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

  • Christina Harprecht
    Affiliations
    Department of Molecular and Cell Biology and Department of Chemistry, University of Connecticut, Storrs, Connecticut 06269
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  • Oghenefejiro Okifo
    Affiliations
    Department of Molecular and Cell Biology and Department of Chemistry, University of Connecticut, Storrs, Connecticut 06269
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  • Kevin J. Robbins
    Affiliations
    Department of Molecular and Cell Biology and Department of Chemistry, University of Connecticut, Storrs, Connecticut 06269
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  • Tina Motwani
    Affiliations
    Department of Molecular and Cell Biology and Department of Chemistry, University of Connecticut, Storrs, Connecticut 06269
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  • Andrei T. Alexandrescu
    Correspondence
    To whom correspondence may be addressed. Tel.: 860-486-4414; Fax: 860-486-4331.
    Affiliations
    Department of Molecular and Cell Biology and Department of Chemistry, University of Connecticut, Storrs, Connecticut 06269
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  • Carolyn M. Teschke
    Correspondence
    To whom correspondence may be addressed. Tel.: 860-486-3992; Fax: 860- 486-4331.
    Affiliations
    Department of Molecular and Cell Biology and Department of Chemistry, University of Connecticut, Storrs, Connecticut 06269
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  • Author Footnotes
    * 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.
Open AccessPublished:March 22, 2016DOI:https://doi.org/10.1074/jbc.M116.716910
      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.

      Introduction

      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 (
      • Caspar D.L.D.
      • Klug A.
      Physical principles in the construction of regular viruses.
      ). 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 (
      • Liu Z.
      • Qiao J.
      • Niu Z.
      • Wang Q.
      Natural supramolecular building blocks: from virus coat proteins to viral nanoparticles.
      ). 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 (
      • Parent K.N.
      • Deedas C.T.
      • Egelman E.H.
      • Casjens S.R.
      • Baker T.S.
      • Teschke C.M.
      Stepwise molecular display utilizing icosahedral and helical complexes of phage coat and decoration proteins in the development of robust nanoscale display vehicles.
      ,
      • Flenniken M.L.
      • Uchida M.
      • Liepold L.O.
      • Kang S.
      • Young M.J.
      • Douglas T.
      A library of protein cage architectures as nanomaterials.
      ), including nanoparticle scaffolds based on phage P22 (
      • Kang S.
      • Uchida M.
      • O'Neil A.
      • Li R.
      • Prevelige P.E.
      • Douglas T.
      Implementation of p22 viral capsids as nanoplatforms.
      ,
      • Patterson D.P.
      • Prevelige P.E.
      • Douglas T.
      Nanoreactors by programmed enzyme encapsulation inside the capsid of the bacteriophage P22.
      ,
      • O'Neil A.
      • Prevelige P.E.
      • Basu G.
      • Douglas T.
      Coconfinement of fluorescent proteins: spatially enforced communication of GFP and mCherry encapsulated within the P22 capsid.
      ).
      P22 coat protein first assembles into a metastable precursor capsid, known as a procapsid, in a process driven by its scaffolding protein, which serves as an assembly chaperone (
      • King J.
      • Lenk E.V.
      • Botstein D.
      Mechanism of head assembly and DNA encapsulation in Salmonella phage P22 II: morphogenetic pathway.
      ). 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 (
      • Prevelige Jr., P.E.
      • Thomas D.
      • King J.
      Scaffolding protein regulates the polymerization of P22 coat subunits into icosahedral shells in vitro.
      ). The procapsid has an overall stability of ∼3000 kcal/mol, although individual coat protein subunits are only marginally stable (
      • Zlotnick A.
      • Suhanovsky M.M.
      • Teschke C.M.
      The energetic contributions of scaffolding and coat proteins to the assembly of bacteriophage procapsids.
      ,
      • Parent K.N.
      • Zlotnick A.
      • Teschke C.M.
      Quantitative analysis of multi-component spherical virus assembly: Scaffolding protein contributes to the global stability of phage P22 procapsids.
      ). 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 (
      • King J.
      • Botstein D.
      • Casjens S.
      • Earnshaw W.
      • Harrison S.
      • Lenk E.
      Structure and assembly of the capsid of bacteriophage P22.
      ). Maturation increases the particle diameter by 10% (
      • Zhang Z.
      • Greene B.
      • Thuman-Commike P.A.
      • Jakana J.
      • Prevelige Jr., P.E.
      • King J.
      • Chiu W.
      Visualization of the maturation transition in bacteriophage P22 by electron cryomicroscopy.
      ), stabilizes the icosahedral shell (
      • Galisteo M.L.
      • King J.
