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1 Biophysics Program, The Ohio State University, Columbus, Ohio 43210, USA
2 Biochemistry Program, The Ohio State University, Columbus, Ohio 43210, USA
3 Protein Research Group, The Ohio State University, Columbus, Ohio 43210, USA
4 Department of Biochemistry, The Ohio State University, Columbus, Ohio 43210, USA
Reprint requests to: Mark P. Foster, Department of Biochemistry, The Ohio State University, 484 W. 12th Ave., Columbus, OH 43210, USA; e-mail: foster.281{at}osu.edu; fax: (614) 292-6773.
(RECEIVED October 12, 2001; FINAL REVISION January 24, 2002; ACCEPTED February 4, 2002)
5 Present address: Heart and Lung Institute, OSU College of Medicine, Columbus, OH 43210, USA. 
Article and publication are at http://www.proteinscience.org/cgi/doi/10.1110/ps.4180102.
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Abstract |
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and FasL receptors. To better understand its role in cell death inhibition, we have sought to characterize the biophysical properties of the protein from adenovirus serotype 5 (Ad5 E3–14.7K, or simply 14.7K) through a variety of approaches. To obtain sufficient quantities of recombinantly expressed protein for biophysical characterization, we explored the use of various expression constructs and chaperones; fusion to MBP was by far the most effective at generating soluble protein. Using limited proteolysis, mass spectrometry, and protein-protein interaction assays, we demonstrate that the C-terminal two-thirds of the protein, predicted to be composed of five ß-strands and one -helix, is highly structured and binds its putative cellular receptors. Furthermore, using atomic absorption and ultraviolet/visible spectroscopies, we have studied the metal binding properties of the protein, providing insight into the observation that cysteine/serine mutants of 14.7K lack in vivo antiapoptotic activity. Lastly, results from size exclusion chromatography, dynamic light scattering, sucrose gradient sedimentation, chemical crosslinking, and electron microscopy experiments revealed that 14.7K exists in a stable high-order oligomeric state (nonamer) in solution. Keywords: Ad5 E3; 14.7K; adenovirus; apoptosis; oligomerization; metal binding; proteolysis
Abbreviations: 14.7K, Ad5 E3-14.7K, a protein encoded in the E3 early region of the genome of adenovirus serotype 5 (gi|58510) • FIPs (1-3), 14.7K-interacting proteins • FLICE/caspase 8, a cysteine protease in the interleukin-1ß • converting enzyme family • DED, death-effector domain • MBP, maltose-binding protein • GST, glutathione-S-transferase • TNF, tumor necrosis factor • ESI-MS, electrospray mass spectrometry • CD, circular dichroism • LB, Luria-Bertani • DTT, dithiothreitol • DMS, dimethyl suberimidate • AEBSF, 4 (2-Aminoethyl)-benzenesulfonyl fluoride hydrochloride
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Introduction |
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TOPAbstractIntroductionResultsDiscussionMaterials and methodsReferences |
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An immunomodulatory adenoviral product that has garnered much attention is the highly conserved 14.7K protein. This protein was initially identified as an inhibitor of TNF-mediated inflammation and cytolysis (Gooding et al. 1990; Zilli et al. 1992; Tufariello et al. 1994a, b) and subsequently was shown to be a general inhibitor of cell death (apoptosis) regulated by the TNF and FasL receptor pathways (Chen et al. 1998; Li et al. 1998). The 14.7K protein is highly conserved across multiple adenovirus serotypes (Horton et al. 1990) (Fig. 1
), underscoring its importance for adenoviral viability. Indeed, deletions or mutations in the 14.7K gene result in adenovirus-infected cells that are very susceptible to cytokine-activated cell death (Gooding et al. 1988, 1990; Horton et al. 1991; Ranheim et al. 1993; Sparer et al. 1996).
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) (Rothwarf et al. 1998; Yamaoka et al. 1998; Li et al. 1999) exhibits some sequence homology to FIP-2 but has been shown to interact with IKKß, RIP, and NIK, affecting NF-
B activity (Li et al. 1999). The identification of FIP-3 as a modulator of NF-B activity and its direct interaction with 14.7K suggest that the adenoviral protein might inhibit cell death by interfering with NF-B–mediated transcription of apoptosis-regulatory genes (Li et al. 1999; Ye et al. 2000). How these putative cellular targets tie into the known apoptotic regulatory pathways remains unclear. In a different study with 14.7K from adenovirus serotype 5, immunoprecipitation experiments revealed an interaction with the DEDs of FLICE/caspase 8 (Chen et al. 1998), a cysteine protease central to the Fas and TNF-R apoptotic regulatory pathways (Muzio et al. 1996). This result suggested an alternate pathway in which 14.7K employs a strategy similar to that of other viruses that inhibit the activation of FLICE via DED-containing proteins, termed FLICE inhibitory proteins (FLIPs) (Thome et al. 1997). However, direct inhibition of FLICE activation has not been demonstrated, and 14.7K lacks a DED sequence or other motif known to mediate cell-death signaling.
Confusion as to the relevant cellular target and absence of a significant homology to proteins with known structures leaves its mechanism of action unclear. To obtain structural insight into its mechanism of cell death inhibition, we sought to characterize 14.7K from adenovirus serotype five by biochemical and biophysical methods (Kim 2000). To that end, we have (1) developed an efficient scheme for expression and purification of soluble protein, (2) found that 14.7K contains a proteolytically-resistant, C-terminal domain that retains the capacity to interact with FIP-1 and the DEDs of FLICE, (3) demonstrated that 14.7K is highly oligomerized, and (4) shown that 14.7K binds zinc with a 1:1 stoichiometry.
