3. CHAPTER 3 – Effect of mutations in and around the TolB box of colicin E9 on
3.1. Introduction
3.1.1. The TolB box of colicin E9
The interaction of colicin E9 with TolB has been demonstrated using the yeast-two hybrid system (Carr et al 2000a). The region of the translocation domain of colicin E9 involved in interacting with TolB was localised to the “TolB box” sequence (DGSGW), from residues 35 to 39 (Carr et al 2000a). Mutation of residues D35, S37 and W39 to alanine in this region abolished the cytotoxic activity of the colicin and it was shown, by yeast two hybrid experiments, that this was due to the loss of
interaction with TolB (Garinot-Schneider et al 1997, Carr et al 2000a). Mutation of residues S34 and S40 to alanine also caused a reduction in activity in a stab test but these mutants were not investigated further (Garinot-Schneider et al 1997).
An interaction between the N-terminal domain of colicin E3 and TolB has also been demonstrated by cross-linking studies and this interaction is abolished if the N- terminal domain of colicin E3 contains the S37F mutation (Bouveret et al 1997).
3.1.2. Structure of the TolB box of colicin E9
3.1.2.1. X-ray structures
The crystal structures of two tol-dependent colicins, colicin N and colicin E3, have now been determined. No electron density for the translocation domain (the first 66 residues) is seen in the 3.1 Ǻ structure of colicin N, despite the domain being present in the initial crystal form, indicating that this region is flexible and unstructured. This lack of structure in the translocation domain is consistent with CD and fluorescence data and the glycine/proline/serine/asparagine-rich composition of the domain (Evans
et al 1996, Raggett et al 1998). Residues 84-315 of the translocation domain of
colicin E3 adopt a jellyroll structure, with three β-sheets flanked by two α-helices (Soelaiman et al 2001). However, no electron density was detected for the first 83 residues, indicating that the N-terminal region of the translocation domain is probably
3.1.2.2. NMR
Sharp signals in the 1H NMR spectra of full-length colicin E9 and the N-terminal 299 and 349 residues have indicated that the translocation domain of colicin E9 is flexible (Collins et al 2002). The narrow 1H chemical shift dispersion in the 1H-15N HSQC spectrum of colicin E9 is also a sign that the protein contains unstructured regions. The observation that this spectrum is significantly different from the spectra produced from the isolated DNase domain or receptor-binding domains, indicates that the translocation domain forms the unstructured region (Collins et al 2002). However, although the consensus chemical shift index for the N-terminal region of colicin E9 indicates that much of the region lacks secondary structure elements, differences in chemical shifts between colicin E9 and those of random coil values suggest that some residues eg S30 and D35 in the N-terminal region are not in random coil
conformations (Collins et al 2002). Relaxation measurements indicate that residues W39 to N44 and L81 to A83 of colicin E9 have more restricted mobility than the rest of the region (Collins et al 2002). The restricted mobility of residue W39 suggests that the TolB box could be more structured than other regions of the translocation domain, although the mobility of D35 does not differ from that seen for the rest of the region and the resonances for residues 36-38 have not yet been assigned (Collins et al 2002).
Differing intensities of the resonances observed for residues S40, S41 and E42 in the NMR spectrum indicate that these residues exist in different conformational states, despite the homogeneity of the protein samples, confirmed with SDS-PAGE and chromatography (Collins et al 2002). Collins et al (2002) suggest that these residues could be involved in side-chain/side-chain interactions, leading to the formation of clusters of interacting residues, with one cluster being involved in the interaction with TolB.
Therefore, colicins appear to be amongst a growing number of recognised proteins with intrinsically disordered domains, involved in macromolecular interactions, which adopt folded structures upon binding to their biological targets (Dyson and Wright 2002). The lack of structure can be functionally advantageous, for example allowing
3.1.3. Extension of the TolB box
The 1H-15N HSQC spectrum of colicin E9 in the presence of excess, unlabelled TolB shows that backbone resonances of residues 33 to 44 of colicin E9, including the TolB box, are perturbed on binding to TolB (Collins et al 2002). It was therefore suggested that the region for binding to TolB should be extended from the recognised
pentapeptide sequence, DGSGW, to twelve amino acids, although it could not be ruled out that the perturbations in resonances could be due to conformational changes in the region flanking the TolB box resulting from the interaction of the TolB box region with TolB, rather than a direct interaction between TolB and these residues. The backbone resonances of N10, H14, H55 and W56 were also affected by addition of TolB (Collins et al 2002). However, mutation of residue W56 to alanine, located in a region of colicin E9 which showed homology to colicin K, did not affect the activity of the colicin in a stab test (Garinot-Schneider et al 1997) and constructs lacking residues 54-164 of colicin E9 have been shown to interact with TolB in yeast two hybrid experiments (Carr et al 2000a).