      Conformational transformations in the protein lattice of phage P22 procapsids.
      ), and induces the release of scaffolding protein (
      • King J.
      • Hall C.
      • Casjens S.
      Control of the synthesis of phage P22 scaffolding protein is coupled to capsid assembly.
      ). The maturation from procapsid to capsid can be recapitulated in vitro by heating the particles (
      • Galisteo M.L.
      • King J.
      Conformational transformations in the protein lattice of phage P22 procapsids.
      ,
      • Teschke C.M.
      • McGough A.
      • Thuman-Commike P.A.
      Penton release from P22 heat-expanded capsids suggests importance of stabilizing penton-hexon interactions during capsid maturation.
      ). Heat expansion can lead to the release of the penton subunits, yielding a “wiffle ball” form of the capsid (
      • Teschke C.M.
      • McGough A.
      • Thuman-Commike P.A.
      Penton release from P22 heat-expanded capsids suggests importance of stabilizing penton-hexon interactions during capsid maturation.
      ,
      • Li Y.
      • Conway J.F.
      • Cheng N.
      • Steven A.C.
      • Hendrix R.W.
      • Duda R.L.
      Control of virus assembly: HK97 “Whiffleball” mutant capsids without pentons.
      ). Phage P22 is an extremely attractive platform for nanomaterial design (
      • Kang S.
      • Uchida M.
      • O'Neil A.
      • Li R.
      • Prevelige P.E.
      • Douglas T.
      Implementation of p22 viral capsids as nanoplatforms.
      ,
      • Patterson D.P.
      • Prevelige P.E.
      • Douglas T.
      Nanoreactors by programmed enzyme encapsulation inside the capsid of the bacteriophage P22.
      ,
      • O'Neil A.
      • Prevelige P.E.
      • Basu G.
      • Douglas T.
      Coconfinement of fluorescent proteins: spatially enforced communication of GFP and mCherry encapsulated within the P22 capsid.
      ) because it is a structurally characterized virus (
      • Suhanovsky M.M.
      • Teschke C.M.
      Nature's favorite building block: deciphering folding and capsid assembly of proteins with the HK97-fold.
      ,
      • Rizzo A.A.
      • Fraser L.C.
      • Sheftic S.R.
      • Suhanovsky M.M.
      • Teschke C.M.
      • Alexandrescu A.T.
      NMR assignments for the telokin-like domain of bacteriophage P22 coat protein.
      ,
      • Parent K.N.
      • Sinkovits R.S.
      • Suhanovsky M.M.
      • Teschke C.M.
      • Egelman E.H.
      • Baker T.S.
      Cryo-reconstructions of P22 polyheads suggest that phage assembly is nucleated by trimeric interactions among coat proteins.
      ,
      • Parent K.N.
      • Khayat R.
      • Tu L.H.
      • Suhanovsky M.M.
      • Cortines J.R.
      • Teschke C.M.
      • Johnson J.E.
      • Baker T.S.
      P22 coat protein structures reveal a novel mechanism for capsid maturation: stability without auxiliary proteins or chemical cross-links.
      ,
      • Cortines J.R.
      • Motwani T.
      • Vyas A.A.
      • Teschke C.M.
      Highly specific salt bridges govern bacteriophage P22 icosahedral capsid assembly: identification of the site in coat protein responsible for interaction with scaffolding protein.
      ,
      • Padilla-Meier G.P.
      • Gilcrease E.B.
      • Weigele P.R.
      • Cortines J.R.
      • Siegel M.
      • Leavitt J.C.
      • Teschke C.M.
      • Casjens S.R.
      Unraveling the role of the C-terminal helix turn helix of the coat-binding domain of bacteriophage P22 scaffolding protein.
      ,
      • Padilla-Meier G.P.
      • Teschke C.M.
      Conformational changes in bacteriophage P22 scaffolding protein induced by interaction with coat protein.
      ,
      • Cortines J.R.
      • Weigele P.R.
      • Gilcrease E.B.
      • Casjens S.R.
      • Teschke C.M.
      Decoding bacteriophage P22 assembly: identification of two charged residues in scaffolding protein responsible for coat protein interaction.
      ), with well understood genetics, assembly, and maturation (
      • Teschke C.M.
      • Parent K.N.
      “Let the phage do the work”: using the phage P22 coat protein structures as a framework to understand its folding and assembly mutants.
      ). 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 (
      • Li Y.
      • Conway J.F.
      • Cheng N.
      • Steven A.C.
      • Hendrix R.W.
      • Duda R.L.
      Control of virus assembly: HK97 “Whiffleball” mutant capsids without pentons.
      ,
      • Capen C.M.
