Major Variations in HIV-1 Capsid Assembly Morphologies Involve Minor Variations in Molecular Structures of Structurally Ordered Protein Segments

Abstract

We present the results of solid state nuclear magnetic resonance (NMR) experiments on HIV-1 capsid protein (CA) assemblies with three different morphologies, namely wild-type CA (WT-CA) tubes with 35-60 nm diameters, planar sheets formed by the Arg(18)-Leu mutant (R18L-CA), and R18L-CA spheres with 20-100 nm diameters. The experiments are intended to elucidate molecular structural variations that underlie these variations in CA assembly morphology. We find that multidimensional solid state NMR spectra of (15)N,(13)C-labeled CA assemblies are remarkably similar for the three morphologies, with only small differences in (15)N and (13)C chemical shifts, no significant differences in NMR line widths, and few differences in the number of detectable NMR cross-peaks. Thus, the pronounced differences in morphology do not involve major differences in the conformations and identities of structurally ordered protein segments. Instead, morphological variations are attributable to variations in conformational distributions within disordered segments, which do not contribute to the solid state NMR spectra. Variations in solid state NMR signals from certain amino acid side chains are also observed, suggesting differences in the intermolecular dimerization interface between curved and planar CA lattices, as well as possible differences in intramolecular helix-helix packing.

Keywords: human immunodeficiency virus (HIV); molecular dynamics; protein assembly; protein structure; solid state NMR.

FIGURE 1.

TEM images of negatively stained HIV-1 capsid protein assemblies. A, WT-CA tubes. B and C, R18L-CA spheres. D, planar R18L-CA sheet. Non-planar material within ellipses may result from disruption or dissolution of R18L-CA sheets during the TEM grid preparation process. E, Fourier transform of the region of panel D enclosed by a square. The pattern of spots with 6-fold symmetry indicates two-dimensional crystallinity of R18L-CA sheets.

FIGURE 2.

A and C, AFM images of R18L-CA sheets. Images were recorded in tapping mode on mica substrates, immersed in assembly buffer within the fluid cell of the AFM instrument. B and D, height profiles along blue and red lines in panels A and C, showing the uniform 6-nm thickness of single R18L-CA sheets. Features with 12 nm height are attributable to double layers.

FIGURE 3.

Two-dimensional ¹³C-¹³C solid state NMR spectra of uniformly ¹⁵N,¹³C-labeled HIV-1 capsid protein assemblies. A, U-WT-CA tubes. B, U-R18L-CA spheres. C, U-R18L-CA sheets. D, one-dimensional slices at 29.3, 51.5, and 65.8 ppm (dashed lines in two-dimensional spectra). The one-dimensional slices are color-coded to match the two-dimensional spectra. DARR mixing periods between t₁ and t₂ periods were 100 ms for U-WT-CA tubes and 25 ms for U-R18L-CA spheres and sheets. Maximum t₁ periods were 5.0–8.9 ms. Total measurement times were ∼18 h. Contour levels increase by factors of 1.5.

FIGURE 4.

Examples of two-dimensional slices from three-dimensional NCACX spectra of uniformly ¹⁵N,¹³C-labeled R18L-CA spheres (blue) and sheets (red). A, slices at 53.4 ppm in the t₂ dimension (i.e. CA dimension). B, slices at 65.5 ppm in the t₂ dimension. Cross-peak assignments are based on analyses of two- and three-dimensional spectra and on previously reported assignments for WT-CA tubes (15). Spectra were recorded with 20 ms DARR mixing periods between t₂ and t₃ periods. Maximum t₁ and t₂ periods were 5.2–5.9 and 6.0–6.7 ms, respectively. Total measurement times were ∼12 days. Contour levels increase by factors of 1.2.

FIGURE 5.

Summary of differences in solid state NMR chemical shifts between R18L-CA assemblies and WT-CA tubes. Residue-specific values of chemical shift differences Δ_rms (defined in the main text) are represented by colors on a schematic representation of a CA hexamer (A, B, D, and E) or monomer (C and F). Hexamer and monomer structures come from PDB file 3J34. Chemical shifts of R18L-CA spheres and WT-CA tubes are compared in panels A–C. Chemical shifts of R18L-CA sheets and WT-CA tubes are compared in panels D–F. Yellow and orange residues have 0.0 ppm < Δ_rms < 0.4 ppm and Δ_rms ≥ 0.4 ppm, respectively. Blue residues do not have assigned chemical shifts. The hexamer is viewed from “above” in panels A and D, and “below” in panels B and E. Helical segments are numbered in panels C and F, and the R18L mutation site is shown.

FIGURE 6.

Two-dimensional NCACX spectra of HIV-1 capsid protein assemblies that are uniformly ¹⁵N-labeled and partially ¹³C-labeled. A, 2-Glyc-WT-CA tubes. B, 2-Glyc,Met-R18L-CA spheres. C, 2-Glyc,Met-R18L-CA sheets. D, color-coded one-dimensional slices at ¹⁵N chemical shifts of 114.8, 123.2, 124.3, and 128.0 ppm (dashed lines in two-dimensional spectra). DARR mixing periods between t₁ and t₂ periods were 25 ms. Maximum t₁ periods were 7.2 ms. Total measurement times were ∼48 h. Contour levels increase by factors of 1.5.

