Characterization of the Dimerization Domain in the FNR Transcription Factor

Abstract

The global anaerobic regulator FNR fromEscherichia coli is a dimeric Fe-S protein that is inactivated by O2 through disruption of its [4Fe-4S] cluster and conversion to a monomeric form. As a first step in elucidating the molecular interactions that control FNR dimerization, we have performed alanine-scanning mutagenesis of a potential dimerization domain. Replacement of many hydrophobic residues (Met-143, Met-144, Leu-146, Met-147, Ile-151, Met-157, and Ile-158) and two charged residues (Arg-140 and Arg-145) with Ala decreased FNR activityin vivo. Size exclusion chromatography and Fe-S cluster analysis of three representative mutant proteins, FNR-M147A, FNR-I151A, and FNR-I158A, showed that the Ala substitutions produced specific defects in dimerization. Because hydrophobic side chains are known to stabilize subunit-subunit interactions between α-helices, we propose that Met-147, Ile-151, and Ile-158 lie on the same face of an α-helix that constitutes a dimerization interface. This alignment would also position Arg-140, Met-144, and Asp-154 on the same helical face. In support of the unusual positioning of a negatively charged residue at the dimer interface, we found that replacing Asp-154 with Ala repaired the defects caused by Ala substitutions of other residues located on the same helical face. These data also suggest that Asp-154 has an inhibitory effect on dimerization, which may be a key element in the control of FNR dimerization by O2 availability. The global anaerobic regulator FNR fromEscherichia coli is a dimeric Fe-S protein that is inactivated by O2 through disruption of its [4Fe-4S] cluster and conversion to a monomeric form. As a first step in elucidating the molecular interactions that control FNR dimerization, we have performed alanine-scanning mutagenesis of a potential dimerization domain. Replacement of many hydrophobic residues (Met-143, Met-144, Leu-146, Met-147, Ile-151, Met-157, and Ile-158) and two charged residues (Arg-140 and Arg-145) with Ala decreased FNR activityin vivo. Size exclusion chromatography and Fe-S cluster analysis of three representative mutant proteins, FNR-M147A, FNR-I151A, and FNR-I158A, showed that the Ala substitutions produced specific defects in dimerization. Because hydrophobic side chains are known to stabilize subunit-subunit interactions between α-helices, we propose that Met-147, Ile-151, and Ile-158 lie on the same face of an α-helix that constitutes a dimerization interface. This alignment would also position Arg-140, Met-144, and Asp-154 on the same helical face. In support of the unusual positioning of a negatively charged residue at the dimer interface, we found that replacing Asp-154 with Ala repaired the defects caused by Ala substitutions of other residues located on the same helical face. These data also suggest that Asp-154 has an inhibitory effect on dimerization, which may be a key element in the control of FNR dimerization by O2 availability. cAMP receptor protein wild type FNR is a global regulator that allows Escherichia colito adapt to anaerobic growth conditions. In the absence of oxygen, FNR is in its active form and binds to specific DNA sites to either activate or repress transcription of target genes (for a review see Refs. 1Guest J.R. Green J. Irvine A.S. Spiro S. Lin E.C.C. Lynch A.S. Regulation of Gene Expression in Escherichia coli. Chapman & Hall, New York1996: 317-342Crossref Google Scholar and 2Patschkowski T. Bates D.M. Kiley P.J. Storz G. Hengee-Aronis R. Bacterial Stress Response. American Society for Microbiology, Washington, D. C.2000: 61-78Google Scholar). A novel feature of FNR is that its DNA binding activity is associated with its ability to dimerize, a property regulated by the presence of an O2-labile [4Fe-4S] cluster (3Lazazzera B. Bates D. Kiley P.J. Genes Dev. 1993; 7: 1993-2005Crossref PubMed Scopus (130) Google Scholar, 4Lazazzera B.A. Beinert H. Khoroshilova N. Kennedy M.C. Kiley P.J. J. Biol. Chem. 1996; 271: 2762-2768Abstract Full Text Full Text PDF PubMed Scopus (268) Google Scholar). When the protein contains a [4Fe-4S] cluster, FNR forms homodimers in solution (4Lazazzera B.A. Beinert H. Khoroshilova N. Kennedy M.C. Kiley P.J. J. Biol. Chem. 1996; 271: 2762-2768Abstract Full Text Full Text PDF PubMed Scopus (268) Google Scholar, 5Khoroshilova N. Popescu C. Munck E. Beinert H. Kiley P.J. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 6087-6092Crossref PubMed Scopus (303) Google Scholar). However, when O2 is present, the [4Fe-4S] clusters are destroyed, and the ability of FNR to dimerize is decreased, therefore reducing its site-specific DNA binding and abolishing its ability to control transcription (5Khoroshilova N. Popescu C. Munck E. Beinert H. Kiley P.J. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 6087-6092Crossref PubMed Scopus (303) Google Scholar, 6Green J. Bennett B. Jordan P. Ralph E.T. Thomson A.J. Guest J.R. Biochem. J. 1996; 316: 887-892Crossref PubMed Scopus (160) Google Scholar). Thus, for FNR, DNA binding and dimerization appear to be tightly coupled processes that provide a mechanism for controlling transcription of specific genes in response to changes in O2 levels. The molecular interactions that promote dimerization of FNR are unknown because there is currently no structural information for this protein. Nevertheless, amino acid residues 140–159 of FNR have been proposed to function in subunit-subunit interactions because the position of these residues corresponds to the α-helix along which the closely relatedE. coli transcription factor, the cAMP receptor protein (CRP),1 dimerizes via a coiled coil interaction (4Lazazzera B.A. Beinert H. Khoroshilova N. Kennedy M.C. Kiley P.J. J. Biol. Chem. 1996; 271: 2762-2768Abstract Full Text Full Text PDF PubMed Scopus (268) Google Scholar, 7Weber I.T. Steitz T.A. J. Mol. Biol. 1987; 198: 311-326Crossref PubMed Scopus (430) Google Scholar) (Fig. 1). Consistent with this region having a role in FNR subunit-subunit interactions, substitution of Asp-154 to Ala (Fig. 1) increases FNR dimerization in the absence of a [4Fe-4S] cluster (3Lazazzera B. Bates D. Kiley P.J. Genes Dev. 1993; 7: 1993-2005Crossref PubMed Scopus (130) Google Scholar, 8Kiley P.J. Reznikoff W.S. J. Bacteriol. 1991; 173: 16-22Crossref PubMed Google Scholar). However, the function of other residues within this putative dimerization domain has not been investigated. In addition, the amino acid side chains of CRP and FNR are not highly conserved in this region (9Shaw D.J. Rice D.W. Guest J.R. J. Mol. Biol. 1983; 166: 241-247Crossref PubMed Scopus (126) Google Scholar), raising the issue of whether this region of FNR will have unique features to attain regulated dimerization. The role of the [4Fe-4S] cluster in promoting subunit-subunit interactions is even less well understood. The [4Fe-4S] cluster is ligated by three Cys residues located within the N-terminal end of FNR, a region that is absent in CRP. The fourth ligand of the cluster (Cys-122) is predicted to fall within a proposed β-roll structure of FNR (10Melville S.B. Gunsalus R.P. J. Biol. Chem. 1990; 265: 18733-18736Abstract Full Text PDF PubMed Google Scholar, 11Sharrocks A.D. Green J. Guest J.R. FEBS Lett. 1990; 270: 119-122Crossref PubMed Scopus (69) Google Scholar, 12Bates D. Lazzazzera B. Kiley P.J. J. Bacteriol. 1995; 177: 3972-3978Crossref PubMed Google Scholar) that precedes the putative dimerization domain (Fig. 1). Given the location of these four cysteine residues, the [4Fe-4S] cluster of FNR is not expected to be very close to the dimerization helix (13Kiley P.J. Beinert H. FEMS Microbiol. Rev. 1999; 22: 341-352Crossref Google Scholar). Thus, it is an open question as to how the presence of a [4Fe-4S] cluster facilitates subunit-subunit interactions within FNR. To understand the molecular interactions that promote FNR dimerization, we have examined the role of residues 140–159 of FNR. Alanine scanning mutagenesis was initially used to identify residues of this region required for FNR activity in vivo. Size exclusion chromatography and Fe-S cluster analysis was subsequently used to characterize the in vitro properties of representative mutant proteins. Our findings suggest that residues 140–159 define a dimerization domain of FNR. In addition, the properties of these mutant FNR proteins support the notion that the [4Fe-4S] cluster of FNR promotes subunit interactions by altering the monomer-dimer equilibrium. Plasmids and strains are listed in Table I. Single alanine substitutions in the dimerization helix (residues 140–159) were constructed by site-directed mutagenesis of pPK821 using oligonucleotide primers (Operon Technologies) as described previously (3Lazazzera B. Bates D. Kiley P.J. Genes Dev. 1993; 7: 1993-2005Crossref PubMed Scopus (130) Google Scholar). Alanine mutants