Structural Alteration in the Pore Motif of the Bacterial 20S Proteasome Homo log HsIV Leads to Uncontrolled Protein Degradation

Abstract

In all cells, ATP-dependent proteases play central roles in the controlled degradation of short-lived regulatory or misfolded proteins. A hallmark of these enzymes is that proteolytic active sites are sequestered within a compartmentalized space, which is accessible to substrates only when they are fed into the cavity by protein-unfolding ATPases. HsIVU is a prototype of such enzymes, consisting of the hexameric HsIU ATPase and the dodecameric HsIV protease. HsIV forms a barrel-shaped proteolytic chamber with two constricted axial pores. Here, we report that structural alterations of HsIV's pore motif dramatically affect the proteolytic activities of both HsIV and HsIVU complexes. Mutations of a conserved pore residue in HsIV (Leu88 to Ala, Gly, or Ser) led to a tighter binding between HsIV and HsIU and a dramatic stimulation of both the proteolytic and ATPase activities. Furthermore, the HsIV mutants alone showed a marked increase of basal hydrolytic activities toward small peptides and unstructured proteins. A synthetic peptide of the HsIU C-terminal tail further stimulated the proteolytic activities of these mutants, even allowing degradation of certain folded proteins in the absence of HsIU. Moreover, expression of the L88A mutant in Escherichia coli inhibited cell growth, suggesting that HsIV pore mutations dysregulate the protease through relaxing the pore constriction, which normally prevents essential cellular proteins from random degradation. Consistent with these observations, an X-ray crystal structure shows that the pore loop of L88A-HsIV is largely disordered. Collectively, these results suggest that substrate degradation by HsIV is controlled by gating of its pores. (C) 2013 Elsevier Ltd. All rights reserved.