Oral Presentation The 46th Lorne Conference on Protein Structure and Function 2021

Probing and modulating the function of the Retromer endosomal trafficking complex through the novel macrocyclic peptides (#26)

Kai-En Chen 1 , Qian Guo 1 , Yi Cui 2 , Amy K. Kendall 3 , Tim A. Hill 1 , Hiroaki Suga 4 , David P. Fairlie 1 , Lauren P. Jackson 3 , Rohan D. Teasdale 2 , Toby Passioura 5 , Brett M. Collins 1
  1. Institute for Molecular Bioscience, the University of Queensland, St. Lucia, Queensland, 4072, Australia
  2. School of Biomedical Sciences, the University of Queensland, St Lucia, Queensland, 4072, Australia
  3. Department of Biological Sciences Center for Structural Biology, Vanderbilt University, Nashville, Tennessee, USA
  4. Department of Chemistry, Graduate School of Science, University of Tokyo, Tokyo, 113-0033, Japan
  5. Sydney Analytical, the University of Sydney, Camperdown, New South Wales 2050, Australia

Maintenance of appropriate levels of endocytic trafficking and subsequent sorting in endosomes is essential for every aspect of cellular life. The evolutionarily conserved Retromer (composed of VPS35-VPS26-VPS29) is a central hub responsible for this process in endosomal compartments in all eukaryotes. It is known that mutations in Retromer can cause late-onset Parkinson’s disease, and can also be hijacked by viral and bacterial pathogens during cellular infection. Seeking tools to modulate Retromer function would provide new avenues in understanding its function and the associated diseases. Here we employed the random nonstandard peptides integrated discovery (RaPID) approach to identify a group of macrocyclic peptides capable of binding to Retromer with high affinity. Our structures show that five of the macrocyclic peptides bind to Vps29 via a di-peptide Pro-Leu sequence. Interestingly, these peptides structurally mimic known interacting proteins including TBC1D5, VARP, and the bacterial effector RidL, and potently inhibit their interaction with Retromer in vitro and in cells. Further analysis using cryo-electron microscopy (CryoEM) and mutagenesis showed that a unique macrocyclic peptide, RT-L4, binds Retromer at the interface between Vps35 and Vps26 subunits and can act as a molecular chaperone to stabilise the complex with minimal disruptive effects on Retromer’s ability to interact with its accessory proteins. Further study using macrocyclic peptide RT-L4 indicates that the tail of Vps26 plays an unexpected autoinhibitory role in the functional interaction of Retromer with the SNX3 cargo adaptor. Finally, using reversible cell permeabilization approach, we demonstrate that both the Retromer inhibiting and stabilizing macrocyclic peptides can specifically co-label Vps35-positive endosomal structures, and can be used as baits for purifying Retromer from cells and subsequent proteomic analyses. We believe these macrocyclic peptides can be used as a novel toolbox for the study of Retromer-mediated endosomal trafficking, and sheds light on developing novel therapeutic modifiers of Retromer function.