Using ion mobility spectrometry-mass spectrometry to decipher the conformational and assembly characteristics of the hepatitis B capsid protein

Abstract

The structural and functional analysis of the core protein of hepatitis B virus is important for a full understanding of the viral life cycle and the development of novel therapeutic agents. The majority of the core protein (CP149) comprises the capsid assembly domain, and the C-terminal region (residues 150-183) is responsible for nucleic acid binding. Protein monomers associate to form dimeric structural subunits, and helices 3 and 4 (residues 50-111 of the assembly domain) have been shown to be important for this as they constitute the interdimer interface. Here, using mass spectrometry coupled with ion mobility spectrometry, we demonstrate the conformational flexibility of the CP149 dimer. Limited proteolysis was used to locate involvement in this feature to the C-terminal region. A genetically fused CP dimer was found to show decreased disorder, consistent with a more restricted C-terminus at the fusion junction. Incubation of CP149 dimer with heteroaryldihydropyrimidine-1, a small molecule known to interfere with the assembly process, was shown to result in oligomers different in shape to the capsid assembly-competent oligomers of the fused CP dimer. We suggest that heteroaryldihydropyrimidine-1 affects the dynamics of CP149 dimer in solution, likely affecting the ratio between assembly active and inactive states. Therefore, assembly of the less dynamic fused dimer is less readily misdirected by heteroaryldihydropyrimidine-1. These studies of the flexibility and oligomerization properties of hepatitis B virus core protein illustrate both the importance of C-terminal dynamics in function and the utility of gas-phase techniques for structural and dynamical biomolecular analysis.

Figure 1

The structure of the HBV core protein (10). (a) The core protein is predominantly α-helical and exists as a dimer. Each monomer contains five helices numbered from the N- to the C-terminus, linked by loop regions. Helices 3 and 4 constitute the four-helix bundle that forms the intermonomer interface. The C-terminus is unstructured in the crystal structure and is preceded by a trypsin cleavage site at R127 (yellow). The monomers in the dimer are covalently bound through a disulphide bond between the two C61 residues (green). (b) The T = 4 capsid of HBV with each quasiequivalent conformer indicated with a different color. The color coding of conformers A (green), B (yellow), C (red), and D (blue) is consistent with that used by others (PDB ID 1QGT).

Figure 2

ESI-MS and ESI-IMS-MS of the CP149 dimer and monomer. (a) m/z mass spectrum of CP149 dimer in 50 mM ammonium acetate (pH 7.5). A bimodal charge state distribution, centered on the 10+ and 17+ charge states, consistent with two conformers with distinct solvent accessible surface areas is observed. (b) Charge state (z) versus CCS (nm²) plot for CP149 dimer showing the ion mobility separation of conformeric families, one compact (squares) and one extended (triangles). (c) m/z mass spectrum of CP149 dimer in 50 mM ammonium acetate (pH 7.5) with 2 mM DTT added. The charge state distribution of the dimer overlaps with monomeric ions (stars). The monomer populates three charge state distributions centered on 7+, 10+, and 14+ ions (monomer charge states underlined). (d) Charge state (z) versus CCS (nm²) plot for CP149 monomer showing the separation of three conformeric families, compact (squares), extended (triangles), and very extended (circles). Error bars indicate the standard deviation from the mean of at least three measurements.

Figure 3

C-terminal cleavage reduces disorder of CP149 dimer. m/z spectra of full- length CP149 dimer (red) undergoing proteolysis observed (a) immediately after addition of 1:35 (molar ratio) trypsin; (b) after 8 h, when the single C-terminal cleavage product, Δ1C, appears (green); (c) after 24 h, when the double C-terminal cleavage product, Δ2C (blue), predominates. (d) Circular dichroism spectra of CP149 dimer before (black) and after (red) cleavage of both C-termini with trypsin. The minima at 208 and 222 nm, characteristic of an α-helical fold, are present in both spectra indicating little change in secondary structure after C-terminal cleavage.

Figure 4

The fused dimer CP149 displays reduced dynamics. (a) A linker of five GGS amino acid repeats links the C-terminus of one monomer (dark gray) to the N-terminus of the second (light gray). (b) Noncovalent ESI-MS m/z spectrum of the fused dimer CP showing a bimodal charge state distribution centered on 11+ and 17+ ions. The high charge state distribution is low in intensity relative to that observed for CP149 dimer, suggesting a reduced propensity to unfold. This is consistent with one of the C-termini being restricted by the C-to-N-terminal linker.

Figure 5

CP149 dimer and fused dimer oligomers. (a) nanoESI-MS m/z spectrum showing CP149 forms oligomers of two (red), three (blue), four (green), five (purple), and six (magenta) dimers in the presence of HAP-1 (10:1 molar ratio, HAP:CP149 dimer) with little change to the unbound CP149 dimer ions remaining in the spectrum. There are also low-intensity signals indicating the presence of species greater in mass than six dimers. (b) TEM analysis of CP149 dimer after incubation with 50:1 HAP:1 showing large, noncapsid structures and (c) a regular CP149 dimer assembly reaction induced by 0.5 M NaCl, displaying capsid particles ∼30 nm in diameter. The scale bars represent 100 nm. (d) Noncovalent ESI-MS m/z spectrum showing fused dimer CP oligomers produced from disassembly of fused dimer CP capsids using 4 M urea. Oligomers of two (red), three (blue), four (green), and five (purple) fused dimers are observed. (e) CCS values of core protein oligomers versus oligomer order. The fused dimer CP oligomers (red triangles) lie on the tiled line (dashed line), whereas the HAP-induced oligomers (black squares) deviate from this, indicating a more compact structure. Error bars indicate the standard deviation from the mean of at least three measurements.