Coiled-coil Coactivators Play a Structural Role Mediating Interactions in Hypoxia-inducible Factor Heterodimerizationby Yirui Guo, Thomas H. Scheuermann, Carrie L. Partch, Diana R. Tomchick, Kevin H. Gardner

Journal of Biological Chemistry

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Year
2015
DOI
10.1074/jbc.m114.632786
Subject
Molecular Biology / Biochemistry / Cell Biology

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Text

Coiled-coil Coactivators Play a Structural Role Mediating

Interactions in Hypoxia-inducible Factor Heterodimerization*

Received for publication,December 15, 2014, and in revised form, January 26, 2015 Published, JBC Papers in Press, January 27, 2015, DOI 10.1074/jbc.M114.632786

Yirui Guo‡, Thomas H. Scheuermann‡, Carrie L. Partch‡1, Diana R. Tomchick‡, and Kevin H. Gardner‡§¶2

From the ‡Departments of Biophysics and Biochemistry, University of Texas SouthwesternMedical Center, Dallas, Texas 75390-8816 and the §Structural Biology Initiative, CUNY Advanced Science Research Center, and ¶Department of Chemistry, City

College of New York, New York, New York 10031

Background: Coiled-coil coactivators can enhance HIF-dependent gene transcription via direct interaction with the HIF/

ARNT heterodimer.

Results: ARNT uses the -sheet of the PAS-B domain to recruit coiled-coil coactivators.

Conclusion: Coiled-coil coactivators bridge HIF and ARNT via the PAS-B domain -sheet contacts to both proteins to form a ternary structure.

Significance: This work reveals the mechanism for assembling a coiled-coil coactivator complex with the HIF-2 transcription factor heterodimer.

The hypoxia-inducible factor complex (HIF-aryl hydrocarbon receptor nuclear translocator (ARNT)) requires association with several transcription coactivators for a successful cellular response to hypoxic stress. In addition to the conventional global transcription coactivator CREB-binding protein/p300 (CBP/p300) that binds to the HIF- transactivation domain, a new group of transcription coactivators called the coiled-coil coactivators (CCCs) interact directly with the second PERARNT-SIM (PAS) domain of ARNT (ARNT PAS-B). These less studied transcription coactivators play essential roles in the

HIF-dependent hypoxia response, and CCC misregulation is associated with several forms of cancer. To better understand

CCC protein recruitment by the heterodimeric HIF transcription factor, we used x-ray crystallography, NMR spectroscopy, and biochemical methods to investigate the structure of the

ARNTPAS-B domain in complex with the C-terminal fragment of a coiled-coil coactivator protein, transforming acidic coiledcoil coactivator 3 (TACC3). We found that the HIF-2 PAS-B domain also directly interacts with TACC3,motivating anNMR data-derived model suggesting a means by which TACC3 could form a ternary complex with HIF-2 PAS-B and ARNT PAS-B via-sheet/coiled-coil interactions. These findings suggest that

TACC3 could be recruited as a bridge to cooperatively mediate between the HIF-2 PAS-BARNT PAS-B complex, thereby participating more directly in HIF-dependent gene transcription than previously anticipated.

Hypoxia-inducible factor (HIF)3 proteins are the central regulators of themammalian hypoxia response (1), consisting of an

O2-regulated  subunit (HIF-1, -2, and -3) and the stably expressed  subunit (ARNT or HIF-) (2). Under hypoxia, stabilized HIF- and ARNT subunits dimerize through the N-terminal basic helix loop helix (bHLH) and two Per-ARNT-Sim (PAS) domains. This heterodimer binds to the hypoxia-response element promoter with its N-terminal bHLH domain and controls the transcription of hundreds of target genes such as pro-angiogenic factors and metabolic enzymes (2, 3).

HIF target gene regulation depends on the participation of several transcription cofactors. The intrinsically unfolded

C-terminal transactivation domain of HIF- subunit plays a major role in this process by directly interacting with the global transcription coactivator CBP/p300 (4). Interestingly, recent studies revealed a group of transcription coactivators involved in cancer development and progression, namely the coiled-coil coactivators (CCCs), could be recruited in a transactivation domain-independent manner (5–7). Three CCC family members have been described to date as follows: coiled-coil coactivator (8), thyroid hormone receptor interacting protein 230 (TRIP230) (9), and transforming acidic coiled-coil 3 (TACC3) (10). Under normal situations, these coactivators play an essential role in the hypoxia response by directly interacting with the

ARNT subunit in a promoter-specific way. However, misregulation by overexpression or activating fusions caused by chromosomal translocations (e.g. FGF receptor-TACC3) is sufficient for transformation and is associated with the development of glioblastoma, renal cell carcinoma, and other cancers (11). * Thisworkwas supported, inwholeor inpart, byNational Institutes ofHealth

Grants R01 GM081875, P01 CA095471, and F32 CA130441. This work was also supported by Cancer Prevention and Research Institute of Texas

Grants RP100846 and RP130513.

The atomic coordinates and structure factors (codes 4LPZ and 4PKY) have been deposited in the Protein Data Bank (http://wwpdb.org/). 1 Present address: Chemistry and Biochemistry Dept., University of California at Santa Cruz, Santa Cruz, CA 95064. 2 To whom correspondence should be addressed: Structural Biology Initiative, CUNY Advanced Science Research Center, 85 St. Nicholas Terrace,

New York, NY 10031. E-mail: Kevin.Gardner@asrc.cuny.edu. 3 The abbreviations used are: HIF, hypoxia inducible factor; ARNT, aryl hydrocarbon receptor nuclear translocator; bHLH, basic helix loop helix; PAS,

Per-ARNT-Sim; CCC, coiled-coil coactivator; TACC, transforming acidic coiled-coil coactivator; FGFR, fibroblast growth factor receptor; PRE, paramagnetic relaxation enhancement; MST, microscale thermophoresis;

MTSL, (1-oxyl-2,2,5,5-tetramethyl-3-pyrroline-3-methyl) methanethiosulfonate; CMTSL, (1-oxyl-2,2,5,5-tetramethylpyrroline-3-yl) carbamidoethyl methanethiosulfonate; PDB, Protein Data Bank; r.m.s.d., root mean square deviation.

THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 290, NO. 12, pp. 7707–7721, March 20, 2015 © 2015 by The American Society for Biochemistry and Molecular Biology, Inc. Published in the U.S.A.

MARCH 20, 2015•VOLUME 290•NUMBER 12 JOURNAL OF BIOLOGICAL CHEMISTRY 7707 at U