NMR Insights Into the Structural Dynamics of p47, A Key Adaptor Protein of p97 in Golgi Reassembly.

The Golgi apparatus undergoes systematic disassembly and reassembly during the cell cycle, a process requiring membrane fusion mediated by the AAA+ ATPase p97/VCP and its adaptor p47. While the p97-p47 complex plays a pivotal role in post-mitotic Golgi reassembly, the exact molecular mechanism underlying its function has not been completely understood. In particular, the conformational flexibility and dynamic feature of p47 hinders its structural characterization by cryo-electron microscopy and X-ray crystallography. Here, using NMR spectroscopy, we characterize the conformational dynamics of p47 and investigate its intra- and intermolecular interactions. p47 consists of three folded domains connected by intrinsically disordered regions (IDRs). We show that p47 adopts a "beads-on-a-string" arrangement and identify several regions that undergo microsecond-to-millisecond timescale motions, which may have functional significance. Using paramagnetic relaxation enhancement (PRE) experiments, we capture transient inter-domain and domain-linker interactions, gaining insights into the structural organization of the domains and linkers in p47. Notably, we identify and characterize an intramolecular interaction between a SEP-interacting motif (SIM), located on the flexible linker, and the SEP domain, suggesting a conserved structural and functional feature among SEP-containing p97 adaptors. Additionally, we observed transient intermolecular interactions between p47 molecules, primarily mediated by the SEP domain. These findings provide a comprehensive view of the structural organization and dynamics of p47, shedding light on how its modular architecture and multivalent interactions may modulate p97 activity and assist Golgi membrane reassembly.