Maintenance of appropriate levels of endocytosis and subsequent endosomal sorting is essential for every aspect of cellular life. Retromer, an evolutionarily conserved protein complex composed of VPS35-VPS26-VPS29, is a central hub in all eukaryotes responsible for trafficking of transmembrane cargo proteins in the endolysosomal system. More specifically, it functions together with a number of cargo adaptors and accessory proteins for membrane recruitment, engagement with cargos, and assembly into tubular or vesicular structures. In humans, mutations or dysfunction of Retromer are implicated in Parkinson’s and Alzheimer’s diseases. Recently, it has been suggested that stabilising the Retromer complex through “molecular chaperones” is a promising approach in treating these neurodegenerative disorders. However, it is challenging to identify molecular chaperones that can specifically bind and enhance the stability of Retromer. Here we employed the random nonstandard peptides integrated discovery (RaPID) approach to identify a group of cyclic peptides capable of binding to Retromer with high affinity. Among the hits, we found one cyclic peptide Rt-L-04 can significantly enhance the stability of the Retromer complex in vitro through binding to the interface of VPS26 and VPS35. Further interaction studies revealed that the presence of Rt-L-04 does not disrupt Retromer’s ability to form complexes with its accessory proteins and cargo adaptors.
Apart from this Retromer stabilizing cyclic peptide, we also identified a group of inhibitory cyclic peptides that bind specifically to the VPS29 subunit. Crystal structures showed that these peptides occlude the hydrophobic pocket of VPS29 that is the binding site for Retromer regulatory proteins such as the Rab GTPase activating protein TBC1D5, as well as the bacterial secreted protein RidL known to hijack Retromer during cell infection. The addition of these high-affinity VPS29 targeting cyclic peptides could effectively compete off TBC1D5 as well as RidL in vitro. Furthermore, we have applied a generalised permeabilisation approach to confirm the activities of these cyclic peptides within the cell. To our knowledge, this is the first set of biochemical probes for the studies of Retromer associated endosomal trafficking. The structural information obtained from this study also provides valuable insight for future structural-based drug design targeting neurodegenerative diseases