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Results |
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). ESI-MS of recombinantly expressed wild-type 14.7K (Kim 2000) showed that the samples contained a mixture corresponding to full-length protein (Met1 through Asn128; 40%), and three posttranslationally modified forms (Bradshaw et al. 1998): methionine-excised protein (Thr2 through Asn128; 40%) and N-acetylated species of both proteins (10% each). Introduction of an additional methionine at the amino terminus effectively eliminated excision of the amino-terminal methionine as well as N-acetylation (Hirel et al. 1989; Kim 2000).
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1 mg/mL. In parallel with investigations on refolded protein, a method to obtain large quantities of solubly expressed 14.7K was pursued. As shown in Figure 2
, wild-type (14.7K), histidine-tagged (H6–14.7K), and GST-tagged (GST-14.7K) proteins are almost exclusively present in the insoluble pellet (P). Fusion to thioredoxin (Trx-Hp-14.7K) or coexpression of thioredoxin (14.7K/Trx) or GroEL/GroES (14.7K/GroE) slightly increased soluble protein expression, although coexpression reduced the overall amounts of protein produced. Only fusion to MBP (MBP-14.7K) caused a dramatic increase in the amount of protein present in the soluble fraction (S), while retaining a high level of expression (Fig. 2). Although MBP-fused 14.7K is soluble at high concentrations (>30 mg/mL), upon removal of MBP by thrombin cleavage, the solubility of 14.7K reverts to that of the unfused protein. Fusing MBP to the DED of FLICE similarly increased its solubility, although upon thrombin cleavage the DED solubility decreased dramatically (data not shown). In addition, while the MBP tag is almost three times the molecular weight of 14.7K, as evidenced by gel filtration (Kim 2000), the fusion protein exhibited the same oligomeric state as unfused 14.7K (see below).
Bacterially expressed, refolded 14.7K is structured
CD spectroscopy was used to examine whether recombinantly expressed 14.7K had well-defined secondary structure. The CD spectrum of 14.7K showed that it contains a significant portion of -helix and ß-strand features (Fig. 3). Deconvolution analyses using the programs K2D (Andrade et al. 1993) and CDNN (Bohm et al. 1992) indicate an approximate composition of 33% -helix, 33% ß-strand, and 33% coil in agreement with PHDsec prediction (Rost and Sander 1993b) (Fig. 1). Solubly expressed (via thioredoxin coexpression) or refolded 14.7K were indistinguishable by CD spectroscopy (Kim 2000) or other measurements (below), indicating that the native secondary structure is regained upon refolding.
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-chymotrypsin. SDS-PAGE analysis of the products of limited trypsin digestion of solubly expressed (via thioredoxin coexpression) or refolded 14.7K revealed two protease-resistant fragments (Fig. 4). A 12-kDa band appeared after initial digestion with 0.1% trypsin (w/w), and an 11-kDa band appeared as the digestion progressed and/or the percentage of trypsin was increased. No further degradation of the 11-kDa fragment was observed, even when cleavage reactions were performed in the presence of 1–2 M urea (Kim 2000).
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-Chymotrypsin also did not further digest the C-terminal fragment, although it possesses 13 potential cleavage sites (W/Y/F/L). This proteolytic pattern indicates that the predicted N-terminal helix (15–42) is disrupted near residue 24, and that residues 31–128 are structured in solution and inaccessible to the enzymes. The CD spectrum of the C-terminal proteolytic fragment showed that the fragment had lost some -helix content, as monitored by ellipticity at 208 and 222 nm, relative to the full length 14.7K (Fig. 3). The solubility of the trypsin-generated, C-terminal fragment was similar to that of full-length 14.7K (or <1 mg/mL).
On the other hand, a recombinantly expressed N-terminal peptide comprising residues 2–43 was highly soluble (>5 mg/mL) and its CD spectrum of exhibited signals consistent with significant -helical content (Fig. 3). However, 2D 1H NOESY NMR spectra (Kim 2000) contained no sequential (i, i+1) backbone amide proton NOEs, indicating the -helical content of the isolated peptide is probably transient.
Recombinant 14.7K and its C-terminal region are capable of interacting with FIP-1 and FLICE
Binding assays were performed to determine whether the refolding procedure yielded recombinant protein capable of binding its putative cellular targets, FIP-1 and FLICE, and to investigate whether the 14.7K proteolytic fragment retained the same capacity. Our results indicate that refolded 14.7K is capable of specifically interacting with FIP-1 (Fig. 5). Further, 14.7K:31–128 is able to bind both FIP-1 and the N-terminal death-effector–containing domain of FLICE (1–271), although it also exhibits weak nonspecific binding to GST. In contrast, the amino-terminal domain (residues 2–43) did not bind itself, GST-FIP-1, GST-FLICE:1–271 (Kim 2000), or indeed to 14.7K:31–128 (Fig. 5).
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). This radius is much too large for a monomeric globular protein of 14.7 kDa, rather it would correspond to a globular protein with a molecular weight of 192 kDa. The Stokes radius of the tryptic fragment (14.7K:31–128) was similarly large (37 Å), corresponding to a 73-kDa globular protein. These data suggested that both 14.7K and the C-terminal fragment were highly oligomerized in solution. Assuming globular shapes and uniform hydration, these apparent masses would be consistent with oligomers with 14 monomers (14 x 14.7 kDa) for the full-length protein and seven monomers (7 x 11.1 kDa) for the 31–128 fragment.
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55 kDa) eluted from a gel filtration column near thyroglobulin (680 kDa) while thrombin cleavage of MBP-14.7K produced monomeric MBP and oligomeric 14.7K, indicating that the latter drives oligomerization of the fusion protein (Kim 2000). However, denaturants had a clear effect on oligomerization, as pretreatment of 14.7K with 1–5 M urea prior to elution on a size exclusion column demonstrated the multimers could be 50% dissociated in 1.5 M urea (Fig. 7a). In contrast, CD spectra and protease resistance data (above) indicate the protein is still folded under these conditions. Urea-induced unfolding of 14.7K, monitored from ellipticity at 220 nm (Fig. 7b) revealed a half-denatured urea concentration, [D]50% of 3.8 M, with the protein remaining >90% folded in 0–2.5 M urea; salt modestly increased the cooperativity of unfolding (mD-N) by 50% over the range of 0.08–0.45 M NaCl. Thus, urea-induced dissolution of the oligomer appears to precede unfolding.
