De novo macrocyclic peptides for inhibiting, stabilizing, and probing the function of the retromer endosomal trafficking complex

Abstract

The retromer complex (Vps35-Vps26-Vps29) is essential for endosomal membrane trafficking and signaling. Mutation of the retromer subunit Vps35 causes late-onset Parkinson's disease, while viral and bacterial pathogens can hijack the complex during cellular infection. To modulate and probe its function, we have created a novel series of macrocyclic peptides that bind retromer with high affinity and specificity. Crystal structures show that most of the cyclic peptides bind to Vps29 via a Pro-Leu-containing sequence, structurally mimicking known interactors such as TBC1D5 and blocking their interaction with retromer in vitro and in cells. By contrast, macrocyclic peptide RT-L4 binds retromer at the Vps35-Vps26 interface and is a more effective molecular chaperone than reported small molecules, suggesting a new therapeutic avenue for targeting retromer. Last, tagged peptides can be used to probe the cellular localization of retromer and its functional interactions in cells, providing novel tools for studying retromer function.

Improved mammalian retromer cryo-EM structures reveal a new assembly interface

“…We obtained an improved heterotrimer reconstruction from 43,808 particles with an average resolution of 4.9 Å. Figure 1 formally compares this structure (panels A, C, E, G; ( 44 )) with the previously published version (panels B, D, F, H; ( 40 )). Critical missing views were acquired from the additional tilted data set (Figure 1E versus 1F).…”

“…To generate improved reconstructions, we combined images from previously published data ( 40 ) with a new third data set in which specimens were tilted to gain additional views (Figure S1; Table 1) ( 44 ). We obtained an improved heterotrimer reconstruction from 43,808 particles with an average resolution of 4.9 Å.…”

“…These smaller 3KE retromer mutant particles resemble the “f-hole” found on the body of a violin (Figure 4). We obtained a tilted data set from 3KE mutant retromer in the presence of a small cyclic peptide called RT-L4 ( 44 ), which has been shown to stabilize retromer structure. The presence of this peptide further enriched our protein sample for 3KE mutant particles.…”

“…The interface buries a substantial amount of surface area (nearly 7,000 Å 2 ), suggesting it is a relatively stable particle in vitro . It is important to note this structure was determined in the presence of RT-L4 ( 44 ), a small molecule shown to stabilize the N-VPS35/C-VPS26 interface. The resolution of our structure is too low to locate or assign RT-L4, but in our hands, the presence of RT-L4 appeared to increase the number of 3KE retromer particles observed on grids.…”

“…Sub-structure maps were deposited for C-VPS35 dimers as EMD-26343 and EMD-26345, and for the 3KE mutant as EMD-26340. The heterotrimer data set was first reported very briefly in ( 44 ), but its refinement and analysis are reported here. Coordinates for this updated retromer heterotrimer were deposited in the Protein Data Bank as XXXX.…”