Journal of Bacteriology, January 2002, p . 200-206, Vol . 184, No . 1

Domain Interactions on the ntr Signal Transduction Pathway: Two-Hybrid Analysis of Mutant and Truncated Derivatives of Histidine Kinase NtrB

Isabel Martínez-Argudo, Paloma Salinas, Rafael Maldonado, and Asunción Contreras^*

División de Genética, Universidad de Alicante, Apartado 99, E-03080 Alicante, Spain

Received 27 July 2001/ Accepted 9 October 2001

Recognition between components of signaling pathways is a problem in organisms having multiple homologous systems . The Escherichia coli genome encodes 62 proteins homologous to one or the other of the components (17) . Specific recognition between pairs of these elements is required for accurate signal channeling, although cross talk between some of these regulators may also be physiologically relevant . In spite of structural conservation in two-component systems and their possible implications for cross talk, the histidine phosphotransfer and receiver domains appear to be responsible for binding and specificity of interactions between histidine kinases and their cognate response regulators (15, 21, 22, 33) . The kinase domain has also been reported to be a target for regulation in histidine kinases (8), and PII interacts in vitro with NtrB fragments containing the kinase domain (11, 23) . Although NtrB homologues are absent from multiple organisms containing one or more PII-like proteins, PII is one of the most ubiquitous signal transduction proteins and genetic complementation is found with glnB from distantly related bacteria (1, 7) . Conservation of kinase domains within histidine kinases and other ATP-binding proteins may facilitate recognition by common regulatory proteins, raising the possibility of cross talk between different signal transduction systems .

Most histidine kinases contain N-terminal transmembrane domains involved in sensory functions . Although there is no evidence of such functions in the case of the N terminus of NtrB, this region of the protein has also been referred to as the sensor domain . The N terminus of NtrB is rather unique and possesses homology to PAS domains (28) . Recently, it has been shown that it contains determinants for dimerization (11, 15) . The region connecting the N terminus of NtrB to the conserved boxes on the transmitter module, known as the linker (32), seems to be critical for regulation and is predicted to form a coiled-coil motif in NtrB and other histidine kinases (26) . Consistent with this regulatory role, in E . coli, amino acids 115 to 138 of NtrB contain most of the constitutive point mutations selected as suppressors of the Ntr^- phenotype of a glnD strain (2) .

In a previous work, we used the yeast two-hybrid system to probe interactions between domains of the NtrB and NtrC proteins, showing specific contacts between the transmitter module of NtrB and the receiver domain of NtrC from Klebsiella pneumoniae (15) . In this study, we used the same in vivo strategy to further analyze interactions between NtrB domains and their interactions with upstream (PII) and downstream (NtrC) components of the nitrogen signal pathway . Results obtained here confirm that interactions between NtrB and NtrC and between NtrB and PII can be mapped, respectively, to the histidine phosphotransfer and kinase domains, validating the yeast two-hybrid approach . In addition, our results suggest that the mutation A129T perturbs interactions with NtrC while not affecting the ability of NtrB to bind PII .

 
Construction of two-hybrid plasmids. All ntrB sequences were derived from plasmid pMD182 . To construct pUAG281 and pUAG282, a pMD182 fragment encompassing the complete ntrB coding sequence was PCR amplified with primers NTRB-1F and NTRB-4R, cut with BamHI, and cloned into pGAD424(+2) and pGBT9(+2), respectively . A pMD182 fragment was PCR amplified with NTRB-2F and NTRB-4R, cut with BamHI, and cloned into pGAD424(+2) and pGBT9(+2), giving plasmids pUAG291 and pUAG292, respectively . A pMD182 fragment was PCR amplified with NTRB-2F and NTRB-5R, cut with BamHI, and cloned into pGAD424(+2) and pGBT9(+2), giving plasmids pUAG301 and pUAG302, respectively . A pMD182 fragment was PCR amplified with primers NTRB-5F and NTRB-1R, cut with BamHI, and cloned into pGAD424(+2) and pGBT9(+2), giving plasmids pUAG311 and pUAG312, respectively . A two-step PCR was used to generate the NtrB^A129T point mutation . The first PCR step was carried out with primers NTRB-1F and NTRB-3R; the second step was done with NTRB-3F and NTRB-1R . Products were used as templates for a third PCR with primers NTRB-1F and NTRB-1R, and the resulting fragment was cut with BamHI and cloned into pGAD424(+2) and pGBT9(+2), giving plasmids pUAG213 and pUAG214, respectively . A fragment from pUAG213 was PCR amplified with primers NTRB-2F and NTRB-1R, cut with BamHI, and cloned into pGAD424(+2) and pGBT9(+2), giving plasmids pUAG341 and pUAG342, respectively . A pMD182 fragment was PCR amplified with primers NTRB-1F and NTRB-5R, cut with BamHI, and cloned into pGAD424(+2) and pGBT9(+2), giving plasmids pUAG351 and pUAG352, respectively .

