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

Abstract

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.

Prediction of quality-control degradation signals in yeast proteins

“…Indeed, the yeast PQC E3 San1 appears to broadly recognize exposed hydrophobicity rather than specific sequence motifs 25, 44 . Other PQC E3s such as Ubr1 45 and STUB1/CHIP 46 have relegated substrate recognition to molecular chaperones, who also display promiscuity in their substrate selection 47, 48 . Second, there is likely extensive redundancy in the components of the PQC system including E3 enzymes, chaperones and adaptor proteins 16, 26, 49–51 .…”

HSP70-binding motifs function as protein quality control degrons

“…A number of studies have aimed at clarifying the sequence features that constitute good Hsp70-binding elements (Clerico et al, 2021; Durme et al, 2009; McCarty et al, 1995; Nordquist et al, 2021; Rüdiger et al, 1997; Zhu et al, 1996). Degron sequences appear to hold similar features, and unsurprisingly, we find that residues flanking the degron motif also critically affect the degron activity.…”

Structural and Kinetic Views of Molecular Chaperones in Multidomain Protein Folding

“…DnaK, a member of the Hsp70 family in E. coli , recognizes ~700 client proteins, including multidomain proteins [ 57 ]. Although several important biochemical and structural studies have been reported for Hsp70/DnaK [ 52 , 53 , 58 , 59 ], an important NMR study, using DNA-binding domain of human telomere repeat-binding factor 1 (hTRF1) as client protein, has reported that hTRF1 exists in equilibrium between unfolded and folded states in solution [ 60 ]. NMR data showed that the conformational landscape of hTRF1 increased upon interaction with DnaK.…”