Assembly of the Bacteriophage T4 Helicase
2002; Elsevier BV; Volume: 277; Issue: 23 Linguagem: Inglês
10.1074/jbc.m111951200
ISSN1083-351X
AutoresFaoud T. Ishmael, Stephen C. Alley, Stephen J. Benkovic,
Tópico(s)Enzyme Structure and Function
ResumoThe bacteriophage T4 59 protein (gp59) plays an essential role in recombination and replication by mediating the assembly of the gene 41 helicase (gp41) onto DNA. gp59 is required to displace the gp32 single-stranded binding protein on the lagging strand to expose a site for helicase binding. To gain a better understanding of the mechanism of helicase assembly, the architecture and stoichiometry of the gp41-gp59 complex were investigated. Both the N and C termini of gp41 were found to lie close to or in the gp41-gp41 subunit interface and interact with gp59. The site of interaction of gp41 on gp59 is proximal to Cys-215 of gp59. Binding of gp41 to gp59 stimulates a conformational change in the protein resulting in hexamer formation of gp59, and gp59 likewise stimulates oligomer formation of gp41. The gp59 subunits in this complex are arranged in a head to head orientation, such that Cys-42 of one subunit is in close proximity to Cys-42 on an adjacent subunit, and Cys-215 on one subunit is close to Cys-215 on a neighboring subunit. As the helicase is loaded onto DNA, a conformational change in the gp41-gp59 complex occurs, which may serve to displace gp32 from the lagging strand and load the hexameric helicase in its place. The bacteriophage T4 59 protein (gp59) plays an essential role in recombination and replication by mediating the assembly of the gene 41 helicase (gp41) onto DNA. gp59 is required to displace the gp32 single-stranded binding protein on the lagging strand to expose a site for helicase binding. To gain a better understanding of the mechanism of helicase assembly, the architecture and stoichiometry of the gp41-gp59 complex were investigated. Both the N and C termini of gp41 were found to lie close to or in the gp41-gp41 subunit interface and interact with gp59. The site of interaction of gp41 on gp59 is proximal to Cys-215 of gp59. Binding of gp41 to gp59 stimulates a conformational change in the protein resulting in hexamer formation of gp59, and gp59 likewise stimulates oligomer formation of gp41. The gp59 subunits in this complex are arranged in a head to head orientation, such that Cys-42 of one subunit is in close proximity to Cys-42 on an adjacent subunit, and Cys-215 on one subunit is close to Cys-215 on a neighboring subunit. As the helicase is loaded onto DNA, a conformational change in the gp41-gp59 complex occurs, which may serve to displace gp32 from the lagging strand and load the hexameric helicase in its place. DNA replication is a multistaged reaction that requires the recruitment of proteins, assembly of replication forks at an origin, and template-directed replication of DNA. The replication machinery in the T4 bacteriophage is composed of three units, the primosome (a complex of the primase (gp61) bound to the helicase (gp41)), the holoenzyme (composed of a DNA polymerase (gp43), a sliding clamp (gp45), and a clamp loader (gp44/62)), and the single-stranded binding protein gp32. An initial event in the replication process is the recruitment and assembly of the primosome at the DNA replication fork. Formation of the primosome on the lagging strand most likely begins with assembly of the helicase. The bacteriophage T4 helicase plays an essential role in DNA replication and recombination during phage infection of Escherichia coli. The helicase is needed to unwind duplex DNA ahead of the holoenzyme to allow nucleotide incorporation during replication and is required to unwind duplex DNA for pairing of single-strand DNA with new partners in recombination (1Benkovic S.J. Valentine A.M. Salinas F. Annu. Rev. Biochem. 