Molecular basis of transcriptional coactivator recruitment by ARNT PAS domains

Abstract

PAS (Per/ARNT/Sim) domains are used throughout all kingdoms of life to monitor diverse environmental stimuli, including light, oxygen, and small molecule ligands. ARNT (Aryl Hydrocarbon Nuclear Translocator) forms a heterodimeric complex with the sensor bHLH‐PAS protein HIF‐α; to mediate hypoxia adaptation ineukaryotes. Aberrant activation of the hypoxia response in solid tumors is a key predictor of poor clinical outcome in cancer patients. We are studying the molecularbasis of transcriptional coactivator recruitment by ARNT to understand how PAS domains coordinate responses to environmental stimuli. Using NMR spectroscopy, we have characterized the binding sites of both ARNT PAS domains on a minimal fragment of one coactivator, TRIP230. The interaction is mediated in part by ahighly conserved LXXLL‐like motif on TRIP230 that targets the a‐helical interface of ARNT PAS‐B. Two other coactivators target a similar interface on the PAS‐Bdomain, suggesting that this interface is critical for coactivator recruitment. Further studies suggest that the minimal TRIP230 peptide makes contacts with both PASdomains that stabilize formation of a proteolytically protected core complex. Using an NMR‐based screen, we have identified exogenous small molecule ligands thatbind internally to ARNT PAS‐B and disrupt the ARNT:TRIP230 interaction in vitro in an effort to develop novel chemotherapeutic agents.