Selective promiscuity in the binding of E. coli Hsp70 to an unfolded protein.

Heat shock protein 70 (Hsp70) chaperones bind many different sequences and discriminate between incompletely folded and folded clients. Most research into the origins of this "selective promiscuity" has relied on short peptides as substrates to dissect the binding, but much less is known about how Hsp70s bind full-length client proteins. Here, we connect detailed structural analyses of complexes between the Escherichia coli Hsp70 (DnaK) substrate-binding domain (SBD) and peptides encompassing five potential binding sites in the precursor to E. coli alkaline phosphatase (proPhoA) with SBD binding to full-length unfolded proPhoA. Analysis of SBD complexes with proPhoA peptides by a combination of X-ray crystallography, methyl-transverse relaxation optimized spectroscopy (methyl-TROSY), and paramagnetic relaxation enhancement (PRE) NMR and chemical cross-linking experiments provided detailed descriptions of their binding modes. Importantly, many sequences populate multiple SBD binding modes, including both the canonical N to C orientation and a C to N orientation. The favored peptide binding mode optimizes substrate residue side-chain compatibility with the SBD binding pockets independent of backbone orientation. Relating these results to the binding of the SBD to full-length proPhoA, we observe that multiple chaperones may bind to the protein substrate, and the binding sites, well separated in the proPhoA sequence, behave independently. The hierarchy of chaperone binding to sites on the protein was generally consistent with the apparent binding affinities observed for the peptides corresponding to these sites. Functionally, these results reveal that Hsp70s "read" sequences without regard to the backbone direction and that both binding orientations must be considered in current predictive algorithms.

A novel method for removing contaminant Hsp70 molecular chaperones from recombinant proteins.

The production of recombinant proteins in bacteria has increased significantly in recent years, becoming a common tool for both research and the industrial production of proteins. One of the requirements of this methodology is to obtain the desired protein without contaminants. However, this goal cannot always be readily achieved. Multiple strategies have been developed to improve the quality of the desired protein product. Nevertheless, contamination with molecular chaperones is one of the recalcitrant problems that still affects the quality of the obtained proteins. The ability of chaperones to bind to unfolded proteins or to regions where the polypeptide chain is exposed make the removal of the contamination during purification challenging to achieve. This work aimed to develop a strategy to remove contaminating DnaK, one of the homologous Hsp70 molecular chaperones found in Escherichia coli, from purified recombinant proteins. For this purpose, we developed a methodology that captures the DnaK from the contaminating proteins by co-incubation with a GST-cleanser protein that has free functional binding sites for the chaperone. The cleanser protein can then be easily removed together with the captured DnaK. Here, we demonstrated the utility of our system by decontaminating a Histidine-tagged recombinant protein in a batch process. The addition of the GST-cleanser protein in the presence of ATP-Mg eliminates the DnaK contamination substantially. Thus, our decontaminant strategy results versatile and straightforward and can be applied to proteins obtained with different expression and purifications systems as well as to small samples or large volume preparations.

How hsp70 molecular machines interact with their substrates to mediate diverse physiological functions.

Hsp70 molecular chaperones are implicated in a wide variety of cellular processes, including protein biogenesis, protection of the proteome from stress, recovery of proteins from aggregates, facilitation of protein translocation across membranes, and more specialized roles such as disassembly of particular protein complexes. It is a fascinating question to ask how the mechanism of these deceptively simple molecular machines is matched to their roles in these wide-ranging processes. The key is a combination of the nature of the recognition and binding of Hsp70 substrates and the impact of Hsp70 action on their substrates. In many cases, the binding, which relies on interaction with an extended, accessible short hydrophobic sequence, favors more unfolded states of client proteins. The ATP-mediated dissociation of the substrate thus releases it in a relatively less folded state for downstream folding, membrane translocation, or hand-off to another chaperone. There are cases, such as regulation of the heat shock response or disassembly of clathrin coats, however, where binding of a short hydrophobic sequence selects conformational states of clients to favor their productive participation in a subsequent step. This Perspective discusses current understanding of how Hsp70 molecular chaperones recognize and act on their substrates and the relationships between these fundamental processes and the functional roles played by these molecular machines.