Fig. 3: Hetero-dimerisation of HSPB7 and FLNC.
A Native MS experiments show that FLNCd24 (red) forms heterodimers (black) with HSPB7, HSPB7ΔN and HSPB7ACD (purple). No complexes between the FLNCd24 homodimer and HSPB7 are observed, revealing how homo- and hetero-dimerisation are directly competitive. Proteins were mixed at a monomer ratio of 1:1, but the instability of full-length HSPB7 means it was somewhat depleted in solution such that FLNCd24 was effectively in excess (and FLNCd24 is visible in the spectrum). B The FLNCd24:HSPB7ACDC131S heterodimer structure reveals an interface that involves the HSPB7 β4, β8 strands and FLNCd24 strands C and D, centred on the parallel pairing of strands C and β4. C The hydrophobic β4-8 groove on HSPB7 accepts M2667 and M2669 from FLNC (top), while the groove between strands C and D on FLNCd24 accepts I102 from HSPB7 (middle). This leads to a network of hydrophobic interactions that stabilise the heterodimer (bottom). D HDX-MS analysis of HSPB7ACD and FLNCd24 in the heterodimer compared to in isolation. Woods plots showing the difference in deuterium uptake of HSPB7ACD (top) and FLNCd24 (bottom) in the two states, at a labelling time of 500 s. The y-axis is calculated as the uptake for the isolated proteins minus that in the heterodimer; negative values denote protection from exchange in the heterodimer. Three technical repeats were carried out, and peptides were considered significantly different if p < 0.01 (coloured: orange or purple). E Plot of deuterium uptake versus exposure time of the most protected peptide in HSPB7ACD (upper) and FLNCd24 (lower) in the heterodimer compared to this same peptide in the isolated protein. Because HSPB7 is monomeric, in the absence of FLNC, this peptide is solvent-exposed and therefore has consistently high uptake values. Error bars refer to the standard deviation of three repeats at each time-point, and the shaded bands 99% confidence. F Mapping the uptake difference at 500 s (as a fraction of the theoretical maximum) onto the heterodimer protein structure shows that protection is centred on the dimer interface that we found in our crystal structure.
