Subunit-specific backbone NMR assignments of a 64 kDa trp repressor/DNA complex: A role for N-terminal residues in tandem binding

Deuterium decoupled, triple resonance NMR spectroscopy was used to analyze complexes of ²H, ¹⁵N, ¹³C labelled intact and (des2-7) trp repressor (Δ2-7 trpR) from E. coli bound in tandem to an idealized 22 basepair trp operator DNA fragment and the corepressor 5-methyltryptophan. The DNA sequence used here binds two trpR dimers in tandem resulting in chemically nonequivalent environments for the two subunits of each dimer. Sequence- and subunit-specific NMR resonance assignments were made for backbone ¹HN, ¹⁵N, ¹³Cα positions in both forms of the protein and for ¹³Cβ in the intact repressor. The differences in backbone chemical shifts between the two subunits within each dimer of Δ2-7 trpR reflect dimer-dimer contacts involving the helix-turn-helix domains and N-terminal residues consistent with a previously determined crystal structure [Lawson and Carey (1993) Nature, 366, 178-182]. Comparison of the backbone chemical shifts of DNA-bound Δ2-7 trpR with those of DNA-bound intact trpR reveals significant changes for those residues involved in N-terminal-mediated interactions observed in the crystal structure. In addition, our solution NMR data contain three sets of resonances for residues 2-12 in intact trpR suggesting that the N-terminus has multiple conformations in the tandem complex. Analysis of Cα chemical shifts using a chemical shift index (CSI) modified for deuterium isotope effects has allowed a comparison of the secondary structure of intact and Δ2-7 tprR. Overall these data demonstrate that NMR backbone chemical shift data can be readily used to study specific structural details of large protein complexes.