Solution Structures of Engineered Vault Particles

Abstract

Prior crystal structures of the vault have provided clues of its structural variability but are non-conclusive due to crystal packing. Here, we obtained vaults by engineering at the N terminus of rat major vault protein (MVP) an HIV-1 Gag protein segment and determined their near-atomic resolution (∼4.8 Å) structures in a solution/non-crystalline environment. The barrel-shaped vaults in solution adopt two conformations, 1 and 2, both with D39 symmetry. From the N to C termini, each MVP monomer has three regions: body, shoulder, and cap. While conformation 1 is identical to one of the crystal structures, the shoulder in conformation 2 is translocated longitudinally up to 10 Å, resulting in an outward-projected cap. Our structures clarify the structural discrepancies in the body region in the prior crystallography models. The vault's drug-delivery potential is highlighted by the internal disposition and structural flexibility of its Gag-loaded N-terminal extension at the barrel waist of the engineered vault.

Keywords: cellular organelle; cryo-EM; drug delivery; human immunodeficiency virus (HIV); imaging; nanoparticle engineering; protein design; single-particle analysis; structural dynamics; vaccine design.

Figure 1. CryoEM single particle analysis result on engineered MVP-only vault

(A) Aligned sum of rat vault raw image stack, showing this dataset has nice orientation distribution. Typical top views are boxed in black square. The scale bar is 50nm. (B) Magnified raw image of top view to show there are ~10 copies in a quadrant of circle, implying close to 40-fold related symmetry. (C) Fourier transform of a sum micrograph. Thon rings can reach to water signal at close to 3.6Å⁻¹. (D) Density map of two vault conformations refined from a single dataset. Conformation 1 (displayed at 4.4 σ) is in pink and conformation 2 (displayed at 4.5 σ) is in yellow. They are all in D39 symmetry. (E) top view of vault density in (D). No diameter change can be observed. (F) FSC curve showing that the resolution (FSC 0.143) of the two conformations are 4.9 Å and 4.7 Å, respectively.

Figure 2. Structural comparison

(A) Conformation 1 and corresponding model. (B) Conformation 2 and corresponding model. (C) Model comparison between conformation 1 (purple) and conformation 2 (olive). One copy of major vault protein (MVP) is colored in rainbow. The back half is hidden for clarity. (D) Overlapped model comparison. R1-R7 has no major conformational change. PDB 4HL8 is colored in aqua to show similarities between conformation 1 model and PDB 4HL8. (E) Near-atomic resolution feature at shoulder and cap-helix domain in both conformations, including an α-helix (red) and β-sheet (green). Large side chains can be identified and is consistent with current resolution estimation. Position is labeled in (D). Contour displayed at 6.7 σ. (F) Magnified view at R1 and R2 domain of two conformations. 4V60 model (grey) are displayed. The mismatch region in 4V60 is colored with black. No significant flexible region can be found at R1 and R2 domain. The major conformational change of cryoEM vault structure is not at waist region. Mesh contour is displayed at 5 σ for conformation 1 (pink) and 6.4 σ for conformation 2 (yellow)

Figure 3. Conformational change diagram

(A) Conformation 1 monomer model as a side view in vault over all model. (B) Magnified view of R7 to cap-helix domain. The missing segment between N428 to S449 locates inside vault. The docking of helix-cap domain of conformation 2 into conformation 1 density shows that cap-helix domain in conformation 2 bends outwards comparing with conformation 1. R8 to shoulder domain can all be roughly divided into two parts, separated by the dashed line. The attachment layer locates inside and the wall layer locates outside. (C) R7 to cap-helix domain of conformation 2. Like conformation 1, the R8 to shoulder domain is double layered. The inner-layer is colored from blue to red, from N-terminus to C-terminus. The outer layer is colored in original olive color. (D, E) Direct overlapping of conformation 1 and conformation 2 model. Position shift is magnified from R8 to shoulder domain. The relative movement of attachment layer is labeled with corresponding color and the movement is larger from R8 to shoulder domain. (F) A diagram to show the cap movement and conformational change between conformation 1 (purple) and conformation 2 (olive). In the refinement result applied D39 symmetry, the relative movement freedom of cap is limited to axial (up and down) and rotational (rotation around 39-fold axis). The movement from conformation 1 to conformation 2 of cap region can be described as “being rotated clockwise by 2 degrees and lifted by 10Å”. There is minor morphing of cap between the two conformations, when conformation 2 is relatively shorter and more twisted at cap region based on shorter translocation distance along axis and more angular rotation at cap-ring region.

Figure 4. Model and density comparison among models and densities via longitudinal section

(A) Conformation 1 model (purple), conformation 1 density (pink, displayed at 3 σ) and docked segmented Gag dimer (PDB 1AFV, red). (B) Conformation 2 model (olive), conformation 2 density (yellow, displayed at 3.7 σ) and docked Gag dimer (red). (C, D) utilizing similar color code with (A) and (B), respectively, with higher visualization threshold (displayed 6.2 σ and 5.1 σ for conformation 1 and 2, respectively). PDB 4V60 (grey) is docked into conformation 1 and conformation 2 density. C-terminus of Gag and N-terminus of MVP is connected by flexible linker, shown as dashed line. (E) Local resolution estimation of conformation 2 density map calculated by Resmap (Kucukelbir et al., 2014). The flexible region is of low resolution and appear blue (fused protein at waist, C terminus near cap and inner surface). The major conformational change takes place at shoulder domain but the resolution is relatively high.