Multiple Functional Roles of the Accessory I-Domain of Bacteriophage P22 Coat Protein Revealed by NMR Structure and CryoEM Modeling

Abstract

SUMMARY Some capsid proteins built on the ubiquitous HK97-fold have accessory domains that impart specific functions. Bacteriophage P22 coat protein has a unique inserted I-domain. Two prior I-domain models from sub-nanometer cryoEM reconstructions differed substantially. Therefore, the NMR structure of the I-domain was determined, which also was used to improve cryoEM models of coat protein. The I-domain has an anti-parallel 6-stranded β-barrel fold, previously not observed in HK97-fold accessory domains. The D-loop, which is dynamic both in the isolated I-domain and intact monomeric coat protein, forms stabilizing salt bridges between adjacent capsomers in procapsids. A newly described S-loop is important for capsid size determination, likely through intra-subunit interactions. Ten of eighteen coat protein temperature-sensitive-folding substitutions are in the I-domain, indicating its importance in folding and stability. Several are found on a positively charged face of the β-barrel that anchors the I-domain to a negatively charged surface of the coat protein HK97-core.

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

“…During capsid maturation, the D-loops are modeled to pull away from one another, breaking some of these interaction while maintaining interactions just at the D-loop tip (Fig. 1B) (19). In this study, we tested our structural model and showed that residues in the D-loop do play a crucial role in coat protein assembly into procapsids.…”

“…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).…”

“…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 as the D-loop (18,19,22).…”

“…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 as the D-loop (18,19,22). Fits of the I domain NMR structure into the cryo-electron microscopy (cryo-EM) density map for both the procapsid and virion suggest that the D-loop could form electrostatic interactions with I domain residues of the adjacent coat protein subunit across an icosahedral 2-fold axis of symmetry.…”

“…Fits of the I domain NMR structure into the cryo-electron microscopy (cryo-EM) density map for both the procapsid and virion suggest that the D-loop could form electrostatic interactions with I domain residues of the adjacent coat protein subunit across an icosahedral 2-fold axis of symmetry. Specifically, residues D244 and D246 at the tip of the D-loop were suggested to form salt bridges with R299 and R269, respectively (19). Furthermore, the interactions between D-loops in the procapsid appear extensive (Fig.…”