New Roles for Conserved Regions within a ς54-dependent Enhancer-binding Protein
2002; Elsevier BV; Volume: 277; Issue: 44 Linguagem: Inglês
10.1074/jbc.m206912200
ISSN1083-351X
Autores Tópico(s)RNA modifications and cancer
Resumo23 amino acid substitutions were made in the C7 and C3 regions of pspFΔHTH, a protein required to convert ς54 closed promoter complexes to open complexes. These mutants were assayed for transcriptional competence, for the ability to hydrolyze ATP, for their multimerization state, and for their ability to interact with ς54 and its holoenzyme. C7 region mutants caused the protein to assume a compact form. This property could be mimicked by the addition of ATP, implying that compaction via C7 and ATP is part of the activation process. A number of C3 mutants were important for energy coupling, as indicated previously for several members of this activator family (1North A.K. Weiss D.S. Suzuki H. Flashner Y. Kustu S. J. Mol. Biol. 1996; 260: 317-331Crossref PubMed Scopus (44) Google Scholar, 2Wang Y.K. Lee J.H. Brewer J.M. Hoover T.R. Mol. Microbiol. 1997; 26: 373-386Crossref PubMed Scopus (46) Google Scholar). However, a patch within C3 influenced oligomerization. The C3 region was especially important in interacting with ς54 during the transition state but not important in inducing ς54holoenzyme to engage the nontemplate strand of the promoter. It is proposed that both regions contain deterrent functions that prevent premature activation. Overall, the results imply unexpected roles for the C7 and C3 regions of this protein family during promoter activation. 23 amino acid substitutions were made in the C7 and C3 regions of pspFΔHTH, a protein required to convert ς54 closed promoter complexes to open complexes. These mutants were assayed for transcriptional competence, for the ability to hydrolyze ATP, for their multimerization state, and for their ability to interact with ς54 and its holoenzyme. C7 region mutants caused the protein to assume a compact form. This property could be mimicked by the addition of ATP, implying that compaction via C7 and ATP is part of the activation process. A number of C3 mutants were important for energy coupling, as indicated previously for several members of this activator family (1North A.K. Weiss D.S. Suzuki H. Flashner Y. Kustu S. J. Mol. Biol. 1996; 260: 317-331Crossref PubMed Scopus (44) Google Scholar, 2Wang Y.K. Lee J.H. Brewer J.M. Hoover T.R. Mol. Microbiol. 1997; 26: 373-386Crossref PubMed Scopus (46) Google Scholar). However, a patch within C3 influenced oligomerization. The C3 region was especially important in interacting with ς54 during the transition state but not important in inducing ς54holoenzyme to engage the nontemplate strand of the promoter. It is proposed that both regions contain deterrent functions that prevent premature activation. Overall, the results imply unexpected roles for the C7 and C3 regions of this protein family during promoter activation. adenosine 5′-O-(thiotriphosphate) adenosine 5′-(β,γ-imino)triphosphate adenasine 3′,5′-diphosphate-aluminum fluoride Transcription of genes with promoters recognized by ς54 holoenzyme requires enhancer-binding activator proteins (3Sasse-Dwight S. Gralla J.D. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 8934-8938Crossref PubMed Scopus (190) Google Scholar). Prior to transcription, both the activator and the holoenzyme are usually inactive, with the latter being promoter-bound in a closed complex. After appropriate signaling pathways are initiated, sites typically located about 100–200 base pairs upstream of the promoter become bound by the modified activator protein (4Wyman C. Rombel I. North A.K. Bustamante C. Kustu S. Science. 