with a second alanine substitution at Asp-154 were constructed in the same manner except using pPK822 (3Lazazzera B. Bates D. Kiley P.J. Genes Dev. 1993; 7: 1993-2005Crossref PubMed Scopus (130) Google Scholar) as the starting template. The FNR coding sequence of the pPK821 or pPK822 derivatives was cloned into pET-11a using the NdeI and BamHI restriction sites. For some mutants, the fnr gene with the normal fnr promoter region was cloned into pACYC184 using the HindIII and BamHI restriction sites. Thefnr gene of each plasmid was sequenced using either a33P thermosequenase terminator cycle or a at the of to the presence of the and used in this or coli strains B. Bates D. Kiley P.J. Genes Dev. 1993; 7: 1993-2005Crossref PubMed Scopus (130) Google Scholar E. in Escherichia of Scholar Kiley P.J. Mol. Microbiol. PubMed Scopus Google Scholar B. Bates D. Kiley P.J. Genes Dev. 1993; 7: 1993-2005Crossref PubMed Scopus (130) Google Scholar promoter and gene NdeI J. Mol. Biol. 1991; PubMed Scopus Google Scholar to of fnr with NdeI at in B. Bates D. Kiley P.J. Genes Dev. 1993; 7: 1993-2005Crossref PubMed Scopus (130) Google Scholar as pPK821 B. Bates D. Kiley P.J. Genes Dev. 1993; 7: 1993-2005Crossref PubMed Scopus (130) Google Scholar of to in B. Bates D. Kiley P.J. Genes Dev. 1993; 7: 1993-2005Crossref PubMed Scopus (130) Google Scholar as B. Bates D. Kiley P.J. Genes Dev. 1993; 7: 1993-2005Crossref PubMed Scopus (130) Google Scholar J. Bacteriol. PubMed Google Scholar in a pET-11a FNR or its mutant derivatives were for because analysis showed that the FNR protein produced the are that the not Thus, the of protein these would of potential dimerization defects at protein that would its promoter on this because FNR is not the control of its these have the of in protein that through a in of FNR S. Guest J. J. Microbiol. 1987; Google Scholar, FEMS Microbiol. Lett. Scopus Google Scholar). also the activity of some mutant proteins at protein by using FNR protein produced the was in that produced fnr on the not FNR activity was by activity of E. coli strains Table that the control of either or promoter as described previously (3Lazazzera B. Bates D. Kiley P.J. Genes Dev. 1993; 7: 1993-2005Crossref PubMed Scopus (130) Google Scholar). were anaerobic in and or was to the when the either pET-11a or pACYC184 In addition, was to the when with the pET-11a to of the A. Gunsalus R.P. J. Bacteriol. 1999; PubMed Google Scholar). the with and are the the of in the presence of to mutants that some activity that found in the absence of FNR. FNR was mutant or FNR anaerobic as described previously D.M. Popescu Khoroshilova N. Beinert H. E. Kiley P.J. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). The and of each was using Kennedy M.C. T.A. H. Beinert H. E. J. Biol. Chem. Full Text PDF PubMed Google Scholar) and Beinert H. Biochem. 1983; PubMed Scopus Google Scholar), Size exclusion chromatography was performed on a to a chromatography within a anaerobic with an of and The were performed in and at a of The FNR were in at a of [4Fe-4S] FNR protein to The of the was to using a The was with and of the mutant proteins in and were in on a a of were in in an The protein were FNR in a of and was in and the was for at each acid residues 140–159 of FNR are to the residues of the CRP that promotes subunit interactions through of a coiled To this acid region an function in FNR, we examined the effect of replacing each residue with Ala on FNR function in vivo. is we would that at a Ala substitution of hydrophobic residues with a of a coiled coil interaction residues FNR FNR activity was by the of strains control of the promoter For this pET-11a its mutant derivatives cloned the promoter were because in the absence of we found that FNR produced this plasmid not of FNR not The of the amino acid substitutions that were found to FNR activity an Ala in of a hydrophobic residue FNR-M147A, FNR-I151A, and In addition, when two charged residues were by Ala and FNR activity was also of the residues that were found to be for FNR activity are three to four residues Met-144, Met-147, Ile-151, and that fall on the same side of an α-helix (Fig. the residues of the helix are to a coiled coil see were using strains to not that the of these mutant proteins are not analysis that the mutant proteins are at to that of wild type not that of these defects be to in FNR levels. To the defects of the Ala substitution mutants subunit-subunit interactions, representative proteins FNR-I151A, and were and The of the mutant proteins were to have an at that is of a [4Fe-4S] cluster (5Khoroshilova N. Popescu C. Munck E. Beinert H. Kiley P.J. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 6087-6092Crossref PubMed Scopus (303) Google Scholar). In addition, the of and in the protein was to whether the of the [4Fe-4S] cluster in the mutant proteins was to that of This analysis showed that and of FNR-M147A, FNR-I151A, and FNR-I158A, a [4Fe-4S] cluster, which is within the of with D.M. Popescu Khoroshilova N. Beinert H. E. Kiley P.J. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). these data suggest that the mutant proteins a [4Fe-4S] cluster at to that of To whether of the mutant proteins defects in dimerization, exclusion chromatography of proteins was anaerobic conditions. To the of the FNR the [4Fe-4S] the of the was on to As expected for FNR protein (4Lazazzera B.A. Beinert H. Khoroshilova N. Kennedy M.C. Kiley P.J. J. Biol. Chem. 1996; 271: 2762-2768Abstract Full Text Full Text PDF PubMed Scopus (268) Google Scholar) the showed that the [4Fe-4S] cluster an expected of a the cluster was in the monomeric the in at of the [4Fe-4S] as a dimer (Fig. exclusion chromatography of FNR-M147A, FNR-I151A, and at protein The of [4Fe-4S] cluster FNR at chromatography of at and at and at and and at and of In the of some mutant FNR proteins was For at a of [4Fe-4S] FNR-I158A, the of the [4Fe-4S] form of as a dimer (Fig. to However, as the of was decreased to (Fig. or (Fig. the and the monomeric A was with (Fig. except that at [4Fe-4S] FNR-M147A, the of the [4Fe-4S] form was between that of a and a dimer (Fig. a dimerization The of the and the and of the suggest that some of the dimeric [4Fe-4S] mutant protein was as it through the These data that of and Ile-158 with Ala not FNR dimer In a because the of as a to an of [4Fe-4S] protein (Fig. This a of a [4Fe-4S] that a [4Fe-4S] cluster be found in the FNR protein even when the protein is not a Because not appear to dimerize well at even very of we the structural features of this protein by the of the protein of and As in the of the two proteins are that the substitution not a in the structure of the protein. is a in the of the at the at which may be to the that the form a these Thus, the in vitro data suggest that the in of FNR-M147A, FNR-I151A, and are by specific defects in dimerization. Because vitro data that dimerization of FNR is not by some Ala substitutions of the dimerization we whether of the mutant proteins would the of FNR activity in vivo. analysis showed that at protein were by mutant and a plasmid control of the normal fnr promoter region by the proteins pET-11a not The of activity strains control of the promoter with FNR a pACYC184 showed at these protein of the FNR mutants and transcription at a to (Fig. a is the effect of the Ala substitutions is to a FNR monomer-dimer equilibrium. by FNR mutant proteins at a increases the of dimeric FNR protein to to the However, two mutants, and FNR-I151A, to and of (Fig. that the in be by these protein levels. In the of FNR-I151A, we found that this protein was also in of (Fig. at these FNR that the in a in DNA binding S. Guest J.R. Mol. Microbiol. PubMed Scopus Google Scholar), an expected for a dimerization The of activity that some dimeric protein form not at to the FNR sites at the This is with the in dimerization by exclusion chromatography that the monomer-dimer is the in In the properties of be by a in transcription because as well as and the other mutants that that the ability of to dimerize and DNA is not This is by the that exclusion analysis of the protein showed that this mutant as well as at an of not FNR dimerizes via a coiled coil interaction Asp-154 is predicted to be on the same side of an α-helix as many of the hydrophobic residues that we found to be for FNR activity (Fig. However, negatively charged residues are found at a coiled coil subunit because the would the interaction Thus, it was that Asp-154 may interactions between the hydrophobic side chains within the dimerization of FNR. To this we whether the at would the activity of FNR proteins that a in dimerization. we found that of Asp-154 for Ala in FNR-M147A, and activity anaerobic to of FNR of Asp-154 with Ala in the mutant with In of Asp-154 for Ala in of the mutants Ala substitutions of residues proposed to be of the and showed or less of FNR to was found when