protein-protein interactions in the complex between the enhancer … · the enhancer binding...

JOURNAL OF BACTERIOLOGY,0021-9193/01/$04.0010 DOI: 10.1128/JB.183.4.1359–1368.2001

Feb. 2001, p. 1359–1368 Vol. 183, No. 4

Copyright © 2001, American Society for Microbiology. All Rights Reserved.

Protein-Protein Interactions in the Complex between theEnhancer Binding Protein NIFA and the Sensor

NIFL from Azotobacter vinelandiiTRACY MONEY, JASON BARRETT, RAY DIXON, AND SARA AUSTIN*

Department of Molecular Microbiology, John Innes Centre, Norwich Research Park,Colney, Norwich, NR4 7UH, Norfolk, United Kingdom

Received 2 October 2000/Accepted 23 November 2000

The enhancer binding protein NIFA and the sensor protein NIFL from Azotobacter vinelandii comprise anatypical two-component regulatory system in which signal transduction occurs via complex formation betweenthe two proteins rather than by the phosphotransfer mechanism, which is characteristic of orthodox systems.The inhibitory activity of NIFL towards NIFA is stimulated by ADP binding to the C-terminal domain of NIFL,which bears significant homology to the histidine protein kinase transmitter domains. Adenosine nucleotides,particularly MgADP, also stimulate complex formation between NIFL and NIFA in vitro, allowing isolation ofthe complex by cochromatography. Using limited proteolysis of the purified proteins, we show here thatchanges in protease sensitivity of the Q linker regions of both NIFA and NIFL occurred when the complex wasformed in the presence of MgADP. The N-terminal domain of NIFA adjacent to the Q linker was also protectedby NIFL. Experiments with truncated versions of NIFA demonstrate that the central domain of NIFA issufficient to cause protection of the Q linker of NIFL, although in this case, stable protein complexes are notdetectable by cochromatography.

In the free-living diazotrophs Klebsiella pneumoniae andAzotobacter vinelandii, activation of expression of genes in-volved in nitrogen fixation by the enhancer binding proteinNIFA is controlled by the sensor protein NIFL in response tochanges in levels of oxygen and fixed nitrogen in vivo. TheNIFA protein activates transcription at the sN-dependent pro-moters for nitrogen fixation (nif) genes, in combination withsN-RNA polymerase holoenzyme. Transcriptional activationby NIFA is repressed by NIFL in response to increases in levelsof fixed nitrogen and extracellular oxygen (reviewed in refer-ence 5). The NIFL proteins from both A. vinelandii and K.pneumoniae have been shown to contain flavin adenine dinu-cleotide (FAD) as the prosthetic group (14, 20). For A. vine-landii NIFL, we have shown that the oxidized form of theprotein inhibits NIFA activity, but when the flavin moiety isreduced, NIFA activity is unaffected (14). In addition to itsability to act as a redox sensor, NIFL is also responsive toadenosine nucleotides in vitro, the inhibitory activity of theprotein being stimulated by the presence of ADP (8). TheNIFL protein is comprised of two domains tethered by a Qlinker (4, 6,7). Q linkers are short (20 to 30 residues), hydro-philic sequences rich in Gln, Glu, Pro, Arg, and Ser, whichserve to tether independently folding domains of some regu-latory proteins (24). The N-terminal domain contains the flavinbinding site and shows some homology to other oxygen- andredox-sensing proteins (4). The C-terminal domain of A. vine-landii NIFL shows significant homology to the histidine proteinkinase transmitter domains, including the conserved histidineresidue (4). We have shown previously that this domain binds

nucleotides, particularly ADP (22). However, phosphotransferbetween NIFL and NIFA has never been demonstrated (2, 15,21), and signal transduction is now known to occur via complexformation between the two proteins. Previous experimentswith cell extracts from K. pneumoniae showed that both pro-teins could be immunoprecipitated by antisera to either NIFAor NIFL, implying the formation of a complex (13). This isconsistent with the requirement for a stoichiometric concen-tration of each protein for effective inhibition of NIFA activityin vivo (10–12) and in vitro (2). Recently we have demon-strated the existence of a complex between A. vinelandii NIFLand NIFA in vitro by cochromatography in the presence ofadenosine nucleotides (17). Using truncated fragments andisolated domains of NIFL and NIFA, we showed that theN-terminal domain of NIFA and the C-terminal domain ofNIFL are involved in the ADP-dependent stimulation ofNIFL-NIFA complex formation. We have now employed pro-tease footprinting experiments to identify amino acid se-quences involved in the interactions occurring between NIFAand NIFL during complex formation in the presence of aden-osine nucleotides. We show here that changes in the proteasesensitivity of the Q linker regions of both proteins occur in theNIFL-NIFA complex. Using isolated domains of NIFA, weprovide evidence that the central domain of NIFA is sufficientto protect the trypsin sites in the NIFL Q linker and that thechanges in trypsin sensitivity in the N-terminal and Q linkerregions of NIFA apparently correlate with the ability of theprotein to form a stable complex with NIFL as detected bycochromatography.

