Crystal structure of bisphosphorylated IGF-1 receptor kinase: insight into domain movements upon kinase activation.

BACKGROUND: The insulin-like growth-factor-1 (IGF-1) receptor, which is widely expressed in cells that have undergone oncogenic transformation, is emerging as a novel target in cancer therapy. IGF-1-induced receptor activation results in autophosphorylation of cytoplasmic kinase domains and enhances their capability to phosphorylate downstream substrates. Structures of the homologous insulin receptor kinase (IRK) exist in an open, unphosphorylated form and a closed, trisphosphorylated form. RESULTS: We have determined the 2.1 A crystal structure of the IGF-1 receptor protein tyrosine kinase domain phosphorylated at two tyrosine residues within the activation loop (IGF-1RK2P) and bound to an ATP analog. The ligand is not in a conformation compatible with phosphoryl transfer, and the activation loop is partially disordered. Compared to the homologous insulin receptor kinase, IGF-1RK2P is trapped in a half-closed, previously unobserved conformation. Observed domain movements can be dissected into two orthogonal rotational components. CONCLUSIONS: Conformational changes upon kinase activation are triggered by the degree of phosphorylation and are crucially dependent on the conformation of the proximal end of the kinase activation loop. This IGF-1RK structure will provide a molecular basis for the design of selective antioncogenic therapeutic agents.

Genetic model organisms in the study of N-glycans.

Recently the genomic sequences of three multicellular eukaryotes, Caenorhabditis elegans, Drosophila melanogaster and Arabidopsis thaliana, have been elucidated. A number of cDNAs encoding glycosyltransferases demonstrated to have a role in N-linked glycosylation have already been cloned from these organisms, e.g., GlcNAc transferases and alpha 1,3-fucosyltransferases. However, many more homologues of glycosyltransferases and other glycan modifying enzymes have been predicted by analysis of the genome sequences, but the predictions of full length open reading frames appear to be particularly poor in Caenorhabditis. The use of these organisms as models in glycobiology may be hampered since they all have N-linked glycosylation repertoires unlike those of mammals. Arabidopsis and Drosophila have glycosylation similar to that of other plants or insects, while our new data from MALDI-TOF analysis of PNGase A-released neutral N-glycans of Caenorhabditis indicate that there exists a range of pauci- and oligomannosidic structures, with up to four fucose residues and up to two O-methyl groups. With all these three 'genetic model organisms', however, much more work is required for a full understanding of their glycobiology.

Molecular cloning and characterization of EndoGlyx-1, an EMILIN-like multisubunit glycoprotein of vascular endothelium.

EndoGlyx-1, the antigen identified with the monoclonal antibody H572, is a pan-endothelial human cell surface glycoprotein complex composed of four different disulfide-bonded protein species with an apparent molecular mass of approximately 500 kDa. Here, we report the purification and peptide analysis of two EndoGlyx-1 subunits, p125 and p140, and the identification of a common, full-length cDNA with an open reading frame of 2847 base pairs. The EndoGlyx-1 cDNA encodes a protein of 949 amino acids with a predicted molecular mass of 105 kDa, found as an entry for an unnamed protein with unknown function in public data bases. A short sequence tag matching the cDNA of this gene was independently discovered by serial analysis of gene expression profiling as a pan-endothelial marker, PEM87. Bioinformatic evaluation classifies EndoGlyx-1 as an EMILIN-like protein composed of a signal sequence, an N-terminal EMI domain, and a C-terminal C1q-like domain, separated from each other by a central coiled-coil-rich region. Biochemical and carbohydrate analysis revealed that p125, p140, and the two additional EndoGlyx-1 subunits, p110 and p200, are exposed on the cell surface. The three smaller subunits show a similar pattern of N-linked and O-linked carbohydrates, as shown by enzyme digestion. Because the two globular domains of EndoGlyx-1 p125/p140 show structural features shared by EMILIN-1 and Multimerin, two oligomerizing glycoproteins implicated in cell-matrix adhesion and hemostasis, it will be of interest to explore similar functions for EndoGlyx-1 in human vascular endothelium.

