Testing homology modeling on mutant proteins: predicting structural and thermodynamic effects in the Ala98-->Val...

Current approaches to homology modeling predict how amino acid substitutions will alter a protein's structure, primarily by modeling sidechain conformations upon essentially immobile backbone frameworks. However, recent crystal structures of T4 lysozyme mutants reveal significant shifts of the mainchain and other potentially serious problems for sidechain rotamer-based modeling. This paper evaluates the accuracy of structural and thermodynamic predictions from two common sidechain modeling approaches to measure errors caused by the fixed-backbone approximation.

Tested on a series of T4 lysozyme mutants, this sidechain rotamer library approach did not handle mainchain shifts well, correctly predicting the sidechain conformations of only two of six mutants. By contrast, allowing sidechains to move more flexibly appeared to compensate for the rigidity of the mainchain and gave reasonably accurate coordinate predictions (rms errors of 0.5-1.0 A for each mutated sidechain), better on average than 90% of possible conformations. The calculated packing energies correlated well with experimental stabilities (r2 = 0.81) and correctly captured the cooperative interactions of several neighboring mutations.

Mutant modeling can be relatively accurate despite the fixed-backbone approximation. Mainchain shifts (0.2-0.5 A) cause increased sidechain coordinate errors of 0.1-0.8 A, torsional errors of 10-30 degrees, and exaggerated strain energy for overpacked mutants, compared with the same calculations performed with the correct mutant backbones.