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). Similarly, DMS crosslinking of 14.7K:31–128 indicates the presence of multimeric states above a hexamer (Fig. 8b). However, the crosslinking data did not enable the unambiguous determination of the native oligomeric state of either protein. Intermolecular disulfide bonds were not found to be responsible for the stability of oligomeric 14.7K complexes, as the absence or presence of 100 mM of the reducing agent DTT had no effect on migration in size exclusion or SDS-PAGE (Kim 2000).
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). Sedimentation coefficients obtained from 5–20% and 10–40% sucrose gradients were reproducibly similar for both 14.7K and 14.7K:31–128 (Table 1). When combined via the Svedberg equation (s = M(1 - v2
)/N0f), the hydrodynamic data indicate that the native molecular weights of 14.7K and 14.7K:31–128 are in the range of 129 and 95 kDa, respectively, corresponding to oligomeric states of 9 for both proteins. These data suggest that the large decrease in Stokes radius upon deletion of the amino-terminal 30 residues results from a significant change in shape and/or hydration (i.e., becoming more compact), rather than from a two-fold change in oligomeric state.
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130 kDa and an effective diameter of 10 nm (14.7K Rs 5 nm), transmission electron microscopic images were obtained to visualize 14.7K. The two-dimensional images of 14.7K on the stained grid revealed regular globular particles (Fig. 9). The resolution of the images was not sufficiently high to observe the subunits of the oligomeric protein; however, measurements of 100 particles yielded a mean diameter of 11.7 +/- 1.3 nm, consistent with the hydrodynamic data.
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). Atomic absorption spectroscopy of MBP-14.7K (and MBP as a control) produced from rich (LB) media and purified without added zinc indicated that the protein binds zinc with a 1:1 stoichiometry (1.23 +/- 0.01 mol zinc/mol protein). To further probe the metal-binding properties of 14.7K, the fusion protein was expressed in minimal media supplemented with cobalt, a useful spectroscopic probe of metal coordination centers (Maret and Vallee 1993). MBP-14.7K isolated from cultures supplemented with 100 mM cobalt (but purified without added metal) was blue and exhibited ultraviolet (UV)/visible spectra (Fig. 10) characteristic of a Co(II)-substituted protein with four thiolate ligands (CoS4) (Maret and Vallee 1993). In addition, the protein exhibited metal-dependent expression, as bacterial cultures expressed and induced in minimal media in the absence of Zn2+ or Co2+ produced negligible amounts of protein (data not shown).
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Discussion |
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The biochemical and biophysical insights reported here represent an important step toward understanding the structure and function of 14.7K. The proteolysis data demonstrate that residues 31–128 are well protected from cleavage, but that Arg24 in the amino terminus is accessible. Secondary structure predictions (PHDSec and PSI-PRED) (Rost and Sander 1993a; Jones 1999) and CD spectra of 14.7K:2–43 suggest a helix extends from Ile13 through His42. However, from a lower PSI-PRED confidence level between Arg23 and Ala26, together with the observed trypsin cleavage at Arg23 (generating 24–128), we infer that the N-terminal helix is disrupted at this point. Because slow cleavage at Arg30 takes place only after cleavage at Arg23, we propose the first cleavage somewhat destabilizes the remaining helix, allowing the protease to access an otherwise structured residue. None of 12 remaining trypsin sites or five chymotrypsin sites is protease accessible, suggesting that with the exception of a few loops, the entire protein adopts a compact, folded structure.
The protein-binding experiments have shown that the determinants for recognition of FIP-1 and FLICE are retained within residues 31–128. The primary sequence of this highly conserved region of the protein is predicted to be composed of ß-strands connected by short coil segments, with an -helix at the extreme C terminus (Fig. 1); CD spectra are consistent with this prediction. Thus, recognition of FIP-1 and FLICE appears to be mediated by the core ß-strands and/or C-terminal helix. However, because in vivo data suggest that the entire protein is required for antiapoptotic activity (Ranheim et al. 1993), it appears that the interaction of 14.7K with FIP-1 and/or FLICE is not sufficient for its antiapoptotic effects. Rather, the amino-terminal helical segment has an important function unrelated to overall protein structure or FIP-1/FLICE binding.
Our discovery that 14.7K binds divalent metals (Zn2+, Co2+) may help explain the observation that the antiapoptotic activity of 14.7K is lost upon mutating to serine the three invariant cysteine residues (Ranheim et al. 1993), which are located in the C-terminal protease-resistant domain of 14.7K (Cys44, Cys50, Cys119; Fig. 1). These mutants, presumed to have compromised metal-binding ability, are poorly expressed in mammalian cells (Ranheim et al. 1993) (M. Horwitz, pers. comm.), consistent with the observed metal-dependent expression in E. coli. While these observations implicate these residues in metal binding, more work is needed to fully characterize the metal-binding site in detail and to investigate the possible structural and/or functional role of the bound metal.