      • Teschke C.M.
      Folding defects caused by single amino acid substitutions in a subunit are not alleviated by assembly.
      ,
      • Foguel D.
      • Teschke C.M.
      • Prevelige Jr., P.E.
      • Silva J.L.
      Role of entropic interactions in viral capsids: single amino acid substitutions in P22 bacteriophage coat protein resulting in loss of capsid stability.
      ,
      • Parent K.N.
      • Suhanovsky M.M.
      • Teschke C.M.
      Polyhead formation in phage P22 pinpoints a region in coat protein required for conformational switching.
      ).
      The P22 coat protein (430 amino acids) has a core structure based on the HK97 fold (
      • Wikoff W.R.
      • Liljas L.
      • Duda R.L.
      • Tsuruta H.
      • Hendrix R.W.
      • Johnson J.E.
      Topologically linked protein rings in the bacteriophage HK97 capsid.
      ) but has an additional genetic insertion, the 123-aminoacyl I-domain (
      • Rizzo A.A.
      • Fraser L.C.
      • Sheftic S.R.
      • Suhanovsky M.M.
      • Teschke C.M.
      • Alexandrescu A.T.
      NMR assignments for the telokin-like domain of bacteriophage P22 coat protein.
      ,
      • Parent K.N.
      • Sinkovits R.S.
      • Suhanovsky M.M.
      • Teschke C.M.
      • Egelman E.H.
      • Baker T.S.
      Cryo-reconstructions of P22 polyheads suggest that phage assembly is nucleated by trimeric interactions among coat proteins.
      ). The I-domain folds very rapidly and stabilizes full-length coat protein, suggesting it serves as an uncleaved intermolecular chaperone and the folding nucleus of the protein (
      • Suhanovsky M.M.
      • Teschke C.M.
      An Intramolecular chaperone inserted in bacteriophage P22 coat protein mediates its chaperonin-independent folding.
      ). Over half of the known temperature-sensitive-folding (tsf) coat protein mutants are localized in the I-domain, attesting to the importance of this module in modulating folding (
      • Rizzo A.A.
      • Suhanovsky M.M.
      • Baker M.L.
      • Fraser L.C.
      • Jones L.M.
      • Rempel D.L.
      • Gross M.L.
      • Chiu W.
      • Alexandrescu A.T.
      • Teschke C.M.
      Multiple functional roles of the accessory I-domain of bacteriophage P22 coat protein revealed by NMR structure and CryoEM modeling.
      ). The NMR structure of the I-domain (
      • Rizzo A.A.
      • Suhanovsky M.M.
      • Baker M.L.
      • Fraser L.C.
      • Jones L.M.
      • Rempel D.L.
      • Gross M.L.
      • Chiu W.
      • Alexandrescu A.T.
      • Teschke C.M.
      Multiple functional roles of the accessory I-domain of bacteriophage P22 coat protein revealed by NMR structure and CryoEM modeling.
      ) 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 (
      • Gordon C.L.
      • King J.
      Genetic properties of temperature-sensitive folding mutants of the coat protein of phage P22.
      ), 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.
      Figure thumbnail gr1
      FIGURE 1Ion-pair interactions in the I-domain. A, stereo diagram of the I-domain NMR structure showing charged residues. Acidic and basic residues that do not participate in ion pairs are colored orange and cyan, respectively. Residues that participate in ion pairs () are labeled red and blue for acidic and basic amino acids, respectively. Hydrogen bonds that link ion pairs in a salt bridge (Asp-302–His-305, Asp-253–Lys-311, Asp-271–Lys-268, Asp-317–Lys-237) are indicated by dashed green lines. B, representative NMR pH titration data for aspartate Hβ protons. Asp-246 is in the disordered D-loop and has a random coil pKa. Asp-316 forms an ion pair with Arg-325 and has the lowest pKa measured in the I-domain. Asp-302 and Asp-317 form hydrogen-bonded salt bridges () and do not shift with pH. C, NMR pH titration of histidine Hϵ1 protons. The unresolved resonances from the His6 tag used for purification gave a pKa of 6.7, in agreement with the random coil histidine value (
      • Croke R.L.
      • Patil S.M.
      • Quevreaux J.
      • Kendall D.A.
      • Alexandrescu A.T.
      NMR determination of pKa values in α-synuclein.
      ). By contrast His-305 had a pKa value shifted up by 2.5 pH units, consistent with the residue forming a stabilizing salt bridge with Asp-302.
      TABLE 1I-domain ion-pairs and pKa values
      ResiduepKa
      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.
      Ion-pair partnerSalt bridge
      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.
      d(O … HN)
      Nearest approach NH to O side-chain distance in the NMR structure of the I-domain closest to the ensemble average.
