B. Tracy Nixon Associate Professor of Biochemistry and Molecular Biology 154 North Frear Laboratory, University Park, PA 16802 Phone: (814) 865-3679 Fax: (814) 863-7024 Email: btn1@psu.edu B.S. in education, University of Missouri, Columbia M.S. in genetics, University of Missouri, Columbia Ph.D. in Cell Biology, Massachusetts Institute of Technology, Boston Post Doctoral in Physiology, Harvard Medical School, Boston Post Doctoral in Molecular Biology, Massachusetts General Hospital, Boston Nixon Lab Web Site

Signal Transduction in Procaryotes: Structure / Function in Rhizobium DctD, enteric NtrC, and NtrC1 of Aquifex aeolicus

Focus

• molecular mechanisms of two component sensory transduction to the transcriptional apparatus in prokaryotes.
  • We examine the structural and functional basis of two component signal transduction in the C4-dicarboxylate transport system of Sinorhizobium meliloti and Rhizobium leguminosarum that fix nitrogen in a symbiotic relationship with the agriculturally important legumes alfalfa and peas. Because dicarboxylic acids are used as energy sources to fuel such nitrogen fixation, the research may lead to improved strains of rhizobium. More importantly, the dct system serves as an example of two-component signal transduction, which is the predominant form of signal transduction in bacteria and also found in archaebacteria, fungi and plants. Despite its obvious importance, the structural basis for this form of signal transduction is only now being elucidated.
  • For comparison, we are also investigating signal transduction in the nitrogen regulation system of Salmonella typhimurium and the NtrC1 protein of Aquifex aeolicus. The function of the NtrC1 protein is unknown, but coming from an extreme thermophile it behaves well in crystallography experiments. This latter work is being conducted in collaboration with Dr. Sydney Kustu and Dr. David Wemmer, both of UC Berkeley.

Working hypothesis

1. sensor protein detects ligand
2. direct binding, or via transport protein in membrane
3. activated sensor phosphorylates transcriptional regulator
4. sensor autophosphorylates on Histidine
5. phosphate transferred to Aspartate on activator protein
6. how phosphorylation activates the activator is not known
7. activated regulator binds upstream of the promoter it regulates
8. binding is to two, tandem sites
9. binding is cooperative
10. the mechanism of cooperative binding is not known
11. DNA-bound activator assembles into an octameric or higher order structure
12. the stoichiometry and mechanism for assembly is not known
13. the activator makes contact with RNA polymerase
14. via a DNA looping mechanism that sometimes involves an additional DNA scaffolding protein
15. the activator binds and hydrolyses ATP, stimulating transcription
16. the polymerase/promoter complex changes from a closed form in which DNA is double stranded to an open one containing the single stranded bubble needed for transcription to initiate
17. the mechanism of linking ATP-hydrolysis to promoter isomerization is unknown

Objectives

• characterize the DNA binding properties of DctD in inactive and active forms
  • determine the intrinsic binding energy for activator / DNA interactions
  • characterize cooperativity in DNA binding by the activator, which has been found to increase upon activation
  • characterizing the biochemical basis for the basal and increased cooperativity

Figure: DNAse 1 footprints for wild type and 5-bp insertion templates reveals cooperativity in DctD binding to the the dctA UAS. Apparently equal affinities for the wild type DNA splits into two differing affinities when the sites are spaced 1/2 turn of the DNA helix apart.

• clarifying the precise role of ATP hydrolysis in the activation process
  • use Mant-ADP and Mant-ATP  in stop-flow experiments to characterize nucleotide binding properties
  • use a variety of techniques to detect conformational and oligomeric changes that are induced by nucleotide binding, hydrolysis or release
    • protease sensitivity assays
    • analytical ultracentrifugation
    • biacore
    • spectroscopic assays and spectrally enhanced forms of DctD
• characterizing the physical basis for signal transduction from the N-terminal domain to the rest of the activator protein
• identification of point mutations that de-regulate the transcriptional activator
  • these mutant proteins activate transcription in the absence of signal transduction
  • they map onto a surface of the N-terminal domain of the activator protein (see figure below)
• determine the structure of the two-component receiver domain and others of DctD or NtrC
  • 1.7 A to 2.9 A X-ray structures have been solved for DctD fragment 2-143 of wild type sequence or bearing substitutions D55C, E121K, K122E, and S54C (see figure below). Contact the lab for details and information about on-going X-ray or NMR experiments.

