Structural Analysis of Gene-Regulatory Protein Complexes:

My laboratory is interested in the way that molecular recognition controls protein function in fundamental molecular processes and in human disease. We use biochemical, biophysical, and structural tools to study chromosomal proteins, DNA modification enzymes and enzymes involved in bacterial pathogenesis. Learning more about structure and mechanism in these systems will advance our understanding of fundamental molecular processes and aid in the development of novel pharmaceuticals.

One area of interest is chromatin structure and function. DNA in chromatin is packaged and condensed into higher order structures, but remains accessible to factors involved in processes, such as transcription, through a complex array of protein-DNA and protein-protein interactions, as well as protein and DNA modifications. The chromosomal proteins that bind DNA directly and that are important for the definition of chromatin structure and regulation of gene expression must be able to bind to many different DNA sequences. This is in contrast to better characterized proteins, such as transcription factors, that recognize a specific sequence of DNA. Histone H1 and the HMG-box proteins are examples of chromosomal proteins that bind to the linker DNA (between nucleosomes) and recognize distinct features of DNA structure, such as shape and flexibility. We are interested in understanding how these proteins recognize DNA and how these complexes are involved in mediating cellular processes.

The Drosophila melanogaster HMG-box protein, HMG-D has been the focus of our study. Through mutagenesis, thermodynamic, and structural analyses, we have learned how HMG-D binds to DNA non-sequence-specifically (Figure A), and determined many of the features of the protein that are important for protein induced DNA bending.

 

 

Figure A. We have determined the structure of the complex of HMG-D bound to linear duplex DNA using X-ray crystallography. HMG-D severely bends the DNA by binding and partially intercalating residues in the DNA minor groove. The structure of this non-sequence- specific protein-DNA complex is similar to homologous sequence-specific complexes, except for the lack of sequence-specific hydrogen bonds. Instead, hydrophobic interactions and water mediated non-specific hydrogen bonds stabilize the complex.

 

 

Our work on the non-sequence-specific HMG-box proteins has contributed to understanding how abundant chromosomal proteins interact with DNA and how they may influence the behavior of other protein-DNA complexes. Future studies will focus on testing these hypotheses through structural and biophysical analyses of multi-protein-DNA complexes important in gene regulation.

A second area of interest is in the mechanism of DNA modification. The RsrI methyltransferase (M.RsrI) is a component of the Rhodobacter sphaeroides restriction-modifcation system that protects bacteria from invasion by bacteriophages and foreign DNA. MoRsrI methylates the N6 position of adenine within the recognition site GAATTC. In collaboration with Dr. Richard Gumport's group at the U. of Illinois at Urbana-Champaign, we have determined the structure of M.RsrI, which suggests a novel mechanism of DNA binding for the methyltransferases (see Figure B). Future studies include analysis of M.RsrI interactions with DNA, and structure determination of other methyltransferases and demethylases important in modulation of DNA structure.

 

Figure B. Structure of M.RsrI: The structure explains how DNA recognition and methylation may occur, when the required functional domains reside on opposite sides of the enzyme monomer. A unique dimer of the enzyme is observed in the crystal. This configuration brings the DNA binding domain of one subunit (aqua) near the enzyme active site, which contains a cofactor analog, 5'methylthioadenosine, bound to the red monomer on the right.

 

 

 

A third research interest is global gene regulation in bacteria. As we enter the post-antibiotic era, it is more important now than ever before to understand the molecular and structural basis of bacterial pathogenicity. Quorum sensing, the ability of the bacteria to sense their local concentration, regulates bacterial pathogenicity by altering gene expression on a global scale. Quorum sensing in gram negative bacteria depends on a simple lipid mediator called acyl-homoserinelactone (AHL) that is synthesized by the AHL-synthase, and is detected by a response regulator transcription factor. We are studying the quorum sensing systems in several pathogenic gram negative bacteria to understand the mechanistic basis for AHL synthesis and specificity. Our structural studies provide the foundation for the development of pharmacological agents for treatment of persistent as well as multi-drug resistant forms of bacterial infection.

Education / Experience

Educational Background

• B.A. in Chemistry, Swarthmore College (1981)
• Ph.D. in Chemistry, The Johns Hopkins University (1988)
  Research Advisor: Professor Thomas D. Tullius
  Dissertation: "Hydroxyl Radical Cleavage of DNA: Structural Studies on the TFIIIA-a5S Gene Complex and on Models for the Holliday Recombination Intermediate"
• Postdoctoral fellow with Sir Aaron Klug. MRC Laboratory of Molecular Biology, Cambridge, U.K. (1987-1993)
  Topic: Structural studies of DNA- protein complexes using
  biochemicaland crystallographic techniques.
• Rockefeller Foundation Academic Exchange Visitor in the laboratory of Dr. Ian A. Wilson, Scripps Research Institute (March-July and October-December, 1991).
  Topic: X-ray crystal structure of an anti-peptide
  Fab-peptide complex

