TY - JOUR

T1 - Mechanism of Tet repressor induction by tetracyclines

T2 - Length compensates for sequence in the α8-α9 loop

AU - Scholz, Oliver

AU - Kintrup, Martin

AU - Reich, Marco

AU - Hillen, Wolfgang

PY - 2001/7/27

Y1 - 2001/7/27

N2 - Natural Tet repressor (TetR) variants are α-helical proteins bearing a large loop between helices 8 and 9, which is variable in sequence and length. We have deleted this loop consisting of 14 amino acid residues in TetR(D) and rebuilt it stepwise with up to 42 alanine residues. All except the mutant with the longest alanine loop show wild-type repression, but none is inducible with tetracycline. This demonstrates the importance of the α8-α9 loop and its amino acid sequence for induction. The induction efficiencies increase with loop length, when the more tightly binding inducer anhydrotetracycline is used. The largest increase of inducibility was observed for TetR mutants with loop lengths between eight and 17 alanine residues. Since loop residues Asp/Glu157 and Arg158 are conserved in the natural TetR sequence variants, we constructed a mutant in which all other residues of the loop were replaced by alanine. This mutant exhibits increased anhydrotetracycline induction compared to the corresponding alanine variant. Thus, these residues are important for induction. Binding constants for the anhydrotetracycline-TetR interaction are below the detection level of 105 M-1 for the mutant with a loop of two alanine residues and increase sharply until a loop size of ten residues is reached. TetR variants with longer loops have similar anhydrotetracycline-binding constants, ranging between 2.6 × 109 M-1 and 8.0 × 109 M-1, about 500-fold lower than wild-type TetR. The increase of the affinity occurs at shorter loop lengths than that of inducibility. We conclude that the induction defect of the polyalanine variants arises from two increments: (i) the loop must have a minimal length to allow efficient inducer binding; (ii) the loop must structurally participate in the conformational change associated with induction. © 2001 Academic Press.

AB - Natural Tet repressor (TetR) variants are α-helical proteins bearing a large loop between helices 8 and 9, which is variable in sequence and length. We have deleted this loop consisting of 14 amino acid residues in TetR(D) and rebuilt it stepwise with up to 42 alanine residues. All except the mutant with the longest alanine loop show wild-type repression, but none is inducible with tetracycline. This demonstrates the importance of the α8-α9 loop and its amino acid sequence for induction. The induction efficiencies increase with loop length, when the more tightly binding inducer anhydrotetracycline is used. The largest increase of inducibility was observed for TetR mutants with loop lengths between eight and 17 alanine residues. Since loop residues Asp/Glu157 and Arg158 are conserved in the natural TetR sequence variants, we constructed a mutant in which all other residues of the loop were replaced by alanine. This mutant exhibits increased anhydrotetracycline induction compared to the corresponding alanine variant. Thus, these residues are important for induction. Binding constants for the anhydrotetracycline-TetR interaction are below the detection level of 105 M-1 for the mutant with a loop of two alanine residues and increase sharply until a loop size of ten residues is reached. TetR variants with longer loops have similar anhydrotetracycline-binding constants, ranging between 2.6 × 109 M-1 and 8.0 × 109 M-1, about 500-fold lower than wild-type TetR. The increase of the affinity occurs at shorter loop lengths than that of inducibility. We conclude that the induction defect of the polyalanine variants arises from two increments: (i) the loop must have a minimal length to allow efficient inducer binding; (ii) the loop must structurally participate in the conformational change associated with induction. © 2001 Academic Press.

KW - Chemistry

KW - Alanine

KW - Anhydrotetracycline

KW - Induction

KW - Loop

KW - Tet repressor

KW - Up-Regulation/drug effects

KW - Amino Acid Sequence

KW - Molecular Weight

KW - Protein Structure, Secondary

KW - Mutation/genetics

KW - Tetracyclines/pharmacology

KW - Models, Molecular

KW - Molecular Sequence Data

KW - Thermodynamics

KW - Conserved Sequence/genetics

KW - Peptides/genetics

KW - Magnesium/metabolism

KW - Repressor Proteins/chemistry

KW - Protein Binding

KW - Bacterial Proteins/chemistry

UR - http://www.scopus.com/inward/record.url?scp=0035958704&partnerID=8YFLogxK

UR - https://www.mendeley.com/catalogue/fea50742-6fa9-3112-90ef-464aba125b86/

U2 - 10.1006/jmbi.2001.4820

DO - 10.1006/jmbi.2001.4820

M3 - Journal articles

C2 - 11502007

AN - SCOPUS:0035958704

VL - 310

SP - 979

EP - 986

JO - Journal of Molecular Biology

JF - Journal of Molecular Biology

SN - 0022-2836

IS - 5

ER -