Visualization of membrane-stabilized SorCS2 cis interactions

Members of the Vps10p receptor family regulate protein trafficking and cellular differentiation in the nervous system. Previous structural studies of the dimeric Vps10p family member SorCS2 have focused on isolated ectodomains, revealing substantial structural plasticity but overlooking the influence of the membrane association on receptor organization. Here we establish two complementary tools for reconstituting the SorCS2 ectodomain on proteoliposomes in its native orientation: non-covalent coupling via a C-terminal His-tag and nickel affinity, and covalent attachment via strain-promoted alkyne-azide cycloaddition using a C-terminal azide. We visualize the SorCS2 membrane-associated protein organization using electron cryo-tomography and obtain a nanometer resolution subtomogram average of the His-tag coupled SorCS2 ectodomain dimer. Four distinct, previously unreported, SorCS2 dimer-of-dimer arrangements are observed. The two most prominent interactions form through "head-to-side" docking of a Vps10p domain to the Vps10p and PKD core of another dimer, and "head-to-head" symmetric interactions between the Vps10p and SoMP domains of two dimers. Two less frequent assemblies comprise "side-by-side" interactions between the beta-propeller and 10CC domains and symmetrical "face-to-face" beta-propeller top face interactions. Together these interactions organize SorCS2 into two distinct helical arrangements and small receptor clusters on liposome surfaces. The promiscuity of membrane-stabilized SorCS2 cis interactions supports a more general mechanism in which the organization of receptor systems is influenced by membrane association. The tools presented here provide a versatile platform for visualizing ectodomain-mediated receptor assemblies in a membrane context.