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Formation of Heterodimers Between Wild Type and Mutant Trp Aporepressor Polypeptides of Eschem'chia Coli Thomas J

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Formation of Heterodimers Between Wild Type and Mutant Trp Aporepressor Polypeptides of Eschem'chia Coli Thomas J PROTEINS: Structure, Function, and Genetics 4:173-181 (1988) Formation of Heterodimers Between Wild Type and Mutant trp Aporepressor Polypeptides of Eschem'chia coli Thomas J. Graddis,' Lisa S. Klig,' Charles Yanofsky,' and Dale L. Oxender' 'Department of Biological Chemistry, University of Michigan, Ann Arbor, Michigan 48109 and 'Department of Biological Sciences, Stanford University, Stanford, California 94305 ABSTRACT Availability of the three-dimen- tophan.1-6 The trp aporepressor is activated by the sional structure of the trp repressor of Escherichia binding of two molecules of its corepressor. Once ac- coli and a large group of repressor mutants has per- tivated, trp repressor binds to the operators of the trp, mitted the identification and analysis of mutants aroH, and trpR operons, regulating transcription ini- with substitutions of the amino acid residues that tiati~n.~,'The aroH and trp operons encode biosyn- form the tryptophan binding pocket. Mutant apore- thetic enzymes, whereas the trpR operon encodes the pressors selected for study were overproduced using trp aporepressor. The trpR gene has been cloned and a multicopy expression plasmid. Equilibrium di- its nucleotide sequence determined.3 The aporepres- alysis with ''C-tryptophan and purified mutant and sor and repressor have been purified and the crystal wild type aporepressors was employed to determine structures of both have been solved at high resolu- tryptophan binding constants. The results obtained ti~n.~-"The trp aporepressor is a dimer of identical indicate that replacement of threonine 44 by methi- 107 residue polypeptides." The existence of the three- onine (TM44) or arginine 84 by histidine (RH84) low- dimensional structures, and the availability of many ers the affinity for tryptophan approximately two- repressor mutants,2i12has facilitated this analysis of and four-fold, respectively. Replacement of arginine the in vivo and in vitro tryptophan-binding activity 54 by histidine (RH84) or glycine 85 by arginine of mutant repressors with changes of those amino (GR85) results in complete loss of tryptophan bind- acid residues predicted to participate in tryptophan ing activity. Purified mutant and wild type apore- binding. pressors were used in in vitro heterodimer studies. A class of trpR mutants was identified that have a The trp repressor of E. coli functions as a stable transdominant effect when present in a strain bear- dimer. A large number of trp repressor mutants pro- ing a chromosomal copy of the wild type repressor duces defective repressors that are transdominant to gene." The transdominant phenotype (trpR-d) is the wild type repressor in vivo. The transdominance thought to result from the formation of heterodimers presumably results from the formation of inactive or defective in repressor activity.13 Analogous results slightly active heterodimers between the mutant and have been observed with mutant forms of other re- wild type polypeptide subunits. An in vitro assay was pressors, such as the lac repres~or.'~,~~The chain ter- developed to detect and measure heterodimer for- minating trpR nonsense mutant Qam68 produces a mation. Heterodimer formation was thermally in- truncated polypeptide lacking the C-terminal 40- duced, and heterodimers were separated on amino acid residues that contain the polypeptide seg- nondenaturing polyacrylamide gels. Aporepressors ment involved in DNA recognition. Since the Qam68 readily formed heterodimers upon treatment at 65°C mutant displays strong transdominance, it is likely for 3 minutes. Heterodimer formation was signifi- that the first 67 residues of the trpR polypeptide are cantly retarded by the presence of the corepressor, sufficient for negative complementation. L-tryptophan. Indole-3-propionic acid, 5-methyl The majority of the trpR-d missense mutations tryptophan, and other analogs of tryptophan, as well (71%) map to a gene segment corresponding to the D- as indole, also inhibited heterodimer formation. helix-turn-E-helix region of the repressor, which is These results indicate that the presence of the indole believed to interact directly with operator DNA. A moiety in the corepressor binding pocket increases second group of transdominant missense mutations the stability of the dimer. results in the substitution of residues that form the surface of the corepressor binding Key words: DNA binding protein, ligand binding, In the present study we have purified four mutant equilibrium dialysis, dimer, stability trp aporepressors that have substitutions of residues believed to comprise the corepressor binding site. Us- INTRODUCTION The trp repressor of Escherichia coli regulates Received December 14, 1987; revision accepted July 28, 1988. expression of genes required for the de novo biosyn- Address reprint requests to Dr. Dale L. Oxender, Department of Biological Chemistry, University of Michigan Medical thesis of tryptophan in response to changes in the School, M-531910606, 1301 Catherine Road, Ann Arbor, MI intracellular concentration of its corepressor, L-tryp- 48109-0606. 