Structure-function relationships of E-type endonuclease colicins

Abstract

Colicin cytotoxicity can take various guises, the most remarkable being degradation of bacterial DNA, requiring the 60 kDa toxin to translocate its C-terminal, 15 kDa enzymatic domain across two membrane boundaries in order to reach the cytoplasm. Current theories concerning colicin translocation envisage that conformational changes, and partly unfolding, are required before the toxin can enter the cell. To date, four colicin DNases have been identified, i.e. E2, E7, E8 and E9, which share high sequence homology. In our study nano-ESI-MS was used to gain insights into metal binding and conformational properties of these metalloproteins. Thermostability and intrinsic trypthophan accessibility measurements were performed and correlated with our ESI-MS data.

ESI-MS analysis of each DNase revealed substantial different folding properties even though they are highly homologous. In the absence of zinc ions, which bind with nM affinity to the enzyme, the E7 DNase mass spectra were dominated by a loosely packed ('unfolded') population of protein conformers, while mass spectra for apo-E8 DNase represented mostly 'folded' conformers. The presence of zinc ions resulted in increased conformational stability for all proteins thereby adopting states that are more folded and uniform. Taken together, the present study demonstrates that ESI-MS is sensitive to changes in the conformational stability in the four DNases in a manner consistent with experimental data deduced from calorimetry and intrinsic Trp fluorescence quenching.