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

J Biol Chem. 2016 May 20;291(21):11359-72. doi: 10.1074/jbc.M116.716910. Epub 2016 Mar 22.


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.

Keywords: bacteriophage; ionization constant; nanoparticle; nuclear magnetic resonance (NMR); procapsid; protein folding; protein stability; virus.

Publication types

  • Research Support, N.I.H., Extramural

MeSH terms

  • Amino Acid Substitution
  • Bacteriophage P22 / genetics
  • Bacteriophage P22 / metabolism*
  • Capsid Proteins / chemistry*
  • Capsid Proteins / genetics
  • Capsid Proteins / metabolism*
  • Hydrogen-Ion Concentration
  • Models, Molecular
  • Mutagenesis, Site-Directed
  • Protein Domains
  • Protein Folding
  • Protein Multimerization
  • Protein Stability
  • Recombinant Proteins / chemistry
  • Recombinant Proteins / genetics
  • Recombinant Proteins / metabolism
  • Sodium Chloride / metabolism
  • Thermodynamics


  • Capsid Proteins
  • Recombinant Proteins
  • Sodium Chloride

Associated data

  • PDB/2M5S