Direct Photoaffinity Labeling of Cysteine-295 of α-Tubulin by Guanosine 5′-Triphosphate Bound in the Nonexchangeable Site
1998; Elsevier BV; Volume: 273; Issue: 16 Linguagem: Inglês
10.1074/jbc.273.16.9894
ISSN1083-351X
AutoresRuoli Bai, Kevin K Choe, John B. Ewell, Nga Y. Nguyen, Ernest Hamel,
Tópico(s)DNA and Nucleic Acid Chemistry
ResumoThe αβ-tubulin heterodimer has two high affinity guanosine 5′-triphosphate binding sites, so that purified tubulin usually contains two molecules of bound guanosine nucleotide. Half this nucleotide is freely exchangeable with exogenous guanine nucleotide, and its binding site has been readily localized to the β-subunit. The remaining nonexchangeable guanosine 5′-triphosphate can only be released from tubulin by denaturing the protein. We replaced the exchangeable site nucleotide of tubulin with 2′-deoxyguanosine 5′-diphosphate, exposed the resulting tubulin to ultraviolet light, degraded the protein, and isolated ribose-containing peptide derived from the nonexchangeable site. A large cyanogen bromide peptide was recovered, and its further degradation with endoproteinase Glu-C established that cysteine-295 of α-tubulin was the major reactive amino acid cross-linked to guanosine by ultraviolet irradiation. The αβ-tubulin heterodimer has two high affinity guanosine 5′-triphosphate binding sites, so that purified tubulin usually contains two molecules of bound guanosine nucleotide. Half this nucleotide is freely exchangeable with exogenous guanine nucleotide, and its binding site has been readily localized to the β-subunit. The remaining nonexchangeable guanosine 5′-triphosphate can only be released from tubulin by denaturing the protein. We replaced the exchangeable site nucleotide of tubulin with 2′-deoxyguanosine 5′-diphosphate, exposed the resulting tubulin to ultraviolet light, degraded the protein, and isolated ribose-containing peptide derived from the nonexchangeable site. A large cyanogen bromide peptide was recovered, and its further degradation with endoproteinase Glu-C established that cysteine-295 of α-tubulin was the major reactive amino acid cross-linked to guanosine by ultraviolet irradiation. Tubulin, the major component of microtubules, is a protein heterodimer containing two tightly bound guanosine nucleotides. Half is readily replaced with exogenous nucleotide and is known as the exchangeable site nucleotide. The other half remains bound to tubulin unless the protein is denatured and is therefore described as being located in the nonexchangeable site (1Weisenberg R.C. Borisy G.G. Taylor E.W. Biochemistry. 1968; 7: 4466-4477Crossref PubMed Scopus (915) Google Scholar, 2Kobayashi T. J. Biochem. (Tokyo). 1974; 76: 201-204Crossref PubMed Scopus (31) Google Scholar, 3Levi A. Cimino M. Mercanti D. Calissano P. Biochim. Biophys. Acta. 1974; 365: 450-453Crossref PubMed Scopus (27) Google Scholar). Besides being hydrolyzed during microtubule assembly, the E site 1The abbreviations used are: E site, the exchangeable nucleotide binding site of tubulin; N site, the nonexchangeable nucleotide binding site of tubulin; dGDP-, dGTP-, GDP-, and [8-14C]GDP-tubulin, tubulin with the indicated nucleotide bound in the E site; EP-GC, endoproteinase Glu-C; PAGE, polyacrylamide gel electrophoresis; HPLC, high performance liquid chromatography; Tricine, N-[2-hydroxy-1,1-bis (hydroxymethyl)ethyl]glycine. 1The abbreviations used are: E site, the exchangeable nucleotide binding site of tubulin; N site, the nonexchangeable nucleotide binding site of tubulin; dGDP-, dGTP-, GDP-, and [8-14C]GDP-tubulin, tubulin with the indicated nucleotide bound in the E site; EP-GC, endoproteinase Glu-C; PAGE, polyacrylamide gel electrophoresis; HPLC, high performance liquid chromatography; Tricine, N-[2-hydroxy-1,1-bis (hydroxymethyl)ethyl]glycine. nucleotide has been proposed as a controlling element in microtubule dynamics (4Mitchison T. Kirschner M.W. Nature. 