Figure: DctD sequence was modeled onto the structure of CheY, and then using MolMol a surface contour was drawn and painted white. The charge potential was then calculated for residues Y100, D101, E121 and K122 and used to color that part of the surface contour; substitutions of these 4 residues have been found to yield constitutive phenotypes.

The structure of DctD residues 2-143, including the receiver domain and adjacent linker that joins it to the central ATPase domain was obtained by X-ray crystallography. The structure reveals a novel dimerization motif for response regulator domains, and the genetic data above indicates that the dimeric state acts to inhibit the ATPase, setting the stage for regulation by phosphorylation. Phosphorylation affects the dimeric state directly by switching between this and a second alternative dimeric state. In the off state, the ATPase domain oligomerization surface is sequestered; phosphorylation stabilizes a second receiver domain dimeric structure that freely permits the ATPase interfaces to find each other (based on NtrC1 structures being prepared for publication by Soek-Yong et al.). OFF State: a) stereo view of the dimer, interface in bold; b) typical in vivo assays for constitutive amino acid substitutions; c) stereo view showing the location of 17 such substitutions (bold).

As alluded to above, solving a crystal structure of BeF-bound receiver domain of DctD culminated in a model for activation in DctD and NtrC1 proteins that starkly contrasts with activation in enteric NtrC. The work shows two distinct mechanisms exist for integrating a common two component signal transduction trigger with different domain-domain interactions to converge on regulated assembly of the AAA+ ATPase domains present in these proteins. The summary figure shown below was used for the cover of the December 2002 issue of FASEB J.

Crystal structure of the combined receiver and ATPase domains of NtrC1, and of the ATPase domain alone, were solved by my sabbatical hosts David Wemmer and Sydney Kustu using X-ray crystallography. I performed functional assays for truncated forms of the protein, and for selected amino acid substitutions, and together with Michaeleen Callahan of the Wemmer lab solved a crystal structure of the phosphorylated receiver domain of NtrC1. Results show that NtrC1 functions like DctD, not NtrC, and provide a model for explaining how the receiver domain alternately represses and derepresses the assembly of the AAA+ ATPase. The off-state dimer of the receiver domain and linker stabilize an inactive dimer of the ATPase. Phosphorylation of the receiver domain stabilizes it in an alternative dimer conformation that repositions the linkers and receiver domains to prevent the contacts needed to maintain the repressed ATPase dimer. This permits the ATPase domain to move from a front-to-front orientation to a back-to-front orientation, which directs self assembly of a heptamer ring. This work was published in Genes & Dev. and J. Mol. Biol. The figure shown below was the G&D journal cover

Amino acid substitutions and Mg2+/BeF3- conditions were obtained that produced monodisperse solutions of activated, full-length NtrC protein from Salmonella typhimurium. Small- and wide angle X-ray scattering data were obtained for the activated enhancer binding protein at the BioCAT beamline 18-ID of the APS at the Argonne National Lab in Argonne, IL. Confirming and extending EM data were also obtained in collaboration with Eva Nogales and Sacha De Carlo (HHMI, UC Berkeley). Low resolution structures were extracted from the SAXS/WAXS data using the GASBOR program of Dmitri Svergun (EMBL), and by image reconstruction of the EM data. The structures revealed for the first first time the juxtaposition of the two-component receiver (yellow), ATPase (red) and DNA binding (blue) domains (the DNA is placed in the picture using artistic license, and it runs below the protein, not through it). Note how the alternating, periphereal receiver domains facilitate ATPase ring assembly. Also, comparing ADP-bound protein with ADP-AlFx bound protein revealed major order-disorder transitions in the GAFTGA-loop regions and the DNA binding domains. Since coupling of hydrolysis, DNA binding and sigma factor remodeling can be dramatically perturbed (Yan and Kustu, 1999), these transitions must be understood to learn how these AAA+ ATPases perform mechanical work to remodel the s54-form of RNA polymerase. This work was published in Genes & Dev. The figure shown below (created by Sacha De Carlo) was adapted for the journal cover

