Oligomerization of the EK18 Mutant of the trp Repressor of Escherichia coli as Observed by NMR Spectroscopy 1 Young Kee Chae,* Frits Abildgaard,* Catherine A. Royer,† ,2 and John L. Markley* ,3 *Department of Biochemistry and National Magnetic Resonance Facility at Madison and †School of Pharmacy, University of Wisconsin—Madison, Madison, Wisconsin 53706 Received April 20, 1999, and in revised form June 14, 1999 The regulation of the trp repressor system of Esche- richia coli is frequently modeled by a single equilib- rium, that between the aporepressor (TR) and the corepressor, L-tryptophan (Trp), at their intracellular concentrations. The actual mechanism, which is much more complex and more finely tuned, involves multi- ple equilibria: TR and Trp association, TR oligomer- ization, specific and nonspecific binding of various states of TR to DNA, and interactions between these various species and ions. TR in isolation exists primar- ily as a homodimer, but the state of oligomerization increases as the TR concentration goes up and/or the salt concentration goes down, leading to species with lower affinity for DNA. We have used multinuclear, multidimensional NMR spectroscopy to investigate structural changes that accompany the oligomeriza- tion of TR. For these investigations, the superrepres- sor mutant EK18 (TR with Glu 18 replaced by Lys) was chosen because it exhibits less severe oligomerization at higher protein concentration than other known variants; this made it possible to study the dimer to tetramer oligomerization step by NMR. The NMR re- sults suggest that the interaction between TR dimers is structurally linked to folding of the DNA binding domain and that it likely involves direct contacts be- tween the C-terminal residues of the C-helix of one dimer with the next dimer. This implies that oligomer- ization can compete with DNA binding and thus serves as a factor in the fine-tuning of gene expression. © 1999 Academic Press Key Words: trp repressor; superrepressor; oligomer- ization; gene regulation. The Escherichia coli trp repressor (apoTR) 4 binds its corepressor, L-tryptophan (Trp), and this complex (holoTR) serves to represses the expression of genes necessary for the synthesis of tryptophan and other aromatic amino acids. Although this repressor system was one of the earliest studied (1–3), the detailed mechanisms underlying many of its interactions re- main to be elucidated. X-ray structures (4 –7) have revealed that Trp serves as a wedge in directing the DNA binding domain into a proper position for inter- action. On the other hand, NMR spectroscopic studies (8 –13) and calorimetric investigations (14, 15) have suggested that ligand binding is coupled to local fold- ing. Fluorescence spectroscopic investigations (16 –19) have shown that TR exists primarily as a dimer but that these dimers can form tetramers and larger olig- omers at high protein concentrations and/or low salt concentrations. Under conditions (protein concentra- tion and salt) where holoTR is dimeric, apoTR may exist as large oligomers. A number of superrepressor 1 This research was supported by NIH Grant GM 35976 (to J.L.M.). NMR studies were carried out at the National Magnetic Resonance Facility at Madison with support from the NIH Biomed- ical Technology Program (RR02301) and additional equipment fund- ing from the University of Wisconsin, NSF Academic Infrastructure Program (BIR-9214394), NIH Shared Instrumentation Program (RR02781 and RR08438), NSF Biological Instrumentation Program (DMB-8415048), and U.S. Department of Agriculture. 2 Present address: Ctr. Biochim. Struct., INSERM U414, 15 Av Charles Flahault 34060 Cedex 01, Montpellier, France. 3 To whom correspondence should be addressed at Department of Biochemistry, University of Wisconsin—Madison, 433 Babcock Dr., Madison, WI 53706. Fax: (608) 262-3453. E-mail: markley@ nmrfam.wisc.edu. 4 Abbreviations used: TR, apo form of the trp repressor from E. coli; EK18, superrepressor mutant of the trp repressor in which Glu 18 is replaced by Lys; HNCA, three-dimensional triple-resonance experiment that correlates signals from backbone 1 H i N , 15 N i , and 13 C i-1  resonances; HN(CO)CA, three-dimensional triple-resonance experiment that correlates signals from backbone 1 H i N , 15 N i , and 13 C i-1  resonances; HSQC, heteronuclear single quantum coherence. 0003-9861/99 $30.00 35 Copyright © 1999 by Academic Press All rights of reproduction in any form reserved. Archives of Biochemistry and Biophysics Vol. 371, No. 1, November 1, pp. 35– 40, 1999 Article ID abbi.1999.1394, available online at http://www.idealibrary.com on