THE STRUCTURE OF FIS MUTANT PRO(61)ALA ILLUSTRATES THAT THE KINK WITHIN THE LONG ALPHA-HELIX IS NOT DUE TO THE PRESENCE OF THE PROLINE RESIDUE

The influence of proline on bending of the alpha-helix was investigate d by replacement of the proline residue located in the middle of the l ong alpha-helix of the Fis protein with alanine, serine, or leucine. E ach of the three substitutions folded into a stable protein with the s ame or higher melting points than the wild-type, but only Pro(61)Ala w as functionally active in stimulating Hin-mediated DNA inversion. Pro( 61)Ala formed crystals that were isomorphous with the wild-type protei n allowing the structure to be determined at 1.9-Angstrom resolution b y x-ray diffraction methods. The structure of the Pro(61)Ala mutant is almost identical to the wild-type protein, consistent with its near w ild-type activity. One of the alpha-helices, the B-helix, is kinked in the wild-type Fis protein by 20 degrees which was previously assumed to be caused solely by the presence of proline 61 in the center of the helix. However, the B-helix is still kinked by 16 degrees when prolin e 61 is replaced by alanine. Local peptide backbone movement around re sidue 57 adjusts the geometry of the helix to accommodate the new main chain hydrogen bond between the -CO group in Glu(57) and the -NH grou p in Ala(61). Thus, the kink of the alpha-helix in Pro(61)Ala does not require the presence of proline.