1997; 275: 1658-1661Crossref PubMed Scopus (209) Google Scholar). Transcription occurs when a DNA loop is formed, allowing activator and holoenzyme to touch and use the ATPase of activator to trigger opening of the DNA by holoenzyme (5Weiss D.S. Batut J. Klose K.E. Keener J. Kustu S. Cell. 1991; 67: 155-167Abstract Full Text PDF PubMed Scopus (238) Google Scholar, 6Wedel A. Weiss D.S. Popham D. Droge P. Kustu S. Science. 1990; 248: 486-490Crossref PubMed Scopus (151) Google Scholar, 7Gralla J.D. Nat. Struct. Biol. 2000; 7: 530-532Crossref PubMed Scopus (9) Google Scholar). These enhancer-binding activator proteins often have three domains, as exemplified by the Salmonella typhimurium NtrC protein (8Morett E. Segovia L. J. Bacteriol. 1993; 175: 6067-6074Crossref PubMed Google Scholar). The C-terminal domain is needed for protein binding to the enhancer. The N-terminal domain receives the metabolic activation signal and begins its processing. The central domain is required for activation, since it contains the information that allows the coupling of ATP hydrolysis to the melting of the DNA by holoenzyme. How ATP hydrolysis is coupled to DNA melting by the activation domain is largely unknown. The central domain contains seven conserved regions termed C1 to C7. The C3 region is believed to be critical for several reasons. First, mutants have been found in this region that have significant levels of ATPase activity and bind DNA normally but fail in activating transcription (1North A.K. Weiss D.S. Suzuki H. Flashner Y. Kustu S. J. Mol. Biol. 1996; 260: 317-331Crossref PubMed Scopus (44) Google Scholar). Second, some mutants in this region appear to be defective in interacting with ς54 (2Wang Y.K. Lee J.H. Brewer J.M. Hoover T.R. Mol. Microbiol. 1997; 26: 373-386Crossref PubMed Scopus (46) Google Scholar, 9Chaney M. Grande R. Wigneshweraraj S.R. Cannon W. Casaz P. Gallegos M.T. Schumacher J. Jones S. Elderkin S. Dago A.E. Morett E. Buck M. Genes Dev. 2001; 15: 2282-2294Crossref PubMed Scopus (111) Google Scholar,10Lee J.H. Hoover T.R. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 9702-9706Crossref PubMed Scopus (84) Google Scholar). Third, there exists a distinct class of proteins that contain domains homologous to the central domain of ς54activators but activate forms of holoenzyme that lack ς54; these have sequences that differ primarily in the C3 region (e.g. Rhodobacter capsulatus NtrC) (11Foster-Hartnett D. Kranz R.G. Mol. Microbiol. 1992; 6: 1049-1060Crossref PubMed Scopus (42) Google Scholar,12Pittard A.J. Davidson B.E. Mol. Microbiol. 1991; 5: 1585-1592Crossref PubMed Scopus (105) Google Scholar). Mutations within C3 generally cause a lack of energy coupling; however, the proposed roles of individual conserved amino acids have varied when different activators were studied (1North A.K. Weiss D.S. Suzuki H. Flashner Y. Kustu S. J. Mol. Biol. 1996; 260: 317-331Crossref PubMed Scopus (44) Google Scholar, 2Wang Y.K. Lee J.H. Brewer J.M. Hoover T.R. Mol. Microbiol. 1997; 26: 373-386Crossref PubMed Scopus (46) Google Scholar). One focus of the current work will be to study the in vitro properties of mutants within this C3 region in a common context. Roles have been proposed for three of the other regions within this central activation domain. Regions C1 and C4 contain the Walker A and B motifs that interact with ATP (13Rombel I. Peters-Wendisch P. Mesecar A. Thorgeirsson T. Shin Y.K. Kustu S. J. Bacteriol. 1999; 181: 4628-4638Crossref PubMed Google Scholar). Region C7 is the site of a number of nonfunctional mutations, but its role is still unknown. Some studies suggest that it is involved in nucleotide binding (13Rombel I. Peters-Wendisch P. Mesecar A. Thorgeirsson T. Shin Y.K. Kustu S. J. Bacteriol. 