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* This work was supported, in whole or in part, by National Institutes of Health Grant R01 GM076661 (to C. M. T. and A. T. A.). The authors declare that they have no conflicts of interest with the contents of this article. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.
The I-domain is a genetic insertion in the phage P22 coat protein that chaperones its folding and stability. Of 11 acidic residues in the I-domain, seven participate in stabilizing electrostatic interactions with basic residues across elements of secondary structure, fastening the β-barrel fold. A hydrogen-bonded salt bridge between Asp-302 and His-305 is particularly interesting as Asp-302 is the site of a temperature-sensitive-folding mutation. The pKa of His-305 is raised to 9.0, indicating the salt bridge stabilizes the I-domain by ∼4 kcal/mol. Consistently, urea denaturation experiments indicate the stability of the WT I-domain decreases by 4 kcal/mol between neutral and basic pH. The mutants D302A and H305A remove the pH dependence of stability. The D302A substitution destabilizes the I-domain by 4 kcal/mol, whereas H305A had smaller effects, on the order of 1–2 kcal/mol. The destabilizing effects of D302A are perpetuated in the full-length coat protein as shown by a higher sensitivity to protease digestion, decreased procapsid assembly rates, and impaired phage production in vivo. By contrast, the mutants have only minor effects on capsid expansion or stability in vitro. The effects of the Asp-302–His-305 salt bridge are thus complex and context-dependent. Substitutions that abolish the salt bridge destabilize coat protein monomers and impair capsid self-assembly, but once capsids are formed the effects of the substitutions are overcome by new quaternary interactions between subunits.
Virus and phage coat proteins requisitely encounter the dilemma of balancing counteracting forces during folding and assembly. For icosahedral particles built from multiple copies of a single coat protein and a triangulation number (T) >1, subunits have to adopt pseudo-symmetric conformations dependent on their position in the capsid (
). Counteracting the needs for flexibility and protection of interaction surfaces until assembly is complete is the requirement that capsid proteins be folded into assembly-competent structures. Despite the potential difficulties inherent in the folding of coat proteins, the resulting capsids are naturally extremely stable and provide excellent platforms for nanoengineering (
). The economical architecture of icosahedral symmetry has been applied to simultaneously display a multiplicity of cargo molecules on the surfaces of capsids, enabling the design of nanomaterials with unique properties (
). In its simplest form, the P22 procapsid has 420 copies of coat protein arranged in a T = 7 (T = triangulation number) icosahedral shell with 100–300 copies of the scaffolding protein bound within its confines with unknown symmetry (
). The large stability of the procapsid is due to an extensive network of non-covalent contacts between coat protein subunits. During phage morphogenesis, procapsids mature concomitant with genome packaging (
). Moreover, procapsids and capsids can be manipulated in vivo and in vitro. To be able to fully exploit P22 capsids for the design of nanomaterials, an understanding the particle's stability properties is critical, as there are many examples where single amino acid substitutions in coat protein can affect self-assembly and/or disassembly (
) consists of a six-stranded β-barrel fold (strands β1-β6) and a smaller sub-domain (strands βi–βiii together with helix αi). Several ion-pair interactions fasten the β-strands in the I-domain structure (Fig. 1A, Table 1). Of these, the Asp-302–His-305 salt bridge is of particular interest because the D302G mutation has been identified as a tsf mutant (
), suggesting this site is particularly important for proper coat protein folding. Here we show the salt bridge between Asp-302 and His-305 contributes significantly to the stability of the I-domain, such that the unfolding free energy change for the I-domain decreases by ∼50% between neutral and basic pH, as His-305 becomes deprotonated.
Uncertainties in pKa values are given as the S.E. of non-linear least square fits of the chemical shift data as a function of pH (e.g. Fig. 1, B and C). The minimum error in the pKa, however, is likely to be limited by the accuracy of the pH meter, which is 0.1 pH units.
We use the terminology “salt bridge” for a hydrogen-bonded ion pair that has the closest approach distance between the side-chain NH hydrogen-bond donor and O carbonyl acceptor atoms shorter than 2.5 Å in the NMR structure closest to the ensemble mean (structure 1 in the ensemble of PDB code 2M5S). The column gives the fraction of structures in the NMR ensemble of 30 structures (32) in which the side chains of the ion-pair are hydrogen-bonded.
