Biomaterial Database

Arginine 197 of lac repressor contributes significant energy to inducer binding. Confirmation of homology to periplasmic sugar binding proteins. 1991

R O Spotts, and A E Chakerian, and K S Matthews

Department of Biochemistry and Cell Biology, Rice University, Houston, TX 77251.

Based on primary sequence homology between the lactose repressor protein and periplasmic sugar-binding proteins (Müller-Hill, B. (1983) Nature 302, 163-164), a hypothetical sugar-binding site for the lac repressor was proposed using the solved x-ray crystallographic structure of the arabinose-binding protein (ABP) (Sams, C. F., Vyas, N. K., Quiocho, F. A., and Matthews, K. S. (1984) Nature 310, 429-430). By analogy to Arg151 in the ABP sugar site, Arg197 is predicted to play an important role in lac repressor binding to inducer sugars. Hydrogen bonding occurs between Arg151 and the ring oxygen and 4-hydroxyl of the sugar ligand, two backbone carbonyls, and a side chain in ABP, and similar interactions in the lac repressor would be anticipated. To test this hypothesis, Arg197 in the lac repressor protein was altered by oligonucleotide-directed site-specific mutagenesis to substitute Gly, Leu, or Lys. Introduction of these substitutions at position 197 had no effect on operator binding parameters of the isolated mutant proteins, whereas the affinity for inducer was dramatically decreased, consistent with in vivo phenotypic behavior obtained by suppression of nonsense mutations at this site (Kleina, L. G., and Miller, J. H. (1990) J. Mol. Biol. 212, 295-318). Inducer binding affinity was reduced approximately 3 orders of magnitude for Leu, Gly, or Lys substitutions, corresponding to a loss of 50% of the free energy of binding. The pH shift characteristic of wild-type repressor is conserved in these mutants. Circular dichroic spectra demonstrated no significant alterations in secondary structure for these mutants. Thus, the primary effect of substitution for Arg197 is a very significant decrease in the affinity for inducer sugars. Arginine is uniquely able to make the multiple contacts found in the ABP sugar site, and we conclude that this residue plays a similar role in sugar binding for lactose repressor protein. These results provide experimental validation for the proposed homology between ABP and the lac repressor and suggest that homology with ABP may be employed to generate additional insight into the structure and function of this regulatory protein.

UI	MeSH Term	Description	Entries
D009875	Operator Regions, Genetic	The regulatory elements of an OPERON to which activators or repressors bind thereby effecting the transcription of GENES in the operon.	Operator Region,Operator Regions,Operator, Genetic,Genetic Operator,Genetic Operator Region,Genetic Operator Regions,Genetic Operators,Operator Region, Genetic,Operators, Genetic,Region, Genetic Operator,Region, Operator,Regions, Genetic Operator,Regions, Operator
D011487	Protein Conformation	The characteristic 3-dimensional shape of a protein, including the secondary, supersecondary (motifs), tertiary (domains) and quaternary structure of the peptide chain. PROTEIN STRUCTURE, QUATERNARY describes the conformation assumed by multimeric proteins (aggregates of more than one polypeptide chain).	Conformation, Protein,Conformations, Protein,Protein Conformations
D012097	Repressor Proteins	Proteins which maintain the transcriptional quiescence of specific GENES or OPERONS. Classical repressor proteins are DNA-binding proteins that are normally bound to the OPERATOR REGION of an operon, or the ENHANCER SEQUENCES of a gene until a signal occurs that causes their release.	Repressor Molecules,Transcriptional Silencing Factors,Proteins, Repressor,Silencing Factors, Transcriptional
D002942	Circular Dichroism	A change from planar to elliptic polarization when an initially plane-polarized light wave traverses an optically active medium. (McGraw-Hill Dictionary of Scientific and Technical Terms, 4th ed)	Circular Dichroism, Vibrational,Dichroism, Circular,Vibrational Circular Dichroism
D001120	Arginine	An essential amino acid that is physiologically active in the L-form.	Arginine Hydrochloride,Arginine, L-Isomer,DL-Arginine Acetate, Monohydrate,L-Arginine,Arginine, L Isomer,DL Arginine Acetate, Monohydrate,Hydrochloride, Arginine,L Arginine,L-Isomer Arginine,Monohydrate DL-Arginine Acetate
D001665	Binding Sites	The parts of a macromolecule that directly participate in its specific combination with another molecule.	Combining Site,Binding Site,Combining Sites,Site, Binding,Site, Combining,Sites, Binding,Sites, Combining
D012689	Sequence Homology, Nucleic Acid	The sequential correspondence of nucleotides in one nucleic acid molecule with those of another nucleic acid molecule. Sequence homology is an indication of the genetic relatedness of different organisms and gene function.	Base Sequence Homology,Homologous Sequences, Nucleic Acid,Homologs, Nucleic Acid Sequence,Homology, Base Sequence,Homology, Nucleic Acid Sequence,Nucleic Acid Sequence Homologs,Nucleic Acid Sequence Homology,Sequence Homology, Base,Base Sequence Homologies,Homologies, Base Sequence,Sequence Homologies, Base
D016297	Mutagenesis, Site-Directed	Genetically engineered MUTAGENESIS at a specific site in the DNA molecule that introduces a base substitution, or an insertion or deletion.	Mutagenesis, Oligonucleotide-Directed,Mutagenesis, Site-Specific,Oligonucleotide-Directed Mutagenesis,Site-Directed Mutagenesis,Site-Specific Mutagenesis,Mutageneses, Oligonucleotide-Directed,Mutageneses, Site-Directed,Mutageneses, Site-Specific,Mutagenesis, Oligonucleotide Directed,Mutagenesis, Site Directed,Mutagenesis, Site Specific,Oligonucleotide Directed Mutagenesis,Oligonucleotide-Directed Mutageneses,Site Directed Mutagenesis,Site Specific Mutagenesis,Site-Directed Mutageneses,Site-Specific Mutageneses
D050260	Carbohydrate Metabolism	Cellular processes in biosynthesis (anabolism) and degradation (catabolism) of CARBOHYDRATES.	Metabolism, Carbohydrate