      Structure contextEnergetics
      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.
      %Å
      Asp-244
      Resonances for Asp-244 were not observed in the NMR spectrum due to conformational exchange line-broadening (19).
      NA
      NA, not applicable.
      None0NAD-loopNA
      Asp-2464.2 ± 0.1None0NAD-loopNull
      Asp-253<2.8Lys-311601.7Links β2-β5Stabilizing
      Asp-271<2.8Lys-268531.7Turn before β3Stabilizing
      Asp-292<2.8Lys-279373.4Links β4-βiStabilizing
      Asp-302<2.8His-305432.2β4-β5 hairpinStabilizing
      His-3059.05 ± 0.04Asp-302See Asp-302See Asp-302see Asp-302Stabilizing
      Glu-3073.9 ± 0.3Arg-299105.3Links β5-β4Slight stabilizing
      Asp-3163.0 ± 0.3Arg-32506.0Links βii-αiSlight stabilizing
      Asp-317< 2.8Lys-237971.7Links βii-β1Stabilizing
      Glu-3233.6 ± 0.1Unknown0NAUnknownSlight stabilizing
      Asp-3364.3 ± 0.3None0NASurface β6Null
      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 (
      • Rizzo A.A.
      • Suhanovsky M.M.
      • Baker M.L.
      • Fraser L.C.
      • Jones L.M.
      • Rempel D.L.
      • Gross M.L.
      • Chiu W.
      • Alexandrescu A.T.
      • Teschke C.M.
      Multiple functional roles of the accessory I-domain of bacteriophage P22 coat protein revealed by NMR structure and CryoEM modeling.
      ) in which the side chains of the ion-pair are hydrogen-bonded.
      c Nearest approach NH to O side-chain distance in the NMR structure of the I-domain closest to the ensemble average.
      d Based on differences of pKa values in the I-domain from the following random-coil values (
      • Croke R.L.
      • Patil S.M.
      • Quevreaux J.
      • Kendall D.A.
      • Alexandrescu A.T.
      NMR determination of pKa values in α-synuclein.
      ): 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 (
      • Rizzo A.A.
      • Fraser L.C.
      • Sheftic S.R.
      • Suhanovsky M.M.
      • Teschke C.M.
      • Alexandrescu A.T.
      NMR assignments for the telokin-like domain of bacteriophage P22 coat protein.
      ).
      f NA, not applicable.
      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.

      Discussion

      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 (
      • Casjens S.
      • Hayden M.
      Analysis in vivo of the bacteriophage P22 headful nuclease.
      ) or ∼1% of the total genome length. Athough P22 mutants have been found that over-package DNA by ∼2000 bp, their capsids are fragile compared with those of WT phage (
      • Casjens S.
      • Wyckoff E.
      • Hayden M.
      • Sampson L.
      • Eppler K.
      • Randall S.
      • Moreno E.T.
      • Serwer P.
      Bacteriophage P22 portal protein is part of the gauge that regulates packing density of intravirion DNA.
      ). 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 (
      • Suhanovsky M.M.
      • Teschke C.M.
      An Intramolecular chaperone inserted in bacteriophage P22 coat protein mediates its chaperonin-independent folding.
      ). Additionally, the I-domain D-loop contributes to procapsid assembly by making critical inter-capsomer interactions across the icosahedral 2-fold symmetry-axis (
      • Rizzo A.A.
      • Suhanovsky M.M.
      • Baker M.L.
      • Fraser L.C.
      • Jones L.M.
      • Rempel D.L.
      • Gross M.L.
      • Chiu W.
      • Alexandrescu A.T.
      • Teschke C.M.
      Multiple functional roles of the accessory I-domain of bacteriophage P22 coat protein revealed by NMR structure and CryoEM modeling.
      ,
      • D'Lima N.G.
      • Teschke C.M.
      A molecular staple: D-loops in the I-domain of bacteriophage P22 coat protein make important intercapsomer contacts required for procapsid assembly.
      ). 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 (
      • Creighton T.E.
      Proteins: Structures and Molecular Properties.
      ). 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 (
      • Shortle D.
      The denatured state (the other half of the folding equation) and its role in protein stability.
      ).
      An analysis of coat protein sequences of phages related to P22 showed that in homologs with ∼60–70% sequence identity, position 302 is occupied by either an aspartate or asparagine.
      S. Casjens, personal communication.
      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 (
      • Tripler T.N.
      • Maciejewski M.W.
      • Teschke C.M.
      • Alexandrescu A.T.
      NMR assignments for the insertion domain of bacteriophage CUS-3 coat protein.
