Hsp90 is a highly conserved molecular chaperone that remodels hundreds of client proteins, many involved in the progression of cancer and other diseases. It functions with the Hsp70 chaperone and numerous cochaperones. The bacterial Hsp90 functions with an Hsp70 chaperone, DnaK, but is independent of Hsp90 cochaperones. We explored the collaboration between E. coli Hsp90 and DnaK and found that the two chaperones form a complex that is stabilized by client protein binding. A J-domain protein, CbpA, facilitates assembly of the Hsp90Ec-DnaK-client complex. We identified E. coli Hsp90 mutants defective in DnaK interaction in vivo and show that the purified mutant proteins are defective in physical and functional interaction with DnaK. Understanding how Hsp90 and Hsp70 collaborate in protein remodeling will provide the groundwork for the development of new therapeutic strategies targeting multiple chaperones and cochaperones.

RpoS, an RNA polymerase s factor, controls the response of Escherichia coli and related bacteria to multiple stress responses. During nonstress conditions, RpoS is rapidly degraded by ClpXP, mediated by the adaptor protein RssB, a member of the response regulator family. In response to stress, RpoS degradation ceases. Small anti-adaptor proteins-IraP, IraM, and IraD, each made under a different stress condition-block RpoS degradation. RssB mutants resistant to either IraP or IraM were isolated and analyzed in vivo and in vitro. Each of the anti-adaptors is unique in its interaction with RssB and sensitivity to RssB mutants. One class of mutants defined an RssB N-terminal region close to the phosphorylation site and critical for interaction with IraP but unnecessary for IraM and IraD function. A second class, in the RssB C-terminal PP2C-like domain, led to activation of RssB function. These mutants allowed the response regulator to act in the absence of phosphorylation but did not abolish interaction with anti-adaptors. This class of mutants is broadly resistant to the anti-adaptors and bears similarity to constitutively activated mutants found in a very different PP2C protein. The mutants provide insight into how the anti-adaptors perturb RssB response regulator function and activation.

Heat shock protein 90 (Hsp90) is a highly conserved ATP-dependent molecular chaperone that is essential in eukaryotes. It is required for the activation and stabilization of more than 200 client proteins, including many kinases and steroid hormone receptors involved in cell-signaling pathways. Hsp90 chaperone activity requires collaboration with a subset of the many Hsp90 cochaperones, including the Hsp70 chaperone. In higher eukaryotes, the collaboration between Hsp90 and Hsp70 is indirect and involves Hop, a cochaperone that interacts with both Hsp90 and Hsp70. Here we show that yeast Hsp90 (Hsp82) and yeast Hsp70 (Ssa1), directly interact in vitro in the absence of the yeast Hop homolog (Sti1), and identify a region in the middle domain of yeast Hsp90 that is required for the interaction. In vivo results using Hsp90 substitution mutants showed that several residues in this region were important or essential for growth at high temperature. Moreover, mutants in this region were defective in interaction with Hsp70 in cell lysates. In vitro, the purified Hsp82 mutant proteins were defective in direct physical interaction with Ssa1 and in protein remodeling in collaboration with Ssa1 and cochaperones. This region of Hsp90 is also important for interactions with several Hsp90 cochaperones and client proteins, suggesting that collaboration between Hsp70 and Hsp90 in protein remodeling may be modulated through competition between Hsp70 and Hsp90 cochaperones for the interaction surface.