2001; 70: 181-208Crossref PubMed Scopus (277) Google Scholar). gp41 forms a hexameric ring that encircles the lagging strand of the replication fork and unwinds the duplex in a 5′–3′ direction by hydrolyzing ATP (1Benkovic S.J. Valentine A.M. Salinas F. Annu. Rev. Biochem. 2001; 70: 181-208Crossref PubMed Scopus (277) Google Scholar). During DNA replication, the gp41 helicase is intimately associated with the gp61 primase, which synthesizes the RNA primers used in discontinuous lagging strand synthesis. The formation of this primosome is needed for efficient unwinding of duplex DNA during replication as well as productive primer synthesis (2Dong F. von Hippel P.H. J. Biol. Chem. 1996; 271: 19625-19631Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar, 3Hinton D.M. Nossal N.G. J. Biol. Chem. 1987; 262: 10873-10878Abstract Full Text PDF PubMed Google Scholar). Loading of the gp41 replicative helicase at the bacteriophage T4 DNA replication fork requires the displacement of single-stranded DNA-binding proteins (gp32) coating the lagging strand. The helicase assembly protein, gp59, is required to load the helicase under these conditions, thus assuming a critical role in bacteriophage T4 DNA replication as well as recombination (4Cunningham R.P. Berger H. Virology. 1977; 80: 67-82Crossref PubMed Scopus (69) Google Scholar, 5Karam J.D. Molecular Biology of Bacteriophage T4. American Society for Microbiology, Washington, D. C.1994: 38-53Google Scholar, 6Mosig G. Molecular Biology of Bacteriophage T4. American Society for Microbiology, Washington, D. C.1994Google Scholar). The gp59 protein is a 26-kDa, basic (pI = 10.18) protein that exists as a monomer in solution (7Lefebvre S.D. Morrical S.W. J. Mol. Biol. 1997; 272: 312-326Crossref PubMed Scopus (33) Google Scholar, 8Yonesaki T. J. Biol. Chem. 1994; 269: 1284-1289Abstract Full Text PDF PubMed Google Scholar). It has a high affinity for DNA and can bind duplex DNA, ssDNA, 1The abbreviations used are: ssDNAsingle-stranded DNADTTdithiothreitolgp41cgp41 with a cysteine residue added to the C terminus (Cys-476)DMFdimethylformamideSA-HRPstreptavidin linked to horseradish peroxidaseSulfo-SBEDsulfosuccinimide-2-[6-(biotinamido)-2-(p-azidobenzamido) hexanoamido]ethyl-1,3′-dithiopropionateBMH1,6 bis- maleimidohexaneAMCAN-6-7-amino-4-methylcoumarin-3-acetamidoAMCA-HPDPN-[6-(7- amino-4-methylcoumarin-3-acetamido)hexyl]-3′-(2′-pyridyldithio)propionamideITCisothermal titration calorimetryTMEAtris(2-maleimidoethyl)amineTCEPtris(2-carboxyethyl)phosphine hydrochlorideMALDImatrix-assisted laser desorption ionizationHPLChigh pressure liquid chromatographyATPγSadenosine 5′-O-(thiotriphosphate) and forked DNA substrates (7Lefebvre S.D. Morrical S.W. J. Mol. Biol. 1997; 272: 312-326Crossref PubMed Scopus (33) Google Scholar, 9Jones C.E. Mueser T.C. Nossal N.G. J. Biol. Chem. 2000; 275: 27145-27154Abstract Full Text Full Text PDF PubMed Google Scholar). The protein is composed of almost all α-helices and contains a C-terminal and an N-terminal domain (10Mueser T.C. Jones C.E. Nossal N.G. Hyde C.C. J. Mol. Biol. 2000; 296: 597-612Crossref PubMed Scopus (44) Google Scholar). Mueser et al. (10Mueser T.C. Jones C.E. Nossal N.G. Hyde C.C. J. Mol. Biol. 2000; 296: 597-612Crossref PubMed Scopus (44) Google Scholar) have shown that gp59 binds to forked DNA substrates with higher affinity than ssDNA. Furthermore, it has been proposed that duplex DNA binds to the N-terminal domain, whereas ssDNA binds to the C-terminal domain based on models generated from the gp59 crystal structure and its similarity to high mobility group proteins (10Mueser T.C. Jones C.E. Nossal N.G. Hyde C.C. J. Mol. Biol. 2000; 296: 597-612Crossref PubMed Scopus (44) Google Scholar). single-stranded DNA dithiothreitol gp41 with a cysteine residue added to the C terminus (Cys-476) dimethylformamide streptavidin linked to horseradish peroxidase sulfosuccinimide-2-[6-(biotinamido)-2-(p-azidobenzamido) hexanoamido]ethyl-1,3′-dithiopropionate 1,6 bis- maleimidohexane N-6-7-amino-4-methylcoumarin-3-acetamido N-[6-(7- amino-4-methylcoumarin-3-acetamido)hexyl]-3′-(2′-pyridyldithio)propionamide isothermal titration calorimetry tris(2-maleimidoethyl)amine tris(2-carboxyethyl)phosphine hydrochloride matrix-assisted laser desorption ionization high pressure liquid chromatography adenosine 5′-O-(thiotriphosphate) gp59 acts via direct contacts with gp41 and has been shown to interact with the helicase both on and off DNA (8Yonesaki T. J. Biol. Chem. 1994; 269: 1284-1289Abstract Full Text PDF PubMed Google Scholar, 11Morrical S.W. Hempstead K. Morrical M.D. J. Biol. Chem. 1994; 269: 33069-33081Abstract Full Text PDF PubMed Google Scholar). gp59 also binds to gp32 in both the presence and absence of DNA (8Yonesaki T. J. Biol. Chem. 1994; 269: 1284-1289Abstract Full Text PDF PubMed Google Scholar, 12Lefebvre S.D. Wong M.L. Morrical S.W. J. Biol. Chem. 1999; 274: 22830-22838Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar). Morrical et al. (13Morrical S.W. Beernink H.T.H. Dash A. Hempstead K. J. Biol. Chem. 1996; 271: 20198-20207Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar) have suggested that the C-terminal, acidic domain (A-domain) of gp32 mediates the interaction between the two proteins. More recent studies have demonstrated that the DNA binding core domain of gp32 also interacts with gp59 (14Ishmael F.T. Alley S.C. Benkovic S.J. J. Biol. Chem. 2001; 276: 25236-25242Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar), and the combination of the two sites of interaction may be necessary for gp32 displacement. Furthermore, gp59 contains distinct sites for gp32 and gp41 binding (11Morrical S.W. Hempstead K. Morrical M.D. J. Biol. Chem. 1994; 269: 33069-33081Abstract Full Text PDF PubMed Google Scholar). As such, the gp59-mediated assembly of gp41 onto gp32-coated DNA most likely involves a ternary complex between the three proteins. Although gp59 exists as a monomer in solution, it can oligomerize in the presence of gp32 or DNA. Cross-linking experiments demonstrated that gp59 could form at least pentamers in the presence of either of these molecules (14Ishmael F.T. Alley S.C. Benkovic S.J. J. Biol. Chem. 2001; 276: 25236-25242Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar). Furthermore, Raney et al. (15Raney K.D. Carver T.E. Benkovic S.J. J. Biol. Chem. 1996; 271: 14074-14081Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar) showed that the enhancement of the ATP-dependent DNA unwinding activity of gp41 was maximal when gp59 was present in a 1:1 molar ratio with gp41 (hexamer of gp59 to hexamer of gp41). As such, gp59 is expected to form higher multimeric states when bound to the helicase. In E. coli, the DnaB helicase is loaded onto DNA by a protein analogous to gp59, DnaC. Cryoelectron microscopy studies indicated that DnaC forms a hexameric ring associated with the hexameric DnaB ring (16San Martin C. Radermacher M. Wolpensinger B. Engel A. Miles C.S. Dixon N.E. Carazo J.M. Structure. 1998; 6: 501-509Abstract Full Text Full Text PDF PubMed Google Scholar). By analogy, we predict a similar oligomeric state and arrangement of gp59 and gp41. To gain a better understanding of the mechanism of helicase assembly, the architecture and stoichiometry of the gp41-gp59 complex was investigated. We have demonstrated that 1) both the N and C terminus of gp41 lie in the gp41-gp41 subunit interface and interact with gp59; 2) the site of interaction of gp41 on gp59 is close to Cys-215 of gp59; 3) conformational changes in the gp41-gp59 complex occur upon gp41 loading on DNA; and 4) gp59 forms a hexamer upon binding gp41, and the gp59 subunits are arranged in a head to head orientation. The importance of these findings is discussed herein. The 41 gene was isolated from bacteriophage T4 genomic DNA (Sigma) by PCR amplification using the primers 5′-GCGGAACATATGGTAGAAATTATTCTTTCT and 5′-GCGGAATTCGCTCTTCCGCAAAATTTTAATTC. The product was digested with NdeI and SapI and ligated into a custom IMPACT vector placed under the control of a T7 promoter. This vector places a self-cleaving intein and chitin binding domain at the C terminus of gp41 and allows purification of the protein on a chitin affinity column followed by cleavage of the intein with dithiothreitol (DTT; Sigma) to yield the unmodified gp41. E. coli BL21(DE3) cells were electrotransformed with the plasmid, and transformants were selected with 50 μg/ml kanamycin on NCZYM (Invitrogen) agar plates. A single colony was grown in NCZYM medium to an A600 of 0.5 and induced for 18 h at 20 °C with 0.1 mmisopropyl-1-thio-β-d-galactopyranoside. The cells were centrifuged at 4,000 × g for 30 min and resuspended in chitin column buffer (20 mm Tris, pH 8.0, 500 mm NaCl, 10% glycerol, 0.1 mm EDTA). The cells were lysed by sonication and centrifuged for 30 min at 20,000 × g, and the supernatant was applied to a 10-ml chitin column. The column was washed with 1 liter of chitin column buffer, and gp41 was cleaved from the column by the addition of 100 mm DTT dissolved in chitin column buffer for 24 h. The protein was eluted from the chitin column and subsequently diluted so that the NaCl concentration was 200 mm. It was then loaded at 0.5 ml/min onto a 15-ml P11 column (Whatman) equilibrated with 20 mm Tris, pH 8.0, 200 mm NaCl, and 10 mm 2-mercaptoethanol. gp41 does not bind to the column under these conditions and was collected in the flow-through fraction. The protein was concentrated to ∼10 μm with an Amicon stirred cell concentrator equipped with a YM30 membrane. For purification of gp41 with a C-terminal cysteine added (Cys-476, denoted as gp41c), the protein was cleaved from the chitin column with 100 mm cysteine dissolved in 20 mm Tris, pH 8.0, 500 mm NaCl, 10% glycerol, 0.1 mm EDTA. The rest of the purification was as described above. The gp41 and gp41c proteins were dialyzed into 20 mm Tris, pH 7.5, 150 mm NaCl, and 10% glycerol and stored at −70 °C. The gp41c, containing a C-terminal cysteine (Cys-476) was applied to a Sephadex G-25 column to remove reducing agents. The protein (100 μl of a 6 μm solution) was then mixed with 10 μl of thiol-reactive trifunctional cross-linker (Fig. 1A) dissolved in dimethylformamide (DMF; 60 μmfinal concentration) for 14 h at 4 °C in the dark. Excess label was removed by applying the solution to a Sephadex G-25 column. gp41c and gp59 (in 20 mm Tris, pH 7.5, 150 mm NaCl, 10% glycerol) were mixed at a final concentration of 1 μm each, in the presence or absence of 3 mmATP and/or 5 nm M13mp18 single-stranded DNA. Cross-linking was initiated by exposure to ultraviolet light as described previously (17Alley S.C. Ishmael F.T. Jones A.D. Benkovic S.J. J. Am. Chem. Soc. 2000; 122: 6126-6127Crossref Scopus (66) Google Scholar). In the samples where reduction of the cross-linker was desired, DTT was added to a final concentration of 100 mm. The products of the cross-linking experiment was separated on a 10% polyacrylamide gel and subjected to a Western blot using streptavidin-horseradish peroxidase (SA-HRP; Invitrogen) as a probe, as described previously (17Alley S.C. Ishmael F.T. Jones A.D. Benkovic S.J. J. Am. Chem. Soc. 2000; 122: 