1999; 181: 4628-4638Crossref PubMed Google Scholar, 14Neuwald A.F. Aravind L. Spouge J.L. Koonin E.V. Genome Res. 1999; 9: 27-43Crossref PubMed Google Scholar), and others suggest a role in activator oligomerization (15Perez-Martin J. de Lorenzo V. Cell. 1996; 86: 331-339Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar). This is an important consideration, because this class of proteins is converted from the dimer to a higher oligomer form as part of the activation process (16Porter S.C. North A.K. Wedel A.B. Kustu S. Genes Dev. 1993; 7: 2258-2273Crossref PubMed Scopus (156) Google Scholar). For this reason, we have also studied the properties of mutations in the C7 region. This study differs from prior ones in a number of significant ways. Prior mutations were studied in a number of different activator proteins and will now be studied in the context of a single protein. The protein chosen, pspFΔHTH, is essentially a pure activation domain; the virally induced protein contains no N-terminal signaling domain, and the natural C-terminal helix-turn-helix DNA binding region has been deleted, but the dimerization region is still intact (17Klose K.E. North A.K. Stedman K.M. Kustu S. J. Mol. Biol. 1994; 241: 233-245Crossref PubMed Scopus (57) Google Scholar, 18Jovanovic G. Rakonjac J. Model P. J. Mol. Biol. 1999; 285: 469-483Crossref PubMed Scopus (66) Google Scholar). This should allow all activation mutants to be studied in the same context in vitro, one that bypasses potential differences due to the differing influences of the unique signaling pathways of the various activators that have been studied previously (19O'Neill E. Wikstrom P. Shingler V. EMBO J. 2001; 20: 819-827Crossref PubMed Scopus (38) Google Scholar, 20Wikstrom P. O'Neill E., Ng, L.C. Shingler V. J. Mol. Biol. 2001; 314: 971-984Crossref PubMed Scopus (46) Google Scholar). In addition, several recently developed assays related to activation (see below) can now be applied to this series of mutant proteins. To do this, we collected data on genetically screened C3 and C7 mutants from several proteins and remade them in pspFΔHTH. A few site-directed C3 mutants were added to extend coverage to every position within this critical region. The proteins were purified and assayed for transcriptional competence, for the ability to hydrolyze ATP, for their multimerization state, and for their ability to interact with ς54 and its holoenzyme. The resulting data yield proposals for the roles of the C3 and C7 regions in the context of a simple protein that acts as a pure activation domain. The plasmid pMJ15 contains His6-pspFΔHTH (18Jovanovic G. Rakonjac J. Model P. J. Mol. Biol. 1999; 285: 469-483Crossref PubMed Scopus (66) Google Scholar). The QuikChange mutagenesis kit (Stratagene) was used for site-directed mutagenesis. Escherichia coli core enzyme was from Epicentre. E. coliς54 was purified as reported (21Wang J.T. Syed A. Gralla J.D. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 9538-9543Crossref PubMed Scopus (53) Google Scholar). Plasmid pHMK3′ contains K. pneumoniae ς54 with a heart muscle kinase tag attached to its 3′-end (22Casaz P. Buck M. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 12145-12150Crossref PubMed Scopus (38) Google Scholar) and was purified as described previously for ς54 (21Wang J.T. Syed A. Gralla J.D. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 9538-9543Crossref PubMed Scopus (53) Google Scholar), except that induction was with 1 mm isopropyl-1-thio-β-d-galactopyranoside and growth was at 37 °C. pspFΔHTH and its mutants are purified as described (18Jovanovic G. Rakonjac J. Model P. J. Mol. Biol. 1999; 285: 469-483Crossref PubMed Scopus (66) Google Scholar), except that a second batch purification was done. This involved elution with 330 mm imidazole and dialysis in the stated buffer without EDTA, followed by a second round of purification using the Ni2+-nitrilotriacetic acid beads. Unless otherwise indicated, proteins were stored in elution buffer. In vitro activated transcription was conducted as described (23Guo Y. Lew C.M. Gralla J.D. Genes Dev. 2000; 14: 2242-2255Crossref PubMed Scopus (63) Google Scholar) with minor changes. Briefly, 75 nm ς54 RNA polymerase was incubated with 0.5 mm GTP, 0.5 mm ATP, and 0.5 mm UTP in 1× buffer B (50 mm Tris-HCl at pH 7.5, 50 mm KCl, 10 mm MgCl2, 0.1 mm EDTA, 2 mm β-mercaptoethanol) (24Dworkin J. Jovanovic G. Model P. J. Bacteriol. 2000; 182: 311-319Crossref PubMed Scopus (78) Google Scholar) for 20 min at 37 °C before 1 μl of CTP mixture (50 μm CTP and 0.2 μCi/μl [α-32P]CTP) was added. This mixture was incubated for an additional 10 min at 37 °C before the sample was prepared for loading on 6% PAGE with urea. Radiolabeled RNA was analyzed with a PhosphorImager. The filter binding assay was done as described (13Rombel I. Peters-Wendisch P. Mesecar A. Thorgeirsson T. Shin Y.K. Kustu S. J. Bacteriol. 1999; 181: 4628-4638Crossref PubMed Google Scholar) with minor changes. Briefly, 5 μm pspFΔHTH and its mutants were incubated in 1× buffer A (25 mm Hepes at pH 7.5, 20 mm MgCl2, 10 mm KCl, 2 mm β-mercaptoethanol) before ATPγS1 mix (0.6 mm ATPγS, 69 μm [γ-35S]ATP) was added to the 20-μl reaction. This was incubated at 37 °C for 4 min. The reaction was applied to a polyvinylidene difluoride membrane (0.45-μm Immobilon P, prepared according to the manufacturer's instructions; Millipore Corp.), which sat on top of a sintered glass filter, and vacuum was quickly applied to remove liquid. 1 ml of wash buffer (20 mm Hepes at pH 7.5, 10 mmMgCl2) was immediately applied to the membrane followed by vacuum. The membrane was dried, and radioactivity was determined by scintillation counting. Each experiment was normalized using a parallel sample of wild type protein. In a standard Pi release assay (13Rombel I. Peters-Wendisch P. Mesecar A. Thorgeirsson T. Shin Y.K. Kustu S. J. Bacteriol. 1999; 181: 4628-4638Crossref PubMed Google Scholar), 200 nm pspFΔHTH and its mutants were incubated in 1× ATPase buffer (25 mm Hepes at pH 7.5, 20 mmMgCl2, 30 mm KCl, 2 mmβ-mercaptoethanol (18Jovanovic G. Rakonjac J. Model P. J. Mol. Biol. 1999; 285: 469-483Crossref PubMed Scopus (66) Google Scholar), 0.3 mm ATP, 20 μg/ml acetylated bovine serum albumin, 0.3 μCi/μl [γ-32P]ATP) for 15 min at 37 °C. A 1-μl aliquot was spotted onto a prerun polyethyleneimine TLC plate, and electrophoresis was with 0.75m phosphate buffer at pH 4.1. Afterward, the plate was dried and analyzed using a PhosphorImager. Sample dye was added to 2.5 μg of protein, sample was loaded onto a 4% stacking, 10% resolving native polyacrylamide gel, and electrophoresis was performed as described (25). In samples that contained ATP, 4 mm ATP was incubated with the protein for 5 min on ice prior to electrophoresis in a system with 4 mm ATP in the gel and electrophoresis buffer. Glutaraldehyde cross-linking was in Buffer G (50 mm sodium phosphate at pH 7, 20 mm NaCl, and 12% glycerol). Where indicated, 4 mm ATP was incubated with 16 μm activator for 5 min at 30 °C. 160 μm glutaraldehyde was added to 16 μm protein in a 6.75-μl reaction volume. This was incubated at 30 °C for 12 min, followed by the addition of 1 μl of 1 m glycine to stop the reaction. 