Modulation of the Vault Protein-Protein Interaction for Tuning of Molecular Release

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 (K Dapp = 24 nM) and dissociates at a slower rate than wild-type INT at pH 6.0.

A 9-Å draft crystal structure of recombinant vaults was published by Anderson et al. (PDB ID 2QZV) RS1 . The domain partitions from the Anderson's structure were used as a reference to identify the site of interaction between MVP and INT.

Confirming interactions between isolated iMVP and INT
The interaction between purified His-iMVP and mCherry-INT was studied using affinity chromatography by monitoring the protein and mCherry absorbance at 280 and 585 nm,     Although monomers and dimers separated as two independent peaks in the SEC, the monomer peak fractions that were run a few hours after the SEC, showed again both species ( Figure S4A, 10-15 ml). Complete separation of monomers from dimers is not achievable using the method.
However, it is clear from Figure S4B that majority of the population in the "monomer" peak (fractions #14 and #15) is monomer. Figure S4C shows that the monomers and dimers are in thermodynamic equilibrium at the given concentration.  Figure S5 and Table S2 show the analysis of peak fractions from SEC of mixture (Fig. 3A). The

Interaction between His-iMVP and His-INT at high immobilization density
The propensity toward 1:1 interaction in Fig. 4 predominates at low immobilization density (410 RU). In contrast, at high His-iMVP immobilization density (12,400 RU), the 1-to-1 model is no longer valid suggesting that more complicated interactions exist between the His-iMVP and the His-INT (Fig. S6). As shown in Fig. S6,  Table S3 summarizes that the key interaction sites on iMVP are negatively charged. Upon introduction of histidines on INT that are protonated and positively charged at acidic pH, the interaction is expected to be enhanced by electrostatic interaction.  Figure S8 shows the SPR sensorgrams of His-iMVP and wild-type His-INT at pH 6.0. His-iMVP was immobilized at 12400 RU and His-INT was injected across the surface as a series of 0.44, 0.87, 1.75, 3.50, or 7.00 µM (assumed as monomer during calculation) in PBS buffer at pH 6.0, respectively. Although affinity constants could not be determined since the sensorgrams could not be fitted with a 1-to-1 Langmuir model, the observations at pH 6.0 seemed to fit bivalent fitting better suggesting 2 INT bind to 1 iMVP. Affinity constants of the bivalent binding was determined to be ka1 = 1.03 × 10 4 M -1 s -1 , kd1 = 0.279 s -1 , ka2 = 6.15 × 10 -6 RU -1 s -1 , and kd2 = 8.26

Interaction between His-iMVP and His-INT at pH 6.0
× 10 -4 s -1 . It has to be noted that these numbers are mathematical representation which may not be compared with affinities of 1-to-1 binding.

Subcloning of the iMVP domains and INT in pET System
To isolate iMVP and construct INTs for investigations, the designed oligonucleotides are shown in Table S4. Table S4. Oligonucleotides for isolated domains constructions.
During the experiments, some other variants of MVP and INT were also investigated. The proteins produced are summarized as Table S5. Table S5. Summary of truncated MVP and INT proteins production.