6126-6127Crossref Scopus (66) Google Scholar). Prestained broad range molecular weight markers (Bio-Rad) were used to assess molecular weights of the species observed. gp41 was dialyzed into 20 mm Hepes, pH 7.0, 150 mm NaCl, 10% glycerol. In the presence of 5 mmADP, pH 7.0, only the N terminus of gp41 is labeled upon the addition of an amine-reactive probe. gp41 (100 μl of a 6 μm solution) was mixed with 10 μl of sulfosuccinimide-2-[6-(biotinamido)-2-(p-azidobenzamido)hexanoamido]ethyl-1,3′-dithiopropionate (Sulfo-SBED; Fig. 1B) dissolved in DMF (10 μm final concentration) for 16 h at 4 °C in the dark. Excess label was removed with a Sephadex G-25 column. gp41 and gp59 were mixed to a final concentration of 1 μm each in the presence or absence of ATP and/or DNA as described above. The samples that required reduction of cross-linker were reduced with 100 mm DTT. The samples were analyzed by a Western blot using SA-HRP as a probe. To verify that only the N terminus of gp41 was labeled, helicase labeled with Sulfo-SBED was digested with trypsin and applied to a 100-μl streptavidin-agarose (Sigma) column. The column was washed with 20 mm Tris, pH 7.5, 1 m NaCl to remove unlabeled peptides. The labeled peptide bound to the column by virtue of the Sulfo-SBED label was then treated with 1 m2-mercaptoethanol for 30 min to cleave the disulfide bond in the cross-linker, thus eluting the peptide. The eluted peptide was dried in a speed vacuum, and the sample was desalted with a zip-tip pipette tip containing C-18 resin (Millipore Corp.) according to the manufacturer's instructions. The peptide was eluted from the zip-tip C-18 resin using 60% acetonitrile, 40% H2O, 0.1% trifluoroacetic acid. The isolated peptide (500 μl) was then mixed with 500 μl of α-cyano-4-hydroxycinnamic acid (15 mg/ml dissolved in 50% acetonitrile, 50% water, 0.1% trifluoroacetic acid), and the mixture was spotted and air-dried on a MALDI target plate. The sample was analyzed by a Voyager MALDI mass spectrometer (Perseptive) in linear mode to confirm that only the N terminus was labeled. Positive ions were detected using a delayed extraction time of 300 ns and 250 laser scans. gp41c was labeled at the N terminus with biotin succinimide (to allow visualization by Western blot using SA-HRP) and mixed with gp59 (to a final concentration of 1 μm) in the presence of 10 μm 1,6-bis- maleimidohexane (BMH; Pierce) with or without 3 mm ATP and/or 5 nm M13mp18 DNA. After 7 min, the reaction was quenched with 5 μl of gel loading buffer containing 60 mm Tris, pH 6.8, 25% glycerol, 2% SDS, 14.4 mm 2-mercaptoethanol, and 0.1% bromphenol blue. The products were separated with a 10% polyacrylamide gel, and a Western blot with SA-HRP as a probe was used to detect the biotinylated protein. In the experiments in which gp41 was cross-linked to gp59 in the presence of gp32, gp32 (2 μm) and gp59 (1 μm) were preincubated for 10 min (in the presence or absence of 2 nm M13 DNA) before the addition of 1 μm gp41c (labeled at the N terminus with biotin). Cross-linking conditions and analyses were performed as described above. Molecular weight markers (precision unstained broad range and/or prestained broad range; Bio-Rad) were used to assess the size of the cross-linked species. gp59 was cross-linked to investigate higher oligomer formation by mixing gp59 and gp41 (without a C-terminal cysteine added) to a final concentration of 5 μm each in a 30-μl reaction volume (with or without 5 mm ATP and/or 20 nm M13 DNA) containing BMH at a concentration of 50 μm. After 7 min, the reaction was quenched with gel loading buffer and subjected to SDS-PAGE. The gel was stained with Gel Code Blue (Pierce). Thiol-thiol cross-linking between gp41c and gp59 could occur between the