σ54 Regulated Two-Component Signal Transduction Systems

Two-component signal transduction systems are a common prokaryotic means for sensing environmental stimulus and providing appropriate cellular response. In NtrC systems the first component (a histidine kinase) senses an environmental change and auto-phosphorylates. The phosphoryl signal is transferred to the three-domain transcriptional activator (also referred to as response regulators and enhancer binding proteins). About half of all transcriptional activators, including most of those studied in our lab, are regulated by an N-terminal receiver domain, which accepts the phosphate signal. They also have a central ATPase domain, which oligomerizes upon activation and, through an ATP hydrolysis coupled mechanism, induces a conformational change in sigma54 that renders it competent for initiating transcription. Finally, they have a C-terminal DNA-binding domain which, among other functions, positions the activator upstream of the desired gene. We study the structure and mechanisms of these transcriptional activators using a combination of NMR spectroscopy, X-ray crystallography, Short Angle X-ray Scattering, and other biophysical techniques, in order to better understand the process of bacterial transcription and the mechanism of these AAA+ ATPases.

Two-component signal transduction pathway in σ54 activation


Adapted from Natasha Keith Vidangos's thesis, 2009
1.  A histidine kinase senses an environmental change and auto-phosphorylates on a conserved histidine. The phosphate is transferred to a conserved aspartate on the receiver domain of one of a number of transcriptional activators that are bound as dimers about 100bp upstream of the transcription initiation site.[1] Other regulatory domains besides the receiver domain have similar activation requirements.

2.  Once the receiver domain receives the signal, the transcriptional activator, whether by a positive or negative regulatory mechanism, no longer favors the dimeric state. The central domain of these transcriptional activators oligomerize into a hexameric or heptameric ring, which functions as a AAA+ ATPase.[2] The activated oligomer, still bound 100bp upstream of the initiation site, is brought into contact with sigma54 by DNA bending.

3.  The transcriptional activator contacts the N-terminal domain of sigma54 near its highly conserved GAFTGA loop. Through ATP hydrolysis and an unknown mechanism, the transcriptional activator interacts with sigma54. Sigma54 then undergoes a conformational change that enables it to melt the dsDNA at the site of transcription (initiation). Once the DNA is melted, core RNA polymerase begins transcribing mRNA off of the DNA (elongation).

Structural studies of transcriptional activators of σ54

The current structural information of these AAA+ ATPase transcriptional activators (also known as enhancer binding proteins) is represented in the figure below:



Circular regulatory domains indicate two-component receiver domains. Star-shaped regulatory domains indicate GAF domains. Color coding is as follows: red indicates a high-resolution structure is available; green indicates multiple high resolution structures are available; blue indicates a low-resolution structure is available.
[3-11]

Recent structures to come out of the Wemmer Lab include the NtrC1 active and inactive state, as well as the NtrC4 inactive state and the NtrC4 DNA binding domain. These structures, along with other biochemical and biophysical studies, have contributed to our understanding of various NtrC-family regulatory mechanisms.

In NtrC1 (right), the presence of its receiver domain represses assembly of the oligomeric protein complex until activated by ATP (or the ATP mimic BeF3) which disrupts the dimeric interface between two regulatory domains thus allowing oligomerization. When removed altogether, the NtrC1-C central domain alone but at high enough concentrations will spontaneously oligomerize into a AAA+ ATPase domain capable of hydrolyzing ATP. Because the receiver domain serves to inhibit formation of the active oligomer, NtrC1 is said to be negatively regulated.[10] This is in contrast to the positively regulated homologue NtrC (left), in which the active AAA+ ATPase oligomer is promoted by the presence of the N-terminal receiver domain and will not assemble at all without it.[13] In NtrC4 (middle), the unactived receiver domain also represses assembly of the hexamer like in NtrC1, but to a much lesser extent. This suggests that NtrC4 represents an evolutionary intermediate between the positive and negative regulatory mechanisms of NtrC and NtrC1 respectively.[11]
NtrC-family regulatory mechanisms



Below are the three recent structures to come out of the Wemmer Lab that have helped explain the regulatory mechanisms of these NtrC-like proteins.
(a) The NtrC4-RC inactive state (PDB: 3DZD)[11], (b) the NtrC1-RC inactive state (PDB: 1NY5)[10] and (c) the NtrC1-C active state (PDB: 1NY6)[10] all solved by X-ray Crystallography in the Wemmer Lab. The inactive state receiver domains both form dimers. The NtrC1 central domain alone enters its active state and forms a heptameric ring when its receiver domain is removed.


Animation of the NtrC1-C heptameriactive state (PDB: 1NY6)[10]

DNA binding in NtrC-family proteins

The C-terminal DNA binding domain in all NtrC-family proteins interacts with a promoter DNA sequence upstream of the transcription start site. One function of the DNA binding domain is to colocalize the transcriptional activators near the sigma54 promoter site, raising the local concentration of the activator and thus increasing the probability of sigma54 activation.[14] A second function is to localize NtrC-family proteins nearby one another to speed up the oligomerization process of the NtrC proteins upon activation.[15,16] The two DNA binding sites exhibit cooperative binding, contributing further to this effect.[17] A third function of the DNA binding domain may be to stabilize the off-state dimer, although this isn't necessary for NtrC1 and NtrC4 dimerization, perhaps because of the strong dimerization interface formed by their negatively regulated receiver domains.

Recent work in the Wemmer Lab has characterized the structure of the DNA binding domain of NtrC4 in complex with DNA.