Finally, we have found that 14.7K adopts a stable multimeric structure in solution. Size exclusion chromatography revealed Stokes radii corresponding to oligomers of 14 and 7 monomers for the full-length protein and C-terminal proteolytic fragment, respectively. This observation led to the preliminary hypothesis that the amino-terminal helical segment was responsible for dimerizing two heptameric assemblies. However, diffusion and sedimentation measurements together suggested that in fact both the full-length and C-terminal fragments adopt nonameric complexes (9-mers) with different overall shapes/degrees of hydration. The 14.7K oligomers were large enough to be observed on negatively stained electron micrographs, with individual particles having an apparent radius of 5 nm. The multimeric state of the protein is independent of whether the protein is isolated in a soluble form through coexpression of or fusion to a protein "chaperone," or refolded after resolubilizing from inclusion bodies with denaturants. Oligomerization of 14.7K has been detected in mammalian cells by cotransfection with vectors expressing wild-type and epitope-tagged 14.7K (J. Bruder, pers. comm.), but the stoichiometry of these oligomers remained unknown. The stability of the 14.7K oligomer and its persistence through a range of protein concentrations, buffers, salts, and reducing agents suggest the oligomeric state identified here is the same as in adenovirus-infected cells.
Although the physiological effects of 14.7K have been recognized for over a decade (Tollefson and Wold 1988; Gooding et al. 1990; Horton et al. 1991) and its potential for use in gene therapy applications is being actively explored (Harrod et al. 1998; Bruder et al. 2000; Doronin et al. 2001; Horwitz 2001), progress toward understanding its mechanism of action has been slow. While several putative cellular targets have been proposed, poor solution characteristics and the lack of a recognizable structural motif that might provide clues to its mechanism of action have contributed to a paucity of structure/function information. Because 14.7K represents a novel class of antiapoptotic proteins and may represent a novel structural fold, continued structure/function analyses are warranted. This work provides a critical methodological and conceptual foundation for a detailed understanding of the structure and function of this interesting protein.
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Materials and methods |
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TOPAbstractIntroductionResultsDiscussionMaterials and methodsReferences |
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Fusions of the N-terminal death-effector–containing domain of FLICE with GST and MBP were constructed by first eliminating an internal EcoR I site from the vector pThioAnFLICE (J. Bruder, GenVec) followed by subcloning into BamH I and EcoR I sites of pGEX-2T and the modified pIH1119 vector, respectively. A plasmid expressing GST-fused FIP-1 was obtained from M. Horwitz (Albert Einstein University) and used without modification (Li et al. 1997).
The sequences of the inserts in all the plasmid constructs were verified by automated DNA sequencing.
Expression and purification
Recombinant 14.7K was expressed in E. coli BL21(DE3) cells (Novagen), grown in a shaker incubator at 37°C in LB media supplemented with the appropriate antibiotics, and induced for 3 h by addition of 1 mM IPTG at an OD600 of 0.6 0.8. To examine expression yield and solubility, cells were sonicated in lysis buffer (100 mM Tris-HCl, pH 8.0 at 4°C, 0.1 M NaCl, 1 mM DTT, 0.2 mM AEBSF, and 0.5 µg/mL leupeptin). Soluble and insoluble fractions were separated by centrifugation and were boiled in Laemmli buffer, alongside equal amounts (from OD600) of uninduced and induced whole-cell pellets, loaded onto 10–20% gradient SDS-PAGE (BioRad) and visualized by staining with Coomassie Brilliant Blue R-250 (Fisher Scientific).
Refolding 14.7K
Cell pellets containing insoluble 14.7K were resuspended in lysis buffer containing 10 mM EDTA and lysed by sonication. Subsequently, inclusion bodies were collected by centrifugation at 12,000g for 30 min at 4°C and solubilized by stirring at 4°C in denaturing buffer (20 mM sodium phosphate, pH 6.4, 6 M urea, 5 mM DTT). The urea-solubilized fraction was clarified by centrifugation (20,000g for 1 h, 4°C) and eluted from an SP-Sepharose column (Pharmacia) by a linear NaCl gradient (0.05–1 M).
Fractions containing 14.7K were refolded as follows: first, denatured 14.7K was diluted and adjusted to pH 8.5 (20 mM Tris-HCl, 140 mM NaCl, 20 mM sodium phosphate, 2 mM MgCl2, and 25 µM ZnSO4), then the urea concentration was decreased slowly (3 h, 4°C) to 2 M by drop-wise addition of dilution buffer (20 mM Tris-HCl [pH 8.5], 140 mM NaCl, 2 mM MgCl2, 25 µM ZnCl2, 2 mM oxidized glutathione, and 4 mM reduced glutathione) (Noel et al. 1991). Next, the sample was brought to room temperature and gently stirred for 8 h. Remaining urea in the sample was removed by sequential dialysis (at 4°C) in 10–12 volumes of dialysis buffer containing decreasing concentrations of urea (1, 0.5, 0 M). Precipitates were removed by centrifugation and the protein was further purified to >95% by elution from a Q-Sepharose column (Pharmacia) with a linear gradient of 0.1–1 M NaCl. Protein concentrations were estimated from extinction coefficients calculated for the denatured state (
276 = 2900 M-1 cm-1; 280 = 2560 M-1 cm-1).
Solubilizing 14.7K by coexpression with TRX or GroES
E. coli BL21(DE3) cells were doubly transformed with plasmids expressing genes encoding 14.7K (pET21-Ad5 14.7K) and thioredoxin or GroES/L (Yasukawa et al. 1995) and cells cultured with the antibiotics appropriate to each vector. The purification procedure was adapted from a method developed for extracting Ad2–14K from HeLa cells (Persson et al. 1978).
Soluble MBP-14.7K
E. coli cells containing overexpressed MBP-14.7K were lysed by sonication and clarified by centrifugation. The supernatant was adsorbed onto amylose-Sepharose resin (New England Biolabs), packed into a gravity column and bound MBP-14.7K was eluted with 20 mM maltose. To remove the MBP tag, thrombin cleavage of MBP-14.7K was carried out with 1% (w/w) thrombin (Sigma) in cleavage buffer (50 mM Tris-HCl (pH 8.4) 0.15 M NaCl, 2.5 mM CaCl2, and 1 mM DTT) and terminated by addition of AEBSF (1 mM).
