Mapping the integrin alpha V beta 3-ligand interface by photoaffinity cross-linking

Integrins are cell surface adhesion molecules involved in mediating cell-extracellular matrix interactions. High-resolution structural data are not available for these heterodimeric receptors. Previous cross-linking studies of integrins aimed at elucidating the nature of the receptor-ligand interface have been limited to identification of relatively large binding domains. To create reagents for "photoaffinity scanning" of the RGD-binding site of human integrin alpha V beta 3, new conformationally constrained ligands were designed. These photoreactive ligands are based on cyclo Ac-[Cys-Asn-Dmt-Arg-Gly-Asp-Cys]-OH, which displays an affinity of 50 nM for alpha V beta 3. This molecular scaffold was modified at the C-terminus by a benzophenone-containing amino acid residue, L-4-benzoylphenylalanine (Bpa). At the N-terminus, a molecular tag was introduced in the form of radioactive iodine or biotin. The newly designed tagged photoreactive RGD-containing ligands display an affinity of 0.5-0.7 microM for alpha V beta 3, and cross-link efficiently and specifically to the receptor. A 100 kDa band corresponding to the beta 3 subunit-ligand conjugate was detected as the major cross-linking product. Cross-linking was dependent upon the presence of Ca2+ and Mg2+ ions, and was competitively inhibited by a nonphotoreactive ligand. Enzymatic and chemical digestions of the radiolabeled photoconjugate enabled identification of a 20-amino acid fragment between positions 99 and 118 in the beta 3 chain of the integrin as the contact domain for ligand at a site adjacent to the C-terminal portion of the RGD triad.     

Fipronil-based photoaffinity probe for Drosophila and human beta 3 GABA receptors.

Modification of the major insecticide fipronil (1) by replacing three pyrazole substituents (hydrogen for both cyano and amino and trifluoromethyldiazirinyl for trifluoromethylsulfinyl) gives a candidate photoaffinity probe (3) of high potency (IC(50) 2-28 nM) in blocking the chloride channel of Drosophila and human beta 3 GABA receptors.     

The cardiac beta-adrenergic receptor. Structural similarities of beta 1 and beta 2 receptor subtypes demonstrated by photoaffinity labeling

The beta-adrenergic receptor photoaffinity ligand p-azido-m-[125I]iodobenzylcarazolol has been used to covalently label the beta 1 and beta 2 adrenergic receptor binding subunits present in left ventricular myocardial membranes derived from mammalian (including human) and nonmammalian species. Covalent incorporation of the photoaffinity ligand into membrane proteins was followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. In the case of the human, canine, porcine, rabbit, and rat left ventricle, all of which contain predominantly or exclusively beta 1-adrenergic receptors, two peptides of Mr approximately equal to 62,000 (major component) and Mr approximately equal to 55,000 (minor component) were specifically labeled and visualized by autoradiography. Photoincorporation into these two bands could be blocked with the appropriate drugs to display a beta 1-adrenergic receptor pharmacological specificity. Simultaneous sodium dodecyl sulfate-polyacrylamide gel electrophoresis of samples from each species revealed that all of the Mr = 62,000 peptides co-migrated suggesting similarity in the beta 1-adrenergic receptor binding subunit peptides in all of these species. The minor component Mr approximately equal to 55,000 appears to be a proteolytic degradation product of the Mr = to 62,000 peptide. Its formation could be decreased by proteinase inhibitors. This suggests that the heterogeneity of the labeling pattern observed in mammalian tissues in this and previous studies may be the result of proteolytic degradation of the receptor subunit which occurs during membrane preparation. Photoaffinity labeling of frog ventricular membranes which contain predominantly beta 2-adrenergic receptors also revealed two peptides of Mr approximately equal to 62,000 (major component) and 55,000 (minor component) with the pharmacological selectivity of a beta 2-adrenergic receptor. These data suggest marked similarities in the beta 1- and beta 2-adrenergic receptor binding subunits of different species and suggest that the pharmacological subtype might be determined by the detailed structure, i.e. amino acid sequence, at the ligand binding sites of the receptor peptide.     

