3-DIMENSIONAL SOLUTION STRUCTURE AND C-13 NUCLEAR-MAGNETIC-RESONANCE ASSIGNMENTS OF THE COLICIN E9 IMMUNITY PROTEIN IM9

The 86-amino acid colicin E9 immunity protein (Im9), which inhibits th e DNase activity of colicin E9, has been overexpressed in Escherichia coli and isotopically enriched with N-15 and C-13. Using the 3D CBCANH and CBCA(CO)NH experiments, we have almost completely assigned the ba ckbone C-13 resonances and extended previously reported N-15/H-1 backb one assignments [Osborne et al. (1994), Biochemistry 33, 12347-12355]. Side chain assignments for almost all residues were made using the 3D C-13 HCCH-TOCSY experiment allied to previous H-1 assignments. Sixty solution structures of Im9 were determined using the DIANA program on the basis of 1210 distance restraints and 56 dihedral angle restraints . The 30 lowest-energy structures were then subjected to a slow-coolin g simulated annealing protocol using XPLOR and the 21 lowest-energy st ructures, satisfying the geometric restraints chosen for further analy sis. The Im9 structure is well-defined except for the termini and two solvent-exposed loops between residues 28-32 and 57-64. The average RM SD about the average structure of residues 4-84 was 0.94 Angstrom for all heavy atoms and 0.53 Angstrom for backbone C-alpha, C=O, and N ato ms. The Im9 fold is novel and can be considered a distorted antiparall el four-helix bundle, in which the third helix is rather short, being terminated close to its N-terminal end by a proline at its C-terminus. The structure fits in well with available kinetic and biochemical dat a concerning the interaction between Im9 and its target DNase, Importa nt residues of Im9 that govern specificity are located on the molecula r surface in a region rich in negatively charged groups, consistent wi th the proposed electrostatically steered association [Wallis et al. ( 1995a), Biochemistry 34, 13743-13750].