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.
journal_name
J Mol Bioljournal_title
Journal of molecular biologyauthors
Spector S,Sauer RT,Tidor Bdoi
10.1016/j.jmb.2004.04.026keywords:
subject
Has Abstractpub_date
2004-07-02 00:00:00pages
253-61issue
2eissn
0022-2836issn
1089-8638pii
S0022283604004619journal_volume
340pub_type
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