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

Christina Harprecht,Oghenefejiro Okifo,Kevin J Robbins,Tina Motwani,Andrei T Alexandrescu,Carolyn M Teschke

doi:10.1074/jbc.m116.716910

Christina Harprecht, Oghenefejiro Okifo + Show 4 more

Open Access

https://doi.org/10.1074/jbc.m116.716910

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Abstract

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.

Highlights

Virus and phage coat proteins requisitely encounter the dilemma of balancing counteracting forces during folding and assembly
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 [8]
In its simplest form, the P22 procapsid has 420 copies of coat protein arranged in a T ϭ 7 (T ϭ triangulation number) icosahedral shell with 100 –300 copies of the scaffolding protein bound within its confines with unknown symmetry [9]

Summary

Experimental Procedures

Materials—D2O (99.96%) for hydrogen exchange studies, DCl and NaOD were from Cambridge Isotope Laboratories (Tewksbury, MA). Preparation of Full-length Coat Protein Monomers—To generate coat protein monomers, empty procapsid shells prepared as previously described [9] were denatured for 30 min at room temperature in 20 mM sodium phosphate buffer (pH 7.6) containing 6.75 M urea. Hydrogen exchange (HX) experiments were performed on 15N-labeled I-domain samples in 20 mM sodium phosphate buffer. For equilibrium denaturation studies of the I-domain, samples were diluted to a final protein concentration of 0.4 mg/ml (28 ␮M) in 20 mM sodium phosphate buffer (pH 7.6) and mixed with 9 M urea using a Hamilton Microlab 50 titrator to generate a range of final urea concentrations between 0 M and 6 M in 0.1 M increments. Bated for 16 h at room temperature before measuring light scattering at 500 nm to detect intact shells [49]

Results

Structure context

Discussion