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 (delta 2-7 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, 13c alpha positions in both forms of the protein and for 13 C beta in the intact repressor. The differences in backbone chemical shifts between the two subunits within each dimer of delta 2-7 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 delta 2-7 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 C alpha chemical shifts using a chemical shift index (CSI) modified for deuterium isotope effects has allowed a comparison of the secondary structure of intact and delta 2-7 tprR. Overall these data demonstrate that NMR backbone chemical shift data can be readily used to study specific structural details of large protein complexes.
Ontario Cancer Institute, University of Toronto, Canada.
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Shan
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Gardner
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Muhandiram
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Kay
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Arrowsmith
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry* / Q000378:metabolism; D002247:Carbon Isotopes; D004268:DNA-Binding Proteins / Q000737:chemistry* / Q000378:metabolism; D003903:Deuterium / Q000378:metabolism; D019281:Dimerization; D004926:Escherichia coli; D007553:Isotope Labeling / Q000379:methods; D008969:Molecular Sequence Data; D009587:Nitrogen Isotopes; D019906:Nuclear Magnetic Resonance, Biomolecular / Q000379:methods*; D011485:Protein Binding; D011487:Protein Conformation; D017433:Protein Structure, Secondary; D012097:Repressor Proteins / Q000737:chemistry* / Q000378:metabolism; D014364:Tryptophan / Q000737:chemistry; D029968:Escherichia coli Proteins
D001426:Bacterial Proteins; D002247:Carbon Isotopes; D004268:DNA-Binding Proteins; D003903:Deuterium; D009587:Nitrogen Isotopes; D012097:Repressor Proteins; D014364:Tryptophan; C027190:TRPR protein, E coli; D029968:Escherichia coli Proteins