Unraveling the role of the C-terminal helix turn helix of the coat-binding domain of bacteriophage P22 scaffolding protein

Abstract

Many viruses encode scaffolding and coat proteins that co-assemble to form procapsids, which are transient precursor structures leading to progeny virions. In bacteriophage P22, the association of scaffolding and coat proteins is mediated mainly by ionic interactions. The coat protein-binding domain of scaffolding protein is a helix turn helix structure near the C terminus with a high number of charged surface residues. Residues Arg-293 and Lys-296 are particularly important for coat protein binding. The two helices contact each other through hydrophobic side chains. In this study, substitution of the residues of the interface between the helices, and the residues in the β-turn, by aspartic acid was used examine the importance of the conformation of the domain in coat binding. These replacements strongly affected the ability of the scaffolding protein to interact with coat protein. The severity of the defect in the association of scaffolding protein to coat protein was dependent on location, with substitutions at residues in the turn and helix 2 causing the most significant effects. Substituting aspartic acid for hydrophobic interface residues dramatically perturbs the stability of the structure, but similar substitutions in the turn had much less effect on the integrity of this domain, as determined by circular dichroism. We propose that the binding of scaffolding protein to coat protein is dependent on angle of the β-turn and the orientation of the charged surface on helix 2. Surprisingly, formation of the highly complex procapsid structure depends on a relatively simple interaction.

FIGURE 1.

Structure of the C-terminal coat protein-binding domain of phage P22 scaffolding protein. The NMR structure of the C-terminal HTH domain (PDB entry 2GP8) (50) was modeled using PyMOL (PyMOL Molecular Graphics System, version 1.3r1, Schrödinger, LLC). a, the labeled residues in (i) helix 1, (ii) the β₁-turn, and (iii) helix 2 were individually substituted with aspartic acid. b, the primary sequence and secondary structure of the C-terminal domain. The residues in orange represent substituted residues.

FIGURE 2.

Kinetic analysis of in vitro procapsid assembly. a, monomeric coat protein (0.5 mg/ml) was mixed with scaffolding protein variants (0.5 mg/ml) in buffer. The progress of procapsid polymerization was monitored by light scattering. Reactions for each of the scaffolding protein mutants are shown as follows: (i) mutations in helix 1, (ii) mutations in the β₁-turn and its periphery, and (iii) mutations in helix 2. b, micrographs of selected in vitro assembled procapsids made with different scaffolding protein variants in 45 mm NaCl as follows: (i) WT scaffolding protein, (ii) N272D, (iii) M280D, and (iv) T291D scaffolding proteins. No partial procapsids are readily evident in these micrographs because normal PCs are generated in 45 mm NaCl. The arrows in b (iii) point to spiral structures. The scale bar represents 100 nm. All micrographs were taken at ×68,000 magnification.

FIGURE 3.

Salt titration assay to assess coat binding affinity of mutant scaffolding proteins. Each panel shows a native agarose electrophoresis gel of an in vitro procapsid assembly reaction. The gels are run with the cathode (+) at the bottom. Samples were prepared by mixing monomeric coat protein (0.5 mg/ml) and scaffolding protein (0.5 mg/ml) at increasing NaCl concentrations from left to right (0, 5, 15, 30, 45, 60, 75, and 100 mm) as indicated by the triangle above the gel. The reactions were incubated for 2 h at room temperature. The indicated labels above the gels are lanes with only monomeric coat protein (MC), in vivo assembled procapsids (PC), and each mutant scaffolding protein (s). The small amount of material that migrates to the position of PC in the assembly reactions with A283D, A284D, G287D, Y292D, or L295D scaffolding proteins is due to uncontrolled assembly of coat protein, and is not generated by scaffolding protein mediated assembly.

FIGURE 4.

Refilling of empty coat protein shells with mutant P22 scaffolding proteins. A mixture of 0.33 mg/ml of empty coat protein shells, 0.2 mg/ml of scaffolding protein, and buffer were incubated overnight at room temperature. The reactions were layered on 20% sucrose cushions and centrifuged to remove unbound scaffolding protein; the proteins in the pellet, which contained the refilled shells, were visualized with 12.5% SDS-PAGE. The lanes are labeled with the scaffolding protein used in the assay. The lane labeled WT (−) shells shows scaffolding protein incubated in buffer with no shells added does not pellet and the lane labeled shells shows the pelleting of the shells without added scaffolding protein.

FIGURE 5.

Circular dichroism spectra of mutant scaffolding proteins. The secondary structure composition of each variant was determined at a protein concentration of 0.5 mg/ml. The panels show CD spectra of the indicated aspartic acid-substituted 6Xhis-Δ1–237 truncated scaffolding proteins as follows: (a) in helix 1, (b) in and adjacent to the β₁-turn, and (c) in helix 2 along with the WT 6Xhis-Δ1–237 protein.

FIGURE 6.

Thermal stability of mutant scaffolding protein HTH domains. The CD signal of 6Xhis-Δ1–237 scaffolding protein variants at 222 nm was monitored with increasing temperature. The samples contained 0.5 mg/ml of protein in 10 mm Na₂HPO₄ buffer. Denaturation curves for proteins with substitutions in (a) helix 1, (b) in and adjacent to the β₁-turn, and (c) helix 2 are compared with the WT 6Xhis-Δ1–237 scaffolding protein.

FIGURE 7.

Replacement of hydrophobic interactions in the HTH core with salt bridges. a, activity assay of full-length N272D/I276R/L299D scaffolding protein. (i) the effect of the N272D/I276R/L299D mutations on the in vitro assembly as measured by light scattering. Samples of 0.5 mg/ml (— —) WT or (solid line) N272D/I276R/L299D scaffolding proteins were mixed with 0.5 mg/ml of coat protein and monitored for 16 min. As negative control, assembly of 0.5 mg/ml of the (— —) coat protein with no scaffolding protein was also measured. (ii) salt titration of PC assembly at various concentrations of NaCl (0, 5, 15, 30, 45, 60, 75, and 100 mm). Lanes with monomeric coat protein (MC), in vivo assembled procapsids (PC), and each scaffolding protein are indicated. (iii) shell refilling assay of the ability of scaffolding protein to bind empty procapsid shells. Solutions of 0.33 mg/ml of procapsid shells and 0.22 mg/ml of scaffolding protein variants were incubated overnight at room temperature. Refilled procapsids were purified by centrifugation through a 20% sucrose cushion and the proteins in the pellet were visualized with 12.5% SDS-PAGE. b, CD measurements of WT and N272D/I276R/L299D mutant 6Xhis-Δ1–237/T265W scaffolding proteins fragments: (i) CD spectra of 0.5 mg/ml of WT (squares) and N272D/I276R/L299D (circles) fragments. (ii) Measurement of molar ellipticity signal at 222 nm with increasing temperature showing WT (squares) and N272D/I276R/L299D (circles).

FIGURE 8.

Scaffolding protein amino acid residues important for coat protein interaction. Opposite side views of the coat protein-binding HTH domain of scaffolding protein showing the informative residues analyzed in this report as space-filling spheres on a ribbon diagram frame (the two important surface residues, red; the three important turn residues, blue; and important zipper core residues, yellow; nonessential residues are at the bottom of HTH, green (figure created with PyMOL)).