C-terminal cysteine on gp41c and one of the two cysteine residues on gp59 (Cys-42 or Cys-215). It was predicted that the site of cross-link on gp59 is Cys-215. To determine whether cross-linking occurred between the C terminus of gp41 and Cys-215 of gp59, Cys-42 was selectively modified covalently, and gp41c was mixed with gp59 in the presence of BMH. Cys-42 of gp59 was blocked by the addition of 100 μl of a 30 μm solution of gp32 labeled with N-[6-(7-amino-4-methylcoumarin-3-acetamido)hexyl]-3′-(2′-pyridyldithio)propionamide (AMCA-HPDP; Pierce) to 50 μl of a 20 μm gp59 solution for 30 min. It was previously shown that under these conditions, complete transfer of the fluorophore occurred via a disulfide exchange between Cys-42 of gp59 and the mixed disulfide between Cys-166 of gp32 and the fluorophore (14Ishmael F.T. Alley S.C. Benkovic S.J. J. Biol. Chem. 2001; 276: 25236-25242Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar). gp32 and excess label were removed from labeled gp59 by subjecting the mixture to a Mono-Q fast protein liquid chromatography column. Proteins were eluted from the Mono-Q column using a linear gradient between 0 and 1 m NaCl in 20 mm Tris, pH 7.5. The amount of labeled gp59 was determined by quantifying the amount of fluorophore based on its absorbance at 345 nm and determination of gp59 amount by Bradford assay. The concentration of fluorophore relative to protein was found to be 1.10:1. The site of label was confirmed by digesting labeled protein with trypsin, separating the fragments by HPLC (14Ishmael F.T. Alley S.C. Benkovic S.J. J. Biol. Chem. 2001; 276: 25236-25242Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar), and identifying the labeled peptide by MALDI mass spectrometry, as described above. Binding of gp41 to gp59 was assessed by isothermal titration calorimetry (ITC) on a MicroCal VP-ITC system (according to the protocol published previously) (18Valentine A.M. Ishmael F.T. Shier V.K. Benkovic S.J. Biocehmistry. 2001; 40: 15074-15085Crossref PubMed Scopus (46) Google Scholar). Proteins of interest were dialyzed into complex buffer (20 mm Tris, pH 7.5, 150 mm KOAc, 10 mm Mg(OAc)2) for at least 8 h to reach dialysis equilibrium. Protein solutions were centrifuged to remove particles before beginning the experiment and quantitated by UV-visible absorbance. All solutions were degassed prior to use. This degassing step was determined in control experiments not to affect protein activity. gp59 (21 μm) was in the syringe and injected in 7-μl volumes into a 2.2 μm solution of gp41 containing 1 mm ATPγS. Data were analyzed by Origin 5.0 (MicroCal). Heats of dilution were subtracted from all data before fitting. Heats of dilution were determined in a separate control experiment by injecting the protein into buffer. Data analysis requires a binding model, and fitting returns values for the binding constant K, ΔH of binding, ΔS of binding, and the binding stoichiometry N. To determine whether gp59 induced higher oligomer formation of gp41, the two proteins were cross-linked in the absence or presence of ATP. A 100-μl solution of gp59 (10 μm) was labeled with biotin by the following procedure. Biotin-maleimide (Sigma) was dissolved in DMF to make a 100 μm solution, and 10 μl of this solution was added to the gp59 solution for 4 h at 4 °C. gp41 (8 μm) was labeled with rhodamine-maleimide (Molecular Probes, Inc., Eugene, OR) by following the same procedure. The two labeled proteins were mixed to a final concentration of 4 μm each in the presence or absence of 1 mmATP. A solution of 0.25% (final concentration) 3,3′-dithio-bis(propionic acid N- hydroxysuccinimide ester (Sigma) was added to each sample for 10 min. The products were separated by SDS-PAGE, and gp41 cross-link