Refining the Dynamic Network of T2SS Endopilus Tip Heterocomplex Combining cw‐EPR and Nitroxide–GdIII Distance Measurements

The type 2 secretion system (T2SS) is a bacterial nanomachine composed of an inner membrane assembly platform, an outer membrane pore and a dynamic endopilus. T2SS endopili are organized into a homo‐multimeric body formed by the major pilin capped by a heterocomplex of four minor pilins. The first model of the T2SS endopilus was recently released, even if structural dynamics insights are still required to decipher the role of each protein in the full tetrameric complex. Here, we applied continuous‐wave and pulse EPR spectroscopy using nitroxide‐gadolinium orthogonal labelling strategies to investigate the hetero‐oligomeric assembly of the minor pilins. Overall, our data are in line with the endopilus model even if they evidenced conformational flexibility and alternative orientations at local scale of specific regions of minor pilins. The integration of different labelling strategies and EPR experiments demonstrates the pertinence of this approach to investigate protein–protein interactions in such multiprotein heterocomplexes. We applied cw‐EPR experiments and DEER measurements with orthogonally spin‐labelled systems to probe dynamics processes in the assembly of the hetero‐tetrameric complex located at the tip of the T2SS endopilus core. This spectroscopic approach appeared particularly appropriate to studying the structural movements and conformational changes occurring during the formation of such heteroprotein complexes.