Transient Contacts on the Exterior of the HK97 Procapsid That Are Essential for Capsid Assembly

Abstract

The G-loop is a 10-residue glycine-rich loop that protrudes from the surface of the mature bacteriophage HK97 capsid at the C-terminal end of the long backbone helix of major capsid protein subunits. The G-loop is essential for assembly, is conserved in related capsid and encapsulin proteins, and plays its role during HK97 capsid assembly by making crucial contacts between the hill-like hexamers and pentamers in precursor proheads. These contacts are not preserved in the flattened capsomers of the mature capsid. Aspartate 231 in each of the ~400 G-loops interacts with Lysine 178 of the E-loop (Extended Loop) of a subunit on an adjacent capsomer. Mutations disrupting this interaction prevented correct assembly, and in some cases induced abnormal assembly into tubes, or small, incomplete capsids. Assembly remained defective when D231 and K178 were replaced with larger charged residues or when their positions were exchanged. Second-site suppressors of lethal mutants containing substitution D231L replaced the ionic interaction with new interactions between neutral or hydrophobic residues of about the same size: D231L/K178V, D231L/K178I, and D231L/K178N. We conclude that it is not the charge, but the size and shape of the side chains of residues 178 and 231 that are important. These two residues control the geometry of contacts between the E-loop and the G-loop, which apparently must be precisely spaced and oriented for correct assembly to occur. We present a model for how the G-loop could control HK97 assembly and identify G-loop-like protrusions in other capsid proteins that may play analogous roles.

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

“…Recently, the coat protein of HK97 and related phages was shown to have a glycine rich “G-loop” that also reaches across capsomers to interact with the E-loop of an adjacent subunit. Unlike the flexible D-loop in P22’s coat protein, the HK97 G-loop appears to be rigid and is proposed to limit subunit conformational flexibility during capsomer assembly (Tso et al, 2014). The role of the D-loop in P22 procapsid assembly is not well defined and is being investigated currently.…”

Flexible Connectors between Capsomer Subunits that Regulate Capsid Assembly

“…SDS-polyacrylamide gels (Figure 3B) confirmed that the bulk of the major capsid protein from the four mutants was in the pellet fractions, where it was present as 42 kDa monomers and as ladders of crosslinked 42 kDa monomers. In normal HK97 assembly, crosslinking only occurs as a late step as Prohead II expands to become Head II [21], well after cleavage of the major capsid protein to 31kDa (Figure 1A), so the presence of crosslinked 42 kDa protein ladders, as observed here, usually indicates that abnormal assembly has occurred [4]. Accordingly, we examined the pellet fractions of the E153 mutants by electron microscopy.…”

“…Under these conditions, expression of HK97 capsid components is so efficient, that they can be readily analyzed by electrophoresis of unpurified extracts [4, 22]. To aid in the interpretation of these experiments, we included mutant K92A as a control that yields a wide spectrum of HK97 assembly intermediates to serve as markers for proheads, heads and capsomers.…”

“…This means that it is the earliest prohead structures that need to be studied in order to understand how capsid size and shape are regulated, although clues may remain in mature capsids. In support of this, we have recently shown that transient inter-capsomer contacts between pairs of residues on the outside of the bacteriophage HK97 prohead (D231 and K178) are essential for correct assembly, even though these same residues move ~20 Å apart in the mature capsid [4]. This was surprising, even though it has long been known from early work on phage T4 [5, 6] that capsid maturation involved large rearrangements of structural elements.…”

“…Structure-informed mutational studies have revealed several residues participating in ionic interactions that are important in regulating assembly [18, 19] [12, 20] [4]. We have focused on identifying additional ionic interactions because they are easily identified and because HK97 Prohead I can be disassembled by treatment with high salt [19], suggesting that ionic interactions are important at the beginning of assembly.…”