Based on differences of pKa values in the I-domain from the following random-coil values (43): histidine = 6.5, aspartate = 4.0; glutamate = 4.4. An interaction is considered stabilizing if the pKa shift is greater than 1 pH unit, slightly stabilizing if the shift is between 0.5 and 1 pH unit and negligible if it is <0.5 pH.
a Uncertainties in pKa values are given as the S.E. of non-linear least square fits of the chemical shift data as a function of pH (e.g.Fig. 1, B and C). The minimum error in the pKa, however, is likely to be limited by the accuracy of the pH meter, which is 0.1 pH units.
b We use the terminology “salt bridge” for a hydrogen-bonded ion pair that has the closest approach distance between the side-chain NH hydrogen-bond donor and O carbonyl acceptor atoms shorter than 2.5 Å in the NMR structure closest to the ensemble mean (structure 1 in the ensemble of PDB code 2M5S). The column gives the fraction of structures in the NMR ensemble of 30 structures (
): histidine = 6.5, aspartate = 4.0; glutamate = 4.4. An interaction is considered stabilizing if the pKa shift is greater than 1 pH unit, slightly stabilizing if the shift is between 0.5 and 1 pH unit and negligible if it is <0.5 pH.
e Resonances for Asp-244 were not observed in the NMR spectrum due to conformational exchange line-broadening (
The role of the salt bridge in the I-domain, full-length coat protein, and assembled P22 procapsids is further examined by substituting alanine at both the Asp-302 and His-305 sites. Substitution of Asp-302 with alanine destabilizes the I-domain, increases the susceptibility of the full-length coat protein monomers to proteolytic digestion, and causes a tsf phenotype leading to impaired phage assembly in vivo. The H305A I-domain mutation has smaller effects than D302A, possibly due to partial compensation of the loss of the Asp-302–His-305 salt bridge by the substitution of the histidine with a smaller alanine side chain. Although the mutations destabilize coat protein monomers leading to assembly defects, they have only minor effects on the heat expansion and urea denaturation of assembled procapsids, as the contributions of the mutations are overcome by new inter-capsomer quaternary interactions.
The size of a virus genome is constrained by the size of its capsid. Thus, there is evolutionary pressure against genome size expansion. The insertion of the I-domain into gene 5 adds ∼375 base pairs (bp) to the 43,400-bp DNA of the P22 genome (
). Thus, we infer the addition of the I-domain must bestow evolutionary advantages that compensate for the higher DNA packing density resulting from its insertion. 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 ΔG of stabilization for monomeric coat protein (
). From these data the I-domain clearly has several critical roles in coat protein folding and assembly. Here, we asked how the I-domain itself is stabilized and the extent to which interactions that stabilize the I-domain module are important for the stability of the full-length coat protein and its resulting capsid assemblies.
Salt bridges and ion-pairs in the I-domain link secondary structure elements that form the six-stranded β-barrel structure of this module (Fig. 1A, Table 1). Based on the present work, the salt bridge between Asp-302 and His-305 is especially important for the stability of the I-domain. However, replacement of the histidine does not destabilize the I-domain as much as substituting the aspartate in the salt bridge. A possible explanation for the greater loss of stability with the D302A mutant is that aspartate is preferred compared with alanine at the first position in a β-turn (
). Thus, the alanine substitution would not be accommodated as well as an aspartate at position 302, perhaps accounting for the increased dynamics observed for the Asp-302–His-305 β-hairpin in the D302A mutant (Fig. 4A). This explanation, however, cannot account for the ∼4-kcal loss in stability experienced by the WT I-domain between physiological and basic pH. The loss of stability with the D302A substitution matches that when the WT salt bridge is disrupted by basic pH. Rather, the 1–2 kcal/mol decrease in stability for the H305A mutation is smaller than what would be expected if the mutation only disrupted the Asp-302–His-305 salt bridge. These observations suggest that the loss of the salt bridge in the H305A mutation is partially compensated by the alanine substitution. His-305 forms close contacts with Thr-258 and Thr-260 from strand β2 in the NMR structure of the I-domain. Unfavorable contacts such as steric clashes with the threonines or other residues in the I-domain may be relieved when the histidine is replaced by a smaller alanine side chain. Alternatively, the alanine substitution could be disfavored in the denatured state, raising its free energy and thereby conferring stability to the native state (
Both residues are favorable for the first position of a reverse turn. The histidine at position 305 is not conserved and is substituted by residues aspartate or asparagine. Thus, smaller side chains may be preferred at position 305 in lieu of a histidine ring. Nevertheless, in all cases the homologous residues have the potential for H-bonding. The fact that His-305 is not conserved suggests that its function is specific to phage P22. In this regard it is interesting to note, however, that recent NMR work on the I-domain from the distantly related phage CUS-3 identified a proton bound to an imidazole nitrogen atom on the aromatic ring of His-277 with an unusual shift of 11.75 ppm (
). For a histidine ring Nδ1/Nϵ2 proton to be protected and to have such an unusual shift strongly suggests that it is involved in an important stabilizing hydrogen-bonding interaction. His-277 in CUS-3 titrates with a pKa of 8.3, which from Equation 4 predicts a stabilizing contribution of ∼2.5 kcal/mol from its charged state, similar to that of His-305 in the I-domain from phage P22. Thus, although His-277 is not a sequence or structural homolog to His-305 in the P22 I-domain, it may nevertheless modulate the pH dependence of the CUS3 I-domain in a similar way, boosting its stability near physiological pH.
