NMR investigation of the interaction of the inhibitor protein Im9 with itspartner DNase

The bacterial toxin colicin E9 is secreted by producing Escherichia coli ce lls with its 9.5 kDa inhibitor protein Im9 bound tightly to its 14.5 kDa C- terminal DNase domain. Double- and triple-resonance NMR spectra of the 21 k Da complex of uniformly C-13 and N-15 labeled Im9 bound to the unlabeled DN ase domain have provided sufficient constraints for the solution structure of the bound Im9 to be determined. For the final ensemble of 20 structures, pairwise RMSDs for residues 3-84 were 0.76 +/- 0.14 Angstrom for the backb one atoms and 1.36 +/- 0.15 Angstrom for the heavy atoms. Representative so lution structures of the free and bound Im9 are highly similar, with backbo ne and heavy atom RMSDs of 1.63 and 2.44 Angstrom, respectively, for residu es 4-83, suggesting that binding does not cause a major conformational chan ge in Im9. The NMR studies have also allowed the DNase contact surface on I m9 to be investigated through changes in backbone chemical shifts and NOEs between the two proteins determined from comparisons of H-1-H-1-C-13 NOESY- HSQC spectra with and without C-13 decoupling. The NMR-defined interface ag rees well with that determined in a recent X-ray structure analysis with th e major difference being that a surface loop of Im9, which is at the interf ace, has a different conformation in the solution and crystal structures. T yr54, a key residue on the interface, is shown to exhibit NMR characteristi cs indicative of slow rotational flipping. A mechanistic description of the influence binding of Im9 has on the dynamic behavior of E9 DNase, which is known to exist in two slowly interchanging conformers in solution, is prop osed.