1984; 312: 237-242Crossref PubMed Scopus (2330) Google Scholar). In this model microtubule stability is determined by whether or not terminal E site GTP has been hydrolyzed, with hydrolysis leading to rapid polymer disassembly. The E site nucleotide has been readily accessible to analysis because it can be replaced with radiolabeled nucleotide from the medium, permitting its precise localization to the β-subunit of tubulin (5Shivanna B.D. Mejillano M.R. Williams T.D. Himes R.H. J. Biol. Chem. 1993; 268: 127-132Abstract Full Text PDF PubMed Google Scholar, 6Hesse J. Thierauf M. Ponstingl H. J. Biol. Chem. 1987; 262: 15472-15475Abstract Full Text PDF PubMed Google Scholar, 7Linse K. Mandelkow E.M. J. Biol. Chem. 1988; 263: 15205-15210Abstract Full Text PDF PubMed Google Scholar, 8Jayaram B. Haley B.E. J. Biol. Chem. 1994; 269: 3233-3242Abstract Full Text PDF PubMed Google Scholar). The N site GTP has eluded investigation, presumably because it is deeply integrated into the structure of tubulin. As an initial approach to the N site GTP, we decided to attempt to locate it within the tubulin heterodimer. Our studies were stimulated by those of Shivanna et al. (5Shivanna B.D. Mejillano M.R. Williams T.D. Himes R.H. J. Biol. Chem. 1993; 268: 127-132Abstract Full Text PDF PubMed Google Scholar). Using boronate column chromatography of tryptic peptides from tubulin bearing [3H]GTP and exposed to UV light, these workers established that a covalent bond had been formed between the E site nucleotide and Cys-12 of β-tubulin. Our strategy is outlined in Fig. 1. Employing dGTP-driven assembly, we prepared tubulin in which the E site GDP/GTP was replaced with dGDP (9Hamel E. Lustbader J. Lin C.M. Biochemistry. 1984; 23: 5314-5325Crossref PubMed Scopus (37) Google Scholar) to minimize formation of guanosine-containing peptides that would interact with the boronate matrix with its affinity forcis-diols. Because dGDP-tubulin still contained covalently bound ribose following photoactivation, we isolated the ribose-enriched peptide(s). We found that a single peptide derived from the N site was retained by the boronate matrix, and the reactive amino acid was Cys-295 of α-tubulin. Bovine brain tubulin was purified as before (10Hamel E. Lin C.M. Biochemistry. 1984; 23: 4173-4184Crossref PubMed Scopus (251) Google Scholar). The boronate matrix (Affi-gel 601) was from Bio-Rad, Texas Red hydrazide was from Pierce, EP-GC (sequencing grade) was from Promega, and 16% polyacrylamide gels, polyvinylidene difluoride, and nitrocellulose membranes were from Novex. Tubulin (1, 800 mg) at 20 mg/ml in 1 m monosodium glutamate (pH 6.6), 2 mmdGTP, and 1 mm MgCl2 was incubated at 37 °C for 20 min. Polymer was harvested by centrifugation at 35,000 rpm for 20 min in a 37 °C rotor, and the polymer pellet was homogenized in 25 ml of 1 m glutamate on ice. Denatured protein was removed by centrifugation at 0 °C (20 min at 35,000 rpm), and another assembly/disassembly cycle was performed with the supernatant. Four cycles were performed, yielding 510 mg of dGDP-tubulin. The reaction mixture contained dGDP-tubulin at 12 mg/ml in 0.2 m4-morpholineethanesulfonate (pH 6.9) and 2 mmMgCl2-EGTA-dithiothreitol. About 2–4 ml of this mixture was spread in plastic weighing boats on ice and irradiated at 254 nm for 15 min (2750 μW/cm2). N-Ethylmaleimide (6 mm) was added to the mixture, which was left at 4 °C overnight. Protein was harvested by centrifugation at 15,000 rpm for 15 min. Residual protein was precipitated with 50% trichloroacetic acid and harvested by centrifugation. The combined pellets were dried by lyophilization. The tubulin (2 mg/ml) was treated with alkaline phosphatase (5Shivanna B.D. Mejillano M.R. Williams T.D. Himes R.H. J. Biol. Chem. 1993; 268: 127-132Abstract Full Text PDF PubMed Google Scholar) for 3 h at 37 °C in 0.1 m Tris-HCl (pH 9) and then with 20 mg/ml CNBr in 70% formic acid for 24 h in the dark. CNBr was removed by repeated lyophilization and resuspension of the peptides in water. For EP-GC digestion, CNBr peptides were dissolved in 1% SDS, diluted 10-fold with 0.1 m phosphate buffer (pH 9), and treated at an enzyme/substrate ratio of 1:50 in 0.1m phosphate buffer (pH 7.8) at 22 °C for 20 h. Peptide