GST-14.7K:2–43 (N-terminal fragment)
Cell pellets containing GST-14.7K:2–43 were lysed in NETN buffer (50 mM Tris-HCl [pH 8 at 4°C], 120 mM NaCl, 1 mM EDTA, 0.5% NP-40, 5 mM DTT, 0.2 mM AEBSF, and 0.5 µg/mL Leupeptin) and adsorbed onto a glutathione-Sepharose 4B column (Pharmacia), cleaved by thrombin, eluted from the column and further purified by gel filtration (TSK G2000SW, 21.5 x 300 mm, 3 mL/min, TosoHaas) in 20 mM sodium phosphate (pH 7.0, 0.1 M NaCl).
Co2+-14.7K
Co2+-MBP-14.7K was obtained by culturing cells in M9 minimal media supplemented with vitamins (GIBCO Eagle Basal Vitamin Mix) and 100 µM CoSO4. Cells were induced and harvested as above and the protein purified as for MBP-14.7K obtained from rich media, with the exception that DTT was excluded from the buffers. UV/visible spectra of the purified protein were recorded at 1.4 mg/mL (25 µM) on an HP-8452A diode array spectrophotometer in a 1-cm path length cell. The water used for these experiments was deionized with a Milli-Q water system (Millipore).
Trypsin proteolysis
Aliquots of 14.7K (0.5 mg/mL, in 20 mM Tris-HCl pH 8.4, or 15 mM sodium borate pH 8.0, containing 0.15–0.45 M NaCl and 1 mM DTT) were mixed at room temperature with 0.1–2% (w/w) trypsin (Sigma) previously treated with 100 µg/mL TCPK (Sigma). Aliquots of proteolyzed 14.7K were subjected to SDS-PAGE or to liquid chromatography-electrospray ionization mass spectrometry (LC-ESI-MS) for mass analysis of trypsin-produced fragments. For detailed biophysical analysis of 14.7K:31–128, the tryptic fragment was further purified on a Superdex-200 HR column (16 x 600 mm; Pharmacia).
CD measurements
Purified proteins were dialyzed overnight at 4°C in 15 mM sodium borate buffer (pH 8.0, 0.15 M NaF, 1 mM DTT). CD spectra were recorded in the range of 190–260 nm at 20°C on an AVIV Model 62A DS. Urea denaturation was monitored by recording ellipticity at 220 nm, 20°C, over a urea concentration of 0–8 M on a Jasco J-500A spectropolarimeter. Urea-induced unfolding thermodynamics were analyzed as a function of NaCl concentration (0.08–0.45 M) by assuming a two-state unfolding model (Clarke and Fersht 1993; Fersht 1999).
Protein-binding assay
Cell pellets containing GST, GST-FIP1, or GST-FLICE:1–271 were lysed in NETN buffer, clarified by centrifugation, and nutated with glutathione-Sepharose 4B resin for 3 h at 4°C; unbound proteins were eluted with NETN buffer. Purified 14.7K, 14.7K:31–128 or 14.7K:1–43 were mixed with aliquots of immobilized GST-FIP1, GST-FLICE:1–271, or GST for 3 h at 4°C. Unbound proteins were removed by extensive washing with NETN buffer containing 0.15 M NaCl (3x), 0.45 M NaCl (2x) and 0.15 M NaCl (2x). Bound proteins were visualized by boiling the resin in Laemmli buffer, separating by SDS-PAGE and staining with Coomassie Brilliant Blue R-250.
Zinc atomic absorption spectroscopy
MBP-14.7K and MBP samples obtained from growth in LB but purified without added zinc were dialyzed overnight in buffer containing 20 mM Tris-HCl (pH 8.4 at 4°C), 0.2 M NaCl, and 1 mM DTT. Using a Perkin Elmer Z5000 graphite furnace atomic absorption spectrometer, zinc atomic absorption of a protein sample was measured at 213.9 nm in the peak area mode during 5 sec after 20 µL injections. Zinc concentrations in serial dilutions of the protein samples were calculated by comparison to solutions of known concentrations (0–40 µg/L) prepared by diluting a zinc atomic absorption standard solution (Perkin Elmer; Cat. No. 766). The zinc content in MBP-14.7K and MBP was measured eight and three times, respectively.
Size exclusion chromatography
The 14.7K, its tryptic fragment, MBP, MBP-14.7K, and thrombin-treated MBP-14.7K (15 mM sodium borate, pH 8.0, 0.45 M NaCl, 2 mM MgCl2, and 1 mM DTT) were subjected to size exclusion chromatography on Superdex-200 or Superdex-75 gel filtration columns (Pharmacia). Diffusion coefficients (D20,w) and Stokes radii (Rs) were determined from regression analysis of Kav versus the hydrodynamic parameters of protein standards (Siegel and Monty 1966; Cantor and Schimmel 1980). The protein standards used were ferritin (450 kDa, Rs 61.0), catalase (240 kDa, Rs 52.2, D20,w 4.1), aldolase (158 kDa, Rs 48.1, D20,w 4.7), BSA (67 kDa, Rs 35.5, D20,w 6.1), ovalbumin (44 kDa, Rs 30.5, D20,w 7.4), and chymotrypsinogen (25 kDa, Rs 20.9, D20,w 9.5) (Pharmacia). The effect of denaturants on oligomerization was assayed by pretreating aliquots of 14.7K (10 µM) with urea (0–5 M) prior to injection on a Superdex-75 column (7.8 x 300 mm; 1 mL/min flow rate).
Chemical crosslinking
Crosslinking experiments with 14.7K or 14.7K:31–128 were performed in 14 mM DMS (dimethyl suberimidate•2 HCl, Pierce) (20 mM sodium borate, pH 8.99, 0.45 M NaCl, 2 mM MgCl2, 1 mM DTT). Aliquots of 20 µL of protein (65 µM) and 5 µL of DMS (70 mM) were nutated at room temperature for 15, 30, 60, 90, or 120 min and analyzed by SDS-PAGE (Fig. 8).