Hsp90 is a widely conserved and ubiquitous molecular chaperone that participates in ATP-dependent protein remodeling in both eukaryotes and prokaryotes. It functions in conjunction with Hsp70 and the Hsp70 cochaperones, an Hsp40 (J-protein) and a nucleotide exchange factor. In E. coli the functional collaboration between Hsp90Ec and Hsp70, DnaK, requires that the two chaperones directly interact. We used molecular docking to model the interaction of Hsp90Ec and DnaK. The top-ranked docked model predicted that a region in the nucleotide-binding domain of DnaK interacted with a region in the middle domain of Hsp90Ec. We then made substitution mutants in DnaK residues suggested by the model to interact with Hsp90Ec. Eleven of the twelve mutants tested were defective or partially defective in their ability to interact with Hsp90Ec in vivo in a bacterial two-hybrid assay and in vitro in a Bio-Layer Interferometry assay. These DnaK mutants were also defective in their ability to function collaboratively in protein remodeling with Hsp90Ec, but retained the ability to act with DnaK cochaperones. Taken together these results suggest that a specific region in the nucleotide-binding domain of DnaK is involved in the interaction with Hsp90Ec and this interaction is functionally important. Moreover, the region of DnaK that we found to be necessary for Hsp90Ec binding includes residues that are also involved in J-protein binding, suggesting a functional interplay between DnaK, DnaK cochaperones and Hsp90Ec.

The DnaK/Hsp70 chaperone system and ClpB/Hsp104 collaboratively disaggregate protein aggregates and reactivate inactive proteins. The teamwork is specific: E. coli DnaK interacts with E. coli ClpB and yeast Hsp70, Ssa1, interacts with yeast Hsp104. This interaction is between the M-domains of hexameric ClpB/Hsp104 and the DnaK/Hsp70 nucleotide-binding domain (NBD). To identify the site on E. coli DnaK that interacts with ClpB, we substituted amino acid residues throughout the DnaK NBD. We found that several variants with substitutions in subdomain IB and IIB of the DnaK NBD were defective in ClpB interaction in vivo in a bacterial two-hybrid assay and in vitro in a fluorescence anisotropy assay. The DnaK subdomain IIB mutants were also defective in the ability to disaggregate protein aggregates with ClpB, DnaJ and GrpE, although they retained some ability to reactivate proteins with DnaJ and GrpE in the absence of ClpB. We observed that GrpE, which also interacts with subdomains IB and IIB, inhibited the interaction between ClpB and DnaK in vitro, suggesting competition between ClpB and GrpE for binding DnaK. Computational modeling of the DnaK-ClpB hexamer complex indicated that one DnaK monomer contacts two adjacent ClpB protomers simultaneously. The model and the experiments support a common and mutually exclusive GrpE and ClpB interaction region on DnaK. Additionally, homologous substitutions in subdomains IB and IIB of Ssa1 caused defects in collaboration between Ssa1 and Hsp104. Altogether, these results provide insight into the molecular mechanism of collaboration between the DnaK/Hsp70 system and ClpB/Hsp104 for protein disaggregation.

Clp ATPases are ring-shaped AAA+ motors in the degradation pathway that perform critical actions of unfolding and translocating substrate proteins (SPs) through narrow pores to deliver them to peptidase components. These actions are effected by conserved diaphragm-forming loops found in the central channel of the Clp ATPase hexamer. Conformational changes, that take place in the course of repetitive ATP-driven cycles, result in mechanical forces applied by the central channel loops onto the SP. We use coarsegrained simulations to elucidate allostery-driven mechanisms of unfolding and translocation of a tagged four-helix bundle protein by the ClpY ATPase. Unfolding is initiated at the tagged C-terminal region via an obligatory intermediate. The resulting nonnative conformation is competent for translocation, which proceeds on a different time scale than unfolding and involves sharp stepped transitions. Completion of the translocation process requires assistance from the ClpQ peptidase. These mechanisms contrast nonallosteric mechanical unfolding of the SP. In atomic force microscopy experiments, multiple unfolding pathways are available and large mechanical forces are required to unravel the SP relative to those exerted by the central channel loops of ClpY. SP threading through a nonallosteric ClpY nanopore involves simultaneous unfolding and translocation effected by strong pulling forces.AAA+ protease | molecular machine | protein translocation I ntracellular regulatory mechanisms for selective destruction of proteins and disassembly of protein aggregates are critical for the maintenance of vital cellular functions (1). Clp macromolecular machines, found in all domains of life from prokaryotes to multicellular eukaryotes (2), perform such protein quality control using powerful ATPase components (3-6) that effect protein unfolding and translocation through narrow pores. Clp (caseinolytic protease) ATPases, are members of the AAA+ (ATPases associated with diverse cellular activities) superfamily of proteins (7, 8) responsible for a variety of functions including intracellular transport, DNA replication and repair, and transcription regulation (9-11). The best understood Clp ATPases are ClpA; ClpX and ClpY (HslU) (12), which enable protein degradation by associating with peptidase subunits ClpP and ClpQ (HslV); and ClpB, which promotes disaggregation (13-15).Functional forms of Clp ATPases are homohexameric assemblies that form in the presence of ATP (16). Monomers include one (ClpX, ClpY) or two (ClpA, ClpB) highly conserved nucleotide binding domains (17), referred to as AAA domains (7,18). Crystal structures (19-21) and oligomeric models (22, 23) based on electron microscopy images (24, 25) reveal the presence of a central channel with a diameter of approximately 9-15 Å at the narrowest point. The channel is occupied by diaphragm-forming loops (one per AAA domain), which contain a conserved motif consisting of an aromatic-hydrophobic dipeptide flanked by glycine amino acids. These loops are implicated in the ti...