The same SAXS/WAXS method was successfully applied to study conformational changes that occur during the hydrolysis cycle of the NtrC1 ATPase domain. Nucleotides and nucleotide analogs were used to trap the ATP-ground, transition, and product states for comparison with the apo state. A large conformational change was seen upon ATP binding that extends the GAFTGA loop region up and away from the plane of the ATPase ring. Though altered, this upward position is retained in the transition state, and collapses back down in the product state. Size-exclusion chromatography showed that the extended GAFTGA loops are able to stably bind to the sigma factor, with the transition state complex being more stable than the ground state one. These results support a model in which stable contact with sigma factor is obtained in the ATP ground state, strenghtened by the transition state, and relaxed after hydrolysis and Pi release. This work was published in Structure (Cell Press). The figures shown below (created by Baoyu Chen) were adapted for the journal cover

Collaborators Michaeleen Doucleff and David E. Wemmer (Chemistry Department, University of California - Berkeley) determined the structure of a fragment of the Aquifex aeolicus s54 protein both free and when bound to the -24 region of a promoter. This work establishes the basis for sigma factor binding to the upstream element of a s54 dependent promoter. Modeling the sigma/DNA complex together with the rest of RNA polymerase suggests novel contact with RNA polymerase which raises hypotheses about how the sigma factor interacts with the core of RNA polymerase to activate transcription. This work was published in the Journal of Molecular Biology, with the editors choosing the summary figure for the cover .

Collaborators

• Dr. Tim Hoover (Microbiology, University of Georgia)
  • examining the physical basis for interaction between the activator and the RNA polymerase
  • isolating cooperativity mutations in DctD
• Dr. Haw Yang (Department of Chemistry, University of California - Berkeley)
  • examining conformational dynamics for NtrC and NtrC1 ATPases
• Dr. Sacha De Carlo (NIH NCRR, 3D-EM Boulder Lab, Molecular, Cellular and Developmental Biology Department, Boulder, CO)
  • using EM image reconstruction to study two component signal transduction and conformational changes involved in AAA+ ATPase function.
• Dr. David Wemmer (Chemistry and Lawrence Berkeley Lab, University of California - Berkeley).
  • Structural and functional studies of DctD, NtrC1, NtrC, and NtrC4
• Dr. Martin Buck and Wendy Cannon (Imperial College, London).
  • Protein-DNA footprinting of activator / s54 complexes.

Representative Publications:

• Nixon, B. T., C. W. Ronson, and F. M. Ausubel. 1986. Two-component regulatory systems responsive to environmental stimuli share strongly conserved domains with the nitrogen assimilation regulatory genes ntrB and ntrC. Proc. Natl. Acad. Sci. USA. 83:7850-7854.
• Miller, K. J., M. W. McKinstry, W. P. Hunt, and B. T. Nixon. 1992. Identification of the diacylglycerol kinase structural gene of Rhizobium meliloti 1021. Mol. Plant Microbe Interactions. 5:363-371.
• Ledebur, H., and B. T. Nixon. 1992. Tandem DctD binding sites of the Rhizobium meliloti dctA UAS are essential for optimal funciton despite a 50 to 100-fold difference in affinity for DctD. Mol. Microbiol. 6:3479-3492.
• Gu, B., J. H. Lee, T. R. Hoover, D. Scholl, and B. T. Nixon. 1994. Rhizobium meliloti DctD, a s⁵⁴-dependent transcriptional activator, may be negatively controlled by a subdomain in the C-terminal end of its two-component receiver module. Mol. Microbiol. 13:51-61.
• Lee, J. H., D. Scholl, B. T. Nixon, and T. R. Hoover. 1994. Constitutive ATP hydrolysis and transcription activation by a stable, truncated form of Rhizobium meliloti DctD, a s⁵⁴-dependent transcriptional activator. J. Biol. Chem. 269:20401-20409.
• Inactive Web Pages
• Nixon, B. T. 1994. Front Page - Department of Biochemistry and Molecular Biology at Penn State
• Nixon, B. T. 1994. Brochure for Department of Biochemistry and Molecular Biology at Penn State
• Nixon, B. T. 1994. Quantitative Image Analysis Workstation: Description of the Molecular Dynamics Phosphorimager and related equipment available to the Life Sciences community at Penn State
• Nixon, B. T. 1995. Discussion Problem: Can modelling of DctD after CheY provide a basis for hypothisizing structure/function relationships of DctD? in Principles of Protein Structure
• Nixon, B. T. 1995. How to modify Brookhaven PDB files for viewing multiple solutions to NMR data with RasMol
• Active Web Pages
• Nixon, B. T. 1995. Quantitative Analysis of DNAse1 Footprint Data - a how to manual available on the Internet.
• Nixon, B. T. 1995. SEQSCAN - a DNA sequence scanning program available on the Internet. Evolved into PromScan, by collaborators David Studholme and Martin Buck.
• Nixon, B. T. 1995. WebTools - a limited set of tools for molecular biologists and biochemists.
• Nixon, B. T. 1995. Computers for Biochemists and Molecular Biologists - an Internet posted course with lessons on  

• R. Ilene Kaufman and B. Tracy Nixon. 1996. Use of PCR to isolate genes encoding s⁵⁴-dependent activators from diverse bacteria. J. Bacteriology 178:3967-3990. 
• Dean Scholl and B. Tracy Nixon. 1996. Cooperative Binding of DctD to the dctA UAS of Rhizobium meliloti is Enhanced in a Constitutively Active Truncated Mutant. J. Biol. Chem. 271:26435-26442.  (pdf)
• Staley, M., Zeringue, L.C., Kidd, R.D., Farber, G.K., Nixon, B.T. 1998. Crystallization and characterization of the Rhizobium meliloti DctD two-component receiver domain. Acta Crystallographica, D 54(2 ( Pt 6)):1416-8. 
• B. Tracy Nixon. 1998. MDL Chime Structural Aids for BMMB 514 - Molecular Biology and Cellular Regulation (course discontinued in 2000, but CHIME pages are still available). 

