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 Å (packaged) and 3.16 Å (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.