Identification of tumor antigens in renal cell carcinoma by serological proteome analysis.

We have investigated the suitability of proteomics for identification of tumor-associated antigens. First, we compared the proteomes of nontumorous kidney and renal cell carcinoma (RCC) by two-dimensional gel electrophoresis (2-DE) and silver staining. Protein patterns were markedly different (approximately 800 spots in RCCs versus approximately 1400 spots in kidney). 2-DE immunoblotting revealed five RCC-specific spots, reproducibly reactive with RCC-patient but not healthy donor control sera. Two of these antigens were isolated by preparative 2-DE, and identified by Edman sequencing of tryptic peptides. The first antigen, smooth muscle protein 22-alpha (SM22-alpha), is an actin-binding protein of unknown function predominantly expressed in smooth muscle cells. In situ hybridization revealed that SM22-alpha is not expressed in the malignant cells but in mesenchymal cells of the tumor stroma. The second antigen represents carbonic anhydrase I (CAI), an isoform usually not expressed in kidney. Interestingly, a different isoform (CAXII) has previously been identified by serological expression cloning as an antigen overexpressed in some RCCs. In additional assays, antibodies to recombinant CAI or SM22-alpha were detected in sera from 3/11 or 5/11 RCC patients, respectively, whereas sera from 13 healthy individuals did not react. In conclusion, serological proteome analysis may be a new tool for the identification of tumor-associated antigens.

Correlation of clinical data with proteomics profiles in 24 patients with B-cell chronic lymphocytic leukemia.

The development of human cancer is caused by complex molecular perturbations leading to variable clinical behavior often even in single disease entities. To prove that expression profiling on the protein level can be correlated with clinical data we systematically compared in a pilot study the protein expression patterns obtained by 2-dimensional gel electrophoresis with clinical features in human B-cell chronic lymphocytic leukemia (B-CLL), a disease characterized by broad clinical variability. Statistical methods were devised to analyze the spot pattern from 24 patient samples. This analysis allowed the identification of proteins that clearly discriminated between the patient groups with defined chromosomal characteristics or whose expression levels did correlate with clinical parameters such as patient survival. This report demonstrates that the correlation of large-scale protein expression profiles with clinical data can be used to gain new insights into molecular aspects of a disease. The data described here show that B-CLL patient populations with shorter survival times exhibit changed levels of redox enzymes, heat shock protein 27 and protein disulfide isomerase. These molecules may be potentially involved in drug resistance.

Molecular cloning and characterization of endosialin, a C-type lectin-like cell surface receptor of tumor endothelium.

Endosialin, the antigen identified with monoclonal antibody FB5, is a highly restricted 165-kDa cell surface glycoprotein expressed by tumor blood vessel endothelium in a broad range of human cancers but not detected in blood vessels or other cell types in many normal tissues. Functional analysis of endosialin has been hampered by a lack of information about its molecular structure. In this study, we describe the purification and partial amino acid sequencing of endosialin, leading to the cloning of a full-length cDNA with an open reading frame of 2274 base pairs. The endosialin cDNA encodes a type I membrane protein of 757 amino acids with a predicted molecular mass of 80.9 kDa. The sequence matches with an expressed sequence tag of unknown function in public data bases, named TEM1, which was independently linked to tumor endothelium by serial analysis of gene expression profiling. Bioinformatic evaluation classifies endosialin as a C-type lectin-like protein, composed of a signal leader peptide, five globular extracellular domains (including a C-type lectin domain, one domain with similarity to the Sushi/ccp/scr pattern, and three EGF repeats), followed by a mucin-like region, a transmembrane segment, and a short cytoplasmic tail. Carbohydrate analysis shows that the endosialin core protein carries abundantly sialylated, O-linked oligosaccharides and is sensitive to O-sialoglycoprotein endopeptidase, placing it in the group of sialomucin-like molecules. The N-terminal 360 amino acids of endosialin show homology to thrombomodulin, a receptor involved in regulating blood coagulation, and to complement receptor C1qRp. This structural kinship may indicate a function for endosialin as a tumor endothelial receptor for as yet unknown ligands, a notion now amenable to molecular investigation.

