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 isolated
DNase domain uniformly labeled with 13C/15N
bound to unlabeled Im9 contain more signals than expected
for a single DNase conformer, consistent with the bound
DNase being present in more than one form. The presence
of chemical exchange cross peaks in 750 MHz
15N–1H–15N
HSQC–NOESY–HSQC spectra for backbone NH groups
of Asp20, Lys21, Trp22, Leu23, Lys69, and Asn70 showed
that the bound DNase was in dynamic exchange. The rate
of exchange from the major to the minor form was determined
to be 1.1 ± 0.2 s−1 at 298 K. Previous
NMR studies have shown that the free DNase interchanges
between two conformers with a forward rate constant of
1.61 ± 0.11 s−1 at 288 K, and that
the bound Im9 is fixed in one conformation. The NMR studies
of the bound DNase show that Im9 binds similarly to both
conformers of the DNase and that the buried Trp22 is involved
in the dynamic process. For the free DNase, all NH groups
within a 9 Å radius of any point of the Trp22 ring
exhibit heterogeneity suggesting that a rearrangement of
the position of this side chain is connected with the conformational
interchange. The possible functional significance of this
feature of the DNase is discussed.
