A Molecular Staple: D-Loops in the I Domain of Bacteriophage P22 Coat Protein Make Important Intercapsomer Contacts Required for Procapsid Assembly

Abstract

Bacteriophage P22, a double-stranded DNA (dsDNA) virus, has a nonconserved 124-amino-acid accessory domain inserted into its coat protein, which has the canonical HK97 protein fold. This I domain is involved in virus capsid size determination and stability, as well as protein folding. The nuclear magnetic resonance (NMR) solution structure of the I domain revealed the presence of a D-loop, which was hypothesized to make important intersubunit contacts between coat proteins in adjacent capsomers. Here we show that amino acid substitutions of residues near the tip of the D-loop result in aberrant assembly products, including tubes and broken particles, highlighting the significance of the D-loops in proper procapsid assembly. Using disulfide cross-linking, we showed that the tips of the D-loops are positioned directly across from each other both in the procapsid and the mature virion, suggesting their importance in both states. Our results indicate that D-loop interactions act as "molecular staples" at the icosahedral 2-fold symmetry axis and significantly contribute to stabilizing the P22 capsid for DNA packaging. Double-stranded DNA (dsDNA) viruses, such as the tailed phages and herpesviruses, have coat proteins that lack sequence homology but possess a common HK97 fold (1, 2). Herpesviruses infect vertebrate hosts and in humans are responsible for a wide spectrum of diseases causing conditions ranging from cold sores and genital sores to chicken pox and shingles. Thus, understanding the assembly of herpesviruses is crucial for identification of novel drug targets. Bacteriophage P22 is a dsDNA virus with a scaffolding protein-mediated assembly pathway, which is similar to that of herpesvirus. Therefore, P22 provides a good simple model system for studying the capsid assembly of dsDNA viruses (3, 4).In bacteriophage P22, capsid assembly involves interaction of 415 coat protein monomers with ϳ100 molecules of scaffolding protein (5-9). This results in the formation of an intermediate structure called the procapsid (10), into which ejection proteins (11, 12) as well as a portal complex are coassembled (13). DNA gets packaged through the portal complex (5), scaffolding protein exits, and the capsid expands in volume (14). The addition of plug, tail needle, and tailspike proteins results in a mature infectious virion (15).Some coat proteins having the HK97 fold are embellished with extra domains (Fig. 1) (16). P22 coat protein is one of these, as it has a nonconserved accessory domain inserted between the A and P domains (10,17,18). This domain, referred to as the insertion domain (I domain) (19), plays an important role in the folding of coat protein by acting as an intramolecular chaperone (20). The I domain is also involved in determination of capsid size (21) and is hypothesized to stabilize procapsids by forming intersubunit interactions between adjacent capsomers (10,19,20). A recent nuclear magnetic resonance (NMR) solution structure of the isolated I domain revealed a large flexible loop, referred to...

Contextual Role of a Salt Bridge in the Phage P22 Coat Protein I-Domain

“…The acidic residues Asp-244 and Asp-246 are in the disordered D-loop and are not involved in ion pairs in the isolated I-domain (32). When coat protein assembles into procapsids, however, Asp-244 and Asp-246 form ion-pair interactions linking capsomer subunits (32,36). Of the 11 basic residues, Lys-249, Arg-255, Arg-269, Lys-272, and Lys-286 are not involved in salt bridges or ion pairs in the I-domain.…”

“…In previous work we showed the I-domain facilitates the folding of full-length coat protein by serving as the folding nucleus and also contributes about half of the ⌬G of stabilization for monomeric coat protein (31). Additionally, the I-domain D-loop contributes to procapsid assembly by making critical inter-capsomer interactions across the icosahedral 2-fold symmetry-axis (32,36). From these data the I-domain clearly has several critical roles in coat protein folding and assembly.…”

“…The pPC plasmid has the genes for scaffolding (gene 8) and coat protein (gene 5) cloned into pET30b (a gift from Peter Prevelige) and is used for purification of procapsids. For complementation experiments to determine phage relative titers, the D302A and H305A mutations were also generated in the pMS11 plasmid (36).…”

NMR assignments for the insertion domain of bacteriophage Sf6 coat protein

“…To date the only structurally characterized I-domain is that of the P22 phage, which has a 6-stranded β-barrel fold, a smaller accessory subdomain consisting of three β-strands, and a one and a half-turn α-helix (Rizzo et al 2014). The P22 I-domain plays critical roles in capsid assembly, stability, viability, and size determination (Rizzo et al 2014, D’Lima and Teschke 2015). Cryo-electron reconstructions show that the Sf6, P22, and CUS-3 I-domains protrude from the surfaces of T = 7 icosahedral particles (Parent et al 2014).…”

“…An important difference for Sf6, is that the D-loop (residues V239 to N254) found in the P22 and CUS-3 I-domains is absent in the Sf6 I-domain. This D-loop forms critical interactions between coat proteins to form proper capsid structures in P22 (D’Lima and Teschke 2015). Its absence in the Sf6 I-domain may account for the differences in morphology observed in cryoEM reconstructions of Sf6 capsids compared to those from P22 and CUS-3 (Parent et al 2014).…”