3 μl of 2× SDS-PAGE dye was added, heated for 2 min at 90 °C, and then loaded onto a 8% SDS-PAGE. The gel was stained with Coomassie Blue to visualize. Promoter probes and electrophoretic mobility shift assay were as described (26Guo Y. Wang L. Gralla J.D. EMBO J. 1999; 18: 3736-3745Crossref PubMed Scopus (50) Google Scholar) with minor modifications. Briefly, 1 nm annealed promoter probe was added to 7.5 nm holoenzyme in 1× STA buffer (25 mm Tris acetate at pH 8, 8 mm magnesium acetate, 10 mm KCl, 1 mm β-mercaptoethanol, 3.5% (w/v) polyethylene glycol 8000 (9Chaney M. Grande R. Wigneshweraraj S.R. Cannon W. Casaz P. Gallegos M.T. Schumacher J. Jones S. Elderkin S. Dago A.E. Morett E. Buck M. Genes Dev. 2001; 15: 2282-2294Crossref PubMed Scopus (111) Google Scholar)). Where indicated, 1.5 μm pspFΔHTH protein or 4 mm ATP concentration was present. The 10-μl reaction was incubated at 37 °C for 10 min followed by the addition of 0.5 μl of 2 mg/ml heparin. This was incubated for an additional 5 min before analysis by 5% PAGE (Minigel system; Bio-Rad) in 1× TBE buffer. The gel was run at 300 V at room temperature. Binding of pspFΔHTH to ς54 in the presence of ADP-aluminum fluoride was as described (9Chaney M. Grande R. Wigneshweraraj S.R. Cannon W. Casaz P. Gallegos M.T. Schumacher J. Jones S. Elderkin S. Dago A.E. Morett E. Buck M. Genes Dev. 2001; 15: 2282-2294Crossref PubMed Scopus (111) Google Scholar). Where indicated, 10 μm activator was added for 5 min at 30 °C to STA buffer with 150 nm32P-labeled heart muscle kinase tagged ς54, 50 ng of α-lactoalbumin, 0.2 mm ADP, 5.0 mm NaF. 0.2 mm AlCl3 was then added and allowed to incubate for an additional 10 min before being loaded onto a native protein gel. Fig.1 (top) shows the alignment of the C3 and C7 regions as well as (bottom) the mutations that were made in pspFΔHTH. Most of these replicated previously identified nonfunctional mutants within Salmonella typhimurium NtrC (1North A.K. Weiss D.S. Suzuki H. Flashner Y. Kustu S. J. Mol. Biol. 1996; 260: 317-331Crossref PubMed Scopus (44) Google Scholar), Rhizobium melioti DctD (2Wang Y.K. Lee J.H. Brewer J.M. Hoover T.R. Mol. Microbiol. 1997; 26: 373-386Crossref PubMed Scopus (46) Google Scholar), and Bradyrhizobium japonicum NifA (27Gonzalez V. Olvera L. Soberon X. Morett E. Mol. Microbiol. 1998; 28: 55-67Crossref PubMed Scopus (35) Google Scholar). A few changes were based on other proteins (15Perez-Martin J. de Lorenzo V. Cell. 1996; 86: 331-339Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar, 28Takata M. Guo L. Katayama T. Hase M. Seyama Y. Miki T. Sekimizu K. Mol. Microbiol. 2000; 35: 454-462Crossref PubMed Scopus (13) Google Scholar), as shown. Finally, four site-directed serine substitutions were made in the intensively studied and well conserved C3 region so that every residue would be covered by mutation. These four positions have not been studied previously in vitro. The C7 region is sparsely covered by previously identified nonfunctional mutants, and we have not attempted to saturate it. The set consisted of 23 individual substitutions in pspFΔHTH. These 23 proteins were purified according to Jovanovic et al. (18Jovanovic G. Rakonjac J. Model P. J. Mol. Biol. 1999; 285: 469-483Crossref PubMed Scopus (66) Google Scholar) with some modifications. They were then tested in anin vitro transcription assay (Fig.2 and not shown) using the ς54-dependent glnAp2promoter. All except one of the proteins, E76Q, were very defective in transcription in vitro. The lack of significant activity of all but one of these is consistent with the original mutant phenotypes (1North A.K. Weiss D.S. Suzuki H. Flashner Y. Kustu S. J. Mol. Biol. 1996; 260: 317-331Crossref PubMed Scopus (44) Google Scholar, 2Wang Y.K. Lee J.H. Brewer J.M. Hoover T.R. Mol. Microbiol. 