      ). 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 (
      • Rizzo A.A.
      • Suhanovsky M.M.
      • Baker M.L.
      • Fraser L.C.
      • Jones L.M.
      • Rempel D.L.
      • Gross M.L.
      • Chiu W.
      • Alexandrescu A.T.
      • Teschke C.M.
      Multiple functional roles of the accessory I-domain of bacteriophage P22 coat protein revealed by NMR structure and CryoEM modeling.
      ,
      • D'Lima N.G.
      • Teschke C.M.
      A molecular staple: D-loops in the I-domain of bacteriophage P22 coat protein make important intercapsomer contacts required for procapsid assembly.
      ). 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 (
      • Rizzo A.A.
      • Suhanovsky M.M.
      • Baker M.L.
      • Fraser L.C.
      • Jones L.M.
      • Rempel D.L.
      • Gross M.L.
      • Chiu W.
      • Alexandrescu A.T.
      • Teschke C.M.
      Multiple functional roles of the accessory I-domain of bacteriophage P22 coat protein revealed by NMR structure and CryoEM modeling.
      ,
      • D'Lima N.G.
      • Teschke C.M.
      A molecular staple: D-loops in the I-domain of bacteriophage P22 coat protein make important intercapsomer contacts required for procapsid assembly.
      ) 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.
      Figure thumbnail gr7
      FIGURE 7The Asp-302–His-305 salt bridge in the context of the capsid structure. A, location of Asp-302–His-305 salt bridge on the capsid surface. The view on the left is from the outside looking at the surface of the capsid. The view on the right is along the width of the capsid shell. Subunits of the icosahedral T = 7 (T = triangulation number) capsid are shown in different colors, with Asp-302 in red and His-305 in blue space-filling spheres. B, view along the 2-fold of the asymmetric unit (corresponding to the blue and brown subunits in A). The Asp-302–His-305 salt bridge is poised to swap partners between subunits related by the dyad axis. The D-loops, which link monomers in the capsid through Asp-244–Arg-299, Asp-246–Arg-269, and Lys-249–Glu-81 salt bridges (
      • Rizzo A.A.
      • Suhanovsky M.M.
      • Baker M.L.
      • Fraser L.C.
      • Jones L.M.
      • Rempel D.L.
      • Gross M.L.
      • Chiu W.
      • Alexandrescu A.T.
      • Teschke C.M.
      Multiple functional roles of the accessory I-domain of bacteriophage P22 coat protein revealed by NMR structure and CryoEM modeling.
      ), are shown with thicker ribbons colored orange and turquoise.

      Author Contributions

      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.

      Acknowledgments

      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.

      References

        • Caspar D.L.D.
        • Klug A.
        Physical principles in the construction of regular viruses.
        Cold Spring Harbor Symp. Quant. Biol. 1962; 27: 1-24
        • Liu Z.
        • Qiao J.
        • Niu Z.
        • Wang Q.
        Natural supramolecular building blocks: from virus coat proteins to viral nanoparticles.
        Chem. Soc. Rev. 2012; 41: 6178-6194
        • Parent K.N.
        • Deedas C.T.
        • Egelman E.H.
        • Casjens S.R.
        • Baker T.S.
        • Teschke C.M.
        Stepwise molecular display utilizing icosahedral and helical complexes of phage coat and decoration proteins in the development of robust nanoscale display vehicles.
        Biomaterials. 2012; 33: 5628-5637
        • Flenniken M.L.
        • Uchida M.
        • Liepold L.O.
        • Kang S.
        • Young M.J.
        • Douglas T.
        A library of protein cage architectures as nanomaterials.
        Curr. Top Microbiol. Immunol. 2009; 327: 71-93
        • Kang S.
        • Uchida M.
        • O'Neil A.
        • Li R.
        • Prevelige P.E.
        • Douglas T.
        Implementation of p22 viral capsids as nanoplatforms.
        Biomacromolecules. 2010; 11: 2804-2809
        • Patterson D.P.
        • Prevelige P.E.
        • Douglas T.
        Nanoreactors by programmed enzyme encapsulation inside the capsid of the bacteriophage P22.
        ACS Nano. 2012; 6: 5000-5009
        • O'Neil A.
        • Prevelige P.E.
        • Basu G.
        • Douglas T.
        Coconfinement of fluorescent proteins: spatially enforced communication of GFP and mCherry encapsulated within the P22 capsid.
        Biomacromolecules. 2012; 13: 3902-3907
        • King J.
        • Lenk E.V.
        • Botstein D.
        Mechanism of head assembly and DNA encapsulation in Salmonella phage P22 II: morphogenetic pathway.