bands were detected with a Fluor-S multiimager, whereas the gp59 cross-link bands were detected with a Western blot procedure using SA-HRP as a probe. gp41 cross-linked in the presence of ATP was used as a control to assess the size of the cross-linked species. The fluorescently labeled thiol-thiol cross-linker shown in Fig. 1C was synthesized from the trifunctional maleimide cross-linker tris(2-maleimidoethyl)amine (TMEA; Pierce) and the fluorescent probe AMCA-HPDP. AMCA-HPDP dissolved in DMF was reduced with 3 equivalents of tris(2-carboxylethyl)phosphine hydrochloride (TCEP; Molecular Probes) and mixed with 10 equivalents of TMEA (dissolved in DMF) for 30 min at 25 °C. The mixture was separated by HPLC as described previously (14Ishmael F.T. Alley S.C. Benkovic S.J. J. Biol. Chem. 2001; 276: 25236-25242Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar). The product was collected by monitoring at 345 nm the absorbance maximum for AMCA and dried in a speed vacuum. MALDI mass spectrometry was used to confirm the identity of the product. gp59 (500 μg) was cross-linked with 10 equivalents of the cross-linker for 7 min in the presence of gp41 (45 μg). The proteins were digested with trypsin as described above, and the peptides were separated by HPLC. Fractions (0.25 ml) were collected, dried in a speed vacuum, and redissolved in 10 μl of 60% acetonitrile, 40% H2O, 0.1% trifluoroacetic acid. The fractions were then placed on the stage of a Fluor-S multiimager, and illuminated in transilluminator mode to identify the fraction that contained the fluorescently labeled cross-linked peptide. This fraction was analyzed by MALDI mass spectrometry to determine the mass of the cross-linked peptide. gp41 contains 4 cysteine residues (amino acids 208, 233, 316, and 319). A DTNB assay determined that one of these cysteines was solvent-accessible, but labeling at this site with a cross-linker yielded no cross-links to gp59, suggesting that it is not close to the interface of interaction between the two proteins. 2F. T. Ishmael, S. C. Alley, and S. J. Benkovic, unpublished data. To determine whether the C terminus of gp41c interacted with gp59, cross-linking between the C terminus of gp41c and gp59 was conducted. A cysteine residue was added to the C terminus of gp41 (Cys-476, with the modified protein denoted as gp41c) by cleavage of a gp41-intein fusion protein with cysteine. The protein was then labeled with the thiol-reactive trifunctional cross-linker shown in Fig. 1A. The labeled gp41c was subsequently mixed with gp59 in the presence or absence of DNA and/or ATP and subjected to ultraviolet light to initiate cross-linking. When the products of the cross-linking reaction were separated by SDS-PAGE and subjected to a Western blot using SA-HRP as a probe, cross-link bands corresponding to gp41c dimers were seen in the absence of gp59, signifying that the C terminus of gp41c is close to or in the homodimeric interface (Fig. 2A, lane 2). When gp41c was mixed with gp59 and exposed to light, in the presence or absence of ATP and/or DNA, high molecular weight species were seen, consistent with gp41c-gp59 cross-links (Fig. 2A, lanes 3–6). Upon the addition of DTT, the cross-link was reduced and the biotin label was transferred to gp59, confirming that a gp41c-gp59 cross-link had occurred (Fig. 2A, lanes 9–12). In order to elucidate a second point of contact between gp41 and gp59, the N terminus of gp41 was labeled as described under "Experimental Procedures" with the amine-reactive trifunctional cross-linker Sulfo-SBED (Fig. 1B). The proteins were mixed in the absence or presence of DNA and/or ATP and then separated by SDS-PAGE followed by a Western blot using SA-HRP as a probe to identify the biotinylated