The structure of the NtrC4 DNA Binding domain in complex with DNA.[12] The NtrC4 DBD dimerizes and folds into a helix-turn-helix motif that binds about 100 base pairs upstream from the transcription start site. The complex reveals specific interactions in the major groove of DNA as well as non-specific interactions with the DNA backbone. The structure shows NtrC4 DBD creates a very slight bending of the DNA, in contrast with related proteins like Fis where the DNA is bent 40-90 degrees. This may reflect an evolutionary trend that has eliminated the need for DNA bending or something specific to the thermophilic nature of NtrC-like proteins in A. aeolicus.

The Wemmer Lab is currently working to fill the gaps in our structural understanding of NtrC family proteins in various states in order to better understand how these proteins interact with each other, the upstream DNA and the sigma54 factor itself.

Other questions about NtrC family transcriptional activators

NtrC-like proteins, NtrC1 and NtrC4, belong to a special subfamily of response regulators that activate transcription via interaction with sigma54 of the RNA polymerase holocomplex. In addition to the structural characterizations, in the Wemmer Lab we are also investigating a number of NtrC-related questions including:
  • How is the activation signal transmitted intramolecularly? How are the DNA promoter sites recognized?
  • How is this recognition different in thermophilic bacteria?
  • How do NtrC-like proteins signal sigma54 and activate it for transcription initiation?
  • How do NtrC homologues and paralogues differ functionally and structurally?

Structural studies of σ54

In the Wemmer Lab, we also study the structure and function of the sigma54 factor, including the structure of its individual domains, the structural requirements for function, and the DNA-binding activity. For more information see our σ54 structural studies structural studies page.

References

[1] Wedel A, Popham, D., Droge, P., Kustu, S. (1990). A bacterial enhancer functions to tether a transcriptional activator near a promoter. Science 248(4954): 486-90.
[2] Tucker, P.A., Sallai, L. (2007) The AAA+ superfamily – a myriad of motions. Curr Opin Struct Biol. 17, 641-52.
[3] Rappas, M., Schumacher, J., Niwa, H., Buck, M., Zhang, X. (2006) Structural basis of the nucleotide driven conformational changes in the AAA+ domain of transcription activator PspF. J Mol Biol. 357, 481-92.
[4] Sallai, L., Tucker, P.A. (2005) Crystal structure of the central and C-terminal domain of the sigma54-activator ZraR. J. Struct. Biol, 151, 160-170.
[5] Park, S., Meyer, M., Jones, A.D., Yennawar, H.P., Yennawar, N.H., Nixon, B.T. (2002) Two-component signaling in the AAA + ATPase DctD: binding Mg2+ and BeF3- selects between alternate dimeric states of the receiver domain. FASEB J. 16, 1964-1966.
[6] Kern, D., Volkman, B.F., Luginbuhl, P., Nohaile, M.J., Kustu, S., Wemmer, D.E. (1997) Structure of a transiently phosphorylated switch in bacterial signal transduction. Nature. 402, 894-898.
[7] Hastings, C.A., Lee, S.Y., Cho, H.S., Yan, D., Kustu, S., Wemmer, D.E. (2003) High- resolution solution structure of the beryllofluoride-activated NtrC receiver domain. Biochemistry. 42, 9081-90.
[8] Lee, S.Y., De La Torre, A., Yan, D., Kustu, S., Nixon, B.T., Wemmer, D.E. (2003) Regulation of the transcriptional activator NtrC1: structural studies of the regulatory and AAA+ ATPase domains. Genes Dev. 17, 2552-63.
[9] Doucleff, M.C., Chen, B., Maris, A.E., Wemmer, D.E., Kondrashkina, E., Nixon, B.T. (2005) Negative regulation of AAA + ATPase assembly by two component receiver domains: a transcription activation mechanism that is conserved in mesophilic and extremely hyperthermophilic bacteria. J Mol Biol. 353, 242-55.
[10] Lee, S.Y., De La Torre, A., Yan, D., Kustu, S., Nixon, B.T., Wemmer, D.E. (2003) Regulation of the transcriptional activator NtrC1: structural studies of the regulatory and AAA+ ATPase domains. Genes Dev. 17, 2552-63.
[11] Batchelor, J.D., Doucleff, M., Lee, C.J., Matsubara, K., De Carlo, S., Heideker, J., Lamers, M.H., Pelton, J.G., Wemmer, D.E. (2008) Structure and regulatory mechanism of Aquifex aeolicus NtrC4: variability and evolution in bacterial transcriptional regulation. J Mol Biol. 384, 1058-75.
[12] Vidangos, N.K. coming soon.
[13] Lee, J., J. T. Owens, et al. (2000). "Phosphorylation-induced signal propagation in the response regulator ntrC." J. Bacteriol. 182(18): 5188-5195.
[14] North A, K. S. (1997). Mutant Forms of the Enhancer-Binding Protein NtrC can Activate Transcription from Solution. JMB 267: 17-36.
[15] Chen, P., Reitzer, L. (1995) Active Contribution of Two Domains to Cooperative DNA Binding of the Enhancer-Binding Protein Nitrogen Regulator I (NtrC) of Escherichia coli: Stimulation by Pohsphorylation and the Binding of ATP. J. Bac. 177, 2490-2496.
[16] Porter, S.C., North, A.K., Kustu S. (1993) Oligomerization of NTRC at the glnA enhancer is required for transcriptional activation. Genes Dev. 7, 2258-73.
[17] Weiss, V., Günter Kramer, Thomas Dünnebier, and Annette Flotho (2002) Mechanism of Regulation of the Bifunctional Histidine Kinase NtrB in Escherichia coli. J Mol Microbiol Biotech. 4, 229-33.