STRUCTURAL ROLE OF A BURIED SALT BRIDGE IN THE 434-REPRESSOR DNA-BINDING DOMAIN

The independently folding 63-residue N-terminal DNA-binding domain of the 434 repressor, 434(1-63), contains a buried Arg10-Glu35 salt bridg e. A corresponding salt bridge is found in a variety of prokaryotic an d eukaryotic DNA-binding proteins with helix-turn-helix motifs. Here, the NMR solution structures of 434(1-63) and the mutant protein 434[R1 0M](1-63) were determined to investigate the structural role of this s alt bridge. Both proteins contain the same type of global fold, with f ive cr-helices and a helix-turn-helix motif formed by the helices II a nd III. The primary structural difference caused by the Arg10 --> Met mutation is a translation of helix I along its axis relative to the he lix II-turn-helix III motif. This limited conformational change is par alleled by a 9 kJ M(-1) decrease of the stability of the folded mutant protein in aqueous solution at pH 4.8. It affects the pK(a) value of Glu19 as well as the population of a hydrogen bond between the backbon e amide proton of Asn16 and the side-chain carboxylate group of Glu19. Using the crystal structure of the 434 repressor dimer complexed with the operator DNA as a basis, model building of the DNA complex with t he NMR structure of 434[R10M](1-63) shows that Asn16, which is located on the protein surface, makes direct contact with the DNA and indicat es that the point mutation Arg10 --> Met should also lead to modificat ions of the protein-protein contacts in the complex. (C) 1996 Academic Press Limited.