cross-links. When gp41 was cross-linked by itself, a band corresponding to a gp41-gp41 homodimer was observed (Fig. 2B, lane 2). When gp41 and gp59 were mixed, cross-links were visualized in both the absence and presence of ATP and/or DNA (Fig. 2B, lanes 3–6). Reduction of the cross-linker with DTT caused transfer of the biotin to gp59, validating that the cross-link occurred between gp41 and gp59 (Fig. 2B, lanes 7 and 8). Morrical et al. (13Morrical S.W. Beernink H.T.H. Dash A. Hempstead K. J. Biol. Chem. 1996; 271: 20198-20207Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar) suggested that gp59 has distinct binding sites for gp41c and gp32. Experiments described previously demonstrated that Cys-42 of gp59 is in close proximity to gp32 (14Ishmael F.T. Alley S.C. Benkovic S.J. J. Biol. Chem. 2001; 276: 25236-25242Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar). gp59 contains a second cysteine residue, Cys-215, that lies on the opposite side of the protein relative to Cys-42. Since it is likely that gp41 interacts on this side of the protein, away from the gp32 binding site, thiol-thiol cross-linking was attempted to cross-link the C terminus of gp41c to gp59. gp41c was N-terminally labeled with biotin to allow easier detection of cross-links by Western blot using SA-HRP as a probe. When mixed with the thiol-thiol cross-linker BMH, gp41c cross-linked to form a homodimer linked at the C-terminal cysteine residues (Fig. 3A). In the presence of gp59, with or without ATP added, bands were seen that correspond to gp41c-gp59 cross-links (Fig. 3A). Typically, two cross-link bands were observed, one that migrated with the expected mobility of a gp41c-gp59 cross-link (molecular mass of about 75 kDa) and a band that migrated with a faster mobility (∼65 kDa). The intensity of the higher mobility band varies from experiment to experiment, whereas the lower mobility band does not vary. In experiments using a cleavable thiol-thiol cross-linker, this pattern of cross-linking between gp41c and gp59 is also seen. Excision of these bands followed by cleavage of the cross-linker and separation of the products on a second gel indicate that both bands are composed of gp41c-gp59 cross-links.2 Most likely, the faster migrating band is due to an intramolecular cross-link of gp41c as well as an intermolecular cross-link to gp59, resulting in higher mobility of the more compact structure than the gp41c-gp59 intermolecular cross-link alone. When DNA was added, no cross-links between gp41c and gp59 were visualized, indicating that some conformational change occurs upon loading of gp41 on DNA (Fig. 3A, lanes 1 and 2). To confirm that gp41c cross-linked to Cys-215 of gp59, a cross-linking experiment was conducted in which the Cys-42 of gp59 was blocked by covalent modification. Previously, we showed that incubation of gp32 labeled with a probe via a mixed disulfide with gp59 resulted in complete transfer of the probe from gp32 to gp59 by a disulfide exchange (Fig. 4A) (14Ishmael F.T. Alley S.C. Benkovic S.J. J. Biol. Chem. 2001; 276: 25236-25242Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar). As described under "Experimental Procedures," a label (AMCA) was placed selectively on Cys-42 of gp59 using this method. The modified gp59 protein contained only one free cysteine residue, Cys-215, as determined by MALDI mass spectrometry. Thiol-thiol cross-linking using BMH showed that gp41c cross-linked to both the modified (Fig. 4B, lanes 2 and 3) gp59 as well as the unmodified gp59 (Fig. 4B, lanes 6 and 7), demonstrating that Cys-42 was not involved in the cross-linking. The interaction between gp41c and gp59 was also analyzed in the presence of gp32. Preincubation o
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