Dynamic light scattering
Right-angle light scattering was performed using a DynaPro-801 dynamic light scattering/molecular sizing instrument (Protein Solutions). The scattering of 14.7K (37 µM in 20 mM Tris-HCl, pH 8.0, 0.3 M NaCl, 2 mM MgCl2, 1 mM DTT) and 14.7K:31–128 (70 µM in 15 mM sodium borate, pH 8.0, 0.45 M NaCl, 2 mM MgCl2, 1 mM DTT) were measured 11 to 18 times at 23°C. Light scattering data were analyzed using the Dynamics and DynalS software programs (Protein Solutions) and were found to be consistent with monodisperse solutions; diffusion coefficients (D) were corrected to 20°C by D20,w = D•(293/T)•(
T,W/20,w), where is solvent viscosity ().
Sucrose gradient sedimentation
The sedimentation coefficients (s20,w) of 14.7K and 14.7K:31–128 were determined by using linear gradients of 5–20% or 10–40% sucrose (v/v) (20 mM Tris-HCl, pH 8.0 at 4°C, 0.45 M NaCl, 2 mM MgCl2, 1 mM DTT). One milliliter of a sample mixture containing protein standards and 14.7K (or trypsin-treated 14.7K) was layered on each sucrose gradient. After sedimentation at 160,000g (L7–55 Ultracentrifuge, Beckman) for 19 h at 4°C, migration distances were obtained by a Gaussian fit of SDS-PAGE band intensities. Sedimentation coefficients of 14.7K and 14.7K:31–128 were obtained by fitting the s20,w values of the standards versus their migration distance (Table 1) (Martin and Ames 1961). Measurements were performed in triplicate.
Oligomeric masses
Oligomeric masses (M) of 14.7K and 14.7K:31–128 were calculated with the Svedberg equation (M = sRT/[D(1 - v2)]) combining the diffusion coefficients (D) obtained from light scattering and sedimentation coefficients (s) from sucrose-gradient sedimentation (Table 1); protein partial specific volumes (v2) were estimated from amino-acid compositions (McMeekin and Marshal 1952; Siegel and Monty 1966; Cantor and Schimmel 1980; Perkins 1986), and (density of medium) was taken to be that of water.
Transmission electron microscopy
Transmission electron microscopy of 14.7K (0.6 mg/mL in 20 mM Tris pH 8.0, 0.33 M NaCl, 2 mM MgCl2, and 1 mM DTT) was performed by sequentially floating FORMVAR-coated copper grids (400 mesh/inch, Ted Tella, Inc.) on a drop of the wetting reagent, bacitracin (50 µg/mL; 1 min), a drop of 14.7K solution (3 min), and then negatively stained with 2% phosphotungstic acid (PTA, pH 7.4, 2 min). After air drying, the sample was observed in a Phillips CM 12 at a magnification of 100,000 and accelerating voltage of 60 kV. The TEM image was photographed with 2.5-fold enlargement and scanned for analysis (Fig. 9).
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Acknowledgments |
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The publication costs of this article were defrayed in part by payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 USC section 1734 solely to indicate this fact.
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Bohm, G., Muhr, R., and Jaenicke, R. 1992. Quantitative analysis of protein far UV circular dichroism spectra by neural networks. Protein Eng. 5: 191–195.
Bradshaw, R.A., Brickey, W.W., and Walker, K.W. 1998. N-terminal processing: the methionine aminopeptidase and N alpha-acetyl transferase families. Trends Biochem. Sci. 23: 263–267.[CrossRef][Medline]
Bruder, J.T., Appiah, A., Kirkman, W.M., 3rd, Chen, P., Tian, J., Reddy, D., Brough, D.E., Lizonova, A., and Kovesdi, I. 2000. Improved production of adenovirus vectors expressing apoptotic transgenes. Hum. Gene Ther. 11: 139–149.[CrossRef][Medline]
Cantor, C.R. and Schimmel, P.R. 1980. Biophysical chemistry, part III: Techniques for the study of biological structure and function 3 vols., Freeman and Co., San Francisco.
Center, R.J., Kobe, B., Wilson, K.A., Teh, T., Howlett, G.J., Kemp, B.E., and Poumbourios, P. 1998. Crystallization of a trimeric human T cell leukemia virus type 1 gp21 ectodomain fragment as a chimera with maltose-binding protein. Protein Sci 7: 1612–1619.[Abstract]
Chen, P., Tian, J., Kovesdi, I., and Bruder, J.T. 1998. Interaction of the adenovirus 14.7-kDa protein with FLICE inhibits Fas ligand-induced apotosis. J. Biol. Chem. 273: 5815–5820.
Clarke, J. and Fersht, A.R. 1993. Engineered disulfide bonds as probes of the folding pathway of barnase: Increasing the stability of proteins against the rate of denaturation. Biochemistry 32: 4322–4329.[CrossRef][Medline]
Doronin, K., Kuppuswamy, M., Toth, K., Tollefson, A.E., Krajcsi, P., Krougliak, V., and Wold, W.S. 2001. Tissue-specific, tumor-selective, replication-competent adenovirus vector for cancer gene therapy. J. Virol. 75: 3314–3324.
Edwards, A.M., Arrowsmith, C.H., Christendat, D., Dharamsi, A., Friesen, J.D., Greenblatt, J.F., and Vedadi, M. 2000. Protein production: Feeding the crystallographers and NMR spectroscopists. Nat. Struct. Biol. 7 Suppl: 970–972.
Fersht, A. 1999. Structure and mechanism in protein science. A guide to enzyme catalysis and protein folding. W.H. Freeman and Company, New York.
