The bacterial toxin colicin E9 is secreted by producing
Escherichia coli cells 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
24 kDa complex of uniformly 13C and 15N
labeled Im9 bound to the unlabeled DNase 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 Å for the backbone atoms and 1.36
± 0.15 Å for the heavy atoms. Representative
solution structures of the free and bound Im9 are highly
similar, with backbone and heavy atom RMSDs of 1.63 and
2.44 Å, respectively, for residues 4–83, suggesting
that binding does not cause a major conformational change
in Im9. The NMR studies have also allowed the DNase contact
surface on Im9 to be investigated through changes in backbone
chemical shifts and NOEs between the two proteins determined
from comparisons of 1H–1H–13C
NOESY-HSQC spectra with and without 13C decoupling.
The NMR-defined interface agrees well with that determined
in a recent X-ray structure analysis with the major difference
being that a surface loop of Im9, which is at the interface,
has a different conformation in the solution and crystal
structures. Tyr54, a key residue on the interface, is shown
to exhibit NMR characteristics 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 proposed.