title: Diverse pore loops of the AAA+ ClpX machine mediate unassisted and adaptor-dependent recognition of ssrA-tagged substrates.
abstract: ClpX, an archetypal proteolytic AAA+ unfoldase, must engage the ssrA tags of appropriate substrates prior to ATP-dependent unfolding and translocation of the denatured polypeptide into ClpP for degradation. Here, specificity-transplant and disulfide-crosslinking experiments reveal that the ssrA tag interacts with different loops that form the top, middle, and lower portions of the central channel of the ClpX hexamer. Our results support a two-step binding mechanism, in which the top loop serves as a specificity filter and the remaining loops form a binding site for the peptide tag relatively deep within the pore. Crosslinking experiments suggest a staggered arrangement of pore loops in the hexamer and nucleotide-dependent changes in pore-loop conformations. This mechanism of initial tag binding would allow ATP-dependent conformational changes in one or more pore loops to drive peptide translocation, force unfolding, and mediate threading of the denatured protein through the ClpX pore.
authors.affiliation: Department of Biology, Massachusetts Institute of Technology, Cambridge, MA 02139, USA.
authors.forename: Andreas
authors.initials: A
authors.lastname: Martin
authors.forename: Tania A
authors.initials: TA
authors.lastname: Baker
authors.forename: Robert T
authors.initials: RT
authors.lastname: Sauer
mesh_terms: D000074183:ATPases Associated with Diverse Cellular Activities; D000251:Adenosine Triphosphatases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D000255:Adenosine Triphosphate / Q000378:metabolism; D003432:Cross-Linking Reagents / Q000378:metabolism; D003545:Cysteine / Q000378:metabolism; D004220:Disulfides / Q000737:chemistry; D049071:Endopeptidase Clp / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D008958:Models, Molecular; D018832:Molecular Chaperones / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D010447:Peptide Hydrolases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D010455:Peptides / Q000235:genetics / Q000378:metabolism; D011485:Protein Binding; D020033:Protein Isoforms / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D020836:Protein Structure, Quaternary; D017434:Protein Structure, Tertiary; D021122:Protein Subunits / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012329:RNA, Bacterial / Q000737:chemistry / Q000235:genetics / Q000378:metabolism
chemical_list: D003432:Cross-Linking Reagents; D004220:Disulfides; D029968:Escherichia coli Proteins; D018832:Molecular Chaperones; D010455:Peptides; D020033:Protein Isoforms; D021122:Protein Subunits; D012329:RNA, Bacterial; C096222:tmRNA; D000255:Adenosine Triphosphate; D010447:Peptide Hydrolases; C483572:ClpP protease, E coli; D049071:Endopeptidase Clp; D000251:Adenosine Triphosphatases; C528384:CLPX protein, human; C088072:ClpX protein, E coli; D000074183:ATPases Associated with Diverse Cellular Activities; D003545:Cysteine