Understanding Conformational Dynamics in E. Coli Protein Tryptophan Repressor

Abstract

E.coli protein tryptophan repressor (TrpR) regulates expression of tryptophan biosynthetic genes in response to the intracellular concentration of L-tryptophan (L-trp). The binding of L-trp by the TrpR dimer is non-cooperative, even though each binding site involves both subunits and their highly dynamic DNA-binding helix-turn-helix (HtH) regions. Despite availability of many X-ray crystal and NMR structures both in presence and absence of L-trp, the mechanism of repressor activation by L-trp and its lack of cooperativity are not understood. Extensive NMR studies have established that the HtH region is helical on a nanosecond but not a millisecond timescale, but the basis of this flexibility remains unexplained and the HtH region has been described as apparently disordered. A recent crystal structure of a temperature-sensitive mutant of TrpR with Phe replacing Leu75 in the HtH region shows a distorted HtH conformation compared to the wildtype protein, and offers a new explanation for HtH flexibility that is corroborated by new NMR data reported here that is analysed by a novel method. The apparent disorder in the old NMR structures can be explained by the fact that the NMR data were used to generate a single structure. A new approach to NMR structure determination that uses the NMR data to solve for multiple structures in molecular dynamics simulations results in an ensemble of structures comprising both wildtype-like and distorted conformations. The data indicate that flexibility in the HtH region in TrpR can be explained by an ensemble of multiple helical conformations, rather than by apparent disorder. The results point out the need to revise current NMR structure determination methods to calculate an ensemble of structures that can distinguish flexibility from apparent disorder.