envZ sequences were derived from pPH006 . To insert an NdeI site, oligonucleotides MD138 (sense) and MD139 (antisense) were introduced into the EcoRI sites of pGAD424(+2) and pGBT9(+2), generating pGAD424(+2)-NdeI and pGBT9(+2)-NdeI, respectively . To construct pUAG501, an NdeI-EcoRI fragment from pPH006 was cloned into pGAD424(+2)-NdeI . An NdeI-BamHI fragment from pUAG501 was cloned into pGBT9(+2)-NdeI, giving plasmid pUAG502 .

glnB sequences were amplified from K . pneumoniae M5a1 chromosomal DNA with primers GLNB-FOR and GLNB-REV, and the corresponding product was cut with BamHI and EcoRI and cloned into pGAD424 and pGBT9, giving plasmids pUAG171 and pUAG172, respectively .

Yeast methods. Saccharomyces cerevisiae Y190 was cotransformed with different pairs of two-hybrid plasmids (1 µg of each), and at least four independent clones were selected for further analysis . The yeast culture and transformation procedures used were previously described (3) . ß-Galactosidase activity was assayed as previously described (25) . Growth on histidine-deficient medium was analyzed on solid YNB medium lacking His, Leu, and Trp in the presence of different concentrations of 3-amino-1,2,4-triazole as previously described (15) .

Western blotting. To obtain protein extracts from Y190 carrying different pairs of fusion proteins, cells from 2- to 3-ml cultures (optical density at 600 nm, 2) were resuspended in sodium dodecyl sulfate-gel loading buffer, boiled, and broken with glass beads in a Minibeadbeater . To increase the protein yield of GAL4AD constructs, 10- to 15-ml cultures (optical density at 600 nm, 10) were resuspended in 5% trichloroacetic acid buffer and broken with glass beads in a Minibeadbeater . After centrifugation, the pellet was resuspended in Laemmli loading buffer . Equivalent amounts of protein extracts were separated by electrophoresis on sodium dodecyl sulfate-12% polyacrylamide gels and electrotransferred onto polyvinylidene difluoride . Membranes were probed with monoclonal antibodies against GAL4AD (sc-1663) and GAL4BD (sc-510) from Santa Cruz Biotechnology . Primary antibodies were detected with alkaline phosphatase-conjugated goat anti-mouse secondary antibodies (Sigma) . Detection was carried out by staining the membrane with 5-bromo-4-chloro-3-indolylphosphate and nitroblue tetrazolium .

 
To investigate the roles of individual domains from the transmitter module in interactions within and between subunits of NtrB, we performed two-hybrid analyses with full-length NtrB and truncated NtrB derivatives in different combinations . The results are summarized in Fig . 3 .

 
In the histidine kinases EnvZ and NtrB, the dimerization interface is provided by the histidine phosphotransfer domain (11, 29) . However, NtrB^HNG does not interact with itself in the two-hybrid system (15) and we wished to investigate whether we could obtain signals from H domains by using different NtrB fragments . To this end, we tested the abilities of truncated NtrB derivatives to interact with themselves and with full-length NtrB . In contrast to the results obtained with constructs bearing the sensor domain, no signals were found when NtrB^HN or NtrB^H was paired with itself or with NtrB (Fig . 3, blocks 1 to 3), indicating that failure to obtain signals from transmitter modules is not due to the presence of the G region in the fusion proteins . To investigate whether lack of interaction between transmitter domains reflects differences between NtrB and other histidine kinases, we tested the ability of the corresponding module of EnvZ to interact with itself . Paralleling results obtained with NtrB^HNG, no signal was found for EnvZ^HNG (Fig . 3, block 1), suggesting that lack of interaction in the yeast system is common to transmitter modules .