Gooding, L.R., Elmore, L.W., Tollefson, A.E., Brady, H.A., and Wold, W.S. 1988. A 14,700 MW protein from the E3 region of adenovirus inhibits cytolysis by tumor necrosis factor. Cell 53: 341–346.[CrossRef][Medline]
Gooding, L.R., Sofola, I.O., Tollefson, A.E., Duerksen-Hughes, P., and Wold, W.S. 1990. The adenovirus E3–14.7K protein is a general inhibitor of tumor necrosis factor-mediated cytolysis. J. Immunol. 145: 3080–3086.[Abstract]
Gooding, L.R. and Wold, W.S. 1990. Molecular mechanisms by which adenoviruses counteract antiviral immune defenses. CRC Crit. Rev. Immunol. 10: 53–71.
Gouet, P., Courcelle, E., Stuart, D.I., and Metoz, F. 1999. ESPript: Analysis of multiple sequence alignments in PostScript. Bioinformatics 15: 305–308.
Harrod, K.S., Hermiston, T.W., Trapnell, B.C., Wold, W.S., and Whitsett, J.A. 1998. Lung-specific expression of adenovirus E3–14.7K in transgenic mice attenuates adenoviral vector-mediated lung inflammation and enhances transgene expression. Hum. Gene Ther. 9: 1885–1898.[Medline]
Higgins, D.G., Bleasby, A.J., and Fuchs, R. 1992. CLUSTAL V: Improved software for multiple sequence alignment. Comput. Appl. Biosci.8: 189–191.
Hirel, P.H., Schmitter, J.M., Dessen, P., Fayat, G., and Blanquet, S. 1989. Extent of N-terminal methionine excision from Escherichia coli proteins is governed by the side-chain length of the penultimate amino acid. Proc. Natl. Acad. Sci. 86: 8247–8251.
Horton, T.M., Ranheim, T.S., Aquino, L., Kusher, D.I., Saha, S.K., Ware, C.F., Wold, W.S., and Gooding, L.R. 1991. Adenovirus E3 14.7K protein functions in the absence of other adenovirus proteins to protect transfected cells from tumor necrosis factor cytolysis. J. Virol. 65: 2629–2639.
Horton, T.M., Tollefson, A.E., Wold, W.S., and Gooding, L.R. 1990. A protein serologically and functionally related to the group C E3 14,700-kilodalton protein is found in multiple adenovirus serotypes. J. Virol. 64: 1250–1255.
Horwitz, M.S. 2001. Adenovirus immunoregulatory genes and their cellular targets. Virology 279: 1–8.[CrossRef][Medline]
Jones, D.T. 1999. Protein secondary structure prediction based on position-specific scoring matrices. J. Mol. Biol. 292: 195–202.[CrossRef][Medline]
Kapust, R. and Waugh, D.S. 1999. Escherichia coli maltose-binding protein is uncommonly effective at promoting the solubility of polypeptides to which it is fused. Protein Sci 8: 1668–1674.[Abstract]
Kim, H.-J. 2000. I. Studies of AD5 E3–14.7K, an adenoviral inhibitor of TNF receptor- and Fas-mediated apoptosis. II. Investigation of interactions between cyclin-dependent kinase 4 and tumor suppressor p16INK4A. Ph.D., The Ohio State University.
Krajcsi, P. and Wold, W.S. 1998. Viral proteins that regulate cellular signalling. J. Gen. Virol. 79: 1323–1335.[Medline]
Li, Y., Kang, J., Friedman, J., Tarassishin, L., Ye, J., Kovalenko, A., Wallach, D., and Horwitz, M.S. 1999. Identification of a cell protein (FIP-3) as a modulator of NF-kappaB activity and as a target of an adenovirus inhibitor of tumor necrosis factor alpha-induced apoptosis. Proc. Natl. Acad. Sci. 96: 1042–1047.
Li, Y., Kang, J., and Horwitz, M.S. 1997. Interaction of an adenovirus 14.7-kilodalton protein inhibitor of tumor necrosis factor alpha cytolysis with a new member of the GTPase superfamily of signal transducers. J. Virol. 71: 1576–1582.[Abstract]
Li, Y., Kang, J., and Horwitz, M.S. 1998. Interaction of an adenovirus E3 14.7-kilodalton protein with a novel tumor necrosis factor alpha-inducible cellular protein containing leucine zipper domains. Mol. Cell. Biol. 18: 1601–1610.
Lukashok, S.A., Tarassishin, L., Li, Y., and Horwitz, M.S. 2000. An adenovirus inhibitor of tumor necrosis factor alpha-induced apoptosis complexes with dynein and a small GTPase. J. Virol. 74: 4705–4709.
Maret, W. and Vallee, B.L. 1993. Cobalt as probe and label of proteins. Methods Enzymol. 226: 52–71.[Medline]
Martin, G. and Ames, B.N. 1961. A method for determining the sedimentation behavior of enzymes: Application to protein mixtures. J. Biol. Chem. 236: 1372–1379.
McMeekin, T.L. and Marshal, K. 1952. Specific volumes of proteins and the relationship to their amino acid contents. Science 116: 142–144.
