Endonuclease colicins are secreted as heterodimers, binding tightly and specifically with an immunity protein on synthesis before being exported into the extracellular medium. Binding of the immunity protein neutralises the endonuclease activity of the colicin and thus protects the host cell from self- destruction. After binding to a sensitive E. coli cell, the immunity protein must be lost from the colicin-immunity protein complex at some stage to ensure that an active endonuclease is imported into the cytoplasm of target cells. Free ColE9 has the same bactericidal activity as ColE9 bound to Im9 (Wallis, Reilly et al. 1992), confirming therefore that the immunity protein is not a requirement for receptor binding or the translocation steps of colicin. Conversely, removal of Im3 from ColE3 leads to substantial loss of bactericidal activity signifying that, in addition to protecting producing cells from ColE3, Im3 may also stabilise ColE3 before entering susceptible cells (Walker, Moore et al. 2003).
The interaction of an endonuclease colicin and its cognate immunity protein (i.e. ColE9 binding to Im9) is one of the highest affinity protein-protein interactions known. This was validated by determining that the equilibrium dissociation constant (Kd) for the binding of Im9 to full-length ColE9, is 9.3 x
10-17M at pH 7 and 25 °C (Wallis, Moore et al. 1995). A similar Kdvalue of
be 7.2 x 10-17 M was found for the interaction between the ColE9 DNase domain and Im9 to, therefore it is assumed that the immunity protein makes energetically essential contacts with the DNase domain of the colicin only (Wallis, Leung et al. 1995). However, binding of the DNase domain to a non- cognate immunity protein (i.e. binding of ColE9 to Im8), resulted in Kdvalues
in the range of 10-4 to 10-16 M (Wallis, Leung et al. 1995). Observation of the in vivo protection provided by the immunity proteins against ColE9 was in the order of Im9 > Im2 >Im8 > Im7 which reflects the difference in their in vitroaffinities (Wallis, Leung et al. 1995).
Structure determination of the three colicin endonuclease domains bound to their cognate immunity proteins (Figure 1-7) has provided insights into how immunity proteins protect bacteria from the cytotoxic activity of endonuclease colicins. The structure of full-length ColE3 in complex with Im3 has been resolved (Soelaiman, Jakes et al. 2001) as well as the structure of only the RNase domain in complex with Im3 (at 2.4 resolution, (Carr, Walker et al. 2000)) were very similar structures. Furthermore, the structures of the DNase domain of ColE7 in complex with Im7 (at 2.3 resolution, (Ko, Liao et al. 1999; Cheng, Shi et al. 2006)) and ColE9 in complex with Im9 (at 2.05 and 1.7 resolution, (Kleanthous, Kuhlmann et al. 1999; Kuhlmann, Pommer et al. 2000)) have also been determined. Comparison of these structures reveals
few similarities; the DNase domains are proteins (Kleanthous, Kuhlmann et al. 1999; Ko, Liao et al. 1999) while the RNase domain consists of predominantly sheet (Carr, Walker et al. 2000; Soelaiman, Jakes et al. 2001). The immunity proteins for the RNase and DNase domains are also structurally unrelated. The DNase immunity proteins are distorted four-helix bundles (Chak, Safo et al. 1996; Osborne, Breeze et al. 1996) whilst the RNase immunity proteins are predominantly sheet proteins (Zakharov, Lindeberg et al. 1999).
However, the DNase and RNase immunity protein complexes share a charge complementarity as a result of the basic nature of the endonuclease domain and the acidic nature of the immunity protein. Moreover, NMR experiments have been used as a guide for alanine scanning mutagenesis of Im9 (Osborne, Wallis et al. 1997; Li, Hamill et al. 1998), and the conserved residues that make the largest relative contribution toward E9 DNase binding were determined (Wallis, Leung et al. 1998).
Figure 1-7: Structures of different endonuclease colicin-immunity complexes. The structures of the colicin E3-Im3 complex (left) (Soelaiman, Jakes et al. 2001), the ColE7-Im7 complex (middle) (Cheng, Shi et al. 2006) and the ColE9 DNase-Im9 complex (right) (Kuhlmann, Pommer et al. 2000) are shown.
The ColE9 DNase-Im9 complex (Figure 1-7) consists of a hydrophobic core of mainly aromatic residues from both the DNase and Im9, with multiple hydrogen bonds and salt bridges surrounding this core. The structure of the ColE7 DNase -Im7 complex shows that there are 2.17 hydrogen bonds per 100 Å2shared between the E7 DNase and Im7, which is substantially more than the average calculated for enzyme-inhibitor complexes of 1.37 hydrogen bonds per 100 Å2, (Jones and Thornton, 1996). This, in conjunction with the fact that
many of the hydrogen bonds involve charged donor and acceptor groups, is likely to contribute to the high affinity of the interaction (Ko, Liao et al. 1999). The structure of the ColE3 RNase domain in complex with Im3 shows that the RNase N-terminal helix wraps around the exposed face of the four-stranded - sheet of Im3 (Kolade, Carr et al. 2002). On binding of the ColE3 RNase domain to Im3, there is a much greater loss of surface area as compared to binding of ColE9 or E7 DNase domains to their respective immunity proteins (2554 Å 2 compared to 1575 Å 2 and 1473 Å 2, respectively) (Kuhlmann, Pommer et al. 2000; Cheng, Shi et al. 2006). Their high affinity of the interaction is a result of the high degree of surface complementarity, which is indicative of a conformational change on formation of the protein-protein complex, but so far no structure of the free RNase is available to confirm this (Kolade, Carr et al. 2002). A recent investigation introduced mutations into Im7 and demonstrated a critical role for three residues, Leu 53, Ile 54 and Tyr 55, to lock Im7 into its unique native structure (Knowling, Bartlett et al. 2011). Leu 53 and Ile 54 were found to provide critical stabilizing interactions in the hydrophobic core of Im7, while Tyr 55 is essential for both stability and function. In contrast, Tyr 56 is crucial for colicin binding and has no role in maintaining a stable native fold (Knowling, Bartlett et al. 2011)