Heterogeneous binding of the SH3 client protein to the DnaK molecular chaperone

Heat shock protein 70 (Hsp70) molecular chaperones play key roles in protein folding and other cellular processes. The effect of Hsp70 on the conformation of its substrate proteins is still largely unknown. This study unveils, for the first time to our knowledge, the effect of the bacterial Hsp70 chaperone DnaK on the structure of the full-length substrate protein SRC homology 3 domain (SH3). We show that multiple largely unstructured conformations of SH3, distinct from the protein’s unfolded state, interact with DnaK. The bound client protein shares a flexible N terminus and multiple slowly interconverting conformations in different parts of the sequence. In all, there is significant structural and dynamical heterogeneity. This result is important because it reveals that proteins may undergo conformational sampling while chaperone-bound.