Transcriptional repressor CopR: Structure model-based localization of the deoxyribonucleic acid binding motif

Abstract

The plasmid pIP501 encoded transcriptional repressor CopR is one of the two regulators of plasmid copy number. CopR binds as a dimer to a nearly palindromic operator with the consensus sequence 5'-CGTG. Intermediate sequence searches revealed a significant structural relationship between CopR and the bacteriophage P22 c2 and the 434 c1 repressors. In this report we describe the experimental verification of a CopR homology model, which is based on a fairly low-sequence identity of 13.8% to P22 c2 repressor. A model for the complex of CopR with the deoxyribonucleic acid (DNA) target was built on the basis of experimental footprinting data, the above-mentioned CopR homology model, and the crystal structure of the 434 c1 repressor-DNA complex. Site-directed mutagenesis was used to test the function of amino acids involved in sequence and nonsequence-specific DNA recognition and amino acids important for correct protein folding. CD measurements were performed to detect structural changes caused by the mutations. Exchanges of residues responsible for sequence-specific DNA recognition reduced binding to a nonspecific level. Mutations of amino acids involved in nonspecific DNA binding lead to decreased binding affinity while maintaining selectivity. Substitution of amino acids necessary for proper folding caused dramatic structural changes. The experimental data support the model of CopR as a helix-turn-helix protein belonging to the lambda repressor superfamily.

Utility of homology models in the drug discovery process

“…The steps involved in the prediction of protein structure by homology modeling. Structure modeling of the bacterial transcriptional repressor CopR is shown [28]. Although the model is based on a low-sequence identity of only 13.8% between CopR and the P22 c2 repressor, several experimental methods support this homology model.…”

“…Typically, the amino acids that are to be modified in these studies are selected on the basis of sequence alignments by focusing on conserved residues. However, if at least some structure information is available, the selection of the amino acids that are to be mutated can be much more precise and successful [28]. This approach is even more powerful when applied in conjunction with pharmacologically active compounds.…”

Transcriptional Repressor CopR: Use of SELEX To Study the copR Operator Indicates that Evolution Was Directed at Maximal Binding Affinity

“…2), termed KS9, had been analyzed previously (28) and was found to be bound at least as efficiently as the wild-type sequence. Our three-dimensional model of the N-terminal 63 amino acids of CopR (30) predicts that this nucleotide position in binding site I is contacted by R34 of the recognition helix (Fig. 2) and that the contact would be stronger with a C instead of a T.…”

“…Furthermore, it was found that CopR binds exclusively as a dimer, and the equilibrium dissociation constants for the CopR dimers and the CopR-DNA complex were calculated to be 0.4 nM and 1.4 M, respectively (29). A three-dimensional model of the Nterminal 63 amino acids of CopR was built and was used to identify amino acids involved in DNA binding and dimerization (30,31,32). By this means, it was found that amino acids R29 and R34, located in the recognition helix (helix III) of the helix-turn-helix motif, make specific contacts to the DNA at G240 (binding site I) and G254 (binding site II) or G242/T243 (site I) and G251 (site II), respectively.…”

“…Water-mediated contacts were suggested for E35 interacting with the outermost C residues in both binding sites. Unspecific DNA contacts via the sugar-phosphate backbone were proposed for K10 in ␣-helix I and S28 in the recognition helix (30). Furthermore, it was established that the structured acidic C terminus of CopR that forms a ␤-strand is necessary for stabilization of the protein (22,23).…”