Structural and thermodynamic analysis of the packing of two α-helices in bacteriophage T4 lysozyme

Abstract

Packing interactions in bacteriophage T4 lysozyme were explored by determining the structural and thermodynamic effects of substitutions for Ala98 and neighboring residues. Ala98 is buried in the core of T4 lysozyme in the interface between two α-helices. The Ala98 to Val (A98V) replacement is a temperature-sensitive lesion that lowers the denaturation temperature of the protein by 15°C (pH3·0, ΔΔG = −4·9 kcal/mol) and causes atoms within the two helices to move apart by up to 0·7 Å. Additional structural shifts also occur throughout the C-terminal domain. In an attempt to compensate for the A98V replacement, substitutions were made for Val149 and Thr152, which make contact with residue 98. Site-directed mutagenesis was used to construct the multiple mutants A98V/T152S, A98V/V149C/T152S and the control mutants T152S, V149C and A98V/V149I/T152S. These proteins were crystallized, and their high-resolution X-ray crystal structures were determined. None of the second-site substitutions completely alleviates the destabilization or the structural changes caused by A98V. The changes in stability caused by the different mutations are not additive, reflecting both direct interactions between the sites and structural differences among the mutants. As an example, when Thr152 in wild-type lysozyme is replaced with serine, the protein is destabilized by 2·6 kcal/mol. Except for a small movement of Val94 toward the cavity created by removal of the methyl group, the structure of the T152S mutant is very similar to wild-type T4 lysozyme. In contrast, the same Thr152 to Ser replacement in the A98V background causes almost no change in stability. Although the structure of A98V/T152S remains similar to A98V, the combination of T152S with A98V allows relaxation of some of the strain introduced by the Ala98 to Val replacement. These studies show that removal of methyl groups by mutation can be stabilizing (Val98 → Ala), neutral (Thr152 → Ser in A98V) or destabilizing (Val149 → Cys, Thr152 → Ser). Such diverse thermodynamic effects are not accounted for by changes in buried surface area or free energies of transfer of wildtype and mutant side-chains. In general, the changes in protein stability caused by a mutation depend not only on changes in the free energy of transfer associated with the substitution, but also on the structural context within which the mutation occurs and on the ability of the surrounding structure to relax in response to the substitution. Inspection of the structures of the wild-type and mutant T4 lysozymes, together with calculations of alternative packing arrangements, show that the dominant effect of the Ala98 → Val replacement is a steric clash with interior main-chain atoms. These contacts can be relieved only partially by the reduction in size of side-chain atoms in the immediate vicinity. The results suggest that mutations that alter contacts of buried main-chain or β-carbon atoms are likely to have greater effects on structure and stability than mutations that perturb only contacts between the distal atoms of buried side-chains. Although interior main-chain atoms are more rigid than interior side-chains, adjustments of backbone atoms do occur in response to amino acid substitutions. Accounting for such backbone movements may improve predictions based on calculations of packing alternatives.