1997; 26: 373-386Crossref PubMed Scopus (46) Google Scholar) and, in the cases studied, with in vitrotranscription studies using the original proteins. The four site-directed mutants in C3 were also nonfunctional, confirming the general importance of residues within this region. Thus, the data confirms that mutations in pspFΔHTH largely reflect the same gross defects seen when the same changes were made in a variety of other proteins containing homologous domains. The ATPase activity of this class of activator proteins is essential for function (5Weiss D.S. Batut J. Klose K.E. Keener J. Kustu S. Cell. 1991; 67: 155-167Abstract Full Text PDF PubMed Scopus (238) Google Scholar). The purified proteins were subjected to an ATPase assay, and the results are shown in Table I. Six of the 23 mutants, A82N, G83D, and G87K in C3, R227H and G224V in C7, and R235H near C7, lacked detectable activity. One C3 mutant, S75F, had activity that was equal to or slightly better than wild type. All of the other mutants had lowered amounts of ATPase activity, ranging as low as 6% of wild type levels.Table IATPase and ATPγS binding assaysPspFΔHTHATPaseATPγS bindingWild type++++S75F+++S75I+/−+E76Q++/−L77S+/−+/−F78S+/−+/−G79S+/−+/−H80R++E81S++/−A82N−+G83D−+/−A84V+/−+F85A+/−+/−F85L++T86A+/−+/−T86M++T86I++G87K−+/−A88T+/−+/−G224D+/−+/−G224V−+/−R227H−−R227C+/−+R235H−+Symbols represent ATP hydrolysis relative to wild type as follows: ++, 100% and greater; + 75–25%; +/− 24–6%; −, below 6%. For ATPγS binding: ++, 100% and greater, +, 75–30%; +/−, 29%–6%; −, below 6%. Open table in a new tab Symbols represent ATP hydrolysis relative to wild type as follows: ++, 100% and greater; + 75–25%; +/− 24–6%; −, below 6%. For ATPγS binding: ++, 100% and greater, +, 75–30%; +/−, 29%–6%; −, below 6%. Thus, only one of the mutants in region C3 (S75F) showed no defect in ATPase activity and yet was transcriptionally deficient. This would be a strict coupling mutant. Six other C3 mutants retained on average 50% of the wild type level of activity (plus symbols in Table I) despite showing little or no transcription. These too, are very strong candidates for coupling mutants in pspFΔHTH. The locus of coupling mutations being in the C3 region is consistent with prior studies using other proteins (1North A.K. Weiss D.S. Suzuki H. Flashner Y. Kustu S. J. Mol. Biol. 1996; 260: 317-331Crossref PubMed Scopus (44) Google Scholar, 2Wang Y.K. Lee J.H. Brewer J.M. Hoover T.R. Mol. Microbiol. 1997; 26: 373-386Crossref PubMed Scopus (46) Google Scholar). However, some differences in ATPase activity are apparent in the data. The adjacent mutants S75F and E76Q failed to show activity in NtrC (13Rombel I. Peters-Wendisch P. Mesecar A. Thorgeirsson T. Shin Y.K. Kustu S. J. Bacteriol. 1999; 181: 4628-4638Crossref PubMed Google Scholar) but do here in pspFΔHTH (Table I), as a different mutation in Ser75did for DctD (S75I) (2Wang Y.K. Lee J.H. Brewer J.M. Hoover T.R. Mol. Microbiol. 1997; 26: 373-386Crossref PubMed Scopus (46) Google Scholar). H80R also showed activity in pspFΔHTH, whereas it did not in DctD (2Wang Y.K. Lee J.H. Brewer J.M. Hoover T.R. Mol. Microbiol. 1997; 26: 373-386Crossref PubMed Scopus (46) Google Scholar). It is possible that the retention of ATPase activity in these three mutants in pspFΔHTH is related to the lack of other domains that might contribute to suppression of activity. On the other hand, G87K has ATPase activity in NtrC but none here. Overall, however, the agreement between pspFΔHTH mutants and that of other proteins is quite good. The C7 region mutants showed varying degrees of ATPase activity, although on average they were more defective than those in the C3 region (Table I). In partial agreement, the C7 mutants studied previously in NtrC were all defective in ATP hydrolysis (13Rombel I. Peters-Wendisch P. Mesecar A. Thorgeirsson T. Shin Y.K. Kustu S. J. Bacteriol. 1999; 181: 4628-4638Crossref PubMed Google Scholar). On the whole, the evidence indicates that the C7 region is required for ATPase activity, although whether this effect is direct or indirect is not established by the existing experimental data. ATP binding was also assayed to learn whether the lack of hydrolysis in some cases was due to a lack of binding. Since ATP binding levels in the membrane-binding assay are low, as also shown previously (13Rombel I. Peters-Wendisch P. Mesecar A. Thorgeirsson T. Shin Y.K. Kustu S. J. Bacteriol. 1999; 181: 4628-4638Crossref PubMed Google Scholar, 20Wikstrom P. O'Neill E., Ng, L.C. Shingler V. J. Mol. Biol. 2001; 314: 971-984Crossref PubMed Scopus (46) Google Scholar), the data obtained varied somewhat for duplicates. For this reason, data from four or five experiments were needed to obtain the average ranges shown in Table I. The procedure relies on the ability of proteins to retain the nonhydrolyzable analogue ATPγS during a membrane-binding assay. Since the assay involves washing filters, the extent of ATP binding is far less than in the equilibrium experiments below. Nonetheless, the mutant data show defects relative to wild type. The results showed that of the six mutants that could not hydrolyze ATP, two bound ATPγS well (A82N and R235H; Table I), and the others bound it either with significantly reduced affinity (G83D, G87K, and G224V) or at a level below the sensitivity of the assay (R227H). We infer that at least some of these mutants cannot hydrolyze ATP because it is poorly bound. In terms of pure coupling effects, A82N and R235H are the only two mutants that can bind ATPγS but could not hydrolyze ATP (Table I). These various ATP-related defects are very widespread in the data, despite the fact that the Walker A and B regions are still intact. This suggests that for a number of mutants the defect could be indirect. For this class of proteins, function requires the formation of higher order oligomers (4Wyman C. Rombel I. North A.K. Bustamante C. Kustu S. Science. 1997; 275: 1658-1661Crossref PubMed Scopus (209) Google Scholar). The central activation domain has been implicated to be involved in oligomerization (29Flashner Y. Weiss D.S. Keener J. Kustu S. J. Mol. Biol. 1995; 249: 700-713Crossref PubMed Scopus (63) Google Scholar); however, it is not known which amino acids in this domain are involved. To assay for these, we next studied the multimerization states of the collection of mutants. PspFΔHTH has been reported to exist predominantly as a dimer (72 kDa) with higher molecular weight oligomers occurring at high concentrations of protein (18Jovanovic G. Rakonjac J. Model P. J. Mol. Biol. 1999; 285: 469-483Crossref PubMed Scopus (66) Google Scholar). We repeated the native gel assay to confirm this under the present conditions and to learn the multimerization status of the collection of mutants. This initial assay was conducted using normal solution concentrations of pspFΔHTH, where dimers are expected to predominate. In this assay, pspFΔHTH runs as a very broad band with an apparent molecular weight somewhat higher than that expected for a dimer (Fig. 3 A,Wt). This broad band has been seen previously (18Jovanovic G. Rakonjac J. Model P. J. Mol. Biol. 1999; 285: 469-483Crossref PubMed Scopus (66) Google Scholar) and may represent the different conformers of pspFΔHTH (30Volkman B.F. Lipson D. Wemmer D.E. Kern D. Science. 