Hedgehog Protein (SHH)
Keith Adler '17 Morgan Miller '18
SHH signaling regulates cell growth and differentiation during
embryogenesis, tissue homeostasis and tumorigenesis. The protein is
made as a precursor that is autocatalytically cleaved; the N-terminal
portion is soluble and contains the signalling activity while the
C-terminal portion is involved in precursor processing. More
importantly, the C-terminal product covalently attaches a cholesterol
moiety to the N-terminal product, restricting the N-terminal product
to the cell surface and preventing it from freely diffusing throughout
the developing embryo.
activator protein (CAP) is a DNA binding protein involved with the
transcription of several genes, including those that code for enzymes
involved in the metabolism of certain sugars (i.e. lactose,
maltose, and arabinose.) Basically, CAP is responsible for the global
regulation of carbon utilization. Upon binding cAMP (adenosine 3', 5'
monophosphate, or cyclic AMP), CAP binds to a conserved DNA sequence
from which it can either activate or repress transcription initiation
from various promoters. In some cases clusters of several promoters
are all controlled by a single cAMP-CAP complex bound to the DNA.
Once CAP has bound cAMP, the protein exhibits a higher affinity for a
specific conserved DNA sequence. When the intracellular level of cAMP
increases, the second messenger is bound by CAP and the cAMP-CAP
complex binds to the DNA. Once bound, it is able to stimulate the
transcription of the aforementioned genes. DNA bound by the CAP-cAMP
complex is bent by ~90 degrees. This DNA bend, coupled with a
protein-protein interaction between CAP and RNA polymerase is thought
to be the mechanism by which CAP regluates transcription initiation on
II. General Structure
CAP is a dimer of 22, 500 molecular weight, composed of two
chemically identical polypeptide chains each 209 amino acids in
The overall structure of the dimer is assymetric; one subunit adopts a
"closed" conformation in which the
amino- and carboxy-termini are closer together than in the more "open" subunit. Each subunit is composed
of two distinct domains connected by a hinge
The N-terminal domain is responsible for
dimerization and cAMP
binding. The carboxy-terminal
domain contains a helix-turn helix DNA
and is also responsible for DNA bending.
III. cAMP Binding
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
interaction in signaling pathway
are two different pathways the SHH protein can be regulated:
one involves the interaction with a protein called Hedgehog
interacting protein (HHIP), the other involves the interaction
with N-terminal Hh signaling domain (HhN).
interactions with HHIP with SHH occur with
residues His140, Asp147 and His182 in SHH and Asp383 from
this interaction leads to the inhibition of
the Hh gene.
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 recognition
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, 158, 159, and 162
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.
Gunasekera, Angelo, Yon W. Ebright, and
Richard H. Ebright. 1992. DNA Sequence Determinants for Binding of
the Escherichia coli Catabolite Gene Activator Protein. The
Journal of Biological Chemistry 267:14713-14720.
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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