Structural Mechanism of the Oxygenase JMJD6 Recognition by the Extraterminal (ET) Domain of BRD4

Tsuyoshi Konuma,Lei Zeng,Rajal Sharma,Di Yu,Ying Ju,Chunyan Ren,Ming-Ming Zhou,Qiang Zhang,Chengcheng Zhao

doi:10.1038/s41598-017-16588-8

Abstract

Jumonji domain-containing protein 6 (JMJD6) is a member of the Jumonji C family of Fe(II) and 2-oxoglutarate (2OG) dependent oxygenases. It possesses unique bi-functional oxygenase activities, acting as both an arginine demethylase and a lysyl-hydroxylase. JMJD6 has been reported to be over-expressed in oral, breast, lung, and colon cancers and plays important roles in regulation of transcription through interactions with transcription regulator BRD4, histones, U2AF65, Luc7L3, and SRSF11. Here, we report a structural mechanism revealed by NMR of JMJD6 recognition by the extraterminal (ET) domain of BRD4 in that a JMJD6 peptide (Lys84-Asn96) adapts an α-helix when bound to the ET domain. This intermolecular recognition is established through JMJD6 interactions with the conserved hydrophobic core of the ET domain, and reinforced by electrostatic interactions of JMJD6 with residues in the inter-helical α1-α2 loop of the ET domain. Notably, this mode of ligand recognition is different from that of ET domain recognition of NSD3, LANA of herpesvirus, and integrase of MLV, which involves formation of an intermolecular amphipathic two- or three- strand antiparallel β sheet. Furthermore, we demonstrate that the association between the BRD4 ET domain and JMJD6 likely requires a protein conformational change induced by single-stranded RNA binding.

Highlights

Human JMJD6 consists of a JmjC (Jumonji C) domain, three apparent nuclear localization signals (NLS), a DNA binding domain (AT-hook domain), a putative sumoylation site, and a polyserine domain[21]
M610-R676) was used in nuclear magnetic resonance (NMR) titration experiments to assess its binding to a JMJD6 peptide that resembles the consensus amphipathic ET domain binding motif found in other effector proteins including NDS3 and Kaposi’s sarcoma-associated herpesvirus (KSHV) LANA33
When the full-length JMJD6 was used in NMR titration, the 15N-HSQC spectrum of the ET domain showed severe line broadening, likely induced by conformation exchange, resulting from the ET domain binding to JMJD6 in solution

Summary

Introduction

Human JMJD6 consists of a JmjC (Jumonji C) domain, three apparent nuclear localization signals (NLS), a DNA binding domain (AT-hook domain), a putative sumoylation site, and a polyserine (polyS) domain[21]. The JMJD6 structure contains a total of 15 short α-helices with α2, α3, α5, α6, α9, α10, and α11 displaying only one-turn and α4 and α8 two-turns These one- and two-turn helices are distributed all over the surface of the protein molecule, are loosely connected by a variety of coil loops, and are likely flexible in a solution. The structures of the BRD4 ET domain bound to MLV Integrase, LANA and NSD3 were recently reported[32,33], and the general ET domain binding motif and specificity were explained. It has remained unclear how the ET domain selectively interacts with JMJD6 considering its conserved β-sheet cupin core and multiple small unique helices spreading around the protein surface. We further show that BRD4/JMJD6 recognition is likely promoted by ssRNA binding to JMJD6 that induces protein conformational change

Methods

Results

Conclusion