The beta 1-adrenergic receptor of the turkey erythrocyte. Molecular heterogeneity revealed by purification and photoaffinity labeling

The beta 1-adrenergic receptor of turkey erythrocytes has been purified by a combination of affinity and high performance steric exclusion chromatography. These procedures provide preparations with specific activities of greater than 15,000 pmol/mg of protein with an overall recovery of approximately 30% of the receptor activity solubilized from membrane preparations. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of radioiodinated purified receptor reveals two bands of labeled protein with apparent Mr = 40,000 +/- 2,000 and 45,000 +/- 3,000 in a 3-4:1 ratio. These same two peptides can also be labeled specifically and in approximately the same ration in both membranes and purified preparations using the photoaffinity probe 125I-labeled p-azidobenzylcarazolol. When the two purified polypeptides are completely separated by high performance liquid chromatography and subjected to detailed ligand binding studies, identical beta 1-adrenergic specificities are found for the two receptor forms. Preliminary characterization of these two proteins by partial protease digestion suggests a large degree of similarity between them, albeit with some significant differences. These results demonstrate that both purification and photoaffinity labeling identify two polypeptides in turkey erythrocyte membranes as containing a beta 1-adrenergic receptor binding site. The functional and structural relationships of these two forms of the receptor remain to be elucidated.     

Photoinactivation and photoaffinity labeling of tryptophan synthase alpha 2 beta 2 complex by the product analogue 6-azido-L-tryptophan

The photoaffinity reagent 6-azido-L-tryptophan was synthesized by chemical methods. It binds reversibly in the dark to the alpha 2 beta 2 complex of tryptophan synthase of Escherichia coli and forms a quinonoid intermediate with enzyme-bound pyridoxal phosphate (lambda max = 476 nm). The absorbance of this chromophore has been used for spectrophotometric titrations to determine the binding of 6-azido-L-tryptophan (the half-saturation value [S]0.5 = 6.3 microM). Photolysis of the quinonoid form of the alpha 2 beta 2 complex results in time-dependent inactivation of the beta 2 subunit but not of the alpha subunit. The extent of photoinactivation is directly proportional to the absorbance at 476 nm of the quinonoid intermediate prior to photolysis. The substrate L-serine is a competitive inhibitor of 6-azido-L-tryptophan binding and photoinactivation. The competitive inhibitors L-tryptophan, D-tryptophan, and oxindolyl-L-alanine also protect against photoinactivation. The results demonstrate that 6-azido-L-tryptophan is a quasi-substrate for the alpha 2 beta 2 complex of tryptophan synthase and that photolysis of the enzyme-quasi-substrate quinonoid intermediate results in photoinactivation. The modified alpha 2 beta 2 complex retains its ability to bind pyridoxal phosphate and to cleave indole-3-glycerol phosphate, a reaction catalyzed by the alpha subunit. 6-Azido-L-tryptophan (side-chain 1,2,3-14C3 labeled) was synthesized enzymatically from 6-azidoindole and uniformly labeled L-[14C]serine by the alpha 2 beta 2 complex of tryptophan synthase on a preparative scale and has been isolated. Incorporation of 14C label from 6-azido-L-[14C]tryptophan is stoichiometric with inactivation. Our finding that most of the incorporated 14C label is bound in an unstable linkage suggests that an active site carboxyl residue is the major site of photoaffinity labeling by 6-azido-L-tryptophan.     

Ligand-integrin alpha v beta 3 interaction determined by photoaffinity cross-linking: a challenge to the prevailing model

Integrin alpha(V)beta(3) plays a crucial role in angiogenesis, apoptosis, and bone remodeling, mainly by interacting with matrix proteins through recognition of an Arg-Gly-Asp (RGD) motif. Recently, a small cyclic RGD-containing alpha(V)beta(3)-ligand possessing a C-terminal photoreactive group was photo-cross-linked within beta(3)[99-118], in the N-terminus of the beta(3) chain [Bitan G et al. (1999) Biochemistry 38, 3414-3420]. In this paper, a photoreactive group at the N-terminus of the RGD-ligand is shown to interact within beta(3)[167-171], approximately 60 residues C-terminal to the previously identified domain. On the basis of these findings, a model of the putative I-like domain of the beta(3) subunit, homologous to alpha(M)-, alpha(L)-, and alpha(2)-I-domains, reveals that the beta(3)[99-118] and beta(3)[167-171] contact sites are close to each other and are on the opposite side relative to the metal ion-dependent adhesion site (MIDAS) motif. These observations contradict the prevailing model that proposes proximity between metal- and RGD-binding sites on the I-like domain. Our data suggest that either the I-like domain structure predicted for beta(3) is incorrect, or there is no spatial proximity between the RGD-binding site and the MIDAS motif in the I-like domain. Our results indicate that the current models for ligand-receptor interaction should be revisited.     

