Research ArticleBIOCHEMISTRY

Designing and defining dynamic protein cage nanoassemblies in solution

See allHide authors and affiliations

Science Advances  14 Dec 2016:
Vol. 2, no. 12, e1501855
DOI: 10.1126/sciadv.1501855
  • Fig. 1 Designed and observed cage symmetry.

    (A) Left: Schematic diagram of the symmetry principles used to design the 12-subunit tetrahedral cage by fusing two oligomeric domains (green and orange) by a semirigid linker (magenta) (14, 15). Right: Single point mutation distinguishing our PCtrip construct from PCquad replaces tyrosine (black sticks) with alanine in the trimeric domain that makes contact with the linker. (B) Side-by-side view of the theoretically designed (perfectly symmetric) model of the protein cage (left) and the most symmetric crystal structure obtained in this work for the PCquad variant (right). (C) Walleyed stereo view of three crystal structures of PCquad (yellow, magenta, and blue) overlaid on the ideal model (green ribbon), showing the agreement of the observed structures and the design.

  • Fig. 2 Cage conformation and assembly in solution from theoretical and experimental SAXS scattering plots.

    (A) X-ray scattering profiles calculated from the most compact crystal structure (PDB ID: 3VDX; black), the most symmetric and open crystal structure (PDB ID: 4QF0; magenta), and the ideal designed model (blue). Scattering plots calculated from snapshots of a morph between the ideal and compact structures are shown in gray. (B) Observed scattering plots from 64 separate experiments involving the two constructs (PCtrip and PCquad profiles are offset vertically for clarity) under various buffer conditions (gray). Extremes of salt for which cages persisted as the dominant structures are shown in color (labeled as x/y = salt concentration [mM]/pH), along with their fits (black). Ratio of SAXS experiments to fits for (C) PCquad and (D) PCtrip.

  • Fig. 3 Comprehensive determination of cage assembly and conformation in response to salt and pH.

    (A) Comparing the agreement of theoretical SAXS data calculated from 21 snapshots of a morph trajectory, between a compact crystal structure (3VDX) and the ideal designed cage, plus a trimer from the crystal representing disassembled cages. Each cell in the heat map matrix compares two SAXS profiles by VR value, with red indicating high similarity and white indicating low similarity. In the lower left part of each panel, the models are arranged in two dimensions in the form of a force plot, where stronger repulsive forces are invoked between models whose calculated SAXS profiles are less similar. The size of each circle reflects the Rg of the corresponding cage structure. The force plot for the trimer and morphed models is included as a “landmark” in subsequent force plots. (B) Heat map and force plots for all eight crystal structures obtained for the cage (PDB code listed). (C and D) Analysis of experimental SAXS data for PCtrip and PCquad, varying salt concentration and pH. Dot color coding is set by pH, and at pH 6 (brown), the micromolar salt concentration is noted. The buffer conditions are labeled as x/y, meaning x mM of salt and pH y.

  • Fig. 4 Solution conditions modulate cage conformation and assembly as determined from fits to SAXS data.

    (A and B) Regions shaded in blue represent conditions where the protein is mostly assembled as a 12-subunit tetrahedral cage as designed (>50% by weight). (A) PCtrip. (B) PCquad. The values for cage abundance are determined from fitting the data with an ensemble of models, including cages, trimers, larger multimers found in crystal structures, and morphs between crystal structures. The quality of fits is shown for a subset of SAXS experiments for (C) PCtrip and (D) PCquad. Subtracting the noncage component for all members within the contour yields modified SAXS curves that represent the average conformation sampled in the cage assemblies for each condition. These are placed in a force plot [insets of (C) and (D)], along with the landmark morph trajectory described in the figures above (blue circles). A subset of conditions is color-coded to draw out the trend observed with the following salt concentrations: 10 mM (red), 100 mM (orange), 300 mM (cyan), and 500 mM (green). Multiple SAXS curves are offset vertically for clarity.

