Assembly and Capsid Expansion Mechanism of Bacteriophage P22 Revealed by High-Resolution Cryo-EM Structures

Abstract

The formation of many double-stranded DNA viruses, such as herpesviruses and bacteriophages, begins with the scaffolding-protein-mediated assembly of the procapsid. Subsequently, the procapsid undergoes extensive structural rearrangement and expansion to become the mature capsid. Bacteriophage P22 is an established model system used to study virus maturation. Here, we report the cryo-electron microscopy structures of procapsid, empty procapsid, empty mature capsid, and mature capsid of phage P22 at resolutions of 2.6 Å, 3.9 Å, 2.8 Å, and 3.0 Å, respectively. The structure of the procapsid allowed us to build an accurate model of the coat protein gp5 and the C-terminal region of the scaffolding protein gp8. In addition, interactions among the gp5 subunits responsible for procapsid assembly and stabilization were identified. Two C-terminal α-helices of gp8 were observed to interact with the coat protein in the procapsid. The amino acid interactions between gp5 and gp8 in the procapsid were consistent with the results of previous biochemical studies involving mutant proteins. Our structures reveal hydrogen bonds and salt bridges between the gp5 subunits in the procapsid and the conformational changes of the gp5 domains involved in the closure of the local sixfold opening and a thinner capsid shell during capsid maturation.

Bacteriophage P22 Capsid as a Pluripotent Nanotechnology Tool

“…A-domain (a.a. 128–221, 346–357, and 417–430) surrounds the center of the oligomer and has a flexible loop exposed to solvents. It is a region susceptible to protease action in the monomer and the PC, but it becomes more resistant after capsid maturation [ 43 , 48 , 51 , 52 ]. The flexibility of the A-domain allows the manipulation of this region with no repercussions to PC assembly or capsid integrity [ 43 ].…”

“…One example is residue T183, which is in an axial position on the capsomer, as seen in structural models of P22, making it an interesting target for mutations, especially because it was shown that T183 modifications did not affect capsid assembly [ 51 ]. The P-domain (a.a. 31–33, 79–127, and 358–416) is positioned at the extremity of the protein and forms an important interaction with the N-terminal arm (N-arm) in (a.a. 2–30) that stabilizes the capsid after expansion [ 44 , 52 ]. The E-loop (a.a. 51–78) is a hinge-shaped region, which is a site of crosslinks in HK97 but is shortened on the P22 [ 34 , 52 ].…”

“…The P-domain (a.a. 31–33, 79–127, and 358–416) is positioned at the extremity of the protein and forms an important interaction with the N-terminal arm (N-arm) in (a.a. 2–30) that stabilizes the capsid after expansion [ 44 , 52 ]. The E-loop (a.a. 51–78) is a hinge-shaped region, which is a site of crosslinks in HK97 but is shortened on the P22 [ 34 , 52 ]. It forms salt bridges with the P-domain of the neighbor subunit, playing a role in capsid stabilization; however, amino acid substitutions in the E-loop apparently did not affect the capsid, according to [ 50 ].…”

“…It forms salt bridges with the P-domain of the neighbor subunit, playing a role in capsid stabilization; however, amino acid substitutions in the E-loop apparently did not affect the capsid, according to [ 50 ]. The I-domain (a.a. 222–345) is inserted into the A-domain and is absent in HK97 [ 34 , 44 , 50 , 52 ]. This domain has a D-loop region forming essential ionic interactions over the local and icosahedral twofold axis that contributes to capsid stabilization.…”

“…The absence of these interactions results in aberrant and tubular particles [ 50 ]. New salt bridges also appear between the N-arm and the F-loop (a.a. 34–50) in the mature capsid [ 52 ]. The low energy of the CP subunits interactions makes the structure flexible, to the point of allowing the substitution of monomers in the PC particle with others in a solution.…”