        J. Mol. Biol. 1973; 80: 697-731
        • Prevelige Jr., P.E.
        • Thomas D.
        • King J.
        Scaffolding protein regulates the polymerization of P22 coat subunits into icosahedral shells in vitro.
        J. Mol. Biol. 1988; 202: 743-757
        • Zlotnick A.
        • Suhanovsky M.M.
        • Teschke C.M.
        The energetic contributions of scaffolding and coat proteins to the assembly of bacteriophage procapsids.
        Virology. 2012; 428: 64-69
        • Parent K.N.
        • Zlotnick A.
        • Teschke C.M.
        Quantitative analysis of multi-component spherical virus assembly: Scaffolding protein contributes to the global stability of phage P22 procapsids.
        J. Mol. Biol. 2006; 359: 1097-1106
        • King J.
        • Botstein D.
        • Casjens S.
        • Earnshaw W.
        • Harrison S.
        • Lenk E.
        Structure and assembly of the capsid of bacteriophage P22.
        Philos. Trans. R. Soc. Lond. B. Biol. Sci. 1976; 276: 37-49
        • Zhang Z.
        • Greene B.
        • Thuman-Commike P.A.
        • Jakana J.
        • Prevelige Jr., P.E.
        • King J.
        • Chiu W.
        Visualization of the maturation transition in bacteriophage P22 by electron cryomicroscopy.
        J. Mol. Biol. 2000; 297: 615-626
        • Galisteo M.L.
        • King J.
        Conformational transformations in the protein lattice of phage P22 procapsids.
        Biophys. J. 1993; 65: 227-235
        • King J.
        • Hall C.
        • Casjens S.
        Control of the synthesis of phage P22 scaffolding protein is coupled to capsid assembly.
        Cell. 1978; 15: 551-560
        • Teschke C.M.
        • McGough A.
        • Thuman-Commike P.A.
        Penton release from P22 heat-expanded capsids suggests importance of stabilizing penton-hexon interactions during capsid maturation.
        Biophys. J. 2003; 84: 2585-2592
        • Li Y.
        • Conway J.F.
        • Cheng N.
        • Steven A.C.
        • Hendrix R.W.
        • Duda R.L.
        Control of virus assembly: HK97 “Whiffleball” mutant capsids without pentons.
        J. Mol. Biol. 2005; 348: 167-182
        • Suhanovsky M.M.
        • Teschke C.M.
        Nature's favorite building block: deciphering folding and capsid assembly of proteins with the HK97-fold.
        Virology. 2015; 479: 487-497
        • Rizzo A.A.
        • Fraser L.C.
        • Sheftic S.R.
        • Suhanovsky M.M.
        • Teschke C.M.
        • Alexandrescu A.T.
        NMR assignments for the telokin-like domain of bacteriophage P22 coat protein.
        Biomol. NMR Assign. 2013; 7: 257-260
        • Parent K.N.
        • Sinkovits R.S.
        • Suhanovsky M.M.
        • Teschke C.M.
        • Egelman E.H.
        • Baker T.S.
        Cryo-reconstructions of P22 polyheads suggest that phage assembly is nucleated by trimeric interactions among coat proteins.
        Phys. Biol. 2010; 7 (045004)
        • Parent K.N.
        • Khayat R.
        • Tu L.H.
        • Suhanovsky M.M.
        • Cortines J.R.
        • Teschke C.M.
        • Johnson J.E.
        • Baker T.S.
        P22 coat protein structures reveal a novel mechanism for capsid maturation: stability without auxiliary proteins or chemical cross-links.
        Structure. 2010; 18: 390-401
        • Cortines J.R.
        • Motwani T.
        • Vyas A.A.
        • Teschke C.M.
        Highly specific salt bridges govern bacteriophage P22 icosahedral capsid assembly: identification of the site in coat protein responsible for interaction with scaffolding protein.
        J. Virol. 2014; 88: 5287-5297
        • Padilla-Meier G.P.
        • Gilcrease E.B.
        • Weigele P.R.
        • Cortines J.R.
        • Siegel M.
        • Leavitt J.C.
        • Teschke C.M.
        • Casjens S.R.
        Unraveling the role of the C-terminal helix turn helix of the coat-binding domain of bacteriophage P22 scaffolding protein.
        J. Biol. Chem. 2012; 287: 33766-33780
        • Padilla-Meier G.P.
        • Teschke C.M.
        Conformational changes in bacteriophage P22 scaffolding protein induced by interaction with coat protein.
        J. Mol. Biol. 2011; 410: 226-240
        • Cortines J.R.
        • Weigele P.R.
        • Gilcrease E.B.
        • Casjens S.R.
