Interaction of substrate-mimicking peptides with the AAA+ ATPase ClpB from Escherichia coli.

A molecular chaperone ClpB disaggregates and reactivates aggregated proteins in cooperation with DnaK, DnaJ, and GrpE. Within a cellular environment, ClpB must distinguish between properly folded and aggregated proteins by recognizing specific physical and/or chemical surface properties of the aggregates. However, the molecular mechanism of substrate binding to ClpB is poorly understood. We hypothesized that ClpB recognizes those polypeptide segments that promote protein aggregation because they are likely present at the surface of growing aggregates. We used an algorithm TANGO (Fernandez-Escamilla et al., Nat. Biotech. 2004, 22, 1302) to predict the aggregation-prone segments within the model ClpB-binding peptides and investigated interactions of the FITC-labeled peptides with ClpB using fluorescence anisotropy. We found that ClpB binds the substrate-mimicking peptides with positive cooperativity, which is consistent with an allosteric linkage between substrate binding and ClpB oligomerization. The apparent affinity towards ClpB for peptides displaying different predicted aggregation propensities correlates with the peptide length. However, discrete aggregation-prone segments within the peptides are neither sufficient nor necessary for efficient interaction with ClpB. Our results suggest that the substrate recognition mechanism of ClpB may rely on global surface properties of aggregated proteins rather than on local sequence motifs.