The 70 kDa heat shock proteins, Hsp70s, sit at the center of cellular protein quality control, binding stretches of exposed hydrophobic sequence on incompletely folded clients and preventing their aggregation. Because their substrate binding domain, SBD, must recognize a vast and chemically diverse clientele, Hsp70s practice what the Gierasch group has coined "selective promiscuity." A structurally conserved cleft in the β-sheet subdomain of the SBD accommodates five to seven residues in an extended conformation, threading the substrate backbone through a series of pockets numbered −3 to +3 around a central, sterically demanding 0th pocket. One of the most unexpected features of this interaction, uncovered in earlier work on Escherichia coli DnaK, is that peptides can bind in either the N- to C- or the reverse C- to N- backbone orientation with nearly equal frequency, raising the immediate question of whether that permissiveness is a universal property of the Hsp70 family or a peculiarity of the bacterial chaperone.
Researchers in the Clerico and Gierasch Groups at the University of Massachusetts Amherst, published in Biochemistry, designed a systematic panel of model peptides to probe orientation preference and binding energetics across three Hsp70 paralogs: E. coli DnaK and the two major human cytoplasmic members, constitutively expressed Hsc70 and stress-inducible HspA1. A "palindromic" peptide, PalP, offered identical side-chain environments upstream and downstream of a central leucine, isolating any intrinsic directional bias of each SBD. A series of central residue variant peptides, CRVPs, systematically substituted Leu, Ile, Val, or Pro at the 0th pocket to parse how that single buried contact shapes orientation. Four peptides derived from natural Hsp70 clients, including sequences from proPhoA, SNAP-25, clathrin, and the glucocorticoid receptor, then tested whether designed-peptide rules translate to physiological substrates. Orientation was resolved at residue-level resolution by 1H–13C methyl-TROSY NMR monitoring the δ1-methyl reporters of Ile438 in DnaK and Ile440 in the human SBDs, supported where interpretable by disulfide-mediated cross-linking between engineered cysteine pairs flanking the binding groove.
The palindromic peptide delivered the clearest statement of chaperone character. DnaK populated both orientations with essentially equal intensity, confirming its backbone agnosticism when side-chain symmetry removes any preference. Hsc70 split the same peptide approximately 80:20 in favor of the C- to N- orientation, an energy difference of less than 1 kcal/mol, showing only a modest directional lean. HspA1, despite sharing 91.2% SBD sequence identity with Hsc70, produced a single dominant NMR signal corresponding to N- to C- binding, a striking reversal of the Hsc70 preference. Among the residues that differ between Hsc70 and HspA1 near the binding cleft, the substitution at position 427/428 stands out: Hsc70 carries a Thr at this position, introducing a hydroxyl capable of additional polar contacts, while HspA1 carries an Ile. These sequence differences, and particularly those proximal to the cleft, appear sufficient to invert backbone orientation preference entirely.
The CRVP series added another dimension by showing that the identity of the residue at the 0th pocket itself steers orientation. In DnaK, a Leu or Ile at the central pocket favors N- to C- binding, Val shows no preference, and Pro weakly favors C- to N-. In both human chaperones, the Ile-containing peptide shifted to C- to N- preference, illustrating that central residue identity and flanking sequence context act together to set orientation. Binding affinities followed a clear hierarchy: Hsc70 and DnaK showed comparable and generally higher affinities across the peptide panel, while HspA1 bound most weakly. This affinity pattern extended to naturally occurring substrate sequences, and the NMR data for those peptides confirmed that the same substrate can be gripped in different orientations and with different central-pocket occupancies depending on which chaperone is presented with it.
The findings reframe how substrate orientation should be treated in predictive models of Hsp70 binding: algorithms that consider only one backbone direction will systematically mischaracterize interactions, particularly for HspA1 and Hsc70 where directional bias is strong. More broadly, the data suggest that despite near-identical folds, the three chaperones navigate distinct binding energy landscapes, with deeper, well-defined wells for DnaK and HspA1 and a more rugged, multi-state landscape for Hsc70. The authors propose that these landscape differences reflect physiological specialization: the lower and more transient affinities of stress-inducible HspA1 may facilitate rapid substrate turnover under acute proteotoxic conditions, whereas the broader landscape of Hsc70 supports its constitutive housekeeping role. Site-directed mutagenesis of the cleft-proximal residues that differ between Hsc70 and HspA1 offers a direct next experiment to assign molecular responsibility for the orientation switch, and extending the analysis to full-length client proteins and nucleotide-modulated full-length chaperones remains an important horizon.