MATERIALS AND METHODS

Protein purification. The native forms of NIFL and NIFA were purified asdescribed previously (2). The C-terminal hexahistidine-tagged forms of NIFLand truncated NIFA derivatives were purified by nickel affinity chromatography

* Department of Molecular Microbiology, John Innes Centre, Nor-wich Research Park, Colney, Norwich NR4 7UH, Norfolk, UnitedKingdom. Phone: 44 (0)1603 450748. Fax: 44 (0)1603 450778. E-mail:[email protected]

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on Hi-Trap chelating columns (Pharmacia) as recommended by the manufac-turer. For the native and histidine-tagged NIFA proteins, 50 mM potassiumthiocyanate was routinely added to the chromatography buffers to inhibit pre-cipitation.

Limited proteolysis. Limited proteolysis was carried out in a mixture contain-ing 50 mM Tris-acetate (pH 8), 100 mM potassium acetate, 8 mM magnesiumacetate, and 1 mM dithiothreitol. Incubations were performed at 20°C in thepresence or absence of 1 mM nucleotide. For the full-length proteins, the C-terminally tagged form of NIFL and nontagged NIFA were used. Truncatedversions of NIFA were histidine tagged at the C terminus. NIFL and NIFA,individually or mixed together in a final volume of 100 ml, were preincubated withor without nucleotide for 5 min before initiation of digestion. Protease/proteinweight ratios of 1:100, 1:20, and 1:5 were used for trypsin, chymotrypsin, and V8protease, respectively. Twenty-five-microliter samples were removed at the timeintervals indicated in the figure legends to tubes containing 12.5 ml of gel loadingbuffer (6% sodium dodecyl sulfate [SDS], 30% glycerol, 0.0003% bromophenolblue, 0.18 M Tris-chloride, 15% b-mercaptoethanol). Trypsin-chymotrypsin in-hibitor from soybean was added in a ratio of 1:2 by weight, and the samples wereheated to 100°C for 4 min before electrophoretic separation. For V8 protease,0.5 mM 3,4-di-isocoumarin was used instead to inhibit proteolysis. Electrophore-sis on 12.5, 14, or 15% polyacrylamide–SDS gels was carried out with either aTris-glycine running buffer or a Tris-tricine buffer system to resolve low-molec-ular-weight peptides.

N-terminal amino acid sequencing. Proteolytic digestion products were elec-trophoresed as described above and electroblotted onto Immobilon-P membrane(Millipore). The membranes were briefly stained with 0.1% Coomassie brilliantblue R250 in 1% acetic acid and 50% methanol and destained in 50% methanol.Stained bands were excised from the dried membrane and subjected to Edmandegradation analysis.

Western blotting. Peptides were electroblotted as described above, and cross-reacting bands were detected with either polyclonal antisera to NIFL or mono-clonal antisera to the six-His tag (Qiagen). Bands were visualized by using eitherthe ECL (enhanced chemiluminescence) system (Amersham) for chemilumines-cent detection or 5-bromo-4-chloro-3-indolyl phosphate–nitroblue tetrazolium(BCIP/NBT) liquid substrate (Sigma) for visual staining.

NIFL-NIFA complex formation assay. NIFL-NIFA complex formation assayswere carried out exactly as described previously (17). Reactions were carried outin Tris-acetate buffer containing 50 mM Tris-acetate (pH 7.9), 100 mM potas-sium acetate, and 8 mM magnesium acetate. NIFA and NIFL and their truncatedderivatives were used at concentrations between 2 and 8 mM, as stated in thefigure legends. Nucleotides were used at 1 mM unless stated otherwise. Reactionmixtures (final volume, 230 ml) were preincubated for 5 min at 30°C and thenloaded onto a 1-ml Hi Trap chelating column (Pharmacia) that had been chargedwith NiCl2 and equilibrated in buffer containing 50 mM Tris-acetate (pH 7.9),300 mM NaCl, 20 mM imidazole, and 5% glycerol. Where nucleotides werepresent in the reaction mixtures, they were added at the same concentrations tothe chromatography buffers to prevent disssociation of NIFL-NIFA complexes.Non-binding protein was washed from the column with the equilibration buffer,and bound material was then eluted with equilibration buffer containing 500 mMimidazole. Aliquots of the fractions were either mixed directly with SDS-poly-acrylamide gel electrophoresis (PAGE) sample buffer or pooled and concen-trated before electrophoresis. Electrophoresis was carried out with 10% polyac-rylamide–SDS gels unless stated otherwise in the text.