Identification of a contact domain between echistatin and the integrin alpha(v)beta(3) by photoaffinity cross-linking.

The integrin alpha(v)beta(3) is the major receptor mediating the attachment of osteoclasts to the extracellular matrix in bone and plays a critical role in bone resorption and bone remodeling. Most of the ligands interacting with the alpha(v)beta(3) receptor contain an Arg-Gly-Asp (RGD) motif. Recently, we have identified two small RGD peptides, containing a benzophenone moiety at either the carboxyl or amino terminus, that photo-cross-linked within the beta(3)[99-118] [Bitan, G., et al. (1999) Biochemistry 38, 3414-3420] or the beta(3)[167-171] [Bitan, G., et al. (2000) Biochemistry 39, 11014-11023] sequence, respectively, of the alpha(v)beta(3) receptor in a selective fashion. Here, we report the synthesis of a photoreactive analogue of echistatin (a 49-amino acid peptide), a potent RGD-containing antagonist of the alpha(v)beta(3) receptor both in vitro and in vivo. This bioactive analogue is substituted at position 45 with a p-benzoyl moiety (pBz(2)), located within the flexible C-terminal domain and removed 20 amino acid residues from the R(24)GD(26) triad. This C-terminal domain was reported to contribute to receptor binding affinity by acting as an auxiliary binding site. The radiolabeled (125)I-[Arg(35),Lys(45)(N(epsilon)-pBz(2))]-echistatin photo-cross-links effectively to a site within the beta(3)[209-220] sequence. Residues in this domain have been reported to be part of the metal ion-dependent adhesion site (MIDAS). Receptor fragments overlapping this domain were reported to bind to fibrinogen and block fibrinogen binding to alpha(IIb)beta(3), the platelet integrin receptor. Taken together, position 45 in echistatin, located within an auxiliary binding site in echistatin, cross-links to a site distinct from the two previously reported sites, beta(3)[99-118] and beta(3)[167-171], which cross-link to photophores flanking the RGD triad. These cross-linking data support the hypothesis that the ligand-bound conformation of the integrin beta(3) subunit differs from the known conformation of I domains.     

Thiols and the diazo group in photoaffinity labels

The photoactivable carbene precursor, 2-diazo-3,3,3-tri-fluoro-propionyloxy group, has been introduced recently for light-induced covalent crosslinking in studies of protein-phospholipid interactions in biomem-branes. The diazo group in this reagent has now been shown to undergo reduction in the dark by a number of thiols (dithiothreitol, 2-mercaptoethanol, cysteine and reduced glutathione) used commonly as protective agents for proteins. In contrast, thioglycolate did not cause significant reduction and, therefore, can be safely used as a protective agent. In vesicles formed from phospholipids containing the above photolabel in the fatty acyl chain, dithiothreitol and 2-mercaptoethanol caused reduction. However, cysteine and reduced glutathione caused insignificant reduction of the diazo group, presumably because of their non-permeant nature.     

Fluorescent and photoaffinity labeling derivatives of rhizoxin

A fluorescent probe (D-RZX) and a photoreactive fluorescent probe (AD-RZX) for studying the rhizoxin binding site on tubulin were prepared by the derivatization of rhizoxin (RZX). D-RZX consists of a rhizoxin moiety and a dansyl moiety. AD-RZX has a 5-azidonaphthalene-1-sulfonyl moiety instead of the dansyl moiety of D-RZX. Both D-RZX and AD-RZX bound tubulin in a mutually competitive manner with rhizoxin, indicating their binding to the rhizoxin site on tubulin. AD-RZX bound the rhizoxin site covalently after UV-irradiation, thus showing its usefulness as a photo-affinity probe for labeling of the rhizoxin site.     