mixtures were applied to Affi-gel 601 (5Shivanna B.D. Mejillano M.R. Williams T.D. Himes R.H. J. Biol. Chem. 1993; 268: 127-132Abstract Full Text PDF PubMed Google Scholar) (column, 1 × 15 cm; flow rate, 1 ml/min). CNBr peptides were dissolved in boiling 1 m Tris-HCl (pH 8.0), and the solution was diluted 10-fold with 50 mmglycine-NaOH buffer (pH 10). CNBr/EP-GC peptides were dissolved in boiling 1% SDS and diluted 10-fold with the 50 mm glycine buffer. After sample application, the column was washed with glycine buffer until elution of unbound peptides was complete. Bound peptides were eluted with 0.1 m formic acid. Peptide-containing fractions were pooled and processed for SDS-PAGE. GDP-tubulin was induced to assemble with dGTP (9Hamel E. Lustbader J. Lin C.M. Biochemistry. 1984; 23: 5314-5325Crossref PubMed Scopus (37) Google Scholar), with four assembly cycles performed. Nucleotide content of the resulting dGDP-tubulin is shown in Fig. 2 C, in comparison with standards (Fig. 2 A) and the nucleotide content of the original GDP-tubulin (Fig. 2 B). Despite the multiple dGTP-driven assembly cycles, there was 5–10% residual GDP, presumably bound to the E site. This could have resulted from incomplete exchange, due to the lower affinity of dGTP for the E site (9Hamel E. Lustbader J. Lin C.M. Biochemistry. 1984; 23: 5314-5325Crossref PubMed Scopus (37) Google Scholar), copolymerization of GDP-tubulin with dGTP-tubulin (11Hamel E. Batra J.K. Lin C.M. Biochemistry. 1986; 25: 7054-7062Crossref PubMed Scopus (18) Google Scholar), and/or slow leaching of N site GTP into the medium from denatured tubulin. Photolabeling conditions were studied with [8-14C]GDP-tubulin (12Grover S. Hamel E. Eur. J. Biochem. 1994; 222: 163-172Crossref PubMed Scopus (34) Google Scholar), and a 15-min exposure to UV light seemed optimum (Fig. 3). To follow putative labeling of the N site, we compared orcinol reactivity (detects ribose but not deoxyribose (13Ashwell G. Methods Enzymol. 1957; 3: 73-105Crossref Scopus (603) Google Scholar)) of dGDP-tubulin and GDP-tubulin following exposure to UV light and recovery of protein by gel filtration in 8 m urea. Both dGDP-tubulin and GDP-tubulin became orcinol-reactive following UV irradiation, and orcinol reactivity of protein did not occur without irradiation. The GDP-tubulin was as much as 3–4-fold more reactive than the dGDP-tubulin, indicating reduced efficiency of the covalent interaction of N site GTP relative to E site GDP. We attempted to quantitate the extent of the orcinol reaction, but the tubulin requirement was prohibitive. Moreover, tubulin quenched color obtained with ribose standards. We proceeded to CNBr digestion of UV-exposed dGDP-tubulin. An additional experiment confirmed that we had a ribose-containing peptide. The entire digest was subjected to SDS-PAGE and transferred to nitrocellulose, and the membrane treated by a method designed to label glycoproteins (periodate oxidation and then reaction with Texas Red hydrazide). Although multiple bands were observed following staining of a duplicate sample with Coomassie Blue (not shown), there was a single prominent band following the periodate/hydrazide reaction (Fig. 4, gel A). This result required that the tubulin be exposed to UV light. We proceeded to removal of ribose-containing peptides by chromatography of the CNBR digest of dGDP-tubulin on the boronate matrix. No peptide bound to the boronate matrix unless the tubulin had been UV irradiated. The bound peptide fraction was eluted with formic acid and subjected to SDS-PAGE. There were two peptide bands (Fig. 4, gel B), both of which were sequenced (Table I). The major b1 peptide yielded an amino acid sequence for 17 cycles consistent with the large CNBr peptide spanning residues 204–302 of α-tubulin (14Ponstingl H. Krauhs E. Little M. Kempf T. Proc. Natl. Acad. Sci. U. S. A. 1981; 78: 2757-2761Crossref PubMed Scopus (248) Google Scholar), with the exception of Cys-213 (cysteine residues cannot be identified by automated Edman degradation). We therefore conclude that this peptide has been