

Homodimeric Structure and DNA-Binding Activity of the Tyrosine Phosphorylated STAT-1 Dimer

Ainsley Lockhart '13 and Kendra Lechtenberg '13


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.

Back to Top