title: Computational and experimental probes of symmetry mismatches in the Arc repressor-DNA complex.
abstract: Arc repressor from bacteriophage P22 is a homodimeric member of the ribbon-helix-helix family of transcription factors. Two dimers bind cooperatively to adjacent sites on operator DNA with the antiparallel beta-sheet of each Arc dimer contacting the major groove. Despite the 2-fold symmetry of the dimer, the sequence of the DNA half-site is not symmetric, and thus there is an inherent symmetry mismatch in the complex. Here, using a combination of computational and experimental methods, we further characterize this symmetry mismatch. Continuum electrostatic calculations show that the DNA-contacting side-chains of Gln9, Asn11 and Arg13 each make better electrostatic interactions in one subunit of the Arc dimer than in the other. Based on the computational results, one or both Gln9 side-chains were substituted with Arg or His and Asn11 was similarly substituted with Ile in the context of a single-chain variant of Arc repressor. Although the double mutants that maintain the symmetry of the protein bind much more weakly than scArc, mutants with single Q9R or N11I substitutions bind operator DNA with affinities close to wild-type. Whereas the effects are nearly additive for the mutations at position 9, the effects of single N11I mutations are non-additive. The calculations thus enabled the identification of functionally tolerated mutations in the DNA-binding surface of Arc repressor, which in turn revealed energetic consequences of asymmetric environments in this protein-DNA interaction.
authors.affiliation: Department of Biology, Massachusetts Institute of Technology, Cambridge, MA 02139, USA.
authors.forename: Shari
authors.initials: S
authors.lastname: Spector
authors.forename: Robert T
authors.initials: RT
authors.lastname: Sauer
authors.forename: Bruce
authors.initials: B
authors.lastname: Tidor
mesh_terms: D020137:Base Pair Mismatch; D001483:Base Sequence; D017931:DNA Primers; D004279:DNA, Viral / Q000235:genetics / Q000378:metabolism*; D008958:Models, Molecular; D015335:Molecular Probes; D009154:Mutation; D012097:Repressor Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D055672:Static Electricity; D014764:Viral Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D054334:Viral Regulatory and Accessory Proteins
chemical_list: D017931:DNA Primers; D004279:DNA, Viral; D015335:Molecular Probes; D012097:Repressor Proteins; D014764:Viral Proteins; D054334:Viral Regulatory and Accessory Proteins; C016649:phage repressor proteins