CURRENT RESEARCH:
1) Chromosomal HMG proteins:
2) Bacterial quorum sensing:
3) DNA modification:
DNA in chromatin is packaged and condensed into higher order structures. Somehow it also remains
accessible to factors involved in processes, such as transcription, through a complex array of protein-DNA interactions,
protein-protein interactions, and covalent modifications. The chromosomal proteins that bind DNA directly and 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 study how these
proteins recognize DNA and how these complexes are involved in mediating cellular processes.
Through mutagenesis, thermodynamic, and structural analyses, we have learned how the Drosophila melanogaster
HMG-box protein HMG-D binds to DNA non-sequence-specifically, and now understand many of the features of the protein that are
important for protein induced DNA bending. 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.
A. Balaeff, M.E.A. Churchill, & K. Schulten “Structure Prediction of a Complex Between the Chromosomal Protein HMG-D and DNA.” (1998) Proteins: Structure Function and Genetics30, 113-135.
F.V. Murphy IV, R. M. Sweet, & M.E.A. Churchill "The Structure of a Chromosomal High-Mobility-Group Protein-DNA Complex Reveals Sequence-neutral Mechanisms Important for Non-sequence-specific DNA Recognition." (1999) EMBO J. 18, 6610-6618.
F.V. Murphy IV & M.E.A. Churchill "Non-sequence-specific DNA recognition: a structural perspective." (2000) Structure 8, R83-89.
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Despite these structural similarities with the site-specific HMG-domain transcription factors, the non-sequence-specific
DNA binding interaction exhibited by the HMG chromosomal proteins has a thermodynamic signature that is distinct from both
the canonical sequence-specific and non-specific modes of DNA binding.
A.I. Dragan, J. Klass, C. Read, M.E.A. Churchill, C., Crane-Robinson, and P.L. Privalov “DNA Binding of a Non-Sequence Specific HMG-D Protein is Entropy Driven with a Substantial Non-Electrostatic Contribution.” (2003) J. Molecular Biology 331, 795-813.
The structural changes that take place with DNA binding can be examined, because the structure of HMG-D was determined by
NMR and the structure of the DNA had been determined crystallographically. We have also studied the interactions of the basic
tail of the protein, which confers high affinity binding and complex stability, and find that it may interact with the DNA
in the major groove.
D.N.M. Jones, M.A. Searles, G.L. Shaw, M.E.A. Churchill, S.S. Ner, J. Keeler, A.A. Travers, & D. Neuhaus “The Solution Structure and Dynamics of the DNA-binding Domain of HMG-D from Drosophila Melanogaster.” (1994) Structure 2, 609-627.
L.K. Dow, D.N.M. Jones, S.A. Wolfe, G.L. Verdine, and M.E.A. Churchill (2000) "Structural Studies of the High Mobility Group Globular Domain and Basic Tail of HMG-D Bound to Disulfide Cross-linked DNA" (2000) Biochemistry, 39, 9725-9736.
J. Klass, F.V. Murphy IV, S. Fouts, M. Serenil,, A. Changela, J. Siple, and M.E.A. Churchill “The Role of Intercalating Residues in Chromosomal High-Mobility-Group Protein DNA Binding, Bending and Specificity.” (2003) Nucleic Acids Research, 31, 2852-64.
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. We are also interested in how the
HMG proteins facilitate the activity of transcription factors, such as Progesterone receptor. In collaboration with the
Dean Edwards laboratory, we are examining the structural role of HMG proteins in PR-DNA
interactions. HMGB proteins appear to interact with the C-terminal extension of the PRDNA binding domain and increase PR DNA
binding affinity.
V.S. Melvin, S.C. Roemer, M.E.A. Churchill, D.P. Edwards “The carboxyl-terminal extension (CTE) of the nuclear hormone receptor DNA binding domain determines interactions and functional response to the HMGB-1/-2 co-regulatory proteins.” (2002) J. Biological Chemistry 277, 25115-24.
V.S. Melvin, C. Harrell, J.S. Adelman, W.L. Kraus, M.E.A. Churchill, D.P. Edwards “The Role of the Carboxyl Terminal Extension (CTE) of the Estrogen Receptor a and b DNA Binding Domain in DNA Binding and Interaction with HMGB.” (2004) J. Biological Chemistry 279, 14763-14771.
Future studies with HMG proteins will focus on further structural and biophysical analyses of multi-protein-DNA complexes
important in gene regulation.
Quorum Sensing. 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 an 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. The figure shows the AHL
synthase EsaI with a substrate model.
W.T. Watson, T. D. Minogue, Dale L. Val, S. Beck von Bodman, and M.E.A. Churchill (2002) “Structural Basis and Specificity of Acyl-homoserine lactone Signal Production in Bacterial Quorum Sensing” (2002) Molecular Cell, 9, 685-694
(with cover photo).
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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. M•RsrI 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.
Future studies include analysis of M•RsrI interactions with DNA, and structure determination of other
methyltransferases and demethylases important in modulation of DNA structure.
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.