        • Teschke C.M.
        Decoding bacteriophage P22 assembly: identification of two charged residues in scaffolding protein responsible for coat protein interaction.
        Virology. 2011; 421: 1-11
        • Teschke C.M.
        • Parent K.N.
        “Let the phage do the work”: using the phage P22 coat protein structures as a framework to understand its folding and assembly mutants.
        Virology. 2010; 401: 119-130
        • Capen C.M.
        • Teschke C.M.
        Folding defects caused by single amino acid substitutions in a subunit are not alleviated by assembly.
        Biochemistry. 2000; 39: 1142-1151
        • Foguel D.
        • Teschke C.M.
        • Prevelige Jr., P.E.
        • Silva J.L.
        Role of entropic interactions in viral capsids: single amino acid substitutions in P22 bacteriophage coat protein resulting in loss of capsid stability.
        Biochemistry. 1995; 34: 1120-1126
        • Parent K.N.
        • Suhanovsky M.M.
        • Teschke C.M.
        Polyhead formation in phage P22 pinpoints a region in coat protein required for conformational switching.
        Mol. Microbiol. 2007; 65: 1300-1310
        • Wikoff W.R.
        • Liljas L.
        • Duda R.L.
        • Tsuruta H.
        • Hendrix R.W.
        • Johnson J.E.
        Topologically linked protein rings in the bacteriophage HK97 capsid.
        Science. 2000; 289: 2129-2133
        • Suhanovsky M.M.
        • Teschke C.M.
        An Intramolecular chaperone inserted in bacteriophage P22 coat protein mediates its chaperonin-independent folding.
        J. Biol. Chem. 2013; 288: 33772-33783
        • Rizzo A.A.
        • Suhanovsky M.M.
        • Baker M.L.
        • Fraser L.C.
        • Jones L.M.
        • Rempel D.L.
        • Gross M.L.
        • Chiu W.
        • Alexandrescu A.T.
        • Teschke C.M.
        Multiple functional roles of the accessory I-domain of bacteriophage P22 coat protein revealed by NMR structure and CryoEM modeling.
        Structure. 2014; 22: 830-841
        • Gordon C.L.
        • King J.
        Genetic properties of temperature-sensitive folding mutants of the coat protein of phage P22.
        Genetics. 1994; 136: 427-438
        • Anderson E.
        • Teschke C.M.
        Folding of phage P22 coat protein monomers: kinetic and thermodynamic properties.
        Virology. 2003; 313: 184-197
        • Teschke C.M.
        • King J.
        Folding of the phage P22 coat protein in vitro.
        Biochemistry. 1993; 32: 10839-10847
        • D'Lima N.G.
        • Teschke C.M.
        A molecular staple: D-loops in the I-domain of bacteriophage P22 coat protein make important intercapsomer contacts required for procapsid assembly.
        J. Virol. 2015; 89: 10569-10579
        • Wishart D.S.
        • Bigam C.G.
        • Yao J.
        • Abildgaard F.
        • Dyson H.J.
        • Oldfield E.
        • Markley J.L.
        • Sykes B.D.
        1H, 13C and 15N chemical shift referencing in biomolecular NMR.
        J. Biomol. NMR. 1995; 6: 135-140
        • Alexandrescu A.T.
        • Shortle D.
        Backbone dynamics of a highly disordered 131 residue fragment of staphylococcal nuclease.
        J. Mol. Biol. 1994; 242: 527-546
        • Watson E.
        • Matousek W.M.
        • Irimies E.L.
        • Alexandrescu A.T.
        Partially folded states of staphylococcal nuclease highlight the conserved structural hierarchy of OB-fold proteins.
        Biochemistry. 2007; 46: 9484-9494
        • Alexandrescu A.T.
        • Jaravine V.A.
        • Dames S.A.
        • Lamour F.P.
        NMR hydrogen exchange of the OB-fold protein LysN as a function of denaturant: the most conserved elements of structure are the most stable to unfolding.
        J. Mol. Biol. 1999; 289: 1041-1054
        • Zhang Y.-Z.
        Protein and peptide structure and interactions studied by hydrogen exchange and NMR. University of Pennsylvania, 1995 (Ph.D. thesis)
        • Sheftic S.R.
        • Croke R.L.
        • LaRochelle J.R.
        • Alexandrescu A.T.
        Electrostatic contributions to the stabilities of native proteins and amyloid complexes.
        Methods Enzymol. 2009; 466: 233-258
        • Croke R.L.
        • Patil S.M.
        • Quevreaux J.
        • Kendall D.A.
        • Alexandrescu A.T.
        NMR determination of pKa values in α-synuclein.
