Evaluation at atomic resolution of the role of strain in destabilizing the temperature-sensitive T4 lysozyme mutant Arg 96 --> His

Abstract

Mutant R96H is a classic temperature-sensitive mutant of bacteriophage T4 lysozyme. It was in fact the first variant of the protein to be characterized structurally. Subsequently, it has been studied extensively by a variety of experimental and computational techniques, but the reasons for the loss of stability of the mutant protein remain controversial. In the crystallographic refinement of the mutant structure at 1.9 A resolution one of the bond angles at the site of substitution appeared to be distorted by about 11( degrees ), and it was suggested that this steric strain was one of the major factors in destabilizing the mutant. Different computationally-derived models of the mutant structure, however, did not show such distortion. To determine the geometry at the site of mutation more reliably, we have extended the resolution of the data and refined the wildtype (WT) and mutant structures to be better than 1.1 A resolution. The high-resolution refinement of the structure of R96H does not support the bond angle distortion seen in the 1.9 A structure determination. At the same time, it does confirm other manifestations of strain seen previously including an unusual rotameric state for His96 with distorted hydrogen bonding. The rotamer strain has been estimated as about 0.8 kcal/mol, which is about 25% of the overall reduction in stability of the mutant. Because of concern that contacts from a neighboring molecule in the crystal might influence the geometry at the site of mutation we also constructed and analyzed supplemental mutant structures in which this crystal contact was eliminated. High-resolution refinement shows that the crystal contacts have essentially no effect on the conformation of Arg96 in WT or on His96 in the R96H mutant.

Figure 1

(a) Stereo figure comparing the structures of WT lysozyme (open bonds) and mutant R96H (black bonds) in the vicinity of the substitution. Amino acids are identified by the single-letter code. To the right of the figure are the residues Asp72 and Arg76 (highlighted with asterisks) that belong to a neighboring molecule in the crystal lattice. Hydrogen bonds are indicated by broken lines. HED166 is a molecule of hydroxyethyl disulfide, used as an aid to crystallization, which also binds in this vicinity. To avoid confusion, water molecules in the vicinity are not shown. (b) Comparison of the structure of WT lysozyme (open bonds) with R96H as seen in the structure of the double mutant D72A/R96H (solid bonds). All conventions are as in (a). In D72A/R96H (but not WT), Asp72 in the neighboring molecule is replaced by an alanine. Notwithstanding this change, the conformation of Arg96 is essentially identical with that seen in (a).

Figure 2

Contour maps showing the difference in C^α—C^α distances between mutants and wildtype T4 lysozyme. The change in the distance between the α-carbon atoms of any two residues i and j can be found at the corresponding coordinates (i,j). The starting contour level is at ±0.16 Å and the contour interval is 0.04 Å. The positive contours (blue) indicate that a pair of atoms has moved further apart in the mutant relative to WT. Likewise, negative contours (red) indicate that a pair of atoms has moved closer together in the mutant. The elements of secondary structure are indicated along each axis: α-helices A to J and β-strands I and II. (a) R96H versus wildtype, (b) D72A/R96H versus wildtype.