Research and Teaching Experience

• American Cyanamid Research Laboratories, Stamford, CT (1980, 1981)
  Scientific Associate in Physical Chemistry and Mining Chemicals Research Divisions.
• Swarthmore College employment (1979-1981)
  Laboratory Assistant: Organic and Advanced Organic Chemistry laboratories.
  Grader and Private Tutor for Introductory, Inorganic, and Organic Chemistry.
• The Johns Hopkins University Chemistry Department employment (1981-1983)
  Teaching Assistant: Inorganic and General Chemistry.
• MRC Laboratory of Molecular Biology, Cambridge, U.K. (1987-1993)
  Non-clinical Scientific Staff Grade I (1989-1993)
• University of Illinois - UIUC (1993-1998)
  Assistant Professor in the Department of Cell and Structural Biology, Biophysics Program, and Department of Biochemistry
• University of Colorado Health Sciences Center - UCHSC (1998-present)
  Assistant Professor, Department of Pharmacology (1998-2001)
  Associate Professor, Department of Pharmacology (2001-present)
  Joint Appointment: Dept. of Biochemistry and Molecular Genetics (1998-present)
  Member: Program in Biomolecular Structure; Colorado Comprehensive Cancer Center; Biomedical Sciences Program; Comprehensive Molecular Biology Program, Medical Scientist Training Program.

Awards, Professional Memberships, and Fellowships

• Associate Member of Sigma Xi (Swarthmore Chapter; 1980)
• American Cancer Society Postdoctoral Fellow (1987-1989)
• Research Fellow, Clare Hall, Cambridge University (1988-1991)
• Life member of Clare Hall, Cambridge University (1991-present)
• Rockefeller Academic Exchange Program (1991)
• NIH Shannon Award (1995-1997)
• Cover Photo and "Hot Paper" Recognition by Nucleic Acids Research (2000)
• Established Investigator Grant of the American Heart Association (2000)

Professional Activities

Professional affiliations

• American Chemical Society
• American Society of Microbiology
• Sigma Xi

Professional Service - Departmental and Institutional Leadership

• Chair of UIUC Biophysics Graduate Admissions Committee (1993-95)
• Course director of Molecular Biophysics UIUC MB320 (1994-95)
• Course director of Molecular Biophysics UIUC MB410zz (1995-98)
• Co-course director of Developmental Biology UIUC CSB301 (1995-98)
• UIUC School of Life Sciences Reorganization Advisory Panel (1996)
• UCHSC Macromolecular X-ray Crystallography Facility Founding-director (1998-2000) Co-director (2000-present)
• Chair of UCHSC Molecular Biology Seminar Program (1999-present)
• Course director of the Graduate Core Course UCHSC IDPT7800 (2001-present)
• Chair of UCHSC Pharmacology Graduate Admissions Committee (2001-present)

Professional Service - Regional and National

• Churchill Scholars review committee (1993-1998)
• American Cancer Society: external grant reviewer. (1996)
• American Heart Association Peer Review Panel (1999-present)
• UCHSC - Howard Hughes Research Grant Review Committee (2000)
• Department of Defense: Breast Cancer Review Panel CET-1 (2001)
• NIH CSR Special Emphasis Panel ZRG1 BM-2(02) section (2001)
• Organizer of "Rocky Mountain RUM 2001" Macromolecular Crystallography meeting
• NSF advisory workshop "Genomics approaches to cis/element TF interactions" (2002)

Peer Review of Journal Articles.

• Biochemistry
• Chemistry and Biology
• European Journal of Biochemistry
• FEBS Letters
• Journal of Biological Chemistry
• Journal of Molecular Biology
• Molecular and Cellular Biology
• Nature Structural Biology
• Nucleic Acids Research
• Proceedings of the National Academy of Sciences
• Trends in Biochemical Sciences

 

Bibliography

1. Churchill ME. Watching flipping junctions. Nat Struct Biol 2003 Feb;10(2):73-5
   

2. Melvin VS, Roemer SC, Churchill ME, Edwards DP.
   The C-terminal extension (CTE) of the nuclear hormone receptor DNA binding domain determines interactions and functional response to the HMGB-1/-2 co- regulatory proteins. J Biol Chem 2002 Jul 12;277(28):25115-24
   

3. W.T. Watson, T. D. Minogue, D. L. Val, S. B. von Bodman, and M.E.A. Churchill (2002) "Structural Basis and Specificity of Acyl-homoserineLactone Signal Production in Bacterial Quorum Sensing" Mol Cell 2002 Mar;9(3):685-94

4. F.V. Murphy IV & M.E.A. Churchill "Non-sequence-specific DNA recognition: a structural perspective." Invited Mini-Review (2000) Structure 8, R83-89.
   