Muzio, M., Chinnaiyan, A.M., Kischkel, F.C., O'Rourke, K., Shevchenko, A., Ni, J., Scaffidi, C., Bretz, J.D., Zhang, M., Gentz, R., Mann, M., Krammer, P.H., Peter, M.E., and Dixit, V.M. 1996. FLICE, a novel FADD-homologous ICE/CED-3-like protease, is recruited to the CD95 (Fas/APO-1) death-inducing signaling complex. Cell 85: 817–827.[CrossRef][Medline]
Noel, J.P., Bingman, C.A., Deng, T.L., Dupureur, C.M., Hamilton, K.J., Jiang, R.T., Kwak, J.G., Sekharudu, C., Sundaralingam, M., and Tsai, M.D. 1991. Phospholipase A2 engineering. X-ray structural and functional evidence for the interaction of lysine-56 with substrates. Biochemistry 30: 11801–11811.[CrossRef][Medline]
Perkins, S.J. 1986. Protein volumes and hydration effects. The calculations of partial specific volumes, neuron scattering matchpoints and 280-nm absorption coefficients for proteins and glycoproteins from amino acid sequences. Eur. J. Biochem. 157: 169–180.[Medline]
Persson, H., Oberg, B., and Philipson, L. 1978. Purification and characterization of an early protein (E14K) from adenovirus type 2-infected cells. J. Virol. 28: 119–139.
Ranheim, T.S., Shisler, J., Horton, T.M., Wold, L.J., Gooding, L.R., and Wold, W.S. 1993. Characterization of mutants within the gene for the adenovirus E3 14.7-kilodalton protein which prevents cytolysis by tumor necrosis factor. J. Virol. 67: 2159–2167.
Rost, B. 1996. PHD: Predicting one-dimensional protein structure by profile-based neural networks. Methods Enzymol 266: 525–539.[CrossRef][Medline]
Rost, B. and Sander, C. 1993a. Improved prediction of a protein secondary structure by use of sequence profiles and neural networks. Proc. Natl. Acad. Sci. 90: 7558–7562.
Rost, B. and Sander, C. 1993b. Prediction of protein secondary structure at better than 70% accuracy. J. Mol. Biol. 232: 584–599.[CrossRef][Medline]
Rothwarf, D.M., Zandi, E., Natoli, G., and Karin, M. 1998. IKK-gamma is an essential regulatory subunit of the IkappaB kinase complex. Nature 395: 297–300.[CrossRef][Medline]
Siegel, L.M. and Monty, K.J. 1966. Determination of molecular weights and frictional ratios of proteins in impure systems by use of gel filtration and density gradient centrifugation, application to crude preparations of sulfite and hydroxylamine reductases. Biochim. Biophys. Acta. 112: 346–362.[Medline]
Sparer, T.E., Tripp, R.A., Dillehay, D.L., Hermiston, T.W., Wold, W.S., and Gooding, L.R. 1996. The role of human adenovirus early region 3 proteins (gp19K, 10.4K, 14.5K, and 14.7K) in a murine pneumonia model. J. Virol. 70: 2431–2439.[Abstract]
Thome, M., Schneider, P., Hofmann, K., Fickenscher, H., Meinl, E., Neipel, F., Mattmann, C., Burns, K., Bodmer, J., Schroter, M., Scaffidi, C., Krammer, P., Peter, M., and Tschopp, J. 1997. Viral FLICE-inhibitory proteins (FLIPs) prevent apoptosis induced by death receptors. Nature 386: 517–521.[CrossRef][Medline]
Tollefson, A.E. and Wold, W.S. 1988. Identification and gene mapping of a 14,700-molecular-weight protein encoded by region E3 of group C adenoviruses. J. Virol. 62: 33–39.
Tufariello, J., Cho, S., and Horwitz, M.S. 1994a. The adenovirus E3 14.7-kilodalton protein which inhibits cytolysis by tumor necrosis factor increases the virulence of vaccinia virus in a murine pneumonia model. J. Virol. 68: 453–462.
Tufariello, J.M., Cho, S., and Horwitz, M.S. 1994b. Adenovirus E3 14.7-kilodalton protein, an antagonist of tumor necrosis factor cytolysis, increases the virulence of vaccinia virus in severe combined immunodeficient mice. Proc. Natl. Acad. Sci. 91: 10987–10991.
Wold, W.S. 1993. Adenovirus genes that modulate the sensitivity of virus-infected cells to lysis by TNF. J. Cell. Biochem. 53: 329–335.[CrossRef][Medline]
Wold, W.S., Doronin, K., Toth, K., Kuppuswamy, M., Lichtenstein, D.L., and Tollefson, A.E. 1999. Immune responses to adenoviruses: Viral evasion mechanisms and their implications for the clinic. Curr. Opin. Immunol. 11: 380–386.[CrossRef][Medline]
Wold, W.S., Hermiston, T.W., and Tollefson, A.E. 1994. Adenovirus proteins that subvert host defenses. Trends Microbiol. 2: 437–443.[CrossRef][Medline]
Yamaoka, S., Courtois, G., Bessia, C., Whiteside, S.T., Weil, R., Agou, F., Kirk, H.E., Kay, R.J., and Israel, A. 1998. Complementation cloning of NEMO, a component of the IkappaB kinase complex essential for NF-kappaB activation. Cell 93: 1231–1240.[CrossRef][Medline]
Yasukawa, T., Kanei-Ishii, C., Maekawa, T., Fujimoto, J., Yamamoto, T., and Ishii, S. 1995. Increase of solubility of foreign proteins in Escherichia coli by coproduction of the bacterial thioredoxin. J. Biol. Chem. 270: 25328–25331.
Ye, J., Xie, X., Tarassishin, L., and Horwitz, M.S. 2000. Regulation of the NF-kappaB activation pathway by isolated domains of FIP3/IKKgamma, a component of the IkappaB-alpha kinase complex. J. Biol. Chem. 275: 9882–9889.
Zilli, D., Voelkel-Johnson, C., Skinner, T., and Laster, S.M. 1992. The adenovirus E3 region 14.7kDa protein, heat and sodium arsenate inhibit the TNF-induced release of arachidonic acid. Biochem. Biophys. Res. Commun. 188: 177–183.[CrossRef][Medline]
, cysteine residues in Ad5–14.7K which when mutated to serine result in loss of cell death protection; •, Cys/Ser mutations with near wild-type phenotype (
220) denaturation [Dstr]50% occurs at 