It has been shown in vivo that the G domain is sufficient to restore positive regulation by the HN fragment (14), suggesting that it folds independently into an active domain and phosphorylates the histidine residue of the H domain in trans . To test whether recognition between the H and G domains could be detected in our assays, NtrB^G was paired with each of the NtrB derivatives . Only NtrB^SHN/NtrB^G gave signals (Fig . 3, blocks 4 and 5), while other proteins containing the H domain did not . While failure of GAL4AD-NtrB^G to promote signals is not surprising and can be attributed to lack of expression, the negative results obtained with most of the pairs providing H and G in different GAL4 fusions suggest very poor recognition between the H and G domains .

 
PII interacts with NtrB to regulate its kinase and phosphatase activities, and NtrB and PII can be cross-linked in vitro to each other (23) . However, the question arose whether the interaction between NtrB and PII could also be detected in the two-hybrid system . As shown in Fig . 4 (blocks 3 and 4), NtrB gave signals with PII, showing that recognition between NtrB and PII also takes place with fusion proteins in yeast . In addition, NtrB^HNG, but not NtrB^S, NtrB^SHN, or NtrB^G, gave signals with PII, in agreement with recent in vitro data showing contacts between PII and a truncated protein containing most of the transmitter module of NtrB (23) . To explore the specificity of the signals obtained with PII, additional fusion proteins were incorporated into the analyses . No signals were found when PII was assayed with EnZ^HNG, CheA, or NtrC, thus supporting the specificity of the contacts between PII and both NtrB and NtrB^HNG (Fig . 4, blocks 3 and 4) .

 
To probe interactions mediated by the histidine kinase and phosphotransfer domains of NtrB, we have analyzed, by using the yeast two-hybrid system, a variety of truncated and mutant NtrB derivatives (Fig . 1) for interactions with nitrogen signal transduction proteins NtrC and PII and also for interactions among NtrB derivatives . Every polypeptide was fused to GAL4AD and GAL4BD in order to get, for each pair of proteins tested, two independent signals for the GAL1::lacZ and GAL1::HIS3 reporters . Interactions between NtrB derivatives and PII were remarkably similar for each pair of interacting proteins (Fig . 4, blocks 3 and 4) . On the other hand, GAL4AD-NtrC always gave a stronger signal for interaction with any given NtrB derivative than did GAL4BD-NtrC (Fig . 4, blocks 1 and 2), following a previously noted pattern (15) . This effect, observed with the four pairs of proteins giving signals with NtrC, was most dramatic with NtrB^H, a result that we attribute to instability of the H domain (14) . GAL4BD-NtrB^H, but not GAL4AD-NtrB^H, could be detected in Western blots (Fig . 2), indicating that fusion to dimeric GAL4BD, but not to GAL4AD, stabilizes the H fragment . Regarding interactions between individual domains of NtrB, only one pair of proteins (NtrB^SHN and NtrB^G; see below) gave signals in just one orientation, and here the same considerations apply to the effects of GAL4AD and GAL4BD on the stability of the G fragment (Fig . 2) .

The phosphotransfer domain provides the dimerization interface in histidine kinase EnvZ (29) . All H domains whose structures are known comprise a four-helix bundle bearing the phosphorylated histidines and formed by two pairs of helices contributed by each of the two subunits . Although there is in vitro evidence of dimerization by the H domains of NtrB (11, 15), NtrB^HNG fusion proteins did not interact with each other in the two-hybrid system (15; Fig . 3) . The smaller polypeptides NtrB^HN and NtrB^H also failed to give signals when paired with themselves or with full-length NtrB, indicating that failure of transmitter modules to interact in the two-hybrid system is not due to a negative effect of the G domain on putative signals provided by interacting H domains . The fact that the NtrB^HNG, NtrB^HN, and NtrB^H fusion proteins specifically gave signals with NtrC (Fig . 4, blocks 1 and 2) suggests appropriate folding of the H domains . It is worth noting that, in our experiments, the transmitter modules of both the NtrB and EnvZ proteins were identical in behavior, that is, in vitro cross-linking of the corresponding His-tagged polypeptides (15) and failure of self-paired NtrB^HNG or EnvZ^HNG to interact in the two-hybrid system (Fig . 3, block 1) . Our interpretation of these results is either that the fusion to GAL4 domains prevents dimerization of the H domains in both proteins or that, in vivo, the association between H domains of class I histidine kinases is not very strong . If this is the case, the helix bundle could be forming and dissociating during the phosphorylation cycle and the role of the N-terminal domain would be to facilitate the association and dissociation between the two pairs of helices, thereby providing the basis of intramolecular signal transduction .

