Substrate specificity of the DnaK chaperone determined by screening cellulose-bound peptide libraries

Abstract

(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

Binding Specificity of an α-Helical Protein Sequence to a Full-Length Hsp70 Chaperone and Its Minimal Substrate-Binding Domain

“…The all-R-helical protein apoMb is employed as a model substrate. Chaperone binding sites are reported to be less common in segments corresponding to helices in native proteins (10). We have, however, found that there are relatively strong binding sites for DnaK along the apoMb sequence.…”

“…The nonpolar character of each amino acid was ranked according to different scales to explore whether the results depend on the choice of the scale and how the nonpolar amino acid nature correlates with chaperone binding affinity ( Figure 9). The binding motif for DnaK is expected to have a three to five residue hydrophobic core (10). Therefore, we assessed the nonpolar character of the five central residues of each peptide.…”

“…Binding site cores are highlighted in bold type and underlined. previous peptide scanning experiments (9,10,34), and additional targeted experiments need to be performed to further test the above hypothesis.…”

Proteoliposome Engineering with Cell‐Free Membrane Protein Synthesis: Control of Membrane Protein Sorting into Liposomes by Chaperoning Systems

“…The molecular chaperone DnaKJE binds to the hydrophobic nascent polypeptide15 with formation of the ribosome–nascent chain/DnaKJE complex, preventing aggregation of nascent polypeptides during cell‐free protein synthesis 16. Addition of DnaKJE resulted in increased solubility of all Cx43–FLAG fusion proteins synthesized in cell‐free synthesis, indicating that DnaKJE acted as a molecular chaperone for reconstitution of Cx43–FLAG to liposomes (Figure S6, Supporting Information).…”

Conformational dynamics of the molecular chaperone Hsp90

“…Hsp60 (GroEL) and Hsp70 (DnaK) represent two well studied members of this class of proteins. Both Hsp60 and Hsp70 interact with nascent polypeptide chains and promote their folding by interacting with hydrophobic surfaces on substrate proteins (Gomez-Puertas et al, 2004;Kurt et al, 2006;Rudiger et al, 1997;Weissman et al, 1995). Once substrates are bound, the chaperone undergoes subsequent rounds of ATP hydrolysis causing conformational changes within the chaperone (Young et al, 2004).…”