cells, with the labeled domain then released by cyanogen bromide cleavage. Capillary electrophoresis with laser-induced fluorescence detection achieved an on-capillary mass sensitivity of 1.6 attomoles (10(-18) mol), with an excellent signal-to-noise ratio. Each of the biologically divergent, but structurally similar probes saturably and specifically labeled the same receptor domain, consistent with conservation of "docking" determinants. This had an apparent mass of 2.9 kDa, most consistent with the first extracellular loop domain. An additional probe having its site of covalent attachment in a different region of the probe (residue 29 of CCK-33) labeled a distinct receptor fragment with differential migration on capillary electrophoresis (third extracellular loop). Identification of the specific receptor residue(s) covalently linked to the amino-terminal probes must await further fragmentation and sequence analysis.
Amino Acid Sequence↗
Characterization of a 2',5'-oligoadenylate (2-5A)-dependent 37-kDa RNase L: azido photoaffinity labeling and 2-5A-dependent activation.
Upregulation of key components of the 2',5'-oligoadenylate (2-5A) synthetase/RNase L pathway has been identified in extracts of peripheral blood mononuclear cells from individuals with chronic fatigue [corrected] syndrome, including the presence of a low molecular weight form of RNase L. In this study, analysis of 2',5'-Oligoadenylate (2-5A) binding and activation of the 80- and 37-kDa forms of RNase L has been completed utilizing photolabeling/immunoprecipitation and affinity assays, respectively. Saturation of photolabeling of the 80- and the 37-kDa RNase L with the 2-5A azido photoprobe, [(32)P]pApAp(8-azidoA), was achieved. Half-maximal photoinsertion of [(32)P]pApAp(8-azidoA) occurred at 3.7 x 10(-8) m for the 80-kDa RNase L and at 6.3 x 10(-8) m for the 37-kDa RNase L. Competition experiments using 100-fold excess unlabeled 2-5A photoaffinity probe, pApAp(8-azidoA), and authentic 2-5A (p(3)A(3)) resulted in complete protection against photolabeling, demonstrating that [(32)P]pApAp(8-azidoA) binds specifically to the 2-5A-binding site of the 80- and 37-kDa RNase L. The rate of RNA hydrolysis by the 37-kDa RNase L was three times faster than the 80-kDa RNase L. The data obtained from these 2-5A binding and 2-5A-dependent activation studies demonstrate the utility of [(32)P]pApAp(8-azidoA) for the detection of the 37-kDa RNase L in peripheral blood mononuclear cell extracts.
Adenine Nucleotides↗
Hormone interactions to Leu-rich repeats in the gonadotropin receptors. III. Photoaffinity labeling of human chorionic gonadotropin with receptor Leu-rich repeat 4 peptide.
Human chorionic gonadotropin (hCG) binds to the extracellular N-terminal domain, exodomain, of its receptor, and the resulting hCG-exodomain complex is thought to modulate the membrane associated domain, endodomain, of the receptor to generate hormone signal. The bulk of the exodomain is speculated to assume a crescent structure consisting of eight to nine Leu-rich repeats (LRRs), which may provide the hormone contact sites. Unfortunately, little experimental evidence is available for the precise hormone contact points in the exodomain and the endodomain. The two preceding articles (Song, Y., Ji, I., Beauchamp, J., Isaacs, N., and Ji, T. (2001) J. Biol. Chem. 276, 3426-3435; Song, Y., Ji, I., Beauchamp, J., Isaacs, N., and Ji, T. (2001) J. Biol. Chem. 276, 3436-3442) show that putative LRR2 and LRR4 are crucial for hormone binding. In particular, the N-terminal region of LRR4 assumes the hydrophobic core of the LRR4 loop, whereas the C-terminal region is crucial for signal generation. However, it is unclear whether LRR4 interacts hCG and the endodomain and how it might be involved in signal generation. In this article, our affinity labeling results present the first evidence that the N-terminal region of LRR4 interacts with hCG, preferentially the hCGalpha subunit and that the hCG/LRR4 complex interacts with exoloop 2 of the endodomain. This interaction offers a mechanism to generate hormone signal.
