The phage L capsid decoration protein has a novel OB-fold and an unusual capsid binding strategy.

The major coat proteins of dsDNA tailed phages (order Caudovirales) and herpesviruses form capsids by a mechanism that includes active packaging of the dsDNA genome into a precursor procapsid, followed by expansion and stabilization of the capsid. These viruses have evolved diverse strategies to fortify their capsids, such as non-covalent binding of auxiliary 'decoration' (Dec) proteins. The Dec protein from the P22-like phage L has a highly unusual binding strategy that distinguishes between nearly identical three-fold and quasi-three-fold sites of the icosahedral capsid. Cryo-electron microscopy and three-dimensional image reconstruction were employed to determine the structure of native phage L particles. NMR was used to determine the structure/dynamics of Dec in solution. The NMR structure and the cryo-EM density envelope were combined to build a model of the capsid-bound Dec trimer. Key regions that modulate the binding interface were verified by site-directed mutagenesis.

NMR assignments for monomeric phage L decoration protein.

Phage L encodes a trimeric 43 kDa decoration protein (Dec) that noncovalently binds and stabilizes the capsids of the homologous phages L and P22 in vitro. At physiological pH Dec was unsuitable for NMR. We were able to obtain samples amenable for NMR spectroscopy by unfolding Dec to pH 2 and refolding it to pH 4. Our unfolding/refolding protocol converted trimeric Dec to a folded 14.4 kDa monomer. We verified that the acid-unfolding protocol did not perturb the secondary structure, or the capsid-binding function of refolded Dec. We were able to obtain complete 1H, 15N, and 13C assignments for the Dec monomer, as well as information on its secondary structure and dynamics based on chemical shift assignments. The assigned NMR spectrum is being used to determine the three-dimensional structure of Dec, which is important for understanding how the trimer binds phage capsids and for the use of the protein as a platform for phage-display nanotechnology.

Mechanism of Protein Denaturation: Partial Unfolding of the P22 Coat Protein I-Domain by Urea Binding.

The I-domain is an insertion domain of the bacteriophage P22 coat protein that drives rapid folding and accounts for over half of the stability of the full-length protein. We sought to determine the role of hydrogen bonds (H-bonds) in the unfolding of the I-domain by examining (3)JNC' couplings transmitted through H-bonds, the temperature and urea-concentration dependence of (1)HN and (15)N chemical shifts, and native-state hydrogen exchange at urea concentrations where the domain is predominantly folded. The native-state hydrogen-exchange data suggest that the six-stranded ß-barrel core of the I-domain is more stable against unfolding than a smaller subdomain comprised of a short α-helix and three-stranded ß-sheet. H-bonds, separately determined from solvent protection and (3)JNC' H-bond couplings, are identified with an accuracy of 90% by (1)HN temperature coefficients. The accuracy is improved to 95% when (15)N temperature coefficients are also included. In contrast, the urea dependence of (1)HN and (15)N chemical shifts is unrelated to H-bonding. The protein segments with the largest chemical-shift changes in the presence of urea show curved or sigmoidal titration curves suggestive of direct urea binding. Nuclear Overhauser effects to urea for these segments are also consistent with specific urea-binding sites in the I-domain. Taken together, the results support a mechanism of urea unfolding in which denaturant binds to distinct sites in the I-domain. Disordered segments bind urea more readily than regions in stable secondary structure. The locations of the putative urea-binding sites correlate with the lower stability of the structure against solvent exchange, suggesting that partial unfolding of the structure is related to urea accessibility.