Domains
N-terminal
The N-terminal domain runs from the 1st residue to the 126th residue. It helps stabilise the dimerisation of STAT proteins to form tetramers. The K37 residue has been proven to be essential for tetramer formation. Dimerisation is insufficient to assure some STAT5B functions. Tetramerization of STAT5B allows it to bind to more DNA-binding sites and reinforces DNA-protein interactions. This was proven in the case of the interleukin-2 promoter site (Dagvadorj et al., 2010).
Cleavage of N-terminal domain is associated with prostate cancer, this was confirmed by western blotting as STA5B proteins in prostate cancer cells lack the N-terminal domain. Lack of N-terminal domain in STAT5B is considered a good predictor of severe clinical outcomes in prostate cancer (Tan and Nevalaien, 2008).
The N-terminal domain also has an important role in the nuclear translocation of STAT5B (Zeng et al., 2004).
Coiled coil
This highly coiled region can act as a dimerization tag after the N terminal domain assists to form a dimer. As the coiled coil structure is predicted to form alpha-helical structures analogous to the leucine zipper (Sheldon and Kingston, 1993)
The coiled-coil (alpha) domain interacts with other proteins
- IRF-9/p48 in STAT1
- c-Jun, StIP1, and GRIM-19 for STAT3
- SMRT with STAT5A and STAT5B.
Coiled coils usually contain a repeated pattern, hxxhcxc. Hydrophobic h and charged c amino-acid residues, referred to as a heptad repeat (Mason and Arndt, 2004). The hydrophobic positions are often being occupied by isoleucine, leucine, or valine. Folding a sequence with this repeating pattern into an alpha-helical secondary structure causes the hydrophobic residues to be presented as a 'stripe' that coils gently around the helix in left-handed fashion, forming an amphipathic structure. The most favourable way for two such helices to arrange themselves in the water-filled environment of the cytoplasm is to wrap the hydrophobic strands against each other sandwiched between the hydrophilic amino acids.
DNA binding
The DNA binding domain is a 166 amino acid long region, spanning between the amino acids at the positions of 330 and 496 (Klammt et al., 2018). STAT5B binds proteins as dimers (Stoecklin et al., 1997). The major site for protein-DNA interactions happens at the loop formed by the amino acids at the positions 470-474. STAT5B interacts with the major groove of the DNA (Stoecklin et al., 1997). The protein recognises the consensus motif of (A/tg)(T/A)TTC(C/T)(T/ca g)(G/a)GAA(T/A)(T/ca) for binding (Soldaini et al., 2000).
SH2
Closer to the C terminus is STAT5B Src Homology 2 (SH2) domain. It is about 100 amino acids (aa.) long, spanning 589-670 aa. according to Pfam database (Waksman, Kumaran and Lubman, 2004). Its structure comprises of a large central beta sheet (made up of beta B,C and D) flanked by 2 alpha-helices (made up of alpha A and B) (de Araujo et al., 2019). SH2 domain is responsible for STAT5B dimerization as the negatively charged phospho-tyrosine (Tyr(P)) of another molecule binds to the positively charged Tyr(P)-binding site of the SH2 domain. In particular, the neighboring 3 C-terminal amino acids of Tyr(P) are critical for primary specificity of SH2 interactions (Ariyoshi et al., 2000). SH2 interactions play an important role in signal transduction from receptors to the nucleus where target genes are transcribed (Scaglia et al., 2012).
References
Ariyoshi, K., Nosaka, T., Yamada, K., Onishi, M., Oka, Y., Miyajima, A. and Kitamura, T. (2000) Constitutive Activation of STAT5 by a Point Mutation in the SH2 Domain *, Journal of Biological Chemistry, Elsevier, 275(32), pp. 24407–24413.
Dagvadorj A, Tan SH, Liao Z, et al. N-terminal truncation of Stat5a/b circumvents PIAS3-mediated transcriptional inhibition of Stat5 in prostate cancer cells. Int J Biochem Cell Biol. 2010;42(12):2037-2046. doi:10.1016/j.biocel.2010.09.008
de Araujo, E. D., Orlova, A., Neubauer, H. A., Bajusz, D., Seo, H.-S., Dhe-Paganon, S., Keserű, G. M., Moriggl, R. and Gunning, P. T. (2019) Structural Implications of STAT3 and STAT5 SH2 Domain Mutations, Cancers, 11(11).
Klammt, J., Neumann, D., Gevers, E.F., Andrew, S.F., Schwartz, I.D., Rockstroh, D., Colombo, R., Sanchez, M.A., Vokurkova, D., Kowalczyk, J., Metherell, L.A., Rosenfeld, R.G., Pfäffle, R., Dattani, M.T., Dauber, A., Hwa, V., 2018. Dominant-negative STAT5B mutations cause growth hormone insensitivity with short stature and mild immune dysregulation. Nat Commun 9, 2105. https://doi.org/10.1038/s41467-018-04521-0
Mason JM, Arndt KM. Coiled coil domains: Stability, specificity, and biological implications. ChemBioChem, vol. 5, 2004, p. 170-6. https://doi.org/10.1002/cbic.200300781.
Tan SH, Nevalainen MT. Signal transducer and activator of transcription 5A/B in prostate and breast cancers. Endocr Relat Cancer. 2008;15(2):367-390. doi:10.1677/ERC-08-0013
Scaglia, P. A., Martínez, A. S., Feigerlová, E., Bezrodnik, L., Gaillard, M. I., Di Giovanni, D., Ballerini, M. G., Jasper, H. G., Heinrich, J. J., Fang, P., Domené, H. M., Rosenfeld, R. G. and Hwa, V. (2012) A Novel Missense Mutation in the SH2 Domain of the STAT5B Gene Results in a Transcriptionally Inactive STAT5b Associated with Severe IGF-I Deficiency, Immune Dysfunction, and Lack of Pulmonary Disease, The Journal of Clinical Endocrinology & Metabolism, 97(5), pp. E830–E839.
Sheldon LA, Kingston RE. Hydrophobic coiled-coil domains regulate the subcellular localization of human heat shock factor 2. 1993.
Soldaini, E., John, S., Moro, S., Bollenbacher, J., Schindler, U., Leonard, W.J., 2000. DNA Binding Site Selection of Dimeric and Tetrameric Stat5 Proteins Reveals a Large Repertoire of Divergent Tetrameric Stat5a Binding Sites. Mol. Cell. Biol. 20, 389–401. https://doi.org/10.1128/MCB.20.1.389-401.2000
Stoecklin, E., Wissler, M., Moriggl, R., Groner, B., 1997. Specific DNA binding of Stat5, but not of glucocorticoid receptor, is required for their functional cooperation in the regulation of gene transcription. Mol. Cell. Biol. 17, 6708–6716. https://doi.org/10.1128/MCB.17.11.6708
Waksman, G., Kumaran, S. and Lubman, O. (2004) SH2 domains: role, structure and implications for molecular medicine, Expert Reviews in Molecular Medicine, 6(3), pp. 1–18.
Zeng R, Aoki Y, Yoshida M, Arai K, Watanabe S. Stat5B shuttles between cytoplasm and nucleus in a cytokine-dependent and -independent manner. J Immunol. 2002 May 1;168(9):4567-75. doi: 10.4049/jimmunol.168.9.4567. PMID: 11971004.