RESULTS

Limited trypsin digestion of NIFA, NIFL, and the NIFL-NIFA complex. Purified NIFA and NIFL proteins were sub-jected to limited trypsin digestion, either individually or mixedtogether in the presence and absence of 1 mM MgADP. Pep-tides from the digested proteins were separated by SDS-PAGEand identified by N-terminal sequence analysis. Western blot-ting was also used to identify changes in the pattern of NIFLpeptides obtained in the presence of NIFA (see Materials andMethods). Digestion of the NIFA protein alone was rapid inthe absence of nucleotide, giving rise to a distinct band (A4 inFig. 1), which migrated at approximately 40 kDa, and minorbands at 35 and 20 kDa (A5 and A6 in Fig. 1). In the presenceof 1 mM MgADP, the rate of digestion was slowed, and the35-kDa fragment, A5, was stabilized without the appearance of

the band A6 at 20 kDa. N-terminal sequencing revealed that allthree fragments arose from a cleavage at Arg-202 in the Qlinker region of NIFA between the N-terminal and centraldomains. The domain structure of full-length NIFA and theschematic diagram of the tryptic digestion products are shownin Fig. 1B. The Q linker region is not well defined in Azoto-bacter vinelandii NIFA, but based on homology to the Q linkerin Klebsiella pneumoniae NIFA (24) and the protease cleavagesites described in this work, we have assigned amino acid res-

FIG. 1. (A) Limited trypsin proteolysis of NIFA in the presenceand absence of 1 mM MgADP. NIFA (final concentration, 3.75 mM)was incubated with trypsin (weight ratio, 1:100) for the times indicated,and reactions were analyzed with 15% polyacrylamide–SDS gels. (B)Schematic map of peptides generated by limited trypsin proteolysis ofNIFA in the presence and absence of MgADP. Open rectangles rep-resent the N-terminal domain of NIFA, where the full-length domaincontains amino acid residues 1 to 190. Grey rectangles represent thecentral domain of NIFA, where the full-length domain contains aminoacid residues 206 to 457. Black rectangles represent the C-terminalDNA binding domain containing amino acid residues 474 to 512. Thinblack lines indicate the linker regions. The trypsin cleavage sites, whichare shown beside each peptide, were determined by N-terminal se-quence analysis, as described in Materials and Methods. Other struc-tural features shown include the proposed Q linker between the N-terminal and central domains of NIFA (amino acid residues 189 to205) and the proposed nucleotide binding site (amino acid residues233 to 255).

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idues 189 to 205 to include this region. From the apparentmolecular masses, the tryptic fragments obtained were as-signed as the central and DNA binding domain, A4; the centraldomain alone, A5; and a central domain subfragment, A6 (Fig.1B). Thus, MgADP binding to the NIFA central domain re-sulted in conformational changes which protected it from fur-ther proteolysis. N-terminal sequencing of some of the higher-molecular-mass fragments that were detected in the presenceof MgADP revealed cleavages at Arg-8, Arg-70, and Arg-165(A1 to -3, respectively, in Fig. 1B). These probably only rep-resent a proportion of the cleavages which occur in the N-terminal domain, which is apparently very protease sensitiveand is rapidly degraded by a series of cleavages at trypsin sitesthroughout the domain. The C-terminal DNA binding domainwas also not observed as a discrete cleavage product. A patternof trypsin digestion of NIFA similar to that obtained with 1mM MgADP was observed with 1 mM MgATPgS, while onlya slight protection of the 35-kDa band was obtained withhigher concentrations (5 mM) of MgGTPgS (data not shown).Concentrations of 1 mM MgGDP, MgCTP, MgUTP, andMgAMP produced a pattern of trypsin digestion indistinguish-able from that obtained in the absence of nucleotide (data notshown).

We have shown previously that adenosine nucleotide bind-ing to NIFL induces conformational changes in the C-terminaldomain of the protein (22). The patterns of trypsin digestionproducts obtained with NIFL in the presence of MgADP aresummarized in Fig. 2A. In the absence of nucleotide, the NIFLN-terminal domain, band L1 in Fig. 2A, was stable in responseto limited trypsin digestion, while the C-terminal domain wasrapidly degraded. The presence of MgADP protected the C-terminal domain with the appearance of a 27-kDa peptidearising from cleavages at Arg-279 and Lys-284 in the Q linker(L2 in Fig. 2A). Lower-molecular-mass C-terminal peptidesarising from cleavages at Arg-359 and Lys-364 were also pro-tected by MgADP, migrating at 18, 15, and 14 kDa, respec-tively. (Fig. 2A, L3 to -5) (22). Bands corresponding to L1 to -5were also detected on Western blots with anti-NIFL serum, asdepicted in Fig. 2C.