was at position This of is to was when the substitution was in with substitutions that defects in either DNA binding (3Lazazzera B. Bates D. Kiley P.J. Genes Dev. 1993; 7: 1993-2005Crossref PubMed Scopus (130) Google Scholar) or transcription Kiley P.J. Mol. Microbiol. PubMed Scopus Google Scholar). In addition, the Ala substitutions that not in FNR activity showed increases in activity when the substitution was of the at position the activity of the mutants Ala substitutions proposed to be at the dimer interface. A of this is that Asp-154 is also at the dimerization in FNR has an inhibitory activity of alanine mutants and are the same as in The are for with on mutant proteins in which the substitution has less the activity of 1) are in as a of are the same as in The are for with on mutant proteins in which the substitution has less the activity of 1) are in in a dimerization of the transcription FNR is known to be regulated by the presence of a [4Fe-4S] cluster (13Kiley P.J. Beinert H. FEMS Microbiol. Rev. 1999; 22: 341-352Crossref Google Scholar), we very the interactions that promote dimerization. To to this we the in and in vitro properties of FNR mutants alanine substitutions along a putative dimerization residues 140–159 of FNR form an α-helix its in of the properties of these mutants is that this region promotes subunit interactions by a hydrophobic dimerization interface. A of residues located on the face of this putative α-helix was also found to be for FNR activity and may a second function for this In addition, that Asp-154 to have an inhibitory role in dimerization may provide into how dimerization of FNR is regulated by the presence of an [4Fe-4S] of the hydrophobic residues found to be required for FNR function are located three to four residues that fall on the same side of an Because the for a hydrophobic is a property to that dimerize as a coiled we this acid region of FNR as a helical of that form coiled A. Biochem. Sci. 1996; Full Text PDF PubMed Scopus Google Scholar, J. Biol. Chem. 1997; Full Text Full Text PDF PubMed Scopus Google Scholar). of the hydrophobic residues be to the first Ile-151, Ile-158) and fourth to as a of the helical The a and are hydrophobic because provide the for the dimer J. Biol. Google Scholar, 1990; PubMed Scopus Google Scholar, T. 1991; PubMed Scopus Google Scholar). we propose that Met-144, Met-147, Ile-151, and Ile-158 form a hydrophobic that promotes FNR dimerization. The proposed alignment of the would also two charged residues, and within the dimerization interface. The of is for FNR function at this position in to FNR replacing with either the or negatively charged residue in an FNR mutant with anaerobic J. and P. J. The of a charged residue at the subunit is not when it is at the of an α-helix A. Proc. Natl. Acad. Sci. U. S. A. 1991; PubMed Scopus Google C. A.S. A. Genes Dev. 1995; PubMed Scopus Google Scholar) as expected for FNR. this side may be an to dimerization through the of as has been predicted with other proteins A. Proc. Natl. Acad. Sci. U. S. A. 1991; PubMed Scopus Google Scholar). many well coiled data suggest that a negatively charged is at the dimerization and has an inhibitory role in dimerization. This inhibitory role of Asp-154 may is for dimerization. In a coiled coil the side of is on the same face of the α-helix and in to This position it that to the of Asp-154 the by of its side we propose that when the hydrophobic side of is dimerization is not because of the of the hydrophobic also because the of Asp-154 in (Fig. Consistent with this we found that the caused by is when Asp-154 was with Ala Thus, when Asp-154 is by may no to function as a for the This may replacing for Ala increases the activity of mutant proteins that defects in dimerization and have substitutions along the proposed interface. is that when Asp-154 is with may have a effect on hydrophobic the of the coiled coil and dimerization PubMed Scopus Google Scholar). that of Asp-154 with Ala would also the activity of an FNR protein anaerobic (3Lazazzera B. Bates D. Kiley P.J. Genes Dev. 1993; 7: 1993-2005Crossref PubMed Scopus (130) Google Scholar) that the promoter used in to be at its for transcription other are to the of substitutions that may dimerization anaerobic conditions. The coiled coil the residues, and Met-157, at the or of an α-helix (Fig. which in a coiled coil stabilize the interactions by and T. 1991; PubMed Scopus Google Scholar). In