NMR detection of slow conformational dynamics in an endonuclease toxin

Sara B M Whittaker, Ruth Boetzel, Colin MacDonald, Lu Yun Lian, Ansgar J. Pommer, Ann Reilly, Richard James, Colin Kleanthous, Geoffrey R. Moore

    Research output: Contribution to journalArticlepeer-review


    The cytotoxic activity of the secreted bacterial toxin colicin E9 is due to a non-specific DNase housed in the C-terminus of the protein. Double-resonance and triple-resonance NMR studies of the 134-amino acid 15N- and 13C/15N-labelled DNase domain are presented. Extensive conformational heterogeneity was evident from the presence of far more resonances than expected based on the amino acid sequence of the DNase, and from the appearance of chemical exchange cross-peaks in TOCSY and NOESY spectra. EXSY spectra were recorded to confirm that slow chemical exchange was occurring. Unambiguous sequence-specific resonance assignments are presented for one region of the protein, Pro65-Asn72, which exists in two slowly exchanging conformers based on the identification of chemical exchange cross-peaks in 3D 1H-1H-15N EXSY-HSQC, NOESY-HSQC and TOCSY-HSQC spectra, together with Cα and Cβ chemical shifts measured in triple-resonance spectra and sequential NH NOEs. The rates of conformational exchange for backbone amide resonances in this stretch of amino acids, and for the indole NH of either Trp22 or Trp58, were determined from the intensity variation of the appropriate diagonal and chemical exchange cross-peaks recorded in 3D 1H-1H-15N NOESY-HSQC spectra. The data fitted a model in which this region of the DNase has two conformers, NA and NB, which interchange at 15 °C with a forward rate constant of 1.61 ± 0.5 s-1 and a backward rate constant of 1.05 ± 0.5 s-1. Demonstration of this conformational equilibrium has led to a reappraisal of a previously proposed kinetic scheme describing the interaction of E9 DNase with immunity proteins [Wallis et al. (1995) Biochemistry, 34, 13743-13750 and 13751-13759]. The revised scheme is consistent with the specific inhibitor protein for the E9 DNase, Im9, associating with both the NA and NB conformers of the DNase and with binding only to the NB conformer detected because the rate of dissociation of the complex of Im9 and the NA conformer, NAI, is extremely rapid. In this model stoichiometric amounts of Im9 convert, the E9 DNase is converted wholly into the NBI form. The possibility that cis-trans isomerisation of peptide bonds preceding proline residues is the cause of the conformational heterogeneity is discussed. E9 DNase contains 10 prolines, with two bracketing the stretch of amino acids that have allowed the NA ⇌ NB interconversion to be identified, Pro65 and Pro73. The model assumes that one or both of these can exist in either the cis or trans form with strong Im9 binding possible to only one form.
    Original languageEnglish
    Pages (from-to)145-159
    Number of pages14
    JournalJournal of Biomolecular Nmr
    Issue number1
    Publication statusPublished - 1998


    • Colicin
    • Conformational dynamics
    • E9 DNase
    • EXSY


    Dive into the research topics of 'NMR detection of slow conformational dynamics in an endonuclease toxin'. Together they form a unique fingerprint.

    Cite this