title: Structural alteration in the pore motif of the bacterial 20S proteasome homolog HslV 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. HslVU is a prototype of such enzymes, consisting of the hexameric HslU ATPase and the dodecameric HslV protease. HslV forms a barrel-shaped proteolytic chamber with two constricted axial pores. Here, we report that structural alterations of HslV's pore motif dramatically affect the proteolytic activities of both HslV and HslVU complexes. Mutations of a conserved pore residue in HslV (Leu88 to Ala, Gly, or Ser) led to a tighter binding between HslV and HslU and a dramatic stimulation of both the proteolytic and ATPase activities. Furthermore, the HslV mutants alone showed a marked increase of basal hydrolytic activities toward small peptides and unstructured proteins. A synthetic peptide of the HslU C-terminal tail further stimulated the proteolytic activities of these mutants, even allowing degradation of certain folded proteins in the absence of HslU. Moreover, expression of the L88A mutant in Escherichia coli inhibited cell growth, suggesting that HslV 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-HslV is largely disordered. Collectively, these results suggest that substrate degradation by HslV is controlled by gating of its pores.
authors.affiliation: School of Biological Sciences, Seoul National University, Seoul 151-742, South Korea.
authors.forename: Eunyong
authors.initials: E
authors.lastname: Park
authors.forename: Jung Wook
authors.initials: JW
authors.lastname: Lee
authors.forename: Hee Min
authors.initials: HM
authors.lastname: Yoo
authors.forename: Byung Hak
authors.initials: BH
authors.lastname: Ha
authors.forename: Jun Yop
authors.initials: JY
authors.lastname: An
authors.forename: Young Joo
authors.initials: YJ
authors.lastname: Jeon
authors.forename: Jae Hong
authors.initials: JH
authors.lastname: Seol
authors.forename: Soo Hyun
authors.initials: SH
authors.lastname: Eom
authors.forename: Chin Ha
authors.initials: CH
authors.lastname: Chung
mesh_terms: D018360:Crystallography, X-Ray; D049071:Endopeptidase Clp / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics / Q000254:growth & development / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D008958:Models, Molecular; D050505:Mutant Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D020125:Mutation, Missense; D011485:Protein Binding; D011487:Protein Conformation; D055503:Protein Multimerization; D059748:Proteolysis
chemical_list: C483566:ClpYQ protease, E coli; D029968:Escherichia coli Proteins; D050505:Mutant Proteins; D049071:Endopeptidase Clp
keywords: AAA; ADEP; ATP-dependent protease; ATPases associated with diverse cellular activities; EDTA; HslU ATPase; MBP; NTA; PDB; Protein Data Bank; WT; Z-GGL-AMC; acyldepsipeptide; bacterial growth inhibition; carbobenzoxy-Gly-Gly-Leu-7-amido-4-methyl coumarin; constricted axial pores; ethylenediaminetetraacetic acid; maltose-binding protein; nitrilotriacetic acid; self-compartmentalized chambers; wild type