Structural stability and domain organization of colicin E1

Thermodynamic properties, stability, and structure of the toxin-like molecu le colicin El were analyzed by differential scanning calorimetry and circul ar dichroism to determine the number of structurally independent domains, a nd the interdomain interactions necessary for colicin import into the Esche richia coli cell. Analysis of denaturation profiles of the 522 residue coli cin E1, together with fragments of 342 and 178 residues that contain subset s of the domains, showed three stable cooperative blocks that differ in the rmal stability and correspond to three major functional domains of the coli cin: (i) the COOH-terminal channel-forming (C) domain with the highest ther mal stability; (ii) the BtuB receptor binding (R) domain; and (iii) the N-t erminal translocation (T) domain that has the smallest stabilization enthal py and thermal stability. Interdomain interactions were described in which T-R interactions stabilize R, and T-C and R-C interactions stabilize R and T, but destabilize C. The R and T domains behaved in a similar way as a fun ction of pH and ionic strength. Interacting extended helices of the R domai n, possibly a coiled-coil, were implied by: (i) the very high (>90%) or-hel ical content of the R domain, (ii) cooperative decreases in a-helical conte nt near the T, of thermal denaturation of the R domain; (iii) a large denat uration enthalpy, implying extensive H-bond and van der Waals interactions. The R domain was inferred, from the extended network of interacting helices , large Delta H, and steep temperature dependence of its stabilization ener gy to have a dominant role in determining the conformation of other domains . It is proposed that cellular import starts with the R domain binding to t he BtuB receptor, followed by unfolding of the R domain coiled-coil and the reby of the T domain, which then interacts with the TolC receptor-transloca tor. (C) 2000 Academic Press.