NMR characterization of novel interactions between p97 AAA+ ATPase and the p47 adaptor revealing insights into substrate delivery mechanism

Abstract

p97/VCP is an essential AAA+ ATPase involved in diverse cellular activities by interacting with an array of protein adaptors that recruit p97 for specific tasks. p47 is one of the adaptors that targets p97 for membrane remodeling by forming a stable complex with p97 through multivalent interactions. Here, we report a pair of previously unidentified interactions between the N-terminal part of p47 (residues 1–94) and the N-terminal domain (NTD) of p97. Using nuclear magnetic resonance (NMR) spectroscopy, we identify two binding sites on p47, one located on the ubiquitin-associated (UBA) domain and the other on the intrinsically disordered linker, that interact with the same basic patch on p97 NTD, driven by electrostatic forces. Reciprocal NMR titration experiments between p47 (residues 1–94) and p97 NTD reveal that these interactions are relatively weak in nature with dissociation constants on the order of hundreds of micromolar to millimolar in trans. Structural models of the two interactions are developed based on NMR chemical shift perturbations, which reveal details of the tentative binding interfaces. Our findings provide new insights into the mechanism by which ubiquitinated substrates are delivered from p47 to p97 for unfolding.