title: Role of the GYVG pore motif of HslU ATPase in protein unfolding and translocation for degradation by HslV peptidase.
abstract: HslVU is an ATP-dependent protease consisting of HslU ATPase and HslV peptidase. In an HslVU complex, the central pores of HslU hexamer and HslV dodecamer are aligned and the proteolytic active sites are sequestered in the inner chamber of HslV. Thus, the degradation of natively folded proteins requires unfolding and translocation processes for their access into the proteolytic chamber of HslV. A highly conserved GYVG(93) sequence constitutes the central pore of HslU ATPase. To determine the role of the pore motif on protein unfolding and translocation, we generated various mutations in the motif and examined their effects on the ability of HslU in supporting the proteolytic activity of HslV against three different substrates: SulA as a natively folded protein, casein as an unfolded polypeptide, and a small peptide. Flexibility provided by Gly residues and aromatic ring structures of the 91st amino acid were essential for degradation of SulA. The same structural features of the GYVG motif were highly preferred, although not essential, for degradation of casein. In contrast, none of the features were required for peptide hydrolysis. Mutations in the GYVG motif of HslU also showed marked influence on its ATPase activity, affinity to ADP, and interaction with HslV. These results suggest that the GYVG motif of HslU plays important roles in unfolding of natively folded proteins as well as in translocation of unfolded proteins for degradation by HslV. These results also implicate a role of the pore motif in ATP cleavage and in the assembly of HslVU complex.
authors.affiliation: NRL of Protein Biochemistry, School of Biological Sciences, Seoul National University, Korea.
authors.forename: Eunyong
authors.initials: E
authors.lastname: Park
authors.forename: Young Min
authors.initials: YM
authors.lastname: Rho
authors.forename: Ohn-Jo
authors.initials: OJ
authors.lastname: Koh
authors.forename: Sung Won
authors.initials: SW
authors.lastname: Ahn
authors.forename: Ihn Sik
authors.initials: IS
authors.lastname: Seong
authors.forename: Ji-Joon
authors.initials: JJ
authors.lastname: Song
authors.forename: Oksun
authors.initials: O
authors.lastname: Bang
authors.forename: Jae Hong
authors.initials: JH
authors.lastname: Seol
authors.forename: Jimin
authors.initials: J
authors.lastname: Wang
authors.forename: Soo Hyun
authors.initials: SH
authors.lastname: Eom
authors.forename: Chin Ha
authors.initials: CH
authors.lastname: Chung
mesh_terms: D000255:Adenosine Triphosphate / Q000737:chemistry; D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D002364:Caseins / Q000737:chemistry; D002845:Chromatography; D003432:Cross-Linking Reagents / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D004591:Electrophoresis, Polyacrylamide Gel; D049071:Endopeptidase Clp / Q000737:chemistry* / Q000502:physiology; D004926:Escherichia coli / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000502:physiology; D005998:Glycine / Q000737:chemistry; D006868:Hydrolysis; D008954:Models, Biological; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016296:Mutagenesis; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D010455:Peptides / Q000737:chemistry; D011485:Protein Binding; D011489:Protein Denaturation; D017510:Protein Folding; D021381:Protein Transport; D017386:Sequence Homology, Amino Acid; D013696:Temperature
chemical_list: D002364:Caseins; C483566:ClpYQ protease, E coli; D003432:Cross-Linking Reagents; D029968:Escherichia coli Proteins; D010455:Peptides; D000255:Adenosine Triphosphate; D049071:Endopeptidase Clp; D005998:Glycine