MYCN oncogene and angiogenesis: down-regulation of endothelial growth inhibitors in human neuroblastoma cells. Purification, structural, and functional characterization.

Angiogenesis, the formation of new blood vessels, is seen during embryonic development and tumor progression, but the mechanisms have remained unclear. Recent data indicate that tumor angiogenesis can be induced by cellular oncogenes, leading to the enhanced activity of molecules stimulating angiogenesis. However, activated oncogenes might also facilitate angiogenesis by down-regulating endogenous inhibitors of angiogenesis. We report here that enhanced expression of the N-myc oncogene in human neuroblastoma cells down-regulates three inhibitors of endothelial cell proliferation. One of them was identified by amino acid sequencing as being identical with activin A, a developmentally-regulated protein. Down-regulation involves interaction of the N-myc protein with the activin A promoter. Work is ongoing to characterize the other two endothelial cell inhibitors. We suggest that the N-myc induced down-regulation of angiogenesis inhibitors could contribute to tumor angiogenesis.

Binding of calmodulin to the D2-dopamine receptor reduces receptor signaling by arresting the G protein activation switch.

Signaling by D(2)-dopamine receptors in neurons likely proceeds in the presence of Ca(2+) oscillations. We describe here the biochemical basis for a cross-talk between intracellular Ca(2+) and the D(2) receptor. By activation of calmodulin (CaM), Ca(2+) directly inhibits the D(2) receptor; this conclusion is based on the following observations: (i) The receptor contains a CaM-binding motif in the NH(2)-terminal end of the third loop, a domain involved in activating G(i/o). A peptide fragment encompassing this domain (D2N) bound dansylated CaM in a Ca(2+)-dependent manner (K(D) approximately 0.1 micrometer). (ii) Activation of purified Galpha(i1) by D2N, and D(2) receptor-promoted GTPgammaS (guanosine 5'-(3-O-thio)triphosphate) binding in membranes was suppressed by Ca(2+)/CaM (IC(50) approximately 0.1 micrometer). (iii) If Ca(2+) influx was elicited in D(2) receptor-expressing HEK293 cells, agonist-dependent inhibition of cAMP formation decreased. This effect was not seen with other G(i)-coupled receptors (A(1)-adenosine and Mel(1A)-melatonin receptor). (iv) The D(2) receptor was retained by immobilized CaM and radiolabeled CaM was co-immunoprecipitated with the receptor. Specifically, inhibition by CaM does not result from uncoupling the D(2) receptor from its cognate G protein(s); rather, CaM directly targets the D(2) receptor to block the receptor-operated G protein activation switch.

Identification of syntenin as a protein of the apical early endocytic compartment in Madin-Darby canine kidney cells.

We used flow cytometry to sort and analyze apical and basolateral endocytic vesicles from filter-grown Madin-Darby canine kidney (MDCK) cells after membrane internalization of the lipophilic fluorescent probe trimethylamino-diphenylhexatriene. Western blot analysis of sorted fractions showed enrichment of the early endosomal markers transferrin receptor and the small GTPase Rab5. Two-dimensional gel analysis indicated that the apical and basolateral early endosomes differed significantly in their protein composition. We found nine polypeptides to be specifically enriched in apical or basolateral endocytic vesicles. An apical protein identified by microsequencing was the adaptor molecule syntenin. This protein contains two PDZ domains (PSD-95, Dlg, and ZO-1 homology) that bind syndecan and ephrin-B2 cytoplasmic domains. In MDCK cells, transiently overexpressed Myc-tagged syntenin localized to both plasma membrane domains and to an intracellular vesicular compartment. Syntenin positive vesicles colocalized with internalized transferrin in the perinuclear region. In addition, syntenin colocalized in the apical supranuclear region with Rab5 and Rab11; the latter is a marker for the apical recycling endosomes in MDCK cells.