A photoaffinity inhibitor of aromatase

Several 7-substituted 4-androstene-3, 17-diones are potent inhibitors of the biosynthesis of estrogens, with the most effective being 7-(4'-amino)phenylthio-4-androstene-3, 17-dione. An azide derivative of this 7-thioether compound has been prepared as a potential photoaffinity inhibitor. The enzyme kinetics of the azide analog were examined under both dark conditions and UV irradiation. In the dark, the azide was a very potent competitive inhibitor, with an apparent K_i of 1.3 nM. Under UV-irradiation, a time-dependent loss of aromatase activity was also observed. These studies indicate that the 7-substituent enhances the affinity of the steroidal analogs for the enzyme site.     

5-Azidoepibatidine: an exceptionally potent photoaffinity ligand for neuronal alpha 4 beta 2 and alpha 7 nicotinic acetylcholine receptors

Racemic 5-azidoepibatidine [(+/-)-1] was synthesized via 5-aminoepibatidine as a candidate photoaffinity ligand with exceptionally high affinity at the mammalian neuronal nicotinic receptors (K(i) values of 0.027 nM for alpha 4 beta 2 and 9.7 nM for alpha 7) and excellent photoreactivity.     

Diazirine based photoaffinity labeling

Diazirines are among the smallest photoreactive groups that form a reactive carbene upon light irradiation. This feature has been widely utilized in photoaffinity labeling to study ligand-receptor, ligand-enzyme and protein-protein interactions, and in the isolation and identification of unknown proteins. This review summarizes recent advances in the use of diazirines in photoaffinity labeling.     

Synthesis and evaluation of oligodeoxyribonucleotides containing an aryl(trifluoromethyl)diazirine moiety as the cross-linking probe: photoaffinity labeling of mammalian DNA polymerase beta.

Photolabile 2'-deoxy- E -5-[4-(3-trifluoromethyl-3 H-diazirin-3-yl)styryl]uridine and its protected phosphoramidite derivatives have been synthesized and introduced into DNA oligomers through solid-phase DNA synthesis. The (trifluoromethyldiazirinyl)stylyl moiety of this nucleoside was found to be sufficiently stable for automated DNA synthesis. In addition, this moiety was found to be stable at 60 degrees C in aqueous solution under the annealing conditions for duplex formation with complementary strands, since >95% of the photolabile nucleoside remained after heating for 1 h. The oligo(dT) 15mer analog bearing the photolabile residue was activated/decomposed by near-UV irradiation. In photoaffinity cross-linking experiments with recombinant rat DNA polymerasebeta, constituted from a 40 kDa polypeptide, using oligo(dT) 15mer analogs bearing the photolabile residue near the 3'-terminus, a covalently bound complex of 45 kDa was obtained in the presence of complementary templates. Thus it was demonstrated that our method for synthesis of photolabile oligodeoxyribonucleotides may be useful for studies of DNA-related enzymes and DNA binding proteins.     

Azidopolynucleotides as photoaffinity reagents.

Polynucleotides containing adenosine and 8-azidoadenosine or inosine and 8-azidoinosine residues have been prepared from mixtures of nucleoside diphosphates using polynucleotide phosphorylase from Escherichia coli. These copolymers can form complexes with polyuridylic or polycytidylic acids respectively. Single stranded poly(adenylic, 8-azidoadenylic acid) [poly(A,z8A)] has been used as a photoaffinity reagent to explore the subunit topography of RNA polymerase from E. coli.     

Identification of the principal binding site for RGD-containing ligands in the alpha(V)beta(3) integrin: a photoaffinity cross-linking study