Amino Acid Motifs↗
pH-dependent changes in photoaffinity labeling patterns of the H1 influenza virus hemagglutinin by using an inhibitor of viral fusion.
The hemagglutinin (HA) protein undergoes a low-pH-induced conformational change in the acidic milieu of the endosome, resulting in fusion of viral and cellular membranes. A class of compounds that specifically interact with the HA protein of H1 and H2 subtype viruses and inhibit this conformational change was recently described (G. X. Luo et al., Virology 226:66-76, 1996, and J. Virol. 71:4062-4070, 1997). In this study, purified HA trimers (bromelain-cleaved HA [BHA]) are used to examine the properties and binding characteristics of these inhibitors. Compounds were able to inhibit the low-pH-induced change of isolated trimers, as detected by resistance to digestion with trypsin. Protection from digestion was extremely stable, as BHA-inhibitor complexes could be incubated for 24 h in low pH with almost no change in BHA structure. One inhibitor was prepared as a radiolabeled photoaffinity analog and used to probe for specific drug interactions with the HA protein. Analysis of BHA after photoaffinity analog binding and UV cross-linking revealed that the HA2 subunit of the HA was specifically radiolabeled. Cross-linking of the photoaffinity analog to BHA under neutral (native) pH conditions identified a stretch of amino acids within the alpha-helix of HA2 that interact with the inhibitor. Interestingly, cross-linking of the analog under acidic conditions identified a different region within the HA2 N terminus which interacts with the photoaffinity compound. These attachment sites help to delineate a potential binding pocket and suggest a model whereby the BHA is able to undergo a partial, reversible structural change in the presence of inhibitor compound.
Amino Acid Sequence↗
Trp82 and Tyr332 are involved in two quaternary ammonium binding domains of human butyrylcholinesterase as revealed by photoaffinity labeling with [3H]DDF.
Purified butyrylcholinesterase (BuChE) was photolabeled by [3H]-p-N, N-dimethylamino benzene diazonium ([3H]DDF) to identify the quaternary ammonium binding sites on this protein [Ehret-Sabatier, L. , Schalk, I., Goeldner, M., and Hirth, C. (1992) Eur. J. Biochem. 203, 475-481]. The covalent photoincorporation occurs with a stoichiometry of one mole of probe per mole of inactivated site and could be fully prevented by several cholinergic inhibitors such as tacrine or tetramethylammonium. After complete deglycosylation of the enzyme using N-glycosidase F, the alkylated protein was trypsinolyzed and the digests were analyzed by HPLC coupled to ES-MS. A direct comparison of tryptic fragments from labeled and unlabeled BuChE allowed us to identify the tryptic peptide Tyr61-Lys103 as carrying the probe. Purification of the labeled peptides by anion-exchange chromatography gave a major radioactive peak which was further fractionated by reversed-phase HPLC leading to three, well-resolved, radioactive peaks. Microsequencing revealed that two of these peaks contained an overlapping sequence starting at Tyr61, while the third peak contained a sequence extending from Thr315. Radioactive signals could be unambiguously attributed to positions corresponding to residues Trp82 and Tyr332. This labeling study establishes the existence of two different binding domains for quaternary ammonium in BuChE and exemplifies additional cation/pi interactions in cholinergic proteins. This work strongly supports the existence of a peripheral anionic site in BuChE, implying residue Tyr332 as a key element.
Amino Acid Sequence↗
Probing the phosphoinositide binding site of the clathrin assembly protein AP-2 with photoaffinity labels.
