XWnt8 in complex with the
cysteine-rich domain of Frizzled 8
Emma Klug '18 and Jess Khrakovsky '18
Wnt proteins are critical, highly conserved mediators of
development in both vertebrates and invertebrates. Wnts are secreted
morphogens that establish gradients of transcription factor expression
in progenitor cells, ultimately affecting cell proliferation,
differentiation, and migration.
Wnt binds to the G-protein coupled receptor Frizzled. The interaction
of Wnt with Frizzled activates down-stream effectors that activate the
transcription of Wnt target genes. Proper Wnt/Frizzled signaling is
required for proper development, and dysregulation of the system is
implicated in many human diseases and cancers. Here we dive into this
crucial interaction via exploration of the structural basis of Xenopus
Wnt8 (XWnt8) in complex with the mouse Frizzled-8 (Fz8) cysteine-rich
II. Complex structure
The Xenopus Wnt (XWnt8) is shown in complex with the cysteine-rich
domain (CRD) of the mouse Frizzled-8. (Fz8) Frizzled receptors are found
exclusively at the plasma membrane on the surface of Wnt-responsive cells.
The extracellular CRD at the amino terminus of Frizzled binds Wnts with hihg affinity.
XWnt8 and Fz8-CRD in complex form a distinct donut shape. XWnt8 makes contact with
Fz8 at two distinct binding sites on opposite sides of the CRD.
III. Wnt Structure
An important recognition site for cAMP within CAP is the
ionic bond formed between the side chain of Arg-82
and the negatively charged phosphate group
of cAMP. In the crystal structure, the two cAMP molecules are buried
deep within the beta roll and the C-helix.
It is unclear how cAMP enters or leaves the binding site, but this
probably requires the separation of the two subunits of the dimer,
or the movement of the beta roll and the C helix away from each
other. Other side-chain interactions between the protein and cAMP
are hydrogen bonds occuring at Thr-127,
Ser-128, Ser-83, and Glu-72.
Additional hydrogen bonding between is seen between cAMP and the
polypeptide backbone at residues 83
IV. DNA Binding
Once CAP has bound cAMP, it is ready to bind to the DNA.
Binding occurs at the conserved sequence of
Hydrogen bonds between the protein and the DNA phsophates occur at the
backbone amide of residue
139, and the side chains of Thr-140,
Ser-179, and Thr-182
In addition to these phosphate interactions, the side chains of Glu-181
and Arg-185, both emanating from the
directly contact the bases within the major groove of the DNA. Because
of the way that the protein binds to the DNA, a kink of ~40
degrees occurs between nucleotide base pairs six
and seven on each side of the dyad
This sequence has been shown to favor DNA flexibility and bending in
other systems as well. Because of this kink, an additional five
ionic interactions and four hydrogen bonds are able to occur
between the protein and the DNA strand. Examples of these new
interactions occur between Lys-26, Lys-166,
His-199 and the DNA sugar-phosphate backbone
The DNA bend is integral to the mechanism of transcription activation.
Not only does it place CAP in the proper orientation for
interaction with RNA polymerase, but wrapping the DNA around the
protein may result in direct contacts between upstream DNA and RNA
V. Activating Regions
Transcription activation by CAP requires more than merely
the binding of cAMP and binding and bending of DNA. CAP contains
an "activating region" that has been proposed to participate in
direct protein-protein interactions with RNA polymerase and/or
other basal transcription factors. Specifically, amino acids 156,
have been proposed to be critical for transcription activation by CAP.
These amino acids are part of a surface loop composed of residues
Researchers have concluded that the third and final step in
transcription activation is this direct protein-protein contact
between amino acids 156-162 of CAP, and RNA polymerase.
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Richard H. Ebright. 1992. DNA Sequence Determinants for Binding of
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Schultz, Steve C., George C. Shields, and
Thomas A. Steitz. 1991. Crystal Structure of a CAP-DNA complex:
The DNA Is Bent by 90 degrees Science 253: 1001-1007.
Vaney, Marie Christine, Gary L. Gilliland,
James G. Harman, Alan Peterkofsky, and Irene T. Weber. 1989.
Crystal Structure of a cAMP-Independent Form of Catabolite Gene
Activator Protein with Adenosine Substituted in One of Two
cAMP-Binding Sites. Biochemistry 28:4568-4574.
Weber, Irene T., Gary L. Gilliland, James
G. Harman, and Alan Peterkofsky. 1987. Crystal Structure of a
Cyclic AMP-independent Mutant of Catabolite Activator Protein. The
Journal of Biological Chemistry 262:5630-5636.
Zhou, Yuhong, Ziaoping Zhang, and Richard
H. Ebright. 1993. Identification of the activating region of
catabolite gene activator protein (CAP): Isolation and
characterization of mutants of CAP specifically defective in
transcription activation. Proceedings of the National
Academy of Sciences of the United States of America
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