        Protein Sci. 2011; 20: 256-269
        • Pace C.N.
        Determination and analysis of urea and guanidine hydrochloride denaturation curves.
        Methods Enzymol. 1986; 131: 266-280
        • Santoro M.M.
        • Bolen D.W.
        Unfolding free energy changes determined by linear extrapolation method. 1. Unfolding of phenylmethanesulfonyl α-chymotrypsin using different denaturants.
        Biochemistry. 1988; 27: 8063-8068
        • Schägger H.
        • von Jagow G.
        Tricine-sodium dodecyl sulfate-polyacrylamide gel electrophoresis for the separation of proteins in the range from 1 to 100 kDa.
        Anal. Biochem. 1987; 166: 368-379
        • Gordon C.L.
        • Sather S.K.
        • Casjens S.
        • King J.
        Selective in vivo rescue by GroEL/ES of thermolabile folding intermediates to phage P22 structural proteins.
        J. Biol. Chem. 1994; 269: 27941-27951
        • Winston F.
        • Botstein D.
        • Miller J.H.
        Characterization of amber and ochre suppressors in Salmonella typhimurium.
        J. Bacteriol. 1979; 137: 433-439
        • Parent K.N.
        • Suhanovsky M.M.
        • Teschke C.M.
        Phage P22 procapsids equilibrate with free coat protein subunits.
        J. Mol. Biol. 2007; 365: 513-522
        • Barlow D.J.
        • Thornton J.M.
        Ion-pairs in proteins.
        J. Mol. Biol. 1983; 168: 867-885
        • Kumar S.
        • Nussinov R.
        Relationship between ion pair geometries and electrostatic strengths in proteins.
        Biophys. J. 2002; 83: 1595-1612
        • Croke R.L.
        • Sallum C.O.
        • Watson E.
        • Watt E.D.
        • Alexandrescu A.T.
        Hydrogen exchange of monomeric α-synuclein shows unfolded structure persists at physiological temperature and is independent of molecular crowding in Escherichia coli.
        Protein Sci. 2008; 17: 1434-1445
        • Thurlkill R.L.
        • Grimsley G.R.
        • Scholtz J.M.
        • Pace C.N.
        pK values of the ionizable groups of proteins.
        Protein Sci. 2006; 15: 1214-1218
        • Lumb K.J.
        • Kim P.S.
        Measurements of interhelical electrostatic interactions in the GCN4 leucine zipper.
        Science. 1995; 268: 436-439
        • Bai Y.
        • Milne J.S.
        • Mayne L.
        • Englander S.W.
        Protein stability parameters measured by hydrogen exchange.
        Proteins. 1994; 20: 4-14
        • Bai Y.
        • Sosnick T.R.
        • Mayne L.
        • Englander S.W.
        Protein folding intermediates: native-state hydrogen exchange.
        Science. 1995; 269: 192-197
        • Newcomer R.L.
        • Fraser L.C.
        • Teschke C.M.
        • Alexandrescu A.T.
        Partial unfolding of the phage P22 I-domain in native state hydrogen exchange experiments is promoted by urea binding.
        Biophys. J. 2015; 109: 2666-2677
        • Prevelige Jr., P.E.
        • King J.
        Assembly of bacteriophage P22: a model for ds-DNA virus assembly.
        Prog. Med. Virol. 1993; 40: 206-221
        • Tripler T.N.
        • Maciejewski M.W.
        • Teschke C.M.
        • Alexandrescu A.T.
        NMR assignments for the insertion domain of bacteriophage CUS-3 coat protein.
        Biomol. NMR Assign. 2015; 9: 333-336
        • Parent K.N.
        • Ranaghan M.J.
        • Teschke C.M.
        A second site suppressor of a folding defect functions via interactions with a chaperone network to improve folding and assembly in vivo.
        Mol. Microbiol. 2004; 54: 1036-1050
        • Casjens S.
        • Hayden M.
        Analysis in vivo of the bacteriophage P22 headful nuclease.
        J. Mol. Biol. 1988; 199: 467-474
        • Casjens S.
        • Wyckoff E.
        • Hayden M.
        • Sampson L.
        • Eppler K.
        • Randall S.
        • Moreno E.T.
        • Serwer P.
        Bacteriophage P22 portal protein is part of the gauge that regulates packing density of intravirion DNA.
        J. Mol. Biol. 1992; 224: 1055-1074
        • Creighton T.E.
        Proteins: Structures and Molecular Properties.
        2nd Ed. W. H. Freeman and Co., New York1993: 225-227
        • Shortle D.
        The denatured state (the other half of the folding equation) and its role in protein stability.
        FASEB J. 1996; 10: 27-34