Crystallogenesis of bacteriophage P22 tail accessory factor gp26 at acidic and neutral pH

Abstract

Gp26 is one of three phage P22-encoded tail accessory factors essential for stabilization of viral DNA within the mature capsid. In solution, gp26 exists as an extended triple-stranded coiled-coil protein which shares profound structural similarities with class I viral membrane-fusion protein. In the cryo-EM reconstruction of P22 tail extracted from mature virions, gp26 forms an approximately 220 angstroms extended needle structure emanating from the neck of the tail, which is likely to be brought into contact with the cell's outer membrane when the viral DNA-injection process is initiated. To shed light on the potential role of gp26 in cell-wall penetration and DNA injection, gp26 has been crystallized at acidic, neutral and alkaline pH. Crystals of native gp26 grown at pH 4.6 diffract X-rays to 2.0 angstroms resolution and belong to space group P2(1), with a dimer of trimeric gp26 molecules in the asymmetric unit. To study potential pH-induced conformational changes in the gp26 structure, a chimera of gp26 fused to maltose-binding protein (MBP-gp26) was generated. Hexagonal crystals of MBP-gp26 were obtained at neutral and alkaline pH using the high-throughput crystallization robot at the Hauptman-Woodward Medical Research Institute, Buffalo, NY, USA. These crystals diffract X-rays to beyond 2.0 angstroms resolution. Structural analysis of gp26 crystallized at acidic, neutral and alkaline pH is in progress.

Figure 1

Crystallization of gp26 at acidic pH. (a) Crystals of P22 tail accessory factor gp26 grew in clusters of shafts joined at the center. Isolated individual crystals have average dimensions of 80 × 100 × 350 µm. (b) Diffraction image recorded from a gp26 crystal oscillated 0.7° with an exposure time of 30 s. The image was measured at SSRL beamline 9-2 using an X-ray wavelength of 0.98 Å. (c) Enlargement of gp26 diffraction pattern showing distinct diffraction spots to 2 Å resolution. Owing to the anisotropic diffraction, X-ray data were only complete to 2.2 Å resolution (Table 1 ▶).

Figure 2

Cryo-annealing dramatically improves the diffraction quality of gp26 crystals. (a) Diffraction image recorded from a gp26 crystal prior to cryo-annealing. Bragg spots are elongated and smeared, with mosaicity spread often higher than 1°. (b) The same diffraction image was recorded after three 10 s rounds of cryo-annealing. In (b), diffraction spots are more circular and the overall quality of the image has improved. (c) and (d), stereographic projections of the κ = 120 and κ = 180° sections of the P22 tail accessory factor gp26 self-rotation function showing the direction of threefold and twofold non-crystallographic symmetry axes, respectively. The maps were contoured at 4σ in steps of 0.25σ. The functions were computed with program GLRF (Tong & Rossmann, 1997 ▶). The polar angles ψ, ϕ and κ follow the Rossmann convention (Rossmann & Blow, 1962 ▶), with the orthogonal y axis aligned with the crystal b axis, the x axis lying in the crystal ab plane and the z axis coinciding with the c axis. The polar angle ψ denotes the inclination of the rotation axis from the y axis, which in space group P2₁ corresponds to the crystallographic screw axis. The polar angle ϕ denotes the inclination of the rotation axis from the x axis.

Figure 3

Crystallization of gp26 fused to maltose-binding protein at neutral and alkaline pH. (a) A chimera of gp26 fused to maltose-binding protein (MBP-gp26) retains full oligomerization on SDS–PAGE. Unboiled MBP-gp26 in lane 1 runs on SDS–PAGE in its native trimeric state (3 × 70 kDa = ∼210 kDa), while an aliquot of MBP-gp26 fusion protein boiled for 5 min migrated as an ∼75 kDa band (lane 2). (b) Light micrograph of a typical MBP-gp26 crystal. The average dimensions of these crystals are 80 × 80 × 500 µm. The morphology of the crystal represents a macroscopic description of the hexagonal unit cell, which has averaged unit-cell parameters a = 43, b = 43, c = 272 Å. (c) Diffraction pattern of MBP-gp26 crystal oscillated by 0.5° with an exposure time of 10 s. The diffraction image was collected at BNLS beamline X6A on an ADSC Quantum-210 CCD detector. (d) and (e), enlargements of MBP-gp26 diffraction pattern showing intense diffraction spots to 1.8 Å resolution.