The relative binding specificities of the subunitsof bovine assembly protein AP-2 for the phosphatidylinositol polyphosphates (PtdInsPn) and inositol polyphosphates (InsPn) were determined by photoaffinitylabeling. Three types of benzophenone-containing photoprobes were employed: (i) the water-solubleP-1- or P-2-tethered p-benzoyldihydrocinnamoyl-InsPn (BZDC-InsPn) analogs, (ii) P-1-linked phosphotriester PtdInsPn analogs that sampled the interface between the water and lipid phases, and (iii) sn-1-O-acyl-linked PtdInsPn analogs that interacted with proteins penetrating the bilayer. The InsPn and PtdInsPn probes bind with highest selectivity and affinity to the two alpha subunit isoforms, with certain probes and conditions resulting in strong labeling of the 50-kDa mu subunit. Three main conclusions were reached: (i) head group recognition predominated over acyl chain recognition, (ii) the PtdInsPn binding site of alpha-AP-2 prefers more highly phosphorylated species, and (iii) the protein-acyl chain interactions showed high capacity but low selectivity.
Adaptor Proteins, Vesicular Transport↗
The design, synthesis, and initial evaluation of benzophenone-containing peptides as potential photoaffinity labels of oligosaccharyltransferase.
The benzophenone photophore was incorporated into protected tripeptides and tetrapeptides as photoactivatable probes to study the multimeric enzyme oligosaccharyltransferase (OST). These peptides contain the -Asn-X-Thr- sequon which is required for OST-catalyzed N-glycosylation. Two tripeptides, Bz-Asn-Bpa-Thr-NH2 (3b) and Bz-Asn-Lys[N epsilon-(4-Bz)Bz]-Thr-NH2 (4b), were found to be good OST substrates. They were competitive inhibitors versus standard peptide substrate [14C]Bz-Asn-Leu-Thr-NH2 and their Ki values were determined to be 41 +/- 6 microM and 21 +/- 6 microM, respectively, using synthetic (GlcNAc)2-PP-dolichol.
Benzophenones↗
alpha-Conotoxin GI benzoylphenylalanine derivatives. (1)H-NMR structures and photoaffinity labeling of the Torpedo californica nicotinic acetylcholine receptor.
alpha-Conotoxins are small peptides from cone snail venoms that function as nicotinic acetylcholine receptor (nAChR)-competitive antagonists differentiating between nAChR subtypes. Current understanding about the mechanism of these selective interactions is based largely on mutational analyses, which identify amino acids in the toxin and nAChR that determine the energetics of ligand binding. To identify regions of the nAChR involved in alpha-conotoxin binding by use of photoactivated cross-linking, two benzoylphenylalanine (Bpa) analogs of alpha-conotoxin GI, GI(Bpa12) and GI(Bpa4), were synthesized by replacing the respective residues with Bpa, and their (1)H-NMR structures were determined. Both analogs preserved the GI conformation, but only GI(Bpa12) displaced (125)I-labeled GI from the Torpedo californica nAChR. (125)I-labeled GI(Bpa12) bound to two sites on the receptor (K(d) 13 and 1800 nM), and on UV irradiation specifically photolabeled the alpha, gamma and delta subunits. Photolabeling sites were mapped by selective proteolysis and enzymatic deglycosylation, combined with SDS/PAGE, HPLC and Edman degradation. In the alpha subunit, cobratoxin-inhibited incorporation was limited to the 22-kDa fragment beginning at alphaSer173 and containing the agonist-binding site segment C. In the gamma subunit, radioactivity was localized to two distinct peptides containing agonist-binding site segments F and D: nonglycosylated 24-kDa and glycosylated 13-kDa fragments starting at gammaAla167 and gammaAla49, respectively. The labeling of these fragments is discussed in terms of a model of GI(Bpa12) bound to the extracellular domain of the Torpedo nAChR homology model derived from the cryo-electron microscopy structure of Torpedo marmorata nAChR and X-ray crystal structures of snail acetylcholine-binding protein complexes with agonists and antagonists.
Amino Acid Sequence↗