Additional changes in the pattern of tryptic peptides wereobserved when NIFA and NIFL were mixed together to forma complex in the presence of 1 mM MgADP. Equimolaramounts of C-terminally tagged NIFL and nontagged NIFAwere used in the incubations as described in Materials andMethods. We have shown previously that the NIFL-NIFAcomplex can be isolated under these conditions by cochro-matography assays (17). Figure 2B and C show Western blotsof NIFL tryptic peptides generated in the presence and ab-sence of NIFA detected with either antiserum to NIFL orantiserum to the six-His tag. The kinetics of partial trypsindigestion are shown in Fig. 2B, where the resulting peptideswere detected with antiserum to the histidine tag. One majorchange in the pattern of NIFL tryptic peptides was observed inthe presence of NIFA. The band corresponding to the NIFLpeptide L2 did not appear when the NIFL-NIFA complex wasformed, indicating that the Arg-279 and Lys-284 cleavages inthe NIFL Q linker had been protected from trypsin cleavage(Fig. 2B). A similar result was obtained when antiserum to thewhole NIFL protein was used to detect NIFL peptides (Fig.2C). No other NIFL cleavage products appeared to be repro-

ducibly altered by the presence of NIFA. Western blotting withTricine gels to detect possible changes in lower-molecular-mass fragments did not reveal any further changes in NIFLpeptides in the presence of NIFA (data not shown).

For NIFA, Western blotting with NIFA antiserum could notbe used to detect tryptic peptides, because the antiserum wasfound to recognize only epitopes present at the C-terminalregion of the protein, which was immediately cleaved on tryp-sin digestion. N-terminal sequence analysis of the NIFA frag-ments obtained after relatively long digestion times was essen-tial to identify any potential changes in the pattern of NIFAtryptic peptides generated in the presence of NIFL. Figure 3Bshows a Coomassie-stained SDS-polyacrylamide gel of thepeptides generated by trypsin digestion of NIFL and NIFAindividually and of the proteins mixed together to form acomplex in the presence of 1 mM MgADP. In the absence ofNIFL, digestion of NIFA produced the 40- and 35-kDa frag-ments A4 and -5, respectively, described in the legend to Fig.1, as a consequence of cleavage at Arg-202 in the Q linkerregion (Fig. 3B, lane2). In the presence of NIFL, a new, larger45-kDa NIFA peptide, A7, was stabilized, arising from a cleav-age at Arg-122 in the NIFA N-terminal domain. (Fig. 3B, lane3). The NIFA peptide A4 was apparently not detectable in thepresence of NIFL, because no sequence corresponding to thispeptide was obtained from N-terminal sequencing of the 45-kDa band. From the apparent molecular mass, A7 consists ofthe last 80 amino acids of the N-terminal domain and thecentral domain of NIFA (Fig. 3E). Thus, both the Q linkerregion of NIFA and the trypsin sites in the adjacent N-terminalregion were protected by NIFL. The change in NIFL identifiedon Western blots in Fig. 2 could also be seen on SDS-poly-acrylamide gels with the absence of the NIFL peptide L2clearly observable in the presence of NIFA (Fig. 3B, comparelanes 1 and 3). In control experiments with the NTRC proteininstead of NIFA, there was no protection of the trypsin sites inthe NIFL Q linker in the presence of NTRC and MgADP(data not shown). The NTRC Q linker, which is normallycleaved by trypsin at Arg-129 (9), was also not protected byNIFL (data not shown). Thus, the changes in trypsin sensitivityin the Q linker regions of both NIFL and NIFA are specific tothe NIFL-NIFA complex.

Effect of ATPgS and GTPgS on limited proteolysis of theNIFL-NIFA complex. We have shown previously that NIFL-NIFA complex formation was also observed with MgATPgSand MgGTPgS, the nonhydrolyzable analogues of ATP andGTP. However, MgATPgS was not as effective as MgADP atlow concentrations, and with MgGTPgS, only low levels ofcomplexes were detected (17). Limited trypsin digestion ofNIFL and NIFA individually with 1 mM MgATPgS produceda pattern of peptides similar to that obtained with MgADP,except that the NIFL C-terminal peptide L2 was not observedunder these conditions, as shown previously (22) (Fig. 3C, lane1). The presence of NIFL caused protection of the same 45-kDa NIFA peptide, A7, as was observed with MgADP (Fig.3C, lane 3). The rate of cleavage of the NIFL protein at the Qlinker to give the NIFL N-terminal peptide L1 was also re-duced in the presence of NIFA, and the full-length protein wasprotected (Fig. 3C, compare lanes 1 and 3). The affinity ofMgGTPgS for NIFL and NIFA was apparently too weak forany protection from proteolysis to be detected, because the

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pattern of trypsin digestion obtained with 1 mM MgGTPgSwas similar to that obtained in the absence of any nucleotide(compare Fig. 3A and D).