the of the we not defects when replacing the residues with The was with the Ala substitution at a residue that is predicted to be in this mutant protein has less activity the mutant with the Ala substitution at the In vitro dimerization that the protein not dimerize as well as not the presence of a hydrophobic residue in this position is highly conserved in the FNR the data suggest that has an its role Nevertheless, the are with the that the and are not as as the hydrophobic residues in the a position B. Sci. 1993; PubMed Scopus Google Scholar). However, these may also suggest that the coiled coil of FNR may not be as as other previously proteins, a property for a regulated the presence of a [4Fe-4S] cluster dimerization is an open two were proposed to how of a [4Fe-4S] cluster FNR. it been that the Fe-S cluster promote dimerization by the through J. Bennett B. Jordan P. Ralph E.T. Thomson A.J. Guest J.R. Biochem. J. 1996; 316: 887-892Crossref PubMed Scopus (160) Google Scholar). [4Fe-4S] clusters have been proposed in other Fe-S cluster as I. 1997; PubMed Scopus Google Scholar), J. Chem. 1997; Scopus Google Scholar), and anaerobic S. E. J. R. P. J. Biol. Chem. 1996; 271: Full Text Full Text PDF PubMed Scopus Google Scholar). it was that the presence of the [4Fe-4S] cluster within each subunit a in the which dimerization (4Lazazzera B.A. Beinert H. Khoroshilova N. Kennedy M.C. Kiley P.J. J. Biol. Chem. 1996; 271: 2762-2768Abstract Full Text Full Text PDF PubMed Scopus (268) Google Scholar). Our that a [4Fe-4S] form of a mutant protein as a in solution the because in the first the protein support a [4Fe-4S] cluster in the dimeric form. the monomeric mutant protein of [4Fe-4S] cluster to that of FNR, the that each [4Fe-4S] cluster is ligated to subunit of FNR. these data support a mechanism by which the [4Fe-4S] cluster a structural within the protein that its ability to dimerize, the of this to be Nevertheless, the presence of the negatively charged Asp-154 within the dimerization it for a residue position may to to dimerization when the protein contains an [4Fe-4S] and are two other because of the mutant substitutions that were found to FNR are located on the face of the a position of expected to a ligand of the [4Fe-4S] and may be in a unique position to the presence of the [4Fe-4S] cluster to the dimerization in to is to that a substitution that FNR is also located on this face of the However, we were to whether has a effect on dimerization or at a step in transcription are to define the role of of the residues to that may function in a in FNR. A key role of Asp-154 in the control of FNR dimerization is also by that dimerizes and transcription when the [4Fe-4S] cluster is absent (3Lazazzera B. Bates D. Kiley P.J. Genes Dev. 1993; 7: 1993-2005Crossref PubMed Scopus (130) Google Scholar, 8Kiley P.J. Reznikoff W.S. J. Bacteriol. 1991; 173: 16-22Crossref PubMed Google Scholar, 12Bates D. Lazzazzera B. Kiley P.J. J. Bacteriol. 1995; 177: 3972-3978Crossref PubMed Google Scholar). The that this in dimerization in the absence of the [4Fe-4S] cluster that Asp-154 an role in dimerization. these it that at role of Asp-154 is to dimerization of FNR when it a [4Fe-4S] This is an for of this putative dimerization helix of FNR to that of other that many of the hydrophobic residues within the proposed dimerization are A alignment of the FNR that and are side chains in of the position is hydrophobic in of the representative of is in This that there may be a for the and it that and may be for dimerization. the at position is conserved in the of FNR that has the same of the Fe-S cluster in the N-terminal region as the E. coli protein J. Mol. Microbiol. 1997; PubMed Scopus Google Scholar). This that dimerization is coupled to the presence of a [4Fe-4S] cluster in these other In other as the N-terminal Cys and and which have a Fe-S cluster binding have an Ala at this position (Fig. are to whether dimerization is regulated in these other FNR In data residues 140–159 as an region for FNR dimerization. In addition, the for many hydrophobic residues within this domain be this region of FNR forms a coiled coil CRP. Nevertheless, data suggest that this proposed coiled coil is the coiled that are with other proteins. This feature may be the key property for of FNR dimerization in response to availability. Beinert for of the and of the proteins. also for with of some The was performed at the at the of with the of