R.D. Scavetta, C.B. Thomas, M.A. Walsh, S. Szegedi, A. Joachimiak, R.I. Gumport, M.E.A. Churchill “Structure of RsrI methyltransferase, a member of the N6-Adenine beta class of DNA methyltransferases” (2000) Nucleic Acids Research, 28, 3950-3961
(with cover photo).
C.B. Thomas, R.D. Scavetta, R.I. Gumport, and M.E.A. Churchill “Structures of liganded and unliganded RsrI N6-Adenine DNA methyltransferase: a distinct orientation for active cofactor binding” (2003) J. Biological Chemistry278, 26094-26101
(with cover photo).
Technical contributions:
S.A. Wolfe, A.E. Ferentz, V. Grantcharova, M.E.A. Churchill, & G.L. Verdine “Modifying the Helical Structure of DNA by Design: Recruitment of an Architecture-Specific Protein to an Enforced DNA Bend.” (1995) Chemistry and Biology 2, 213-221.
L.K. Dow, A. Changela, H.E. Hefner, & M.E.A. Churchill “Oxidation of a Critical Methionine Modulates DNA Binding of the Drosophila melanogaster High-Mobility-Group Protein, HMG-D” (1997) FEBS Lett.414, 514-520.
M.E.A. Churchill, A. Changela, L.K. Dow, & A.J. Krieg “Interactions of HMG-box Proteins with DNA and Chromatin” (1999) Methods in Enzymology304, 99-131.
F.V. Murphy IV, J. V. Sehy, Y.G. Gao, L.K. Dow & M.E.A. Churchill “Co-crystallization and Preliminary Crystallographic Analysis of the HMG Domain of HMG-D Bound to DNA.” (1999) Acta CrystallographicaD55, 1594-1597.
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.
T.A. Gould, W.T. Watson, K.-H. Choi, H.P. Schweizer, and M.E.A. Churchill “Crystallization of Pseudomonas aeruginosa AHL synthase LasI using beta-turn crystal engineering.” (2004) Acta Cryst.D60, 518-520.
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Selected Publications
Balaeff, A., Churchill, M.E.A., and Schulten, K. (1998) Structure prediction of a complex between the chromosomal protein
HMG-D and DNA. Proteins: Structure Function and Genetics 30, 113-135.
Kuo, M-H., Zhou, J., Jambeck, P., Churchill, M.E.A., and Allis, C.D. (1998) Histone acetyltransferase
activity of yeast Gcn5p is required for the
activation of target genes in vivo. Genes Dev. 12,
627-639
M.E.A. Churchill, A.
Changela, L.K. Dow, & A.J. Krieg ³Interactions of
HMG-box Proteins with DNA and Chromatin² (1999)
Methods in Enzymology 304, 99-131.
F.V. Murphy IV, J. V. Sehy,
Y.G. Gao, L.K. Dow & M.E.A. Churchill
³Co-crystallization and Preliminary Crystallographic
Analysis of the HMG Domain of HMG-D Bound to DNA.²
(1999) Acta Crystallographica D55, 1594-1597.
F.V. Murphy IV, R. M.
Sweet, & M.E.A. Churchill ³The Structure of a
Chromosomal High-Mobility-Group Protein-DNA Complex
Reveals Sequence-neutral Mechanisms Important for
Non-sequence-specific DNA Recognition.² (1999) EMBO
J. 18, 6610-6618.
L.K. Dow, D.N.M. Jones, S.A.
Wolfe, G.L. Verdine, and M.E.A. Churchill
³Structural Studies of the High Mobility Group
Globular Domain and Basic Tail of HMG-D Bound to
Disulfide Cross-linked DNA² (2000) Biochemistry 39,
9725-9736.
R.D. Scavetta, C.B. Thomas,
M.A. Walsh, S. Szegedi, A. Joachimiak, R.I. Gumport,
M.E.A. Churchill ³Structure of RsrI
methyltransferase, a member of the N6-Adenine b
class of DNA methyltransferases² (2000) Nucleic
Acids Research, 28, 3950-3961.
F.V. Murphy IV & M.E.A.
Churchill ³Non-sequence-specific DNA recognition: a
structural perspective.² Invited Mini-Review (2000)
Structure 8, R83-89.
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
³Crystallization and Rhenium MAD Phasing of the
Acyl-homoserinelactone Synthase EsaI.² (2001) Acta
Crystallographica, D57, 1945-1949.
W.T. Watson, T. D. Minogue,
Dale L. Val, S. Beck von Bodman, and M.E.A.
Churchill (2002) ³Structural Basis and Specificity
of Acyl-homoserine lactone Signal Production in
Bacterial Quorum Sensing² Molecular Cell, 9, 685-694.
J. Klass, F.V. Murphy
IV, S. Fouts, M. Serenil,, A. Changela, J. Siple,
and M.E.A. Churchill ³The Role of Intercalating
Residues in Chromosomal High-Mobility-Group Protein
DNA Binding, Bending and Specificity.² (2003)
Nucleic Acids Research, in press.
M.E.A. Churchill ³Watching Flipping Junctions² News
and Views Report (2003) Nature Structural Biology 10,
73-75.
CU Faculty PubMed Publications |
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Other Links
Department of Pharmacology web page
Biomolecular Structure web page
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