Observation of unphosphorylated STAT3 core protein binding to target dsDNA by PEMSA and X-ray crystallography

• Published in: FEBS letters Vol. 587; no. 7; pp. 833 - 839
• Main Authors: Nkansah, Edwin, Shah, Rahi, Collie, Gavin W., Parkinson, Gary N., Palmer, Jonathan, Rahman, Khondaker M., Bui, Tam T., Drake, Alex F., Husby, Jarmila, Neidle, Stephen, Zinzalla, Giovanna, Thurston, David E., Wilderspin, Andrew F.
• Format: Journal Article
• Language: English
• Published: England Elsevier B.V 02-04-2013
• Subjects: Animals; Binding Sites; Binding, Competitive; Circular Dichroism; Crystallography, X-Ray; DNA - chemistry; DNA - genetics; DNA - metabolism; Electrophoretic Mobility Shift Assay - methods; Humans; Luminescent Proteins - genetics; Luminescent Proteins - metabolism; M67; Models, Molecular; Nucleic Acid Conformation; PEMSA; Phosphopeptides - chemistry; Phosphopeptides - metabolism; Phosphorylation; Protein Binding; Protein Structure, Secondary; Protein Structure, Tertiary; SH2; Signal transducer and activator of transcription 3; src homology 2 domain; STAT3; STAT3 Transcription Factor - chemistry; STAT3 Transcription Factor - genetics; STAT3 Transcription Factor - metabolism; STAT3βtc; the STAT3 core consisting of amino acid residues 127–722; Tyrosine - chemistry; Tyrosine - genetics; Tyrosine - metabolism; X-ray crystallography; yellow variant of eGFP, the enhanced green fluorescent protein; YFP; X-ray crystallography; PEMSA; YFP; STAT3; Signal transducer and activator of transcription 3; SH2; STAT3βtc; M67
• ISSN: 0014-5793; 1873-3468; 1873-3468
• DOI: 10.1016/j.febslet.2013.01.065

► Stoichiometric uSTAT3 interaction with M67 DNA. ► Interaction is weaker compared to pSTAT3 M67 DNA interaction. ► Report of a novel protein electrophoretic mobility shift assay (PEMSA).
The STAT3 transcription factor plays a central role in a wide range of cancer types where it is over-expressed. Previously, phosphorylation of this protein was thought to be a prerequisite for direct binding to DNA. However, we have now shown complete binding of a purified unphosphorylated STAT3 (uSTAT3) core directly to M67 DNA, the high affinity STAT3 target DNA sequence, by a protein electrophoretic mobility shift assay (PEMSA). Binding to M67 DNA was inhibited by addition of increasing concentrations of a phosphotyrosyl peptide. X-ray crystallography demonstrates one mode of binding that is similar to that known for the STAT3 core phosphorylated at Y705.
pSTAT3βtcandpSTAT3βtcbindbymolecular sieving(View interaction)