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p97/VCP (Cdc48 in yeast) is an essential and abundant member of the AAA+ family of ATPases and is involved in a number of diverse cellular pathways through interactions with different adaptor proteins. The two most characterized adaptors for p97 are p47 and the Ufd1 (ubiquitin fusion degradation 1)-Npl4 (nuclear protein localization 4) complex. p47 directs p97 to membrane fusion events and has been shown to be involved in protein degradation. The Ufd1-Npl4 complex directs p97 to an essential role in endoplasmic reticulum-associated degradation and an important role in mitotic spindle disassembly postmitosis. Here we describe the structural features of the Ufd1-Npl4 complex and its interaction with p97 with the aid of EM and other biophysical techniques. The Ufd1-Npl4 heterodimer has an elongated bilobed structure that is [almost equal to]80 x 30 Å in dimension. One Ufd1-Npl4 heterodimer is shown to interact with one p97 hexamer to form the p97-Ufd1-Npl4 complex. The Ufd1-Npl4 heterodimer emanates from one region on the periphery of the N-D1 plane of the p97 hexamer. Intriguingly, the p97-p47 and the p97-Ufd1-Npl4 complexes are significantly different in stoichiometry, symmetry, and quaternary arrangement, reflecting their specific actions and their ability to interact with additional cofactors that cooperate with p97 in diverse cellular pathways.

Activators of bacterial [sigma]⁵⁴-RNA polymerase holoenzyme are mechanochemical proteins that use adenosine triphosphate (ATP) hydrolysis to activate transcription. We have determined by cryogenic electron microscopy (cryo-EM) a 20 angstrom resolution structure of an activator, phage shock protein F [PspF[subscript (1-275)]], which is bound to an ATP transition state analog in complex with its basal factor, [sigma]⁵⁴. By fitting the crystal structure of PspF[subscript (1-275)] at 1.75 angstroms into the EM map, we identified two loops involved in binding [sigma]⁵⁴. Comparing enhancer-binding structures in different nucleotide states and mutational analysis led us to propose nucleotide-dependent conformational changes that free the loops for association with [sigma]⁵⁴.

p97/VCP (Cdc48 in yeast) is an essential and abundant member of the AAA+ family of ATPases and is involved in a number of diverse cellular pathways through interactions with different adaptor proteins. The two most characterized adaptors for p97 are p47 and the Ufd1 (ubiquitin fusion degradation 1)-Np14 (nuclear protein localization 4) complex. p47 directs p97 to membrane fusion events and has been shown to be involved in protein degradation. The Ufd1-Np14 complex directs p97 to an essential role in endoplasmic reticulum-associated degradation and an important role in mitotic spindle disassembly postmitosis. Here we describe the structural features of the Ufd1-Np14 complex and its interaction with p97 with the aid of EM and other biophysical techniques. The Ufd1-Np14 heterodimer has an elongated bibbed structure that is [asymptotically =]80 x 30 Å in dimension. One Ufd1-Np14 heterodimer is shown to interact with one p97 hexamer to form the p97-Ufd1-Np14 complex. The Ufd1-Np14 heterodimer emanates from one region on the periphery of the N-D1 plane of the p97 hexamer. Intriguingly, the p97-p47 and the p97-Ufd1-Np14 complexes are significantly different in stoichiometry, symmetry, and quaternary arrangement, reflecting their specific actions and their ability to interact with additional cofactors that cooperate with p97 in diverse cellular pathways. [PUBLICATION ABSTRACT]

Activators of bacterial σ54-RNA polymerase holoenzyme are mechanochemical proteins that use ATP hydrolysis to activate transcription. We have determined a 20 Å resolution structure of an activator, PspF(1-275), bound to an ATP transition state analog (ADP.AlFx), in complex with its basal factor σ54 by cryo-electron microscopy. By fitting the crystal structure of apo PspF(1-275) at 1.75 Å into the EM map we identify two loops involved in binding σ54. By comparing enhancer-binding structures in different nucleotide states and mutational analysis, we propose nucleotide dependent conformational changes that free the loops for association with σ54.

p47 is a major adaptor molecule of the cytosolic AAA ATPase p97. The principal role of the p97–p47 complex is in regulation of membrane fusion events. Mono‐ubiquitin recognition by p47 has also been shown to be crucial in the p97–p47‐mediated Golgi membrane fusion events. Here, we describe the high‐resolution solution structures of the N‐terminal UBA domain and the central domain (SEP) from p47. The p47 UBA domain has the characteristic three‐helix bundle fold and forms a highly stable complex with ubiquitin. We report the interaction surfaces of the two proteins and present a structure for the p47 UBA–ubiquitin complex. The p47 SEP domain adopts a novel fold with a βββααβ secondary structure arrangement, where β4 pairs in a parallel fashion to β1. Based on biophysical studies, we demonstrate a clear propensity for the self‐association of p47. Furthermore, p97 N binding abolishes p47 self‐association, revealing the potential interaction surfaces for recognition of other domains within p97 or the substrate.