Metal-dependent nucleotide binding to the Escherichia coli rotamase SlyD.

Upon expression and purification of the first catalytic domain of mammalian adenylate cyclase type 1 (IC1), a 27 kDa contaminant was observed, which was labelled by three radioactive ATP analogues (8-azido-ATP, 3'-O-(4-benzoyl)benzoyl-ATP and 2',3'-dialdehyde-ATP); the protein was purified separately and identified as Escherichia coli SlyD by N-terminal amino acid sequence determination. SlyD is the host protein required for lysis of E. coli upon infection with bacteriophage PhiX174 and has recently been shown to display rotamase (peptidylproline cis-trans-isomerase) activity. The covalent incorporation of ATP analogues into SlyD was promoted by bivalent transition metal ions (Zn(2+)>/=Ni(2+)>Co(2+)>Cu(2+)) but not by Mg(2+) or Ca(2+); this is consistent with the known metal ion specificity of SlyD. ATP, ADP, GTP and UTP suppressed labelling of SlyD with comparable potencies. Similarly, SlyD bound 2',3'-O-(-2,4, 6-trinitrophenyl)-ATP with an affinity in the range of 10 microM, as determined by fluorescence enhancement. This interaction was further augmented in the presence of Zn(2+) (K(d)= approximately 2 microM at saturating Zn(2+)) but not of Mg(2+). Irrespective of the assay conditions, hydrolysis of nucleotides by SlyD was not detected. Upon gel filtration on a Superose HR12 column, SlyD (predicted molecular mass=21 kDa) migrated with an apparent molecular mass of 44 kDa, indicating that the protein was a dimer. However, the migration of SlyD was not affected by the presence of Zn(2+) or of Zn(2+) and ATP. Thus we concluded that SlyD binds nucleotides in the presence of metal ions. These findings suggest that SlyD serves a physiological role that goes beyond that accounted for by its intrinsic rotamase activity, which is observed in the absence of metal ions.

Molecular characterization of Dau c 1, the Bet v 1 homologous protein from carrot and its cross-reactivity with Bet v 1 and Api g 1.

BACKGROUND: Up to 70% of patients with birch pollen allergy exhibit the so-called oral allergy syndrome, an IgE-mediated food allergy. The most frequent and therefore best characterized pollen-fruit syndrome is apple allergy in patients suffering from tree pollen-induced pollinosis. The occurrence of adverse reactions to proteins present in vegetables such as celery and carrots in patients suffering from pollen allergy has also been reported. cDNAs for Bet v 1 homologous proteins have been cloned from celery, apple and cherry. Objective The aim of the study was to identify Bet v 1 homologues from carrot (Daucus carota), to isolate the respective cDNA, to compare the IgE-binding capacity of the natural protein to the recombinant allergen and determine the cross-reactivity to Api g 1 and Bet v 1. METHODS: Molecular characterization of the carrot allergen was performed using IgE-immunoblotting, cross-inhibition assays, N-terminal sequencing, PCR-based cDNA cloning and expression of the recombinant protein in Escherichia coli. RESULTS: A 16-kDa protein from carrot was identified as a major IgE-binding component and designated Dau c 1. Sequencing corresponding cDNAs revealed three extremely similar sequences (Dau c 1.1, 1.2 and 1.3) with an open reading frame of 462 bp coding for 154 amino acid residues. CONCLUSIONS: Purified recombinant Dau c 1.2 was tested in immunoblots displaying IgE-binding capacity comparable to its natural counterpart. Cross-inhibition assays verified the existence of common B-cell epitopes present on Dau c 1, Api g 1 as well as on Bet v 1.

Biochemical characterization of Pru a 2, a 23-kD thaumatin-like protein representing a potential major allergen in cherry (Prunus avium).