Limited proteolysis using chymotrypsin and V8 protease.Chymotrypsin and V8 protease were employed to try to iden-tify other potential changes in the pattern of peptides gener-ated in the NIFL-NIFA complex compared to the patterns ofthe proteins alone. Figure 4A shows the pattern of proteolysiswith chymotrypsin in the presence of 1 mM MgADP. Thepattern of chymotrypsin digestion of NIFA was similar to thatobtained with trypsin. A central domain fragment, which arose

from a cleavage at Tyr-205 in the Q linker, was observed in thepresence of MgADP (Fig. 4A and B, band A8). In the presenceof NIFL, a larger NIFA peptide was generated, which arosefrom a cleavage at Tyr-126 in the N-terminal domain and wasanalogous to the 45-kDa fragment produced under the sameconditions by trypsin (Fig. 4A and B, band A9). For NIFL, themajor chymotryptic products in the presence of MgADP weretwo C-terminal domain peptides from cleavages at Phe-218and Phe-252 (Fig. 4A and B, bands L6 and -7) and a 27-kDaamino-terminal peptide with the same N-terminal sequence asthe full-length protein (Fig. 4A and B, L8). None of the NIFL

FIG. 2. Analysis of NIFL tryptic peptides generated in the presence and absence of NIFA. Incubation mixtures containing 1 mM MgADP andNIFA or NIFL individually or mixed together to form a complex (final concentration, 1 mM) were digested with trypsin as described in Materialsand Methods. (A) Schematic map of peptides generated by limited trypsin proteolysis of NIFL in the presence and absence of MgADP. Dottedrectangles represent the N-terminal domain of NIFL containing amino acid residues 1 to 274. The striped rectangles represent the C-terminaldomain of NIFL, where the full-length domain contains amino acid residues 300 to 519. The thin black line represents the proposed Q linkerbetween the N- and C-terminal domains of NIFL (amino acid residues 275 to 299). The proposed nucleotide binding sites (amino acid residues445 to 456 and 478 to 482) are marked with an arrow. The trypsin cleavage sites, which are shown beside each peptide, were determined byN-terminal sequence analysis as described in Materials and Methods. (B). Western blot analysis of NIFL tryptic peptides detected with antiserumto the six-His tag (Anti histag). Twenty-five-microliter samples were removed at the times indicated, and the peptides were separated on 12.5%polyacrylamide–SDS gels. Western blotting was carried out as described in Materials and Methods with BCIP/NBT liquid substrate to detect thecross-reacting bands. (C) Western blot analysis of NIFL tryptic peptides detected with antiserum to NIFL. Samples were digested for 60 min andthen processed as described for panel A. Lane 1, NIFL alone; lane 2, NIFL plus NIFA. The unlabeled arrows in panels B and C denote thepositions of NIFL peptides not generated in the presence of NIFA. Bands labeled L1 to -5 are NIFL tryptic peptides as defined for panel A.



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peptides was altered by the presence of NIFA, either whenvisualized on stained gels (Fig. 4A, compare lanes 1 and 3) orby Western blotting with NIFL antiserum (data not shown).

NIFA digestion by V8 protease in the presence of MgADPgave rise to two major digestion products (Fig. 4C, lane 2,bands A10 and -11): a 55-kDa peptide, A11, with an N-termi-nal sequence identical to that of the full-length protein; and a35-kDa fragment, A10, arising from a cleavage in the Q linkerat Glu-200. From the apparent molecular masses, these corre-

sponded to the amino terminus plus the central domain andthe NIFA central domain alone (Fig. 4D). In the presence ofNIFL, the Q linker in NIFA was again protected from cleav-age, resulting in accumulation of NIFA peptide A11, while theNIFA central domain peptide A10 was not observed (Fig. 4C,compare lanes 1 and 3). NIFL remained quite resistant to V8protease digestion under the conditions of the assay, and nochanges in NIFL peptides in the presence of NIFA were ob-served by Western blotting with NIFL antiserum.

FIG. 3. SDS-PAGE of NIFL and NIFA tryptic peptides generated in the presence and absence of nucleotides. Incubations were carried outas described in the legend to Fig. 2, and the reactions were analyzed on 12.5% polyacrylamide gels. Peptides were generated by digestion withtrypsin (weight ratio, 1:100) for 45 min at 20°C. (A) No nucleotide. (B) MgADP (1 mM). (C) MgATPgS (1 mM). (D) MgGTPgS. (E) Schematicrepresentation of the NIFA peptide A7 protected in the presence of NIFL. Bands labeled A4 to -6 and L1 and -2 are shown in Fig. 1B and 2A,respectively. In each case, lane 1 is NIFL, lane 2 is NIFA, and lane 3 is NIFL plus NIFA.



