Uncovering a cryptic allosteric hub governing energy landscape plasticity within the Nur77 ligand-binding domain

Nur77 (NR4A1) is an orphan nuclear receptor whose allosteric mechanism departs fundamentally from that of classical nuclear receptors. Its ligand-binding pocket is constitutively occupied by endogenous hydrophobic side chains, obscuring the structural and allosteric basis of its conformational regulation. As a result, drug design targeting Nur77 has always posed greater challenges. Using NMR spectroscopy, we identified two conformational substates defined by a pocket enclosed by the H1-H3 loop of the ligand-binding domain. These substates interconvert slowly beyond the NMR detection timescale, reflecting a substantial free energy barrier. Solvent-induced isotope shift (SIIS) experiments reveal differential hydrophobic exposure of these two substates, with the minor substate displaying enhanced solvent accessibility. Integrating multiple sequence alignment (MSA) with mutational dissection, we identified a network of long-range weak interactions that acts as a cryptic allosteric hub governing substate equilibrium. Notably, the two substates exhibit distinct ligand-binding affinities, implying functional specialization. Therefore, this study reveals a non-classical allosteric framework of Nur77, providing a structural basis for the design of drugs that target allosteric regulation.