BACKGROUND: The prevalence of allergy to fruits and vegetables increased with pollinosis over the last 10 years. So far, clusters of hypersensitivity have been established and corroborated by the molecular characterization of individual cross-reacting allergens. Several case studies demonstrated the existence of allergic reactions to fruits of the subfamily Prunoideae (apricots, cherries, plums and peaches). Here, we present the characterization of a major allergen in cherry. METHODS: Characterization was performed using IgE immunoblotting and immunoblot inhibition, N-terminal sequencing, mass spectroscopy analysis and PCR-based cDNA cloning. RESULTS: A 23-kD protein was identified as IgE-binding component. As all cherry-extract-reactive sera displayed IgE-binding to this band, it was designated a major allergen from Prunus avium (Pru a 2). Sequencing the corresponding cDNA identified Pru a 2 as a thaumatin-like protein belonging to the group 5 of pathogenesis-related proteins. CONCLUSIONS: A thaumatin-like protein in cherry has been identified as a major allergen (Pru a 2). Homologous proteins from the thaumatin family share sequence similarities and should therefore be checked for the capability to elicit an IgE-mediated allergic reaction.

Dual localization of squalene epoxidase, Erg1p, in yeast reflects a relationship between the endoplasmic reticulum and lipid particles.

Squalene epoxidase, encoded by the ERG1 gene in yeast, is a key enzyme of sterol biosynthesis. Analysis of subcellular fractions revealed that squalene epoxidase was present in the microsomal fraction (30,000 x g) and also cofractionated with lipid particles. A dual localization of Erg1p was confirmed by immunofluorescence microscopy. On the basis of the distribution of marker proteins, 62% of cellular Erg1p could be assigned to the endoplasmic reticulum and 38% to lipid particles in late logarithmic-phase cells. In contrast, sterol Delta24-methyltransferase (Erg6p), an enzyme catalyzing a late step in sterol biosynthesis, was found mainly in lipid particles cofractionating with triacylglycerols and steryl esters. The relative distribution of Erg1p between the endoplasmic reticulum and lipid particles changes during growth. Squalene epoxidase (Erg1p) was absent in an erg1 disruptant strain and was induced fivefold in lipid particles and in the endoplasmic reticulum when the ERG1 gene was overexpressed from a multicopy plasmid. The amount of squalene epoxidase in both compartments was also induced approximately fivefold by treatment of yeast cells with terbinafine, an inhibitor of the fungal squalene epoxidase. In contrast to the distribution of the protein, enzymatic activity of squalene epoxidase was only detectable in the endoplasmic reticulum but was absent from isolated lipid particles. When lipid particles of the wild-type strain and microsomes of an erg1 disruptant were mixed, squalene epoxidase activity was partially restored. These findings suggest that factor(s) present in the endoplasmic reticulum are required for squalene epoxidase activity. Close contact between lipid particles and endoplasmic reticulum may be necessary for a concerted action of these two compartments in sterol biosynthesis.

High-level expression and purification of the major birch pollen allergen, Bet v 1.

Bet v 1, the single major allergen from birch pollen, shares IgE epitopes with all major tree pollen allergens from closely related species such as alder, hazel, hornbeam, beech, and European chestnut. Because of high sequence homologies among these allergens and the well-studied cross-reactivities on B cell epitopes, Bet v 1 is a representative model protein which can be used for in vitro studies. The cDNA coding for Bet v 1, the single major allergen from birch pollen, was cloned into the T7-based Escherichia coli expression system pMW 175/BL21(DE3) and synthesized as a nonfusion protein. In contrast to other E. coli systems (e.g., pKK233-2/JM105), this system produces high levels of readily extractable proteins corresponding to 5-10% of E. coli total protein, the percentage varying with culture conditions. The overall yield was 8-10 mg of purified recombinant protein per liter of culture medium. The recombinant allergen was purified by several steps, including ion-exchange and hydrophobic interaction chromatography. The purified recombinant allergen showed identical immunological properties with the respective natural counterpart. The use of recombinant allergens of high purity is expected to result in more accurate diagnostic procedures, but possibly also in a superior immunotherapy of Type I allergic diseases when compared with methods using crude allergen extracts containing various amounts of allergen concentrations.