cross-linked to the N site GTP by UV irradiation. The minor b2 peptide yielded a sequence for 13 cycles (except for Cys-12) consistent with the CNBr peptide spanning residues 2–72 of β-tubulin (15Krauhs E. Little M. Kempf T. Hofer-Warbinek R. Ade W. Ponstingl H. Proc. Natl. Acad. Sci. U. S. A. 1981; 78: 4156-4160Crossref PubMed Scopus (275) Google Scholar). This peptide includes the Cys-12 residue that cross-links to the E site nucleotide (5Shivanna B.D. Mejillano M.R. Williams T.D. Himes R.H. J. Biol. Chem. 1993; 268: 127-132Abstract Full Text PDF PubMed Google Scholar), and it presumably derives from the residual E site GDP in the dGDP-tubulin (Fig. 2 C).Table IAmino acid sequence analysis of the major guanosine-containing peptides derived from dGDP-tubulinCycleCNBr peptidesCNBr/EP-GC peptidesb1b2c1c2 1Val (α-204) (Pro)Arg (β-2)Ile (α-291) (Gly)Ile (β-4) 2AspGluThrVal 3AsnIleAsnHis 4GluValAlaIle 5AlaHisX (α-295 = Cys)Gln 6IleIlePheAla 7TyrGlnGluGly 8AspAlaProGln 9IleGlyAlaX (β-12 = Cys)10X (α-213 = Cys)GlnAsnGly11ArgX (β-12 = Cys)GlnAsn12ArgGlyMetGln13AsnAsnIle14LeuGly15AspAla16IleLys17Glu Open table in a new tab To further define the reactive N site amino acid in the α-204–302 peptide, we employed several proteases. The best results were obtained with EP-GC, which cleaves at the carboxyl side of glutamate and, to a lesser extent, aspartate residues. A CNBr digest of dGDP-tubulin was further digested with this protease and applied to the boronate matrix. The acid eluate on SDS-PAGE yielded two peptide bands (Fig. 4,gel C), which were sequenced (Table I). The c1 peptide spanned residues 291–302 of α-tubulin, with the exception of Cys-295. The c2 peptide spanned residues 3–19 (except Cys-12) of β-tubulin, again including the E site Cys-12 residue (5Shivanna B.D. Mejillano M.R. Williams T.D. Himes R.H. J. Biol. Chem. 1993; 268: 127-132Abstract Full Text PDF PubMed Google Scholar). Because a modified amino acid residue would not be identified by sequential Edman degradation and because every expected amino acid except Cys-295 was identified in peptide c1, we conclude that it is α-tubulin Cys-295 that reacts covalently with the N site GTP during UV irradiation. Presumably the same photoreaction described by Shivannaet al. (5Shivanna B.D. Mejillano M.R. Williams T.D. Himes R.H. J. Biol. Chem. 1993; 268: 127-132Abstract Full Text PDF PubMed Google Scholar) that occurs between the E site GTP and β-Cys-12 occurs between the N site GTP and α-Cys-295. This mechanism included loss of the C-6 carbonyl from guanine, with the covalent bond formed between the S atom of cysteine and the C-5 atom of guanosine. We sought evidence for this by micro-HPLC coupled to mass spectrometry. The boronate-bound samples used to generate the b1, c1, and c2 peptides were examined by this technique, and each HPLC peak was subjected to high resolution mass spectrometry. Mass spectral peaks were obtained corresponding to peptides cross-linked to the guanosine fragment predicted by the mechanism of Shivanna et al. (5Shivanna B.D. Mejillano M.R. Williams T.D. Himes R.H. J. Biol. Chem. 1993; 268: 127-132Abstract Full Text PDF PubMed Google Scholar) for α-residues 204–302 and 291–302 and β residues 4–22 (Table II).Table IILC-MS molecular masses of ribose-containing peptides derived from dGDP-tubulin following digestion with CNBr or with CNBr and EP-GCPeptideCalculated weightsObtained weight (±S.D.)No adduct derived from guanosine+ Guanosine (−2H)2-aAssumes a reaction between the S atom of cysteine and C-8 of guanosine.