• B. Tracy Nixon. 1998. Tutorial on Making HTML-Chime Pages 
• John Sojda, III, Baohua Gu, Joon Lee, Timothy R. Hoover, B. Tracy Nixon. 1999. A rhizobial homolog of IHF stimulates transcription of dctA in Rhizobium leguminosarum but not in Sinorhizobium meliloti. Gene 238:489-500. 
• David J. Studholme, Martin Buck and B. Tracy Nixon. 2000. Identification of potential s^N-dependent promoters in bacterial genomes. Microbiol. 146:3021-3023. (pdf) 
• B. Tracy Nixon. 2000. A structural sub-family of two-component receiver domains sharing a coiled-coil dimer motif as seen in DctD. 
• Matthew Meyer, Sungdae Park, Lori Zeringue, Mark Staley, Mike McKinstry, R. Ilene Kaufman, Hong Zhang, Dalai Yan, Neela Yennawar, Hemant Yennawar, Greg Farber, and B. Tracy Nixon. 2001. A dimeric two-component receiver domain inhibits the s⁵⁴-dependent ATPase in DctD. FASEB J.. The FASEB Journal Express Article 10.1096/fj.00-0516fje. Published online March 20, 2001, (pdf at FASEB J) 
• B. Tracy Nixon. 2001. A 1 credit course in crystallography methods.
• Sungdae Park, Hong Zhang, A. Daniel Jones, and B. Tracy Nixon. 2002. Biochemical evidence for multiple dimeric states of the Sinorhizobium meliloti DctD receiver domain. Biochemistry 41:10934-10941.
• Sungdae Park, Matthew Meyer, A. Daniel Jones, Hemant P. Yennawar, Neela H. Yennawar, and B. Tracy Nixon. 2002. Two-component signaling in the AAA+ ATPase DctD: binding Mg²⁺ and BeF₃^- selects between alternative dimeric states of the receiver domain. The FASEB Journal Express Article 10.1096/fj.02-0395fje, October 4, 2002.  (pdf at FASEB J)
• Ying-Kai Wang, Sungdae Park, B. Tracy Nixon, and Timothy R. Hoover. 2003. Nucleotide-induced structural changes in the sigma54-dependent activator DctD. J. Bacteriol. 189: 6215-6219.
• Seok-Yong Lee, Armando de la Torre, Dalai Yan, Sydney Kustu, B. Tracy Nixon, and David Wemmer. 2003. Regulation of the transcriptional activator NtrC1: structural studies of the regulatory and AAA+ ATPase domains. Genes and Dev. 17:2552-2563. Cover image local jpeg file. or publisher's site.
• Hao Xu, Baohua Gu, B. Tracy Nixon, and Timothy R. Hoover. 2004. Purification and characterization of the AAA+ domain of Sinorhizobium meliloti DctD, a s⁵⁴-dependent transcriptional activator. J. Bacteriol. 186: 3499-3507.
• Hao Xu, Mary T. Kelly, B. Tracy Nixon, and Timothy R. Hoover. 2004. Novel substitutions in the s⁵⁴-dependent transcriptional activator DctD that uncouple ATP hydrolysis from transcriptional activation. Mol. Microbiol. 54: 32-44. pdf This is an electronic version of an article published in Molecular Microbiology. Complete citation information for the final version of the paper, as published in the print edition of Molecular Microbiology, is available on the Blackwell Synergy online delivery service, accessible via the journal's website at www.mol-micro.com or www.blackwell-synergy.com.
• Michaeleen Doucleff, Baoyu Chen, Ann E. Maris, David E. Wemmer, Elena Kondrashkina and B. Tracy Nixon. 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: 242-255.
• B. Tracy Nixon, Hemant P. Yennawar, Michaeleen Doucleff, Jeffrey G. Pelton, David E. Wemmer, Susan Krueger, and Elena Kondrashkina. 2005. SAS Solution Structures of the Apo and Mg2+/BeF3--bound Receiver Domain of DctD from Sinorhizobium meliloti. Biochemistry 44: 13962-13969.
• Sacha De Carlo, Baoyu Chen, Timothy R. Hoover, Elena Kondrashkina, Eva Nogales, and B. Tracy Nixon. 2006. The structural basis for regulated assembly and function of the transcriptional activator NtrC. Genes & Dev. 20: 1485-1495. Cover image local jpeg file. or publisher's site.
• Baoyu Chen, Michaeleen Doucleff, David E. Wemmer, Sacha De Carlo, Hector H. Huang, Eva Nogales, Timothy R. Hoover, Elena Kondrashkina, Liang Guo and B. Tracy Nixon. 2007. ATP ground- and transition states of bacterial enhancer binding AAA+ ATPases support complex formation with their target protein, s⁵⁴, Structure,15: 429-440. Cover image local gif file or publisher's site.
• Michaeleen Doucleff, Jeffrey G. Pelton, Peter S. Lee, B. Tracy Nixon and David E. Wemmer. Structural basis of DNA recognition by the alternative sigma factor, s54. Cover image local jpeg file. or publisher's site. J. Mol. Biol., in press (2007).

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