0 1988 ALAN R. LISS, INC. 174 T.J.GRADDIS ET AL. ing equilibrium dialysis analyses, we showed that yields were obtained with each repressor, indicating these mutant aporepressors are partially or totally that none was particularly labile. Dialyzed aporepres- defective in L-tryptophan binding. We also developed sor preparations were concentrated using an Amicon an in vitro procedure for generating heterodimers ultrafiltration apparatus and P10 membranes. Pro- from purified wild type and mutant trp aporepressors. tein concentration was determined spectrophotomet- The net charge differences between wild type apore- ri~a1ly.l~The repressor was stored frozen at -20°C pressor and some mutant species, and their hetero- in buffer containing 0.2M NaCl at a protein concen- dimers, allowed us to separate heterodimers elec- tration of 4-7 mg/ml. trophoretically on nondenaturing polyacrylamide gels, and to quantitate their formation. We have dem- Equilibrium Dialysis onstrated that L-tryptophan and certain tryptophan The affinity of L-tryptophan for pure wild type and analogs inhibit heterodimer formation. mutant trp aporepressors was determined using equi- librium dialysis measurements with L-[methylene- MATERIALS AND METHODS 14C] tryptophan (sp. act. 53.5 mCi/mmol, Amersham). Chemicals Strains Plasmids, and Media An eight-place Hoeffer equilibrium microdialyzer L-tryptophan, D-tryptophan, L-phenylalanine, D,L- module with EMD104 membranes (6,000-8,000 dal- tryptophan, D,L-5-methyltryptophan,tryptamine, in- ton cutoff),driven by a rotary motor, was used in this dole, and indole-3-propionic acid were purchased from study. Each chamber had a capacity of 100 pl on each Sigma. Overexpressing plasmids were constructed by side of the dialysis membrane. The dialysis solution incorporating BamHI fragments containing the var- contained 10 mM Tris-HC1, pH 7.8, and 0.1 M NaCl. Aporepressor at a final concentration of 40 pM was ious mutant trpR alleles into expression plasmid ptacterm, as describe9.16 In this plasmid, the tac pro- placed on one side of the chamber, and various con- moter drives trpR mRNA production. The recipient centrations of L-[methylene-14C] tryptophan were placed on the other side. Dialysis was carried out strain used for overproduction of the trp aporepressor overnight (16-18 hours) at 4°C. All chambers were was CY15071 (W31 10 tnaAZAtrpR thr lac@). This emptied, and 10 p1 aliquots of each were counted in a strain has trpR deleted and carries lacZQ for control scintillation counter. of the tac promoter of the expression plasmid. Each expression plasmid was introduced into CY15071, and trp aporepressor synthesis was induced by addition of Samples for Heterodimer Formation IPTG, as described.I6 Standard conditions for formation of heterodimers Repression was measured in vivo using a lysogenic were as follows: two purified trp aporepressors, at the recipient, strain CY15058 (W3110 tnaA2 trpR thr- same concentration, were combined, so that 0.15 AlacU169/XTLFl),in which the single integrated copy nmoles of total dimeric protein (3.75 pg protein) were of the lambda genome contains a trp promoter/opera- present in a final volume of 3-6 pl(25-50 pM) in 100 tor trpL'-'lacZ gene fusion. Production of hybrid (3- mM Tris buffer (pH 6.81, 50-100 mM NaC1. Where galactosidase by this strain is controlled by repres- indicated, L-tryptophan was added. Samples were sion of transcription initiation at the trp promoter of then heated in water baths at various temperatures XTLF1. To measure in vivo repression by any of the for various times. After heating, samples were cooled mutant repressors, derivatives of pRLK13" contain- rapidly in an ice bath. Three microliters of 80% glyc- ing the various mutant alleles were introduced into erol with 0.0025% marking dye were added to each CY15058, and bacterial cultures were grown in min- sample before loading the entire sample on a gel. The imal mediurnl7 containing 0.2% glucose, 0.2% casein rate of heterodimer formation was not affected by hydrolysate, with or without 40 pg/ml tryptophan, as changing the pH of the Tris buffer from 6.8 to 8.8. indicated. Culture media generally contained either Samples to be examined for heterodimer formation 30 pg/ml chloramphenicol or 100 pg/ml ampicillin to induced by ethanol were prepared essentially as stabilize the appropriate plasmid. Plasmid pRLK13 above, except that tryptophan was added prior to the is a derivative of pACYC184 in which trpR expression ethanol,
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