Subcellular fractionation of polarized epithelial cells and identification of organelle-specific proteins by two-dimensional gel electrophoresis.

Protein targeting and sorting is accomplished by complex vesicular transport processes that are tightly regulated within a cell. This is especially important for epithelial cells because correct delivery of newly synthesized proteins as well as recycling and sorting of internalized membrane proteins is essential for the establishment and preservation of cellular polarity. Many transport events, linking various subcellular compartments, have been analyzed, but many transport mechanisms still remain unresolved. In this study we attempted to identify proteins specifically associated with distinct organelles in murine mammary epithelial cells (EpH4). We isolated subcellular compartments by continuous sucrose gradient centrifugation in order to further analyze their protein composition by high-resolution two-dimensional gel electrophoresis (2-DE). The successful separation of late endosomes (LE), early endosomes (EE) and most of the rough endoplasmic reticulum (RER) was confirmed by subsequent analysis of gradient fractions for compartment-specific enzymes and marker proteins. Both Golgi and plasma membrane (PM) were found to partially co-purify with EE in such gradients. Characteristic polypeptide patterns were revealed on 2-DE gels for fractions enriched in membranes of different origin. Based on improved sample preparation and loading techniques (this issue, C. Pasquali et al., Electrophoresis, 1997, 18, 2573-2581), we were able to identify several proteins by immunoblotting or microsequencing of Coomassie-stained spots. This will be the basis for a further characterization of organelle-specific molecules in epithelial cells as well as for the establishment of a 2-DE reference map of membrane proteins from murine mammary epithelium.

Characterization of a high-affinity monoclonal antibody specific for CD44v6 as candidate for immunotherapy of squamous cell carcinomas.

Variant isoforms of CD44, a family of cell-surface glycoproteins generated by alternative splicing and post-translational modifications, are expressed in a variety of human tumors and play important roles in tumor progression and metastasis formation. The murine monoclonal IgG1 antibody VFF18, specific for an epitope encoded by human CD44 variant exon 6, binds with high affinity to the recombinant protein (Kd = 1.7 x 10(-10) M) as well as to tumor cell lines in vitro, and is suitable for immunohistochemical analysis of human tumors. Screening of more than 500 tumor samples of different histogenesis showed that VFF18 most strongly and uniformly reacts with squamous cell carcinomas (SCC). Detailed analysis of 185 SCC (head and neck, lung, skin) confirmed reactivity of the antibody with 99% of the samples, with intense and homogeneous staining of the tumor cells in the majority of cases, whereas reactivity of VFF18 with normal tissues is limited to certain epithelia and activated lymphocytes. When radiolabelled VFF18 was administered to nude mice bearing human epidermoid carcinoma (A-431) xenograft, fast and selective tumor uptake of the radioimmunoconjugate with a maximum of 18% of the injected dose per gram of tissue was observed. Taken together, these data suggest that mAb VFF18 is a promising targeting vehicle for radioimmunotherapy of squamous cell carcinomas in humans.

Molecular characterization of Api g 1, the major allergen of celery (Apium graveolens), and its immunological and structural relationships to a group of 17-kDa tree pollen allergens.

Individuals suffering from immediate hypersensitivity (type-I allergy) to a particular pollen frequently display intolerance to several foods of plant origin. In this respect, individuals sensitized to birch pollen and/or mugwort pollen frequently display type-I allergic symptoms after ingestion of celery. In this study, we expressed the major allergenic protein of celery, Api g 1, which is responsible for the birch-celery syndrome, in the form of a non-fusion protein. The open reading frame of the cDNA of Api g 1 codes for a protein of 153 amino acids with a molecular mass of 16.2 kDa and 40% identity (60% similarity) to the major allergen of birch pollen, Bet v 1. Furthermore, Api g 1 exhibited similar characteristics to (a) two proteins in parsley induced by fungal infection, (b) the major tree pollen allergens and (c) pathogenesis-related and stress-induced proteins in other plant species. The reactivity of recombinant Api g 1 with IgE antibodies present in sera from celery intolerant patients was comparable to that of the natural celery allergen. Cross-reactivity with Bet v 1 was proven by cross-inhibition experiments, which provides further support for the existence of the birch-celery syndrome and for the suggestion that allergies to some vegetable foods are epiphenomena to allergies caused by inhalation of tree pollen.