5. W.T. Watson, F.V. Murphy IV, T.A. Gould, P. Jambeck, D. L. Val, J. E. Cronan,Jr., S. Beck von Bodman, and M.E.A. Churchill "Crystallization and Rhenium MAD Phasing of the Acyl-homoserinelactone Synthase EsaI." (2001) Acta Crystallographica, D57, 1945-1949.

6. Scavetta RD, Thomas CB, Walsh MA, Szegedi S, Joachimiak A, Gumport RI, Churchill ME. Structure of RsrI methyltransferase, a member of the N6-adenine beta class of DNA methyltransferases. Nucleic Acids Res. 2000 Oct 15;28(20):3950-61.

7. Dow LK, Jones DN, Wolfe SA, Verdine GL, Churchill ME. Structural studies of the high mobility group globular domain and basic tail of HMG-D bound to disulfide cross-linked DNA. Biochemistry. 2000 Aug 15;39(32):9725-36.

8. Murphy FV 4th, Churchill ME. Nonsequence-specific DNA recognition: a structural perspective. Structure Fold Des. 2000 Apr 15;8(4):R83-9. Review.

9. Murphy FV 4th, Sweet RM, Churchill ME. The structure of a chromosomal high mobility group protein-DNA complex reveals sequence-neutral mechanisms important for non-sequence-specific DNA recognition. EMBO J. 1999 Dec 1;18(23):6610-8.

10. Murphy FV 4th, Sehy JV, Dow LK, Gao YG, Churchill ME. Co-crystallization and preliminary crystallographic analysis of the high mobility group domain of HMG-D bound to DNA. Acta Crystallogr D Biol Crystallogr. 1999 Sep;55 ( Pt 9):1594-7.

11. Churchill ME, Changela A, Dow LK, Krieg AJ. Interactions of high mobility group box proteins with DNA and chromatin. Methods Enzymol. 1999;304:99-133. No abstract available.

12. Kuo MH, Zhou J, Jambeck P, Churchill ME, Allis CD. Histone acetyltransferase activity of yeast Gcn5p is required for the activation of target genes in vivo. Genes Dev. 1998 Mar 1;12(5):627-39.

13. Balaeff A, Churchill ME, Schulten K. Structure prediction of a complex between the chromosomal protein HMG-D and DNA. Proteins. 1998 Feb 1;30(2):113-35.

14. Dow LK, Changela A, Hefner HE, Churchill ME. Oxidation of a critical methionine modulates DNA binding of the Drosophila melanogaster high mobility group protein, HMG-D. FEBS Lett. 1997 Sep 15;414(3):514-20.

15. Lampe DJ, Churchill ME, Robertson HM. A purified mariner transposase is sufficient to mediate transposition in vitro. EMBO J. 1996 Oct 1;15(19):5470-9.

16. Churchill ME. The latest on DNA form and function. Chem Biol. 1996 Sep 1;3(9):729-30. No abstract available. [Record as supplied by publisher]

17. Churchill ME, Jones DN, Glaser T, Hefner H, Searles MA, Travers AA. HMG-D is an architecture-specific protein that preferentially binds to DNA containing the dinucleotide TG.

18. Wolfe SA, Ferentz AE, Grantcharova V, Churchill ME, Verdine GL. Modifying the helical structure of DNA by design: recruitment of an architecture-specific protein to an enforced DNA bend. Chem Biol. 1995 Apr;2(4):213-21.

19. Churchill ME, Jones DN, Glaser T, Hefner H, Searles MA, Travers AA. HMG-D is an architecture-specific protein that preferentially binds to DNA containing the dinucleotide TG. EMBO J. 1995 Mar 15;14(6):1264-75.

20. Churchill ME, Stura EA, Pinilla C, Appel JR, Houghten RA, Kono DH, Balderas RS, Fieser GG, Schulze-Gahmen U, Wilson IA. Crystal structure of a peptide complex of anti-influenza peptide antibody Fab 26/9. Comparison of two different antibodies bound to the same peptide antigen. J Mol Biol. 1994 Aug 26;241(4):534-56.

21. Jones DN, Searles MA, Shaw GL, Churchill ME, Ner SS, Keeler J, Travers AA, Neuhaus D. The solution structure and dynamics of the DNA-binding domain of HMG-D from Drosophila melanogaster. Structure. 1994 Jul 15;2(7):609-27.

22. Ner SS, Travers AA, Churchill ME. Harnessing the writhe: a role for DNA chaperones in nucleoprotein-complex formation. Trends Biochem Sci. 1994 May;19(5):185-7. Review. No abstract available.

23. Travers AA, Ner SS, Churchill ME. DNA chaperones: a solution to a persistence problem? Cell. 1994 Apr 22;77(2):167-9. Review. No abstract available.

24. Churchill ME, Gemmell MA, Woloschak GE. Detection of retinoblastoma gene deletions in spontaneous and radiation-induced mouse lung adenocarcinomas by polymerase chain reaction. Radiat Res. 1994 Mar;137(3):310-6.