+ Guanosine fragment2-bThe mechanism proposed by Shivanna et al. (5) included elimination of the C-6 carbonyl group of guanosine, with the cross-link being between the S atom of cysteine and C-5 of guanosine.CNBr digestion α-Tubulin 204–302 (b1)2-cThe calculated masses for this peptide include oneN-ethylmaleimide moiety, presumably at position 213. The unmodified peptide has a calculated molecular weight of 11,090.6.11,215.711,496.911,471.911,472.8 ± 1.4CNBr/EP-GC digestion α-Tubulin 291–302 (c1)1338.521619.741594.761595.40 ± 4.80 β-Tubulin 4–22 (c2)2099.402380.622355.642355.73 ± 5.112-a Assumes a reaction between the S atom of cysteine and C-8 of guanosine.2-b The mechanism proposed by Shivanna et al. (5Shivanna B.D. Mejillano M.R. Williams T.D. Himes R.H. J. Biol. Chem. 1993; 268: 127-132Abstract Full Text PDF PubMed Google Scholar) included elimination of the C-6 carbonyl group of guanosine, with the cross-link being between the S atom of cysteine and C-5 of guanosine.2-c The calculated masses for this peptide include oneN-ethylmaleimide moiety, presumably at position 213. The unmodified peptide has a calculated molecular weight of 11,090.6. Open table in a new tab Analysis of sequence homologies in proteins has been invaluable in predicting protein function and ligand binding sites, including nucleotide sites. Tubulin sequences, however, are sufficiently different from those of other GTP binding proteins to have been a theoretical challenge for precise identification of GTP binding sites (16Sternlicht H. Yaffe M.B. Farr G.W. FEBS Lett. 1987; 214: 226-235Crossref PubMed Scopus (60) Google Scholar, 17Burns R.G. J. Cell Sci. 1995; 108: 2123-2130PubMed Google Scholar). Nonetheless, the extensive sequence homology between α- and β-tubulin, combined with localization of the E site to β-tubulin (5Shivanna B.D. Mejillano M.R. Williams T.D. Himes R.H. J. Biol. Chem. 1993; 268: 127-132Abstract Full Text PDF PubMed Google Scholar, 6Hesse J. Thierauf M. Ponstingl H. J. Biol. Chem. 1987; 262: 15472-15475Abstract Full Text PDF PubMed Google Scholar, 7Linse K. Mandelkow E.M. J. Biol. Chem. 1988; 263: 15205-15210Abstract Full Text PDF PubMed Google Scholar, 8Jayaram B. Haley B.E. J. Biol. Chem. 1994; 269: 3233-3242Abstract Full Text PDF PubMed Google Scholar), led to the widespread assumption that the N site is on α-tubulin. Our studies confirm this prediction and represent the first successful attempt at defining tubulin amino acid residues near the N site GTP by cross-linking experiments. Based on the proposed mechanism of the photoinduced covalent bond between a cysteine residue and GTP (5Shivanna B.D. Mejillano M.R. Williams T.D. Himes R.H. J. Biol. Chem. 1993; 268: 127-132Abstract Full Text PDF PubMed Google Scholar), this reaction should occur between the S atom of α-Cys-295 and C-5 of the guanine moiety. It is particularly interesting that the homology between α- and β-tubulin in the region of α-Cys-295 is not extensive, and that there are no obvious sequence homologies between the α-tubulin peptide we have isolated and the β-tubulin E site peptides previously reported (5Shivanna B.D. Mejillano M.R. Williams T.D. Himes R.H. J. Biol. Chem. 1993; 268: 127-132Abstract Full Text PDF PubMed Google Scholar, 6Hesse J. Thierauf M. Ponstingl H. J. Biol. Chem. 1987; 262: 15472-15475Abstract Full Text PDF PubMed Google Scholar, 7Linse K. Mandelkow E.M. J. Biol. Chem. 1988; 263: 15205-15210Abstract Full Text PDF PubMed Google Scholar, 8Jayaram B. Haley B.E. J. Biol. Chem. 1994; 269: 3233-3242Abstract Full Text PDF PubMed Google Scholar). This may reflect the profoundly different properties of nucleotide bound at the E and N sites. Nogales et al. (18Nogales E. Wolf S.G. Downing K.H. Nature. 1998; 391: 199-203Crossref PubMed Scopus (1791) Google Scholar) presented a detailed model of the tubulin α-β-dimer based on electron crystallographic analysis of paclitaxel-stabilized sheets of antiparallel protofilaments induced by zinc. In this model each subunit had a bound guanine nucleotide in a Rossmann-type fold, confirming the prediction that the α-subunit contained the N site. There was little difference in the overall conformation of the two subunits. In the zinc sheets all GTP binding sites were shielded by the adjacent subunit, explaining the nonexchangeability of all polymer-bound nucleotide in both E (19Weisenberg R.C. Deery W.J. Dickinson P.J. Biochemistry. 1976; 15: 4248-4254Crossref PubMed Scopus (200) Google Scholar) and N sites. Although specific αβ pairs in soluble dimers could not be identified, Nogales et al. (18Nogales E. Wolf S.G. Downing K.H. Nature. 