Recently an alanine mutation at residue 42 has been shown to be abolish activity of colicin E9 and this mutant protein does not interact with TolB in SPR experiments (Holland, 2003).
The three residues after the TolB box pentapeptide sequence of colicins E2-E9 and A are conserved (see Figure 3.1), providing further evidence that residues directly downstream of the TolB box may also be involved in the interaction with TolB (Bouveret et al 1998). Colicin E1, which does not require TolB for translocation, contains a pentapeptide sequence homologous to the TolB box but the region
immediately following this TolB box is not homologous to the endonuclease colicins, supporting the possibility that residues downstream of the TolB box are involved in the colicin-TolB interaction. Although the TolB box of colicin A is predicted to lie between residues 11 and 15, a colicin A mutant with residues 16-30 deleted does not interact with TolB, whilst a mutant with residues 21-29 deleted does interact with TolB, indicating that residues between 15 and 20 could also be involved in the interaction of colicin A with TolB (Bouveret et al 1998). However, no mutational data has been published to confirm the role of any of these six residues in binding to TolB.
Colicin E9
34
SDGSGWSSENNPW
46
Colicin E7
34
SDGSGWSSENNPW
46
Colicin E3
34
SDGSGWSSENNPW
46
Colicin E2
34
SDGSGWSSENNPW
46
Colicin E1
29
PDGSGSGGGGGKG
41
Colicin A
10
GDGTGWSSERGSG
22
Colicin K
16
MGGTGANLNQQGG
28
Colicin N
25
TSGAGSNGSASSN
37
Figure 3.1 Alignment of known and predicted TolB boxes (black) and flanking residues (red) of TolB-dependent colicins (adapted from Garinot-Schneider 1997).
3.1.4. TolB interaction with colicin E9
Although the region of colicin E9 involved in interacting with TolB has been
localised to the TolB box, the region of TolB involved in interacting with colicin E9 is less well-defined. As discussed in Chapter 1, the crystal structure of TolB shows that the protein consists of an N-terminal mixed αβ domain and a C-terminal β-propeller domain (Carr et al 2000a, Abergel et al 1999).
β-propeller proteins usually use their central tunnel or the entrance to the tunnel to coordinate a ligand or to carry out a catalytic function that is preserved by the structural rigidity of the propellers. It was therefore proposed that the translocation domains of colicins interact with the β-propeller domain of TolB (Carr et al 2000a). N-terminal and C-terminal deletions have been made in the tolb gene and the effect of the deletions on the TolB protein on binding to colicin E9 has been investigated using the yeast-two hybrid technique (Carr et al 2000a). Deletion of the first 119 residues of TolB had no effect on the interaction with the translocation domain of colicin E9. However, deletion of residues 1-203, 205-431 or 304-431 abolished the TolB-colicin E9 interaction (Carr et al 2000a). This suggests that the region of TolB involved in the interaction with colicin E9 is located between residues 119 and 431. As this region encompasses the β-propeller domain, it has been suggested that it is the β- propeller domain of TolB that is involved in the interaction with colicin E9 (Carr et al
of TolB does not affect the interaction with colicin, whereas insertion of two residues (SS) after residue P87 affected the ability of the colicin to enter cells (Abergel et al 1999). An E.coli tolbmutant strain, when transformed with a cloned tolb mutant gene, lacking most of blades V and VI of the β-propeller domain, was shown to be sensitive to colicins A and E1 (Bénédetti et al 1991b).
Yeast-two hybrid experiments have shown that the translocation domain of colicin A does not interact directly with either the isolated β-propeller domain or N-terminal domain, in contrast to the yeast-two hybrid result for colicin E9, which detected an interaction between residues 120-413 of TolB and colicin E9 (Wallburger et al 2002, Carr et al 2000a). It is suggested by Wallburger et al (2002) that the discrepancy could be due to differing translocation mechanisms between the pore-forming colicin A and the endonuclease colicin E9.
3.1.5. Aims of this chapter
The work described in this chapter further characterises the role of the residues in the recognised pentapeptide TolB box. The technique of surface plasmon resonance has been used to directly compare binding of TolB to wild-type colicin E9/Im9 and to colicin E9/Im9 mutants with an alanine mutation in the TolB box. The SPR technique has also been used to investigate the effect of mutating the three TolB box residues, known to abolish biological activity and TolB binding, to residues other than alanine. Additionally, this chapter further investigates the hypothesis that the region of the translocation domain of colicin E9, involved in interaction with TolB, is longer than the pentapeptide sequence initially proposed.