By superimposing data obtained by photo-cross-linking RGD-containing ligands to the human alpha(V)beta(3) integrin onto the crystal structure of the ectopic domain of this receptor (Xiong et al. (2001) Science 294, 339-345), we have identified the binding site for the RGD triad within this integrin. We synthesized three novel analogues of the 49-amino acid disintegrin, echistatin: [Bpa(21),Leu(28)]-, [Bpa(23),Leu(28)]-, and [Bpa(28)]echistatin. Each contains a photoreactive p-benzoyl-phenylalanyl (Bpa) residue in close proximity to the RGD motif which spans positions 24-26; together, the photoreactive positions flank the RGD motif. The analogues bind with high affinity to the purified recombinant alpha(V)beta(3) integrin, but very poorly to the closely related human alpha(IIb)beta(3) platelet integrin. While echistatin analogues containing Bpa in either position 23 or 28 cross-link specifically and almost exclusively to the beta(3) subunit of alpha(V)beta(3), [Bpa(21),Leu(28)]echistatin cross-links to both the alpha(V) and the beta(3) subunits, with cross-linking to the former favored. [Bpa(23),Leu(28)]echistatin cross-links 10-30 times more effectively than the other two analogues. We identified beta(3)[109-118] as the domain that encompasses the contact site for [Bpa(28)]echistatin. This domain is included in beta(3)[99-118] (Bitan et al. (2000) Biochemistry 39, 11014-11023), a previously identified contact domain for a cyclic RGD-containing heptapeptide with a benzophenone moiety in a position that is similar to the placement of the benzophenone in [Bpa(28)]echistatin relative to the RGD triad. Recently, we identified beta(3)[209-220] as the contact site for an echistatin analogue with a photoreactive group in position 45, near the C-terminus of echistatin (Scheibler et al. (2001) Biochemistry 40, 15117-14126). Taken together, these results support the hypothesis that the very high binding affinity of echistatin to alpha(V)beta(3) results from two distinct epitopes in the ligand, a site including the RGD triad and an auxiliary epitope at the C-terminus of echistatin. Combining our results from photoaffinity cross-linking studies with data now available from the recently published crystal structure of the ectopic domain of alpha(V)beta(3), we characterize the binding site for the RGD motif in this receptor.     

Probing the salmeterol binding site on the beta 2-adrenergic receptor using a novel photoaffinity ligand, [(125)I]iodoazidosalmeterol.

Salmeterol is a long-acting beta2-adrenergic receptor (beta 2AR) agonist used clinically to treat asthma. In addition to binding at the active agonist site, it has been proposed that salmeterol also binds with very high affinity at a second site, termed the "exosite", and that this exosite contributes to the long duration of action of salmeterol. To determine the position of the phenyl ring of the aralkyloxyalkyl side chain of salmeterol in the beta 2AR binding site, we designed and synthesized the agonist photoaffinity label [(125)I]iodoazidosalmeterol ([125I]IAS). In direct adenylyl cyclase activation, in effects on adenylyl cyclase after pretreatment of intact cells, and in guinea pig tracheal relaxation assays, IAS and the parent drug salmeterol behave essentially the same. Significantly, the photoreactive azide of IAS is positioned on the phenyl ring at the end of the molecule which is thought to be involved in exosite binding. Carrier-free radioiodinated [125I]IAS was used to photolabel epitope-tagged human beta 2AR in membranes prepared from stably transfected HEK 293 cells. Labeling with [(125)I]IAS was blocked by 10 microM (-)-alprenolol and inhibited by addition of GTP gamma S, and [125I]IAS migrated at the same position on an SDS-PAGE gel as the beta 2AR labeled by the antagonist photoaffinity label [125I]iodoazidobenzylpindolol ([125I]IABP). The labeled receptor was purified on a nickel affinity column and cleaved with factor Xa protease at a specific sequence in the large loop between transmembrane segments 5 and 6, yielding two peptides. While the control antagonist photoaffinity label [125I]IABP labeled both the large N-terminal fragment [containing transmembranes (TMs) 1-5] and the smaller C-terminal fragment (containing TMs 6 and 7), essentially all of the [125I]IAS labeling was on the smaller C-terminal peptide containing TMs 6 and 7. This direct biochemical evidence demonstrates that when salmeterol binds to the receptor, its hydrophobic aryloxyalkyl tail is positioned near TM 6 and/or TM 7. A model of IAS binding to the beta 2AR is proposed.     

New photoaffinity analogs of paclitaxel.

Two new photoreactive paclitaxel analogs bearing [3H2]-3-(4-benzoyl)phenylpropanoyl group as the photophore as well as radiolabeling unit at the 7 and 10 positions, respectively, are developed. These new photoreactive analogs showed excellent preliminary results on the photoaffinity labeling of tubulin and P-glycoprotein.     