  • Table 1 X-ray diffraction and crystallographic refinement data.
    Space groupI222P21212P212121
    Unit cell dimensions123.51, 165.52,
    167.67
    175.50, 147.73,
    167.63
    155.50, 156.52,
    325.03
    Resolution (Å)4.196.497.81
    Measured reflections16,720256,95834,715
    Unique reflections12,9149,0018,723
    Completeness99.7% (96.4%)99.6% (96.1%)92.1% (77.4%)
    Rsym12.7% (88.5%)8.6% (81.9%)14.2% (59.7%)
    I/σ(I)14.2 (3.3)14.2 (2.3)10.7 (2.3)
    Refinement
    Resolution83.83–4.1993.71–6.4991.27–7.81
    Asymmetric unit3 molecules6 molecules12 molecules
    Matthews coefficient2.853.613.29
    Solvent content56.85%65.97%62.61%
    Data used for
    refinement
    11,6228,1007,849
    Data used for Rfree646451437
    Final Rwork0.2510.2710.288
    Final Rfree0.2950.3240.339
    RMSD
      Bonds (Å)0.0090.0070.010
      Angles (°)1.441.011.44
    PDB ID4QES4QF04QFF
  • Table 2 Compared structural features among cage crystal structures.

    The degree of asymmetry was taken as the largest difference between elliptical diameters divided by the largest of the three values. The rows are color-grouped by different cage variants: red, original cage structures (14); blue, PCtrip crystal structures (17); green, PCquad structures (this study), including the most symmetric structure obtained to date (4QF0).

    PDB codeSpace
    group
    Elliptical
    diameters (Å)
    AsymmetryRMSD (Å)
    3VDXI222121, 112, 10315%17.5
    4D9JP2221115, 108, 1068%17.4
    4IVJI222135, 125, 10919%12.8
    4IQ4P21212123, 112, 10316%17.6
    4ITVP212121130, 118, 10023%16.3
    4QESI222128, 122, 1169.5%12.0
    4QF0P21212130, 129, 1263.4%7.1
    4QFFP212121138, 121, 11913.8%11.6

Supplementary Materials

  • Supplementary material for this article is available at http://advances.sciencemag.org/cgi/content/full/2/12/e1501855/DC1

    fig. S1. Position of the Y51A mutation in PCquad.

    fig. S2. Crystallographic density maps of PCquad as found in PDB entry 4QF0.

    fig. S3. Testing the concentration dependence of the cage systems.

    fig. S4. Scattering profiles and their fits.

    fig. S5. Heat map and force plot analysis using VR and χ2.

    fig. S6. Comparing RMSD changes measured from atomic models to the similarity of SAXS calculated from the same atomic models.

    fig. S7. Purity of PCtrip and PCquad after affinity purification and SEC.

    fig. S8. PCtrip multimerization as a function of pH as probed by SEC.

    fig. S9. Minimalist ensembles with nearly equivalent fits to SAXS data.

    table S1. SAXS-related parameters estimated from crystal structures.

    table S2. Summary of buffer effects on SAXS parameters of PCtrip.

    table S3. Summary of buffer effects on SAXS parameters of PCquad.

    table S4. Multimeric composition from fitting SAXS results from PCtrip.

    table S5. Multimeric composition from fitting SAXS results from PCquad.

    movie S1. Structural morphing between idealized symmetric structure and most compact asymmetric crystal structure.

  • Supplementary Materials

    This PDF file includes:

    • fig. S1. Position of the Y51A mutation in PCquad.
    • fig. S2. Crystallographic density maps of PCquad as found in PDB entry 4QF0.
    • fig. S3. Testing the concentration dependence of the cage systems.
    • fig. S4. Scattering profiles and their fits.
    • fig. S5. Heat map and force plot analysis using VR and χ2.
    • fig. S6. Comparing RMSD changes measured from atomic models to the similarity of SAXS calculated from the same atomic models.
    • fig. S7. Purity of PCtrip and PCquad after affinity purification and SEC.
    • fig. S8. PCtrip multimerization as a function of pH as probed by SEC.
    • fig. S9. Minimalist ensembles with nearly equivalent fits to SAXS data.
    • table S1. SAXS-related parameters estimated from crystal structures.
    • table S2. Summary of buffer effects on SAXS parameters of PCtrip.
    • table S3. Summary of buffer effects on SAXS parameters of PCquad.
    • table S4. Multimeric composition from fitting SAXS results from PCtrip.
    • table S5. Multimeric composition from fitting SAXS results from PCquad.

    Download PDF

    Other Supplementary Material for this manuscript includes the following:

    • movie S1 (.mp4 format). Structural morphing between idealized symmetric structure and most compact asymmetric crystal structure.

    Files in this Data Supplement: