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.
Publication types
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Research Support, N.I.H., Extramural
MeSH terms
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ATPases Associated with Diverse Cellular Activities
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Adenosine Triphosphatases / chemistry*
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Adenosine Triphosphatases / genetics
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Adenosine Triphosphatases / metabolism*
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Adenosine Triphosphate / metabolism
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Cross-Linking Reagents / metabolism
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Cysteine / metabolism
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Disulfides / chemistry
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Endopeptidase Clp / chemistry*
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Endopeptidase Clp / genetics
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Endopeptidase Clp / metabolism*
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Escherichia coli Proteins / chemistry*
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Escherichia coli Proteins / genetics
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Escherichia coli Proteins / metabolism*
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Humans
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Models, Molecular
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Molecular Chaperones / chemistry*
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Molecular Chaperones / genetics
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Molecular Chaperones / metabolism*
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Peptide Hydrolases / chemistry*
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Peptide Hydrolases / genetics
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Peptide Hydrolases / metabolism*
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Peptides / genetics
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Peptides / metabolism
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Protein Binding
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Protein Isoforms / chemistry
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Protein Isoforms / genetics
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Protein Isoforms / metabolism
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Protein Structure, Quaternary
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Protein Structure, Tertiary*
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Protein Subunits / chemistry
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Protein Subunits / genetics
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Protein Subunits / metabolism
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RNA, Bacterial* / chemistry
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RNA, Bacterial* / genetics
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RNA, Bacterial* / metabolism
Substances
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Cross-Linking Reagents
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Disulfides
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Escherichia coli Proteins
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Molecular Chaperones
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Peptides
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Protein Isoforms
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Protein Subunits
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RNA, Bacterial
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tmRNA
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Adenosine Triphosphate
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Peptide Hydrolases
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ClpP protease, E coli
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Endopeptidase Clp
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Adenosine Triphosphatases
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CLPX protein, human
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ClpX protein, E coli
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ATPases Associated with Diverse Cellular Activities
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Cysteine