Exofacial photoaffinity labelling of the human erythrocyte sugar transporter

The 4-azidosalicylate derivative of 1,3-bis(D-mannos-4'-yloxy)-2-[2-3H]propylamine (ASA-[2-3H]BMPA) has been tested as a photoaffinity label for the sugar transporter in human erythrocytes. When photolysed in the presence of intact erythrocytes, ASA-[2-3H]BMPA covalently binds to the exofacial surface of the transporter. This labelled protein appears as a broad band in the 4.5 region in sodium dodecyl sulphate (SDS)-polyacrylamide gel electrophoresis. The peak of radiolabel incorporation gives an apparent Mr of approx. 50 000 on 5-20% acrylamide gels. The binding is 80% inhibitable by 320 mM 4,6-O-ethylidene-D-glucose, by 320 mM D-glucose and by 50 microM cytochalasin B. Photoirradiation of a saturating concentration of ASA-BMPA in the presence of erythrocytes results in a 25-30% loss of D-galactose transport activity. From transport inactivation data and estimations of the amount of ASA-[2-3H]BMPA binding to the transporter it is calculated that there are approx. 220 000 exofacial hexose-transport binding sites per erythrocyte. The labelling of the transporter has been carried out using freshly drawn blood and 4-weeks-old transfusion blood. No change in the binding profile on SDS-polyacrylamide gel electrophoresis was observed. Proteolytic digestion of the ASA-[2-3H]BMPA-labelled transporter with either trypsin or alpha-chymotrypsin results in the appearance of a labelled 19 kDa fragment on SDS-polyacrylamide gel electrophoresis.     

Design and synthesis of biotin-tagged photoaffinity probes of jasmonates

Jasmonates (JAs) are a class of oxylipin compounds that play diverse roles in plant defense and development. The F-box protein coronatine insensitive1 (COI1) plays a crucial role in the JA signaling pathway. To determine whether COI1 binds directly to jasmonates, three biotin-tagged photoaffinity probes for JAs, a jasmonic acid photoaffinity probe (PAJA), a JAIle photoaffinity probe (PAJAIle), and a coronatine photoaffinity probe (PACOR), were designed and synthesized based on analysis of JA structure–activity relationships and molecular modeling of the interaction between COI1 and JAs. Among them, PACOR exhibited the most significant biological activity in inhibiting root growth, promoting accumulation of JA-responsive proteins, and triggering COI1–JAZ1 interaction in Arabidopsis seedlings. PACOR is an effective tool for elucidating the interaction between COI1 and JA.     

Development and utilization of 8-azidopurine nucleotide photoaffinity probes

The 8-azidopurine analogs of adenosine and guanine nucleotides have proved to be very useful probes for nucleotide-binding sites. In most systems they have proved to be effective mimics of the natural compounds with 1) both 8-azidoadenosine-3',5'-monophosphate and 8-azidoguanosine-3',5'-monophosphate activating their respective kinases, 2) 8-azidoguanosine-5'-triphosphate effecting tubulin polymerization and activation of adenylate cyclase, and 3) 8-azidoadenosine-5'-triphosphate appearing to be a substrate for a large number of ATPases and several kinases. As photoprobes they have been used to 1) isolate and study active site peptides; 2) determine the membrane sidedness and cellular location of binding sites; 3) detect the availability of various nucleotide-binding sites as cells progress through development, maturation, infectious stages, etc.; 4) study membrane-soluble partitioning of binding sites relative to nucleotide regulation of a biochemical process; 5) detect nucleotide-binding sites exposed by small molecules such as Ca2+ and calmodulin; and 6) detect potential catalytic and regulatory subunits of protein kinases found in preparations that actively phosphorylate endogenous substrates. The difference between the gamma-32P-labeled 8-azidopurine nucleotide triphosphate and the alpha-32P-labeled species has been used to study the in situ hydrolysis of the nucleotides on specific protein receptors and determine the fate of the produced nucleotide diphosphate. Such factors are important in studying the molecular dynamics of such systems as tubulin polymerization, G-actin to F-actin conversions, and GTP activation of adenylate cyclase. A review of techniques used and data obtained with these probes is presented.