Homodimeric
Structure and DNA-Binding Activity of the Tyrosine Phosphorylated
STAT-1 Dimer
Ainsley Lockhart '13 and Kendra Lechtenberg '13
Contents:
I.
Introduction
Signal transducers and
activators of transcription (STAT) proteins are a family of cytoplasmic
proteins that mediate polypeptide ligand-mediated alterations in gene
transcription. STATs dock at ligand-activated receptors and become
phosphorylated on tyrosine, which allows them to dimerize and
translocate to the nucleus. They are then able to activate or suppress
gene transcription, depending on the identity of the specific STAT
protein and which receptor they interacted with. STAT-1 is activated by
interferons alpha and gamma (IFN-α and IFN-γ).
Binding of these ligands at their respective receptors activates the
JAK kinase, which phosphorylates STAT-1 and causes it to form either
a homodimer or a complex with STAT-2 and p48, which bind the promoters
of IFN-activated genes (1). This tutorial will examine the structure,
DNA-binding activity, and dimerization of the phosphorylated DNA-bound
STAT-1 homodimer.
II.
General Structure
The
STAT-1 core has 4 major structural domains: the
coiled-coil domain
(residues 136-317), the
DNA binding domain (residues
318-488),
the linker domain (residues
488-576),
and the SH2 domain
(residues 577-683).
The
the C-terminal tail at the end of the SH2 domain is phosphorlyated
at
Tyr-701
,
which allows the two STAT-1 monomers to form a homodimer
.
The STAT-1 homodimer is shown here co-crystallized with an 18-mer duplex DNA
at the DNA binding domain.
III.
Coiled-Coil Domain
The
STAT-1 coiled-coil
domain is thought to
facilitate interactions of STAT-1 with other proteins. It is comprised
of 4 α-helices arranged in a predominantly hydrophilic
coiled-coil
structure
that
projects out from the protein core. The coiled-coil domain is rich in
charged amino acids, providing a likely strategy for specific
interactions with other proteins. In total the surface of the
coiled-coil domain contains 19 lysines,
16
glutamates, 11 aspartates,
7
arginines, and 4 histidines
.
The number of acidic and basic side chains on the surface of the
α-helices suggest that the coiled-coil domain interacts with
other
helical proteins, such as p48 (1).
IV.
DNA Binding Domain
The
STAT-1 protein contacts DNA in a semi-sequence-specific manner, via an
immunoglobulin fold binding domain. This domain consists of 11
β-strands and 2 α-helices which run perpendicular to
the DNA
axis, allowing loops at one end of the β-sheet to contact the
DNA
(2).
The DNA-binding
domain also has a 2-fold axis of symmetry, defined
by the interaction between the two STAT-1 monomers and the
nearly-symmetric DNA sequence
.
The optimal DNA consensus sequence for STAT-1 binding is 15 base pairs
long (3'-ACAGTTTCCCGTAAATG C-5'),
and the central cysteine is numbered as 0. There is ambiguity in the
bases at the -4, -2, 0, and 2 positions (3).
Contacts
between the STAT-1 molecule and DNA base pairs are mediated by water
molecules and hydrogen bonding. Lys-336
,
located between β2 and β3, is positioned in the major
groove, and makes
contacts with the phosphate backbone as well as water-mediated
interactions with cytosine and guanine bases at the 0 and 1 positions.
The Asn-460
side chain
,
which is located between β11 and α6, is particularly
important to DNA
recognition and makes hydrogen bonds with guanine and thymine bases at
positions 1
and 2, and water-mediated contacts with thymine at position 3. Glu-421
interacts with a position 7 guanine via hydrogen bonding through the
minor groove
.
Phosphate
backbone contacts are made by the Arg-378
side chain, which is located between a5 and B5, Thr-459,
located between β11 and α6, Thr-327,
and His-328,
which are both located between β1 and β3
.
Additionally, several side chains located between β8 and
β9 make
water-mediated contacts with the phosphate backbone, including Glu-411,
Lys-413,
Lys-410,
and Thr-427
.
There
are few sequence-specific contacts between DNA and STAT-1, suggesting
that STAT-1 binding is not based on interaction with a well-defined
consensus sequence. Instead, interactions with other DNA-bound proteins
and STAT dimers may facilitate the specificity of the DNA-binding
activity of STAT-1. Additionally, hydrogen bonding between Pro-465
in a6 and Trp-555
couple the DNA-binding
domain and the SH2 domain,
suggesting that the SH2 domain may also modulate DNA binding
.
V.
SH2 Domain and STAT-1 Homodimer Formation
The
STAT-1
SH2 domain
is the site of tyrosine phosphorylation and dimer formation. The core
SH2 domain is comprised of 4 α-helices, aA,
aB',
αB,
and
αC,
and 2 β-sheets,
βB and βC.
.
The core SH2 domain is connected to the phosphorylated C-terminal tail
by a flexible linker (not shown). The C-terminal tail
projects away to contact
the SH2 domain of the other monomer, forming a two-stranded
antiparallel β-sheet with the other C-terminal tail that passes
between
αB and αB'
,
and
providing the basis for dimer formation. pTyr-701
from
the C-terminal tail of one monomer interacts with and Arg-602
and
Lys-584 from the SH2 domain
of the other monomer
.
The structure of the domain prevents pTyr-701 from contacting these
residues on its own monomer (4). Leu-706,
Ile-707,
and Ser-708
on the C-terminal tail also contribute to dimer formation by
interacting with a hydrophobic site formed by the proximity of the two αB'
helices
.
VI.
References
1.
Chatterjee-Kishore, M., van den Akker, F., and G.R. Stark. 2000.
Association of STATs with relatives and friends. Trends
in Cell Biology 10: 106-111.
2.
Chen, X., Vinkemeir, U., Zhao, Y., Jeruzalmi, D., Darnell, J.E., and J.
Kuriyan. 1998. Crystal Structure of a Tyrosine Phosphorylated STAT-1
Dimer Bound to DNA. Cell
93: 827-839.
3.
Hovarth CM, Stark GR, Kerr IM, and JE Darnell. 1995. A STAT protein
domain that determines DNA sequence recognition suggests a novel
DNA-binding domain. Genes and
Development 9: 984-994.
4.
Shuai K, Ziemiecki A, Wilks AF, Harpur AG, Sadowski HB, Gilman MZ, et
al. 1993. Polypeptide signaling
to the nucleus through tyrosine phosphorylation of Jak and Stat
proteins. Nature 366, 580-583.
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