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
   • direct binding, or via transport protein in membrane
2. activated sensor phosphorylates transcriptional regulator
   • sensor autophosphorylates on Histidine
   • phosphate transferred to Aspartate on activator protein
   • how phosphorylation activates the activator is not known
3. activated regulator binds upstream of the promoter it regulates
   • binding is to two, tandem sites
   • binding is cooperative
   • the mechanism of cooperative binding is not known
4. DNA-bound activator assembles into an octameric or higher order structure
   • the stoichiometry and mechanism for assembly is not known
5. the activator makes contact with RNA polymerase
   • via a DNA looping mechanism that sometimes involves an additional DNA scaffolding protein
6. the activator binds and hydrolyses ATP, stimulating transcription
   • 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
   • 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, have been solved by X-ray crystallography. Functional assays have been performed for truncated forms of the protein, and for selected amino acid substitutions. 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 will be published in October in Genes & Dev. The figure shown below is to be the journal 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. Dale Kaiser (Department of Microbiology, Stanford)
  • examining the role of similar activator proteins in controlling the starvation response of Myxococcus xanthus
    
• Dr. Karen Miller (Department of Food Science, Penn State)
  • investigating the role of cyclic-a-glucans in the nodulation process that leads to effective symbiosis between rhizobium species and legumes.
    
• Drs. Sydney Kustu (Plant and Microbial Biology, UC Berkeley), David Wemmer (Chemistry and LBL, UC Berkeley), A. Daniel Jones (Chemistry, Penn State), and Juliette T. Lecomte and Christopher Falzone (Chemistry, Penn State).
  • Structural studies of DctD, NtrC1, and NtrC
• Dr. Martin Buck and David Studholme (Imperial College, London).
  • Genome analysis of sigma-54 dependent promoters.

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.
• 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
• 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.
• 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

1. HTML - How easy it is! 
2. Finding those interesting WWW sites... 
3. Forms & Scripts 
4. Learning About RasMol 
5. Learning About KineMage 
6. Sequence Alignments 
7. Phylogenetic Trees 
8. 3D Protein Models by E-mail 
9. 3D Protein Models by InsightII 
10. Quantitative Analysis of Data 

Two-Component Signal Transduction
DNA Double Helix Structure
Lambda Repressor cI / DNA Interactions
Eukaryotic Transcription Factor TFIIIA - A Zinc Finger Protein
Leucine Zipper bzip DNA Complex
TBP
The a Subunit of E. coli RNA Polymerase
Sigma factors and promoters 
Polymerase III b Subunit - DNA Clamp

Search the MEDLINE database at PubMed for articles by B.T. Nixon

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