title: A single ClpS monomer is sufficient to direct the activity of the ClpA hexamer.
abstract: ClpS is an adaptor protein that interacts with ClpA and promotes degradation of proteins with N-end rule degradation motifs (N-degrons) by ClpAP while blocking degradation of substrates with other motifs. Although monomeric ClpS forms a 1:1 complex with an isolated N-domain of ClpA, only one molecule of ClpS binds with high affinity to ClpA hexamers (ClpA(6)). One or two additional molecules per hexamer bind with lower affinity. Tightly bound ClpS dissociates slowly from ClpA(6) with a t((1/2)) of approximately 3 min at 37 degrees C. Maximum activation of degradation of the N-end rule substrate, LR-GFP(Venus), occurs with a single ClpS bound per ClpA(6); one ClpS is also sufficient to inhibit degradation of proteins without N-degrons. ClpS competitively inhibits degradation of unfolded substrates that interact with ClpA N-domains and is a non-competitive inhibitor with substrates that depend on internal binding sites in ClpA. ClpS inhibition of substrate binding is dependent on the order of addition. When added first, ClpS blocks binding of both high and low affinity substrates; however, when substrates first form committed complexes with ClpA(6), ClpS cannot displace them or block their degradation by ClpP. We propose that the first molecule of ClpS binds to the N-domain and to an additional functional binding site, sterically blocking binding of non-N-end rule substrates as well as additional ClpS molecules to ClpA(6). Limiting ClpS-mediated substrate delivery to one per ClpA(6) avoids congestion at the axial channel and allows facile transfer of proteins to the unfolding and translocation apparatus.
authors.affiliation: Laboratory of Cell Biology, Center for Cancer Research, NCI, National Institutes of Health, Bethesda, Maryland 20892-4256, USA.
authors.forename: Gian Marco
authors.initials: GM
authors.lastname: De Donatis
authors.forename: Satyendra K
authors.initials: SK
authors.lastname: Singh
authors.forename: Sarada
authors.initials: S
authors.lastname: Viswanathan
authors.forename: Michael R
authors.initials: MR
authors.lastname: Maurizi
mesh_terms: D002352:Carrier Proteins / Q000378:metabolism*; D018833:Chaperonins / Q000737:chemistry; D004305:Dose-Response Relationship, Drug; D049071:Endopeptidase Clp / Q000737:chemistry / Q000378:metabolism / Q000502:physiology*; D004926:Escherichia coli / Q000201:enzymology*; D029968:Escherichia coli Proteins / Q000378:metabolism*; D015964:Gene Expression Regulation, Bacterial; D007700:Kinetics; D010447:Peptide Hydrolases / Q000737:chemistry; D010957:Plasmids / Q000378:metabolism; D011485:Protein Binding; D011487:Protein Conformation; D011489:Protein Denaturation; D025941:Protein Interaction Mapping; D013997:Time Factors
chemical_list: D002352:Carrier Proteins; C454820:ClpS protein, E coli; D029968:Escherichia coli Proteins; D010447:Peptide Hydrolases; C483568:ClpA protease, E coli; D049071:Endopeptidase Clp; D018833:Chaperonins