[Relation between structure and anti-viral activity in interferon system].

The interferons (IFN) were discovered in 1957 as biological agents interfering with viral replication. IFNs were initially classified according to their sources as leukocyte, fibroblast and immune IFNs. Both leukocyte and fibroblast IFNs are designated as Type I IFNs and immune IFN as a Type II IFN. There are almost twenty related IFNs which belong to the group of Type I IFN. IFN-alpha 2 and IFN-omega 1 are described as members of the large group of Type I IFN. Both natural IFN-alpha 2 and IFN-omega 1 are isolated from human leukocytes. IFN-alpha 2 carries an O-linked carbohydrate moiety, whereas IFN-omega 1 has an N-linked complex oligosaccharide. Recombinant IFN-alpha 2 expressed in E coli. and Sf-9 cells have been characterized. Moreover, recombinant IFN-omega 1 expressed in CHO cells and Sf-9 cells have also been characterized. In the present study, the recombinant human IFN-alpha 2 and IFN-omega 1 were analyzed for their anti-viral activity compared with native products.

Structural and functional characterization of the interaction between 2',3'-dialdehyde guanine nucleotide analogues and the stimulatory G protein alpha-subunit.

We have searched for irreversible ligands which target the guanine nucleotide binding pocket of G protein alpha-subunits by testing the ability of periodate-oxidized 2',3'-dialdehyde guanine nucleotide analogues of GTP (oGTP) and GTP gamma S (oGTP gamma S) to bind to the recombinant alpha-subunit of the stimulatory G protein, rGs alpha-s. oGTP and oGTP gamma S bind to rGs alpha-s in a quasi-irreversible manner via formation of a Schiff's base, which can be reduced with borhydrid resulting in covalent incorporation of [alpha-32P]oGTP and [35S]oGTP gamma S into rGs alpha-s. When bound to rGs alpha-s, oGTP is hydrolyzed and traps the protein in the inactive conformation, while oGTP gamma S persistently activates rGs alpha. Thus, oGTP and oGTP gamma S act as irreversible G protein antagonist and agonist, respectively, and represent a pair of nucleotide analogues suitable as functional and structural tools. Cleavage of covalently labeled rGs alpha-s with cyanogen bromide generates several labeled fragments. Labeled fragments were assigned to the G1 and G4 region of the guanine nucleotide binding pocket using sequence-specific antisera. An additional, labeled fragment was identified by amino-terminal sequencing and corresponded to the helix alpha A in the recently determined crystal structure of the transducin alpha-subunit (Noel, J. P., Hamm, H. E., and Sigler, P. B. (1993) Nature 366, 654-663). In the oGDP-liganded conformation, incorporation occurs predominantly into the G1-fragment, while [35S]oGTP gamma S labels the additional fragments to a similar extent indicating tight packing around the guanine nucleotide binding pocket in the active conformation. Furthermore, rGs alpha-s contains a single acid cleavable bond (Asp317-Pro318), such that formic acid releases a carboxyl-terminal fragment from [alpha-32P]oGTP- and [35S]oGTP gamma S-liganded rGs alpha-s. This fragment contains a single lysine residue (Lys324) which is only labeled by [35S]oGTP gamma S. Lys324 is unique to Gs alpha and lies within its effector binding region. Hence, during the switch from the inactive to the active state, this region undergoes a major conformational change that moves it closer to the nucleotide binding pocket.