Figure 3

Mapping of the Hsp70 client peptides to the RRM and RNA-binding interface of TDP-43. (A) Binding of HspA5 on immobilized 15-mer TDP-43 peptides, in overlapping five amino acid steps. The blots were scanned, and spot intensities were quantified and represented as a normalized signal. Highly scored LIMBO predicted client peptides of TDP-43 are shown in red and orange, respectively. (B) Accessibility of the client peptides from the peptide array at the TDP-43 surface (ASA) were calculated using Areaimol as implemented in the CCP4 suite³⁵ on the free form structure of the tethered RRM domains (PDB ID 4bs2²²). To note, there is no significant difference in accessibility values when RNA is present. The percentage of accessibility represents the ASA of the motif compared to the total surface of TDP-43. (C,D) Mapping of the client peptides on TDP-43 or NMR structures (cartoon representation) (C) or surface (D) of the RRM domain (PDB code: 4bs2²²). The predicted client peptides were color coded as described in (A). (E) Microscale thermophoresis of NTA-labelled TDP-43_102–269 interaction with HspA5 in the absence or in the presence of increasing concentrations of UG₆ RNA. The presence of RNA shifted the Kd of the TDP-43_102–269/HspA5 interaction from 0.89 ± 0.25 µM (red curve) to 28.3 ± 23.7 µM (black curve). Data is presented as Mean ± SD (n = 3).