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The central domain of NIFA, NIFA(191–457), is sufficient toprotect NIFL from trypsin cleavage in the Q linker. Previousexperiments with a truncated form of NIFA lacking the N-terminal domain, NIFA(191–522), showed that this proteinwas unable to form a complex with full-length NIFL detectableby cochromatography (17). However, transcriptional activationby this truncated form of NIFA is inhibited by high concen-trations of NIFL in the presence of ADP in vitro, implying thatan interaction between the two proteins must still occur underthese conditions (A. Sobczyk and R. Dixon, unpublished ob-servations). We examined the ability of NIFA(191–522) andthe isolated central domain of NIFA, NIFA(191–457), to pro-tect the trypsin sites in the NIFL Q linker in the presence ofMgADP. In the presence of both truncated NIFA proteins,protection of the trypsin cleavage sites Arg-279 and Lys-284 inNIFL could be observed in Western blotting experiments (Fig.

5A and B) and on Coomassie-stained SDS gels (Fig. 5C) (datanot shown). NIFA(191–522) appeared to have a somewhathigher affinity for NIFL than did NIFA(191–457) and causedcomplete protection of the trypsin sites at the same proteinconcentration as the full-length protein (Fig. 5A, comparelanes 2 and 3). In the presence of NIFA(191–457), a low levelof NIFL peptide L2 was still observed (Fig. 5B, lane 2). Anapparent enhancement of the C-terminal NIFL peptide L3 wasalso observed in the presence of NIFA(191–457), although thiswas not detected on the Coomassie-stained gel. Full-lengthNIFL did not protect the trypsin cleavage site in the NIFA Qlinker region present in both the truncated NIFA proteins,since the same NIFA central domain peptide, A5*, derivedfrom cleavage at Arg-202, was observed in the presence andabsence of NIFL with both NIFA proteins (Fig. 5C) (data notshown). Thus, the N-terminally deleted forms of NIFA are

FIG. 4. SDS-PAGE of NIFL and NIFA peptides generated by digestion with chymotrypsin or V8 protease in the presence of 1 mM MgADP.(A) Digestion with chymotrypsin (weight ratio, 1:20) was for 45 min at 20°C. (B) Schematic representation of NIFL and NIFA chymotrypticpeptides L6 to -8 and A8 and -9. (C) Digestion with V8 protease (weight ratio, 1:5) was for 60 min at 20°C. The bands labeled F derive from V8protease peptides. (D) Schematic representation of NIFA V8 peptides A10 and -11. Protein domains are represented as described for Fig. 1B and2A. In panels A and C, lane 1 is NIFL, lane 2 is NIFA, and lane 3 is NIFL plus NIFA.



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able to interact with NIFL and protect the NIFL Q linker fromtrypsin cleavage, even though such complexes are not suffi-ciently stable to allow detection by cochromatography.

Role of the amino-terminal domain of NIFA in the forma-tion of stable complexes with NIFL detectable by cochromatog-raphy. The formation of NIFL-NIFA complexes sufficientlystable to be detected by cochromatography may depend oncontacts or conformational changes in the N-terminal and Qlinker regions of NIFA in addition to the interaction of thecentral domain of NIFA with NIFL described above. We in-vestigated the ability of various N-terminal fragments of NIFAto form a complex with NIFL detectable by cochromatographyas well as the ability of NIFL to protect the Q linker andadjacent N-terminal region of the NIFA fragments from tryp-sin digestion. The isolated N-terminal domain of NIFA alone,NIFA(1–203), or a longer fragment consisting of the N-termi-nal region, Q linker, and first 70 amino acids of the centraldomain, NIFA(1–275), did not form a complex with NIFL andwas not protected by NIFL from trypsin cleavage. Neither didthey protect the trypsin sites in the NIFL linker region (datanot shown). However, a C-terminally deleted version of NIFA,NIFA(1–457), with its DNA binding domain deleted, but withan intact central domain, was competent to form a stablecomplex with NIFL in the presence of MgADP (Fig. 6C).However, with equimolar concentrations of the proteins, lessNIFA(1–457) was bound to NIFL than was obtained with wild

type NIFA, indicating a reduced affinity of NIFA(1–457) forNIFL (data not shown). NIFA(1–457) also protected the tryp-sin sites in the NIFL linker (Fig. 6A). In addition, NIFL pro-tected the Q linker and the adjacent N-terminal region of thisform of NIFA from trypsin cleavage (Fig. 6B, compare lanes 2and 3).

DISCUSSION

The A. vinelandii NIFA and NIFL proteins have Q linkerregions that are protease sensitive and nucleotide binding do-mains that are protected from protease digestion by the pres-ence of adenosine nucleotides. Presumably nucleotide bindinginduces conformational changes in the respective domains,which result in the protease cleavage sites being less exposed tosolvent. Both MgATPgS and MgADP protect the central do-main of NIFA from protease digestion, as we have observedpreviously for the NTRC protein, a homologous sN-dependentactivator (9). However, the NIFA N-terminal domain is verytrypsin sensitive compared to that of NTRC and may thereforebe a more loosely folded domain. Under conditions in whichNIFL and NIFA have been shown to form a complex in thepresence of MgADP, the trypsin sensitivity of both Q linkerregions changes. In addition, the N-terminal region of NIFAimmediately adjacent to the Q linker is also protected by

FIG. 5. Ability of amino-terminally truncated versions of NIFA to protect the Q linker of NIFL from trypsin cleavage. (A) Western blot analysisof NIFL tryptic peptides generated in the presence and absence of NIFA(191–522). Incubations were carried out as described for Fig. 2 with 1 mMMgADP. Proteins were present at a final concentration of 1 mM, and digestion was for 60 min at 20°C. Western blots were probed with antiserumto NIFL and detected with the ECL system. Lane 1, NIFL; lane 2, NIFL plus NIFA; lane 3, NIFL plus NIFA(191–522). The band labeled witha dot is due to a cross-reacting signal in the NIFA(191–522) preparation that is recognized by the NIFL antiserum. (B) Western blot analysis ofNIFL tryptic peptides generated in the presence and absence of NIFA(191–457). Incubations and Western blotting were carried out as for panelA. Lane 1, NIFL; lane 2, NIFL plus NIFA(191–457). (C) SDS-PAGE of tryptic peptides generated by digestion of NIFL and NIFA(191–457).Incubation conditions were as for panel A. Samples were analyzed on 12.5% polyacrylaminde gels. Lane 1, NIFL; lane 2, NIFA(191–457); lane3, NIFL plus NIFA(191–457). In each case, the unlabeled arrows denote the positions of NIFL peptides not generated in the presence of NIFA.NIFL tryptic peptides labeled L1 to -3 are shown in Fig. 2A. The NIFA tryptic peptide labeled A5* is analogous to peptide A5 shown in Fig.1,except that the C terminus has a six-His tag.



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NIFL. The regions protected from trypsin digestion in theNIFL-NIFA complex are summarized in Fig. 7A.

The changes in trypsin sensitivity may be due to the linkerregions undergoing a conformational change in response toprotein contacts elsewhere in the proteins or may be due todirect protein-protein contacts involving these surfaces ofNIFL and NIFA. Q linker regions have been identified at theboundaries of distinct structural domains in a number of bac-terial signal transduction proteins (24). Whether the linkerregions themselves have an active role in signal transduction ormerely act as a tether between interacting domains of theproteins remains to be determined. In K. pneumoniae, exten-sion of the NIFA Q linker region by four and eight amino acidsdid not affect the transcriptional activity of the protein in vivo,but regulation by NIFL was not tested (24). Linker regions inother multidomain response regulator proteins have beenidentified as having a potential role in signal transduction fromone domain to another. Changes in protease sensitivity of thelinker region of the response regulator OmpR have recentlybeen reported in response to both phosphorylation of the N-terminal domain and DNA binding by the C-terminal domain(1).

The region of NIFL protected by NIFA appears to be local-ized to sites close to the NIFL Q linker. The chymotrypsin siteslocated at Phe-252 and Phe-218, 27 and 61 amino acids, re-spectively, upstream of the trypsin sites in the linker, appearunaffected by the presence of NIFA, as do the trypsin sites atArg-359 and Lys-364, 80 amino acids downstream of the linkersites. In A. vinelandii NIFL, the histidine at position 305 isanalogous to the highly conserved histidine residue, which isautophosphorylated in bona fide members of the family ofhistidine protein kinase (19). Although the NIFL-NIFA pairare not typical members of the two-component signal trans-duction protein family, the location of the conserved histidineresidue near the Q linker suggests that this region may be acandidate for interaction with NIFA, since in orthodox sys-tems, the modified histidine is likely to approach the receiverdomain of the response regulator to effect phosphotransfer(18). However, mutations in His-305 in A. vinelandii NIFLresulted in proteins that were still able to inhibit NIFA activityin vivo, implying that this residue is not itself crucial for NIFLfunction, including interaction with NIFA (23). The regionsurrounding His-305 contains a number of residues conservedin the known NIFL proteins, and it is possible that this regionof NIFL may have evolved the function of interaction with theactivator. Using cochromatography assays in vitro, we showedpreviously that a C-terminal subdomain of A. vinelandii NIFL,NIFL(360–519), was unable to bind to NIFA in the presence ofMgADP, but that a longer derivative, NIFL(147–519), whichincluded the linker and surrounding region, was competent tobind NIFA (17). Recently an interaction between the C-termi-nal domain of A. vinelandii NIFL containing sequences fromGlu-257 to the end of the protein and the central domain ofNIFA has been demonstrated in vivo by using the yeast two-hybrid system (16). Taken together, these results indicate thatthe site of NIFA interaction is likely to be located betweenamino acids 260 and 360 in A. vinelandii NIFL. Experimentswith a subdomain of NIFL incorporating this region, NIFL(256–356), were unable to demonstrate any interaction with NIFA(T. Money and S. Austin, unpublished observations). However,

FIG. 6. Ability of NIFA(1–457) to interact with NIFL. (A) Westernblot analysis of NIFL tryptic peptides generated in the presence andabsence of NIFA(1–457). Incubations were carried out as described forFig. 2 with 1 mM MgADP. Proteins were present at a final concentra-tion of 1 mM, and digestion was for 60 min at 20°C. Western blots wereprobed with antiserum to NIFL and detected with the ECL system.Lane 1, NIFL; lane 2, NIFL plus NIFA(1–457). The unlabeled arrowdenotes the peptide in NIFL not generated in the presence of NIFA.(B) SDS-PAGE of tryptic peptides generated by NIFL and NIFA(1–457). Incubations were carried out as described for panel A, exceptthat digestion was for 30 min at 20°C. Samples were analyzed onSDS-polyacrylamide gels (12.5% acrylamide). Lane 1, NIFL; lane 2,NIFA(1–457); lane 3, NIFL plus NIFA(1–457). NIFL tryptic peptidesL1 to -3 are shown in Fig. 2A. NIFA peptides A5* and A7* areanalogous to peptides A5 and A7, except they contain a six-His tag atthe C terminus. (C) Complex formation between NIFL and NIFA(1–457). Complexes were formed and chromatographed as described pre-viously (16). The nontagged form of NIFL (2.3 mM) was used withC-terminally tagged NIFA(1–457) (2.7 mM). MgADP was used at 1mM. Electrophoresis was carried out on SDS-polyacylamide gels(12.5% polyacylamide). Lanes 2 to 4 and 9 to 11, wash fractions; lanes5 to 7 and 12 to 14, fractions containing bound protein which elutedwith 0.5 M imidazole.



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this is not unexpected, because NIFL-NIFA complexes areonly observed in our assays in the presence of nucleotide bind-ing, and this NIFL fragment lacks the nucleotide binding sites.

Evidence to date suggests that NIFL contacts the centraldomain of NIFA. The central domains of both A. vinelandiiand K. pneumoniae NIFA activate transcription in vitro, andthis activity is inhibited by NIFL, indicating that an interactionis taking place (3; J. Barrett and R. Dixon, unpublished obser-vations). As described above, the interaction demonstrated invivo with the yeast two-hybrid system was obtained with thecentral domain of NIFA alone (16). We show here that theisolated central domain of NIFA is competent to protect the Qlinker of NIFL from trypsin digestion, indicating that NIFLcontacts this domain of NIFA. There are no indications atpresent as to where the contact site(s) on the central domainmight be located. The NIFA construct that contained the N-terminal domain and 70 amino acids of the central domain wasunable to interact with NIFL, as determined by both trypsinprotection and cochromatography assays, implying that thepresence of this region is not sufficient to allow contact withNIFL.

The N-terminal domain of NIFA is required for a stablecomplex, detectable by cochromatography, to be formed withNIFL, and the changes in trypsin sensitivity of the NIFA Qlinker and adjacent N-terminal domain appear to correlatewith the ability of NIFA to form a stable complex with NIFL.The NIFA N-terminal domain is also required for NIFL toinhibit the ATPase activity of NIFA, indicating that this do-main has a key regulatory role in controlling the catalyticdomain of the protein (A. Sobczyk and R. Dixon, unpublisheddata). A model for the interaction of NIFA with NIFL incor-porating these observations is presented in Fig. 7B. In thismodel, MgADP binding to the C-terminal domain of NIFLresults in a conformational change in this domain and/or linkerof NIFL, allowing it to interact with the central domain ofNIFA, thereby inhibiting transcriptional activation. This con-tact results in the protection of the trypsin sites in the NIFL Qlinker and is independent of the other domains of NIFA.When the NIFA N-terminal domain is present, further con-tacts or conformational changes can occur in the NIFA Qlinker and adjacent amino terminus, giving rise to altered pro-tease sensitivity. These appear to be dependent on the centraldomain contact with NIFL and may be responsible for theinhibition of NIFA ATPase activity, possibly by blocking thenucleotide binding site in the central domain. The affinity ofthe proteins in this complex is strong enough for it to bedetected by cochromatography. In the absence of the N-termi-nal domain of NIFA, the central domain contact with NIFLcan still be made and transcription is inhibited. However, with-out the extra contacts or conformational changes via the NIFAN-terminal and Q linker regions, the affinity of the complex isnot strong enough to be detected by cochromatography.

ACKNOWLEDGMENTS

We are very grateful to Mike Naldrett for N-terminal sequenceanalysis of the protease cleavage products. We also thank Gary Sawersand Mike Merrick for comments on the manuscript.

This work was supported by the Biotechnology and Biological Sci-ence Research Council.

FIG. 7. (A) Summary of regions protected from trypsin digestion inthe NIFL-NIFA complex formed in the presence of MgADP. Thehatched areas signify protection from protease treatment. (B) Modelof the interaction of NIFL with NIFA in the presence of MgADP.



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