ABSTRACT
Cell survival under severe thermal stress requires the activity of the ClpB (Hsp104) AAA+ chaperone that solubilizes and reactivates aggregated proteins in concert with the DnaK (Hsp70) chaperone system. How protein disaggregation is achieved and whether survival is solely dependent on ClpB-mediated elimination of aggregates or also on reactivation of aggregated proteins has been unclear. We engineered a ClpB variant, BAP, which associates with the ClpP peptidase and thereby is converted into a degrading disaggregase. BAP translocates substrates through its central pore directly into ClpP for degradation. ClpB-dependent translocation is demonstrated to be an integral part of the disaggregation mechanism. Protein disaggregation by the BAP/ClpP complex remains dependent on DnaK, defining a role for DnaK at early stages of the disaggregation reaction. The activity switch of BAP to a degrading disaggregase does not support thermotolerance development, demonstrating that cell survival during severe thermal stress requires reactivation of aggregated proteins.
Subject(s)
Escherichia coli Proteins/metabolism , Escherichia coli/metabolism , HSP70 Heat-Shock Proteins/metabolism , Heat-Shock Proteins/metabolism , Heat-Shock Response/genetics , Molecular Chaperones , Protein Folding , Cell Survival/genetics , Endopeptidase Clp , Escherichia coli/genetics , Escherichia coli Proteins/genetics , Escherichia coli Proteins/isolation & purification , HSP70 Heat-Shock Proteins/genetics , Heat-Shock Proteins/genetics , Heat-Shock Proteins/isolation & purification , Molecular Chaperones/genetics , Molecular Chaperones/metabolism , Molecular Motor Proteins , Molecular Sequence Data , Multiprotein Complexes/genetics , Multiprotein Complexes/metabolism , Protein Engineering , Protein Transport/physiology , Sequence Homology, Amino Acid , Sequence Homology, Nucleic AcidABSTRACT
Adhesive type 1 pili from uropathogenic Escherichia coli strains have a crucial role during infection by mediating the attachment to and potentially the invasion of host tissue. These filamentous, highly oligomeric protein complexes are assembled by the 'chaperone-usher' pathway, in which the individual pilus subunits fold in the bacterial periplasm and form stoichiometric complexes with a periplasmic chaperone molecule that is essential for pilus assembly. The chaperone subsequently delivers the subunits to an assembly platform (usher) in the outer membrane, which mediates subunit assembly and translocation to the cell surface. Here we show that the periplasmic type 1 pilus chaperone FimC binds non-native pilus subunits and accelerates folding of the subunit FimG by 100-fold. Moreover, we find that the FimC-FimG complex is formed quantitatively and very rapidly when folding of FimG is initiated in the presence of both FimC and the assembly-competent subunit FimF, even though the FimC-FimG complex is thermodynamically less stable than the FimF-FimG complex. FimC thus represents a previously unknown type of protein-folding catalyst, and simultaneously acts as a kinetic trap preventing spontaneous subunit assembly in the periplasm.
