Slow conformational dynamics of an endonuclease persist in its complex with its natural protein inhibitor

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 isol ated DNase domain uniformly labeled with C-13/N-15 bound to unlabeled Im9 c ontain more signals than expected for a single DNase conformer, consistent with the bound DNase being present in more than one form. The presence of c hemical exchange cross peaks in 750 MHz N-15-H-1-N-15 HSQC-NOESY-HSQC spect ra for backbone NH groups of Asp20, Lys21, Trp22, Leu23, Lys69, and Asn70 s howed that the bound DNase was in dynamic exchange. The rate of exchange fr om the major to the minor form was determined to be 1.1 +/- 0.2 s(-1) at 29 8 K. Previous NMR studies have shown that the free DNase interchanges betwe en two conformers with a forward rate constant of 1.61 +/- 0.11 s(-1) at 28 8 K, and that the bound Im9 is fixed in one conformation. The NMR studies o f the bound DNase show that Im9 binds similarly to both conformers of the D Nase and that the buried Trp22 is involved in the dynamic process. For the free DNase, all NH groups within a 9 Angstrom radius of any point of the Tr p22 ring exhibit heterogeneity suggesting that a rearrangement of the posit ion of this side chain is connected with the conformational interchange. Th e possible functional significance of this feature of the DNase is discusse d.