Vault Packaging Dynamics: Cargo Loading Stabilizes MVP Conformation for High-Payload Cytokine Delivery in Cancer Therapy

Recombinant vaults are dynamic protein cages with a large interior volume that can encapsulate therapeutic cargo. Using integrative cryogenic electron microscopy (cryoEM), cryogenic electron tomography (cryoET) and charge-detection mass spectrometry (CDMS), we characterized recombinant empty and interleukin-2 (IL-2)-packaged human vaults produced in yeast to define assembly, packaging heterogeneity and conformational dynamics relevant to optimized cancer and immune therapeutics. CryoEM maps at 2.79 angstrom (packaged) and 3.16 angstrom (empty) reveal a D39-symmetric barrel built from 78 major vault protein (MVP) subunits each with an interaction domain (INT) that anchors a cargo on the inner surface at MVP repeats R3-R4. CDMS deconvolution of empty versus packaged particles yields two IL-2-INT vault populations centered near 100 and 400 molecules per vault, with a maximum of ~660 copies/vault (i.e., many more copies than the 78 available INT anchors), matching cryoET estimates of ~150-600. Both methods show that highly packaged vaults are enlarged and distorted. Empty particles are conformationally flexible, which promotes initial cargo entry; loading then stabilizes the barrel and condenses cargo, producing vaults that each contain many hundreds of IL-2 molecules. These data establish the structural basis of vault encapsulation and a quantitative framework for engineering vault-based delivery vehicles.