Fine-tuning function: correlation of hinge domain interactions with functional distinctions between LacI and PurR.

Abstract

LacI and PurR are highly homologous proteins. Their functional units are homodimers, with an N-terminal DNA binding domain that comprises the helix-turn-helix (HTH), N-linker, and hinge regions from both monomers. Hinge structural changes are known to occur upon DNA dissociation but are difficult to monitor experimentally. The initial steps of hinge unfolding were therefore examined using molecular dynamics simulations, utilizing a truncated, chimeric protein comprising the LacI HTH/N-linker and PurR hinge. A terminal Gly-Cys-Gly was added to allow "dimerization" through disulfide bond formation. Simulations indicate that differences in LacI and PurR hinge primary sequence affect the quaternary structure of the hinge x hinge' interface. However, these alternate hinge orientations would be sterically restricted by the core domain. These results prompted detailed comparison of recently available DNA-bound structures for LacI and truncated LacI(1-62) with the PurR structure. Examination revealed that different N-linker and hinge contacts to the core domain of the partner monomer (which binds effector molecule) affect the juxtapositions of the HTH, N-linker, and hinge regions in the DNA binding domain. In addition, the two full-length repressors exhibit significant differences in the interactions between the core and the C-linker connection to the DNA binding domain. Both linkers and the hinge have been implicated in the allosteric response of these repressors. Intriguingly, one functional difference between these two proteins is that they exhibit opposite allosteric response to effector. Simulations and observed structural distinctions are correlated with mutational analysis and sequence information from the LacI/GalR family to formulate a mechanism for fine-tuning individual repressor function.