Exploring the role of alanine in the structure of the Lac repressor tetramerization domain, a ferritin-like Alacoil.

We are interested in the determinants that specify the structure of antiparallel coiled coils. Antiparallel coiled coils often contain alanine as an important interfacial packing residue; structures containing alanine at certain well-defined positions in the heptad-repeating unit are referred to as Alacoils. Two types have been identified, containing alanine at either the g position of the heptad repeating unit (defined as the d position in the Richardson nomenclature), referred to as a rop-like Alacoil, or the e position (a position in the Richardson nomenclature), referred to as a ferritin-like Alacoil. The Lac repressor tetramerization domain forms an antiparallel four-chain coiled coil, which falls into the second class of Alacoils based on recent crystal structures. The role of alanine in such structures has not yet been explored experimentally. We test the importance of alanine at the e positions on the oligomeric state and stability of the isolated coiled-coil domain of Lac repressor by testing the effect of mutations at this position. We find that mutation to leucine is tolerated and its moderately stabilizing effect is most likely a consequence of plasticity of this motif. The effects on stability of the mutations to either serine or glutamine can be largely accounted for by helix propensity differences between these residues and alanine. The ability of the helices to adjust to such mutations, while maintaining the basic fold of this coiled coil, was further tested by making the same changes at the more highly exposed g position. Leucine at the g positions also causes an increase in stability, presumably by subtle rearrangement of the helices to allow partial desolvation of this side-chain. Copyright 2002 Elsevier Science Ltd.