FIGURE 7.

Intermolecular and intramolecular side chain-side chains interactions in HIV-1 capsid protein assemblies. A, intermolecular dimerization interface, involving helix 9 segments of interacting CTD domains from different CA molecules (green and cyan) around local 2-fold symmetry axes. B, intramolecular contacts of helices 1, 2, and 3 within a single NTD domain (purple). Structures in panels A and B are present in crystalline WT-CA (PDB file 4XFX), and may be somewhat different in WT-CA tubes, R18L-CA spheres, and R18L-CA sheets.

FIGURE 8.

Aromatic regions of two-dimensional ¹³C-¹³C and two-dimensional ¹⁵N-¹³C spectra of HIV-1 capsid protein assemblies that are uniformly ¹⁵N labeled and partially ¹³C labeled. A and C, two-dimensional spectra of R18L-CA spheres (blue), overlaid on two-dimensional spectra of WT-CA tubes (black). Assignments of cross-peaks from tryptophan residues and one tyrosine residue are shown. B and D, two-dimensional spectra of R18L-CA sheets (red), overlaid on two-dimensional spectra of WT-CA tubes (black). Spectra in panels A and B were obtained with U-R18L-CA and U-WT-CA. Spectra in panels C and D were obtained with 2-Glyc,Met-R18L-CA and 2-Glyc-WT-CA. DARR mixing periods were 500 ms in two-dimensional ¹³C-¹³C spectra and 140 ms in two-dimensional ¹⁵N-¹³C spectra. Maximum t₁ periods were 6.9 ms in two-dimensional ¹³C-¹³C spectra and 6.4 ms in two-dimensional ¹⁵N-¹³C spectra. The ¹³C carrier frequency during ¹⁵N-¹³C cross-polarization was set to 127 ppm in the two-dimensional ¹⁵N-¹³C spectrum of 2-Glyc-WT-CA tubes and 174 ppm in two-dimensional ¹⁵N-¹³C spectra of 2-Glyc,Met-R18L-CA assemblies, accounting for differences in signals below 115 ppm in these spectra. Contour levels increase by factors of 1.2.

FIGURE 9.

Selected regions of two-dimensional ¹³C-¹³C spectra of HIV-1 capsid protein assemblies in which methionine residues are uniformly ¹⁵N,¹³C labeled. A, Met-WT-CA tubes. B, 2-Glyc,Met-R18L-CA spheres. C, 2-Glyc,Met-R18L-CA sheets. Orange lines indicate intra-residue correlations to methionine methyl signals. The dashed line in panel C indicates the absence of signals from Met¹⁸⁵. Inter-residue cross-peaks in two-dimensional spectra of 2-Glyc,Met-R18L-CA assemblies, which were also partially ¹³C labeled at non-methionine residues by expression with [2-¹³C]glycerol as the carbon source, are indicated by purple labels and arrows. D, color-coded one-dimensional slices at 31.8 and 59.9 ppm (dashed lines in two-dimensional spectra). Spectra were recorded with 500-ms DARR mixing periods and maximum t₁ periods of 6.9 ms. Contour levels increase by factors of 1.2.

FIGURE 10.

Two-dimensional ¹³C-¹³C INEPT-TOBSY spectra of uniformly ¹⁵N,¹³C-labeled HIV-1 capsid assemblies, showing signals from protein segments that remain highly dynamic in the assemblies. Two-dimensional spectra are shown for U-WT-CA tubes (A), U-R18L-CA spheres (B), and U-R18L-CA sheets (C), with color-coded one-dimensional slices (D) at 3.18, 42.3, and 53.4 ppm, corresponding to the dashed lines in the two-dimensional spectra. Residue-type assignments, based on random-coil chemical shifts, are shown in panel A. Maximum t₁ periods were 7.2–8.9 ms. Total measurement times were ∼24 h. Contour levels increase by factors of 1.4.

FIGURE 11.

Predicted chemical shift differences between HIV-1 CA structural models. A, values of Δ_rms between engineered hexameric (PDB code 3MGE) and pentameric (PDB code 3P05) CA mutants, based on ¹⁵N and ¹³C chemical shift predictions from SHIFTS (red triangles) and ShiftX (blue squares). For comparison, experimental values of Δ_rms between CA tubes and R18L-CA sheets are shown as solid black circles. B, superposition of C_α traces for the hexameric (magenta) and pentameric (green) CA mutants, for which the r.m.s. deviation between C_α coordinates in residues 10–140 and 150–200 is 1.72 Å. C, values of Δ_rms between CA chains m and A from a cryo-EM-based model for CA tubes (PDB 3J34), based on chemical shift predication from SHIFTS (red triangles) and ShiftX (blue squares). D, superposition of C_α traces for chains A (magenta) and m (green), for which the r.m.s. deviation between C_α coordinates in residues 10–140 and 150–200 is 2.53 Å.