When different NtrB derivatives were paired with each other to detect possible interactions between different domains, no signals were found, except for NtrB^SHN and NtrB^G in a given orientation (Fig . 3, blocks 4 and 5) . Although caution should be used in interpreting the NtrB^SHN/NtrB^G signal, it is tempting to speculate that it may be due to the ability of the G domains to weakly recognize either an S or an H domain in the particular conformation adopted by the truncated protein . Lack of signals from the reciprocal NtrB^G/NtrB^SHN pair can be explained by the poorer stability of GAL4AD-NtrB^G versus GAL4BD-NtrB^G (Fig . 2) .

Mutation A129T in NtrB was identified by screening for glnD suppressors, and its phenotypic characterization suggested that the mutant protein was altered in PII binding or in the ability to bring about a conformational change responsible for the conversion into a phosphatase (2) . Our results indicate that mutation A129T produced, in truncated proteins and in particular pairs, a conformational change large enough to be detected in our assays since it impaired signals between NtrB^HNG and NtrC and between GAL4AD-NtrB^SHN and GAL4BD-NtrB^G but not the remaining signals (Table 3) . The lack of impact of the mutation on interactions with PII suggests that A129T affects not the ability of NtrB to bind to PII but rather its ability to interact with NtrC as a phosphatase .

Protein-protein interactions detected here with NtrB derivatives took place only between components of the nitrogen signal pathway, and evidence is provided for specific recognition between domains of the NtrB transmitter module and both PII and NtrC (Fig . 4) . Analysis of the different NtrB derivatives confirms that the H domain is responsible for the signals obtained with NtrC and indicates that, in the NtrBC system, the histidine phosphotransfer domain is sufficient for receiver recognition (Fig . 4, blocks 1 and 2) . In agreement with results obtained with full-length NtrB (15), NtrB^HNG did not give signals with the heterologous response regulator PhoP . In addition, EnvZ^HNG did not interact with NtrC or PhoP . Specificity is also observed for interactions between the NtrB transmitter module and PII . NtrB^HNG, but not EnvZ^HNG or histidine kinase CheA, gave a signal when paired with PII (Fig . 4, blocks 3 and 4) . Similar results have been obtained with purified components (A . Ninfa, unpublished data) . Our failure to detect binding of any noncognate pair of signaling proteins strongly suggests that the signals we did observe are physiologically relevant, providing a rationale for the use of yeast two-hybrid approaches to investigate cross talk among signal transduction proteins and to set up screens to find, for a given component, the cognate partner(s) acting in the same signal transduction pathway .

Regarding determinants for interactions with PII, our data indicate that only constructs retaining the NG region gave signals, while the S domain did not seem to contribute (Fig . 4, blocks 3 and 4), in accordance with recent results showing in vitro cross-linking between PII and a C-terminal fragment of NtrB (amino acids 190 to 349) and suggesting that PII controls NtrB by interaction with the kinase domain of the transmitter module (11, 23) . Although the G domain has in vivo activity (14), it is clearly not sufficient to promote signals with PII in the two-hybrid system . A larger region, including the N domain and perhaps part of the H domain, seems to be required for contacts with PII . For EnvZ, it has recently been proposed that physical linkage between the phosphotransfer and kinase domains is critical for regulation and enables the two domains to maintain their correct spatial arrangements in a dimer configuration (34) . It is worth noting that the same experimental approaches (cross-linking and two-hybrid system) showing interactions between PII and polypeptides containing the kinase domain failed to show contacts between NtrB^S and PII . This highlights the present lack of knowledge of the role of the unique sensor domain of NtrB . Its homology to PAS domains, often involved in protein-protein interaction or ligand binding, is most intriguing . The possibility of additional regulators acting on this domain is currently being explored by two-hybrid screening of genomic libraries .

We thank I . Fuentes for excellent technical assistance, M . Drummond and M . Inouye for strains and plasmids, R . Dixon and I . Luque for critical reading of the manuscript, and A . Ninfa for unpublished information and helpful comments .

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