2001; 291: 2429-2433Crossref PubMed Scopus (529) Google Scholar) undergoing kinetic exchange. Although at this concentration the dimer is the predominant form in solution, it is difficult to say with certainty what forms exist within the broad band. At this protein concentration, no specific bands that would clearly represent higher order oligomers are seen. When the mutant proteins are assayed, many show smears with mobility altered slightly from wild-type or with lesser intensity. These and other results were reproducible using two different preparations of proteins. The mobility changes may represent alterations in the equilibrium population of different oligomers undergoing kinetic exchange during the native gel electrophoresis. Two patches of mutants, however, show bands that are more discrete than wild-type and also have altered mobility. One of these consists of the adjacent amino acids Leu77, Phe78, and Gly79 in the C3 region. In these cases, the broad band observed with wild type is replaced by a series of discrete bands beginning with an apparent dimer and including apparent higher molecular weight forms (Fig. 3 A, L77S, F78S, and G79Sversus wild type). These forms probably correspond to tetramers and hexamers, since the pattern resembles that seen for wild-type protein at high concentration (18Jovanovic G. Rakonjac J. Model P. J. Mol. Biol. 1999; 285: 469-483Crossref PubMed Scopus (66) Google Scholar). Mutation of the adjacent residue Glu76 shows a sharpened band but little evidence of oligomerization (Fig. 3 A). No other mutations caused this pattern of band sharpening. We infer that this patch within the C3 region contains determinants that strongly affect the propensity of pspFΔHTH to form higher order multimers. The (E)LFG sequence is highly conserved within the ς54 family of activators (8Morett E. Segovia L. J. Bacteriol. 1993; 175: 6067-6074Crossref PubMed Google Scholar), so this phenomenon could apply to other proteins. Alanine substitutions within this sequence in DctD led to some small defects in expression in vivo, but in vitro transcription and oligomerization state were not tested (2Wang Y.K. Lee J.H. Brewer J.M. Hoover T.R. Mol. Microbiol. 1997; 26: 373-386Crossref PubMed Scopus (46) Google Scholar). Other mutants that differed in mobility from wild type were restricted to the C7 region. In fact, all C7 mutants tested had altered mobility on native gels (Fig. 3 B). The broad band characteristic of wild type protein was converted into more discrete bands by the C7 mutations. In four of the five mutants, this band ran significantly lower on the gel (Fig. 3 B, e.g. G224D versus Wt), reducing the apparent molecular mass from 90 to 66 kDa (the theoretical dimer molecular mass is 72 kDa). Thus, the C7 region plays a role in influencing the conformational state of the pspFΔHTH dimer. In most cases, mutations convert a conformational diverse collection of states (a broad band with low mobility, Wt in Fig. 3 B) to unique species with apparently compacted conformations (narrow bands with high mobility; e.g. G224D in Fig. 3 B). Although the concentration used in this assay may be higher than found in vivo, the result shows that certain mutants alter the propensity to assume these various states. Most of the mutants show some evidence of discrete bands of differing mobility, particularly in the case of R227C. Moreover, not all the bands have precisely identical mobility. Thus, it appears that the C7 mutations create predominantly more compact forms of the protein, but many conformational states may still be accessible. We hypothesized that the C7 region mutations might be partially mimicking the effect of ATP, and so the experiments were repeated in the presence of ATP. The sample, gel, and buffer reservoir contained ATP to maximize its occupancy of the proteins during electrophoresis. The results for selected mutants of interest are shown in Fig. 3 C. The effect of ATP on the native gel mobility of the wild-type protein was dramatic. The broad band was converted to a narrow band with much greater mobility (Fig. 3, compare Wt in A withWt in C). In fact, the effect of ATP on wild-type closely mimicked the effects of C7 mutants (Fig. 3, compareWt in C and G224D in B). It appears that ATP converts a mixtur
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