Abstract
Vaults are naturally occurring ovoid nanoparticles constructed from a protein shell that is composed of multiple copies of major vault protein (MVP). The vault-interacting domain of vault poly(ADP-ribose)-polymerase (INT) has been used as a shuttle to pack biomolecular cargo in the vault lumen. However, the interaction between INT and MVP is poorly understood. It is hypothesized that the release rate of biomolecular cargo from the vault lumen is related to the interaction between MVP and INT. To tune the release of molecular cargos from the vault nanoparticles, we determined the interactions between the isolated INT-interacting MVP domains (iMVP) and wild-type INT and compared them to two structurally modified INT: 15-amino acid deletion at the C terminus (INTΔC15) and histidine substituted at the interaction surface (INT/DSA/3 H) to impart a pH-sensitive response. The apparent affinity constants determined using surface plasmon resonance (SPR) biosensor technology are 262 ± 4 nM for iMVP/INT, 1800 ± 160 nM for iMVP/INTΔC15 at pH 7.4. The INT/DSA/3 H exhibits stronger affinity to iMVP (KDapp = 24 nM) and dissociates at a slower rate than wild-type INT at pH 6.0.
Highlights
The major vault protein (MVP) contributes up to 70% of the total mass of the vault, while poly(ADP-ribose)polymerase (VPARP), telomerase-associated protein 1 (TEP1)[15] and several copies of non-protein-coding vault RNA[5,16] are found to associate inside the particles making up the remaining mass
In our model (Fig. 1), interacting domain of vault poly(ADP-ribose)polymerase (INT) is predicted to dock at the lumen side of MVP, ranging over MVP domains 3, 4, and 5 indicating that the interacting MVP domains (iMVP) is located between domain 3 and 5
As determined by surface plasmon resonance (SPR) technique, His-INT binds to His-iMVP in a 1:1 interaction with an apparent affinity constant of ~260 nM
Summary
The major vault protein (MVP) contributes up to 70% of the total mass of the vault, while poly(ADP-ribose)polymerase (VPARP), telomerase-associated protein 1 (TEP1)[15] and several copies of non-protein-coding vault RNA (vRNA)[5,16] are found to associate inside the particles making up the remaining mass. A domain located at the C-terminus of VPARP (INT, GenBank accession No AF158255; residues 1563–1724) interacts with MVP near the waist of the vault, at the luminal side. Leveraging on the specific interactions between INT and MVP, the INT domain can serve as a shuttle to transport molecules into the cavity of the vault[24]. We hypothesize that modification of this interaction by truncation or surface engineering of INT will result in modulated binding and release profile. INT-interacting MVP domains (iMVP) which bind to the INT.
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