Structural basis for DNA recognition by STAT6

Jing Li; Jose Pindado Rodriguez; Fengfeng Niu; Mengchen Pu; Jinan Wang; Li-Wei Hung; Qiang Shao; Yanping Zhu; Wei Ding; Yanqing Liu; Yurong Da; Zhi Yao; Jie Yang; Yongfang Zhao; Gong-Hong Wei; Genhong Cheng; Zhi-Jie Liu; Songying Ouyang

doi:10.1073/pnas.1611228113

New Research In

Edited by Wei Yang, National Institutes of Health, Bethesda, MD, and approved October 10, 2016 (received for review July 9, 2016)

Significance

STAT6 is a transcription factor and plays a predominant role in IL-4/IL-13 and virus-mediated signaling pathways. Extensive studies have linked malfunctions of STAT6 to pathological features of asthma and cancer. Targeting the function of STAT6 has become an attractive therapy. Understanding the molecular mechanisms of STAT6 transcriptional regulation is still scarce. Here, we report the atomic-level structures of the phosphorylated STAT6 core fragment homodimer, both in DNA-free and complexed with N4 or N3 site DNA, uncovering both a larger dimer interface intersection angle and the unique residue H415 of STAT6 as important factors for discrimination of N4 from N3 site DNA. This study uncovers a dramatic conformational change in STAT6 dimer for recognizing and preferring N4 site DNA.

Abstract

STAT6 participates in classical IL-4/IL-13 signaling and stimulator of interferon genes-mediated antiviral innate immune responses. Aberrations in STAT6-mediated signaling are linked to development of asthma and diseases of the immune system. In addition, STAT6 remains constitutively active in multiple types of cancer. Therefore, targeting STAT6 is an attractive proposition for treating related diseases. Although a lot is known about the role of STAT6 in transcriptional regulation, molecular details on how STAT6 recognizes and binds specific segments of DNA to exert its function are not clearly understood. Here, we report the crystal structures of a homodimer of phosphorylated STAT6 core fragment (STAT6^CF) alone and bound with the N3 and N4 DNA binding site. Analysis of the structures reveals that STAT6 undergoes a dramatic conformational change on DNA binding, which was further validated by performing molecular dynamics simulation studies and small angle X-ray scattering analysis. Our data show that a larger angle at the intersection where the two protomers of STAT meet and the presence of a unique residue, H415, in the DNA-binding domain play important roles in discrimination of the N4 site DNA from the N3 site by STAT6. H415N mutation of STAT6^CF decreased affinity of the protein for the N4 site DNA, but increased its affinity for N3 site DNA, both in vitro and in vivo. Results of our structure–function studies on STAT6 shed light on mechanism of DNA recognition by STATs in general and explain the reasons underlying STAT6’s preference for N4 site DNA over N3.

Footnotes

↵¹J.L. and J.P.R. contributed equally to this work.
↵²To whom correspondence should be addressed. Email: ouyangsy{at}ibp.ac.cn.

Author contributions: J.L., Y.L., Y. Zhao, and S.O. designed research; J.L., J.P.R., F.N., J.W., L.-W.H., Y. Zhu, Y.L., Y.D., Z.Y., and J.Y. performed research; J.L., M.P., J.W., L.-W.H., Q.S., Y. Zhu, W.D., Y. Zhao, G.-H.W., G.C., Z.-J.L., and S.O. analyzed data; and J.L. and S.O. wrote the paper.
The authors declare no conflict of interest.
This article is a PNAS Direct Submission.
Data deposition: Atomic coordinates and structure factors have been deposited in the Protein Data Bank, www.pdb.org (PDB ID codes 4Y5U, 4Y5W, and 5D39).
This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1611228113/-/DCSupplemental.

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