(Flynn et al., 1991; Landry et al., 1992; Hsp70 chaperones assist protein folding by ATP- Blond-Elguindi et al., 1993;Gragerov et al., 1994). With dependent association with linear peptide segments of respect to the binding motif and its amino acid composition, a large variety of folding intermediates. The molecular they yielded results in conflict with each other (Flynn et al., basis for this ability to differentiate between native 1991; Blond-Elguindi et al., 1993;Gragerov et al., 1994) and non-native conformers was investigated for the and the structure of the DnaK substrate binding pocket DnaK homolog of Escherichia coli. We identified bind- (Blond-Elguindi et al., 1993). Furthermore, they were not ing sites and the recognition motif in substrates by aimed at identifying Hsp70 binding sites in biologically screening 4360 cellulose-bound peptides scanning the relevant protein substrates. sequences of 37 biologically relevant proteins. DnaKThis study was performed to identify the binding sites binding sites in protein sequences occurred statistically within protein sequences and the substrate binding motif of every 36 residues. In the folded proteins these sites are the DnaK chaperone. For this purpose we screened cellumostly buried and in the majority found in β-sheet lose-bound peptide scans (Reineke et al., 1995) representing elements. The binding motif consists of a hydrophobic complete protein sequences for DnaK binding. This novel core of four to five residues enriched particularly in approach offers the advantages of (i) avoiding precipitation, Leu, but also in Ile, Val, Phe and Tyr, and two flanking in particular, of DnaK binding peptides anticipated to be regions enriched in basic residues. Acidic residues are hydrophobic, (ii) allowing identification of DnaK binding excluded from the core and disfavored in flanking sites in natural substrate sequences, (iii) allowing direct regions. The energetic contribution of all 20 amino identification of the recognition motif by sequence alignacids for DnaK binding was determined. On the basis ment of neighboring binding peptides and (iv) providing a of these data an algorithm was established that predicts large data set for binding as well as non-binding peptides. DnaK binding sites in protein sequences with highIt allowed the identification of the substrate binding motif accuracy.of DnaK and the establishment of an algorithm predicting Keywords: cellulose-bound peptide libraries/heat shock DnaK binding sites within protein sequences. proteins/Hsp70/protein folding/spot synthesis

We systematically analyzed the capability of the major cytosolic chaperones of Escherichia coli to cope with protein misfolding and aggregation during heat stress in vivo and in cell extracts. Under physiological heat stress conditions, only the DnaK system efficiently prevented the aggregation of thermolabile proteins, a surprisingly high number of 150-200 species, corresponding to 15-25% of detected proteins. Identification of thermolabile DnaK substrates by mass spectrometry revealed that they comprise 80% of the large (≥90 kDa) but only 18% of the small (≤30 kDa) cytosolic proteins and include essential proteins. The DnaK system in addition acts with ClpB to form a bi-chaperone system that quantitatively solubilizes aggregates of most of these proteins. Efficient solubilization also occurred in an in vivo order-of-addition experiment in which aggregates were formed prior to induction of synthesis of the bi-chaperone system. Our data indicate that large-sized proteins are most vulnerable to thermal unfolding and aggregation, and that the DnaK system has central, dual protective roles for these proteins by preventing their aggregation and, cooperatively with ClpB, mediating their disaggregation.

Summary Protein folding in the cell relies on the orchestrated action of conserved families of molecular chaperones, the Hsp70 and Hsp90 systems. Hsp70 acts early and Hsp90 late in the folding path, yet the molecular basis of this timing is enigmatic, mainly because the substrate specificity of Hsp90 is poorly understood. Here we obtained a structural model of Hsp90 in complex with its natural disease-associated substrate, the intrinsically disordered Tau protein. Hsp90 binds to a broad region in Tau that includes the aggregation-prone repeats. Complementarily, a 106 Å long substrate-binding interface in Hsp90 enables many low affinity contacts. It allows recognition of scattered hydrophobic residues in late folding intermediates that remain after early burial of the Hsp70 sites. Our model resolves the paradox of how Hsp90 specifically selects for late folding intermediates but also for some intrinsically disordered proteins – through the eyes of Hsp90 they look the same.

Neurodegeneration in Parkinson's disease is correlated with the occurrence of Lewy bodies, intracellular inclusions containing aggregates of the intrinsically disordered protein (IDP) α-Synuclein 1 . The aggregation propensity of α-Synuclein in cells is modulated by specific factors including posttranslational modifications 2,3 , Abelson-kinase-mediated phosphorylation 4,5 and interactions with intracellular machineries such as molecular chaperones, although the underlying mechanisms are unclear [6][7][8] . Here, we systematically characterize the interaction of molecular chaperones with α-Synuclein in vitro as well as in cells at the atomic level. We find that six vastly different molecular chaperones commonly recognize a canonical motif in α-Synuclein, consisting Users may view, print, copy, and download text and data-mine the content in such documents, for the purposes of academic research, subject always to the full Conditions of use:http://www.nature.com/authors/editorial_policies/license.html#terms *

Determination of the structure of the substrate binding domain of the Escherichia coli Hsp70 chaperone, DnaK, and the biochemical characterisation of the motif it recognizes within substrates provide insights into the principles governing Hsp70 interaction with polypeptide chains. DnaK recognizes extended peptide strands composed of up to five consecutive hydrophobic residues within and positively charged residues outside the substrate binding cavity.