In summary, we have shown that the stability contributions of the Asp-302–His-305 salt bridge are context-dependent in the hierarchy of structural complexity going from the I-domain module to the coat protein monomer building block to the very stable capsid that protects the phage genome. Loss of the Asp-302–His-305 salt bridge at high pH as well as the D302A substitution destabilizes the I-domain module (Fig. 2B) and thereby the coat protein monomer (Fig. 5B), leading to impaired procapsid assembly (Fig. 5C) and a decrease in production of phage in vivo (Fig. 5D). By contrast, once icosahedral particles are assembled, loss of the salt bridge has relatively minor effects on the stability of procapsids to heat expansion (Fig. 6A) or urea denaturation (Fig. 6B). The position of the Asp-302–His-305 salt bridge in the context of the procapsid structure is illustrated in Fig. 7A, which shows the asymmetric unit of the phage P22 T = 7 (T = triangulation number) icosahedron. The salt bridge is located on the surface of the procapsid and is isolated from other coat-protein monomers related by the 6-fold symmetry axis. Interestingly, the salt bridges of adjacent monomers come in close proximity along the dyad 2-fold symmetry axis illustrated by the blue and brown coat protein monomers in Fig. 7B, with the Asp-302 and His-305 residues seemingly poised to form subunit-swapped salt bridges between coat protein monomers. The D-loops, which occur just below the Asp-302–His-305 salt bridge in the view of the I-domain structure shown in Fig. 1A, form intercapsomer salt bridges that stabilize the procapsid along the same 2-fold symmetry axis, shown in Fig. 7B (
). Clearly the network of charged residues brought together in the assembled procapsids would lead to more complex electrostatic interactions than in coat protein monomers or the isolated I-domain. The stability of the capsid shell will depend on the totality of tertiary and quaternary interactions formed by its coat protein subunits. New interactions formed in the procapsid, including the intersubunit salt bridges formed by the D-loops (
) as well as other types of polar and non-polar interactions, apparently override the loss in stability caused by disruption of the Asp-302–His-305 salt bridge. Thus the effects of the D302A mutation on procapsid denaturation are not commensurate with those on procapsid assembly. For the design of nanomaterials based on phage particles or other types of supramolecular assemblies, this implies that perturbations in the building blocks may not be additive in the assembled product, and that changes in subunits could have different consequences on assembly and disassembly pathways.
C. H., K. J. R., and A. T. A. conducted the NMR experiments, C. H. performed the I-domain urea denaturation experiments, and O. O. and T. M. did the phage and coat protein experiments. All authors contributed to the analysis of the data. C. M. T. and A. T A. wrote the manuscript and designed the project.
We thank Latasha C. R. Fraser and Dr. Margaret Suhanovsky for data on the pH dependence of ΔGu0 for the WT I-domain, Therese Tripler for an NMR sample of the CUS-3 I-domain, and the students of MCB5896, Practicum in NMR Spectroscopy, for performing the NMR pH titrations of histidines in the I-domains of phage P22 and CUS-3.
Physical principles in the construction of regular viruses.
Cold Spring Harbor Symp. Quant. Biol.1962; 27: 1-24