1998; 391: 199-203Crossref PubMed Scopus (1791) Google Scholar) proposed that in the heterodimer the N site on α-tubulin remained shielded by β-tubulin, explaining its inaccessibility, whereas the E-site on β-tubulin would become exposed to the medium, explaining the rapid equilibration of bound with free nucleotide. This model agreed with observations indicating that the β-subunit was at the plus end of microtubules (20Mitchison T.J. Science. 1993; 261: 1044-1077Crossref PubMed Scopus (141) Google Scholar). This “steric hindrance” explanation of N site properties, however, fails to explain the total nonexchangeability of nucleotide bound to α-tubulin, because the αβ-heterodimer readily dissociates into its subunits (21Detrich III, H.W. Williams Jr., R.C. Biochemistry. 1978; 17: 3900-3907Crossref PubMed Scopus (145) Google Scholar, 22Sackett D.L. Zimmerman D.A. Wolff J. Biochemistry. 1989; 28: 2662-2667Crossref PubMed Scopus (22) Google Scholar, 23Mejillano M.R. Himes R.H. Biochemistry. 1989; 28: 6518-6524Crossref PubMed Scopus (27) Google Scholar, 24Sackett D. Lippoldt R.E. Biochemistry. 1991; 30: 3511-3517Crossref PubMed Scopus (49) Google Scholar, 25Panda D. Roy S Bhattacharyya B. Biochemistry. 1992; 31: 9709-9716Crossref PubMed Scopus (33) Google Scholar, 26Shearwin K.E. Perez-Ramirez B. Timasheff S. Biochemistry. 1994; 33: 885-893Crossref PubMed Scopus (23) Google Scholar). Yet N site GTP remains totally nonexchangeable in cells (27Spiegelman B.M. Penningroth S.M. Kirschner M.W. Cell. 1977; 12: 587-600Abstract Full Text PDF PubMed Scopus (94) Google Scholar) and through multiple cycles of assembly with radiolabeled GTP (9Hamel E. Lustbader J. Lin C.M. Biochemistry. 1984; 23: 5314-5325Crossref PubMed Scopus (37) Google Scholar, 12Grover S. Hamel E. Eur. J. Biochem. 1994; 222: 163-172Crossref PubMed Scopus (34) Google Scholar) or GTP analogs (Ref. 28Hamel E. Lin C.M. Biochemistry. 1990; 29: 2720-2729Crossref PubMed Scopus (12) Google Scholar and Fig. 2 C). Moreover, no evidence for nucleotide exchange into the N site of dissociated tubulin monomer could be found when such exchange was specifically sought (26Shearwin K.E. Perez-Ramirez B. Timasheff S. Biochemistry. 1994; 33: 885-893Crossref PubMed Scopus (23) Google Scholar). Thus, the N site remains inaccessible in the dissociated α-subunit, as well as in α-tubulin bound to the β-subunit in heterodimer. In terms of the current studies, in the model of Nogales et al. (18Nogales E. Wolf S.G. Downing K.H. Nature. 1998; 391: 199-203Crossref PubMed Scopus (1791) Google Scholar) Cys-295 of α-tubulin appears to be too distant from the N site GTP to account for the covalent interaction we have observed. Because the reaction occurs rapidly and only a single amino acid reacts with the GTP, it seems unlikely that the reaction is nonspecific or results from tubulin denaturation. Most likely there is a conformational change in soluble heterodimer relative to zinc polymer that brings Cys-295 close enough to the guanine residue for the photoreaction to occur. An alternative possibility, particularly in view of the apparently low efficiency of the α-Cys-295 reaction as compared with the β-Cys-12 reaction, is that the reactive residue is close to the guanine moiety only when the αβ-heterodimer dissociates. A substantial conformational change could occur in the α-monomer so that additional portions of the polypeptide chain prevent access to the N site. This could explain the observed total nucleotide nonexchangeability in steric terms despite reversible subunit dissociation. Finally, photoaffinity studies do not identify amino acid residues that are essential components of ligand binding sites, but only residues that are close to ligands occupying such sites. Our work demonstrates that in soluble tubulin a significant proportion of α-tubulin Cys-295 is in close proximity to the guanine residue occupying the N site. We thank Dr. R. H. Himes for helpful discussions regarding use of the boronate affinity technique for isolation of guanosine-containing peptides.
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