27 Jun 2019

Abstract

The Cdc48 ATPase (p97 or VCP in mammals) and its cofactor Ufd1/Npl4 extract poly-ubiquitinated proteins from membranes or macromolecular complexes for subsequent degradation by the proteasome. How Cdc48 processes its diverse and often well-folded substrates is unclear. Here, we report cryo-EM structures of the Cdc48 ATPase in complex with Ufd1/Npl4 and poly-ubiquitinated substrate. The structures show that the Cdc48 complex initiates substrate processing by unfolding a ubiquitin molecule. The unfolded ubiquitin molecule binds to Npl4 and projects its N-terminal segment through both hexameric ATPase rings. Pore loops of the second ring form a staircase that acts as a conveyer belt to move the polypeptide through the central pore. Inducing the unfolding of ubiquitin allows the Cdc48 ATPase complex to process a broad range of substrates.

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Fig. 3 Binding of unfolded ubiquitin to a conserved Npl4 groove.

(A) Surface model of the Npl4 tower with residues colored according to the degree of their conservation, as calculated by the ConSurf Server. The conserved groove to which unfolded ubiquitin binds is enclosed by a broken line. The positions of the β-strand finger and Zn2+-finger 1 (ZF-1) are indicated.

(B) The highlighted conserved residues in the Npl4 groove, colored according to their position, were mutated to test their effect on substrate unfolding by the Cdc48 ATPase complex.

(C) UN cofactor containing wild-type Ufd1 and either wild-type (WT) or the indicated Npl4 mutant was purified (fig. S4) and tested together with Cdc48 for unfolding of poly-ubiquitinated, photo-converted Eos, measured as loss of fluorescence. The curves show the mean and standard deviation of three replicates.

(D) Experiments as in (C) were done with selected Npl4 mutants and controls. The initial unfolding rates were determined and normalized to that of WT. Shown are the mean and standard deviation of three replicates.

(E) Regions of Npl4 protected by substrate against HDX in the Cdc48/UN complex (in blue). HDX MS was performed with Cdc48/UN/ADP/BeFx in the absence or presence of poly-ubiquitinated substrate at different time points (fig. S5).

(F) As in (E), but in the absence of Cdc48.