Members of the Hsp90 and Hsp70 families of molecular chaperones are important for the maintenance of protein homeostasis and cellular recovery following environmental stresses, such as heat and oxidative stress. Moreover, the two chaperones can collaborate in protein remodeling and activation. In higher eukaryotes, Hsp90 and Hsp70 form a functionally active complex with Hop (Hsp90-Hsp70 organizing protein) acting as a bridge between the two chaperones. In bacteria, which do not contain a Hop homolog, Hsp90 and Hsp70, DnaK, directly interact during protein remodeling. Although yeast possess a Hop-like protein, Sti1, Hsp90 and Hsp70 can directly interact in yeast in the absence of Sti1. Previous studies showed that residues in the middle domain of Escherichia coli Hsp90 are important for interaction with the J-protein binding region of DnaK. The results did not distinguish between the possibility that i) these sites were involved in direct interaction and ii) the residues in these sites participate in conformational changes which are transduced to other sites on Hsp90 and DnaK that are involved in the direct interaction. Here we show by crosslinking experiments that the direct interaction is between a site in the middle domain of Hsp90 and the J-protein binding site of Hsp70 in both E. coli and yeast. Moreover, J-protein promotes the Hsp70-Hsp90 interaction in the presence of ATP, likely by converting Hsp70 into the ADP-bound conformation. The identification of the protein-protein interaction site is anticipated to lead to a better understanding of the collaboration between the two chaperones in protein remodeling.

Metallo-β-lactamases (MBLs) are a growing clinical threat because they inactivate nearly all β-lactam-containing antibiotics, and there are no clinically-available inhibitors. A significant number of variants have already emerged for each MBL subfamily. To understand the evolution of IMiPenemase (IMP) genes (blaIMP) and their clinical impact, twenty clinically-derived IMP-1 like variants were obtained using site-directed mutagenesis and expressed in a uniform genetic background in Escherichia coli DH10B. Strains of IMP-1-like variants harboring S262G or V67F substitutions exhibited increased resistance towards carbapenems and decreased resistance towards ampicillin. Strains expressing IMP-78 (S262G/V67F) exhibited the largest changes in MIC values as compared to IMP-1. In order to understand the molecular mechanisms of increased resistance, biochemical, biophysical, and molecular modeling studies were conducted to compare IMP-1, IMP-6 (S262G), IMP-10 (V67F), and IMP-78 (S262G/V67F). Finally, unlike most NDM and VIM variants, the IMP-1 like variants do not confer any additional survival advantage if zinc availability is limited. Therefore, the evolution of MBL subfamilies (i.e. IMP-6, -10, and -78) appears to be driven by different selective pressures.