Competitive recruitment of the periplasmic translocation portal TolB by a natively disordered domain of colicin E9

Abstract

The natively disordered N-terminal 83-aa translocation (T) domain of E group nuclease colicins recruits OmpF to a colicin-receptor complex in the outer membrane (OM) as well as TolB in the periplasm of Escherichia coli, the latter triggering translocation of the toxin across the OM. We have identified the 16-residue TolB binding epitope in the natively disordered T-domain of the nuclease colicin E9 (ColE9) and solved the crystal structure of the complex. ColE9 folds into a distorted hairpin within a canyon of the six-bladed ␤-propeller of TolB, using two tryptophans to bolt the toxin to the canyon floor and numerous intramolecular hydrogen bonds to stabilize the bound conformation. This mode of binding enables colicin side chains to hydrogen-bond TolB residues in and around the channel that runs through the ␤-propeller and that constitutes the binding site of peptidoglycan-associated lipoprotein (Pal). Pal is a globular binding partner of TolB, and their association is known to be important for OM integrity. The structure is therefore consistent with translocation models wherein the colicin disrupts the TolB-Pal complex causing local instability of the OM as a prelude to toxin import. Intriguingly, Ca 2؉ ions, which bind within the ␤-propeller channel and switch the surface electrostatics from negative to positive, are needed for the negatively charged T-domain to bind TolB with an affinity equivalent to that of Pal and competitively displace it. Our study demonstrates that natively disordered proteins can compete with globular proteins for binding to folded scaffolds but that this can require cofactors such as metal ions to offset unfavorable interactions.native disorder ͉ thermodynamics ͉ toxin

Structural basis of toxicity and immunity in contact-dependent growth inhibition (CDI) systems

“…The N-terminal regions are exceptionally labile to proteolysis, suggesting these domains are flexible and perhaps partially disordered. Intrinsically flexible domains are critical for colicin toxin import (23,24), so perhaps the N-terminal region mediates CdiA-CT transport across the target cell envelope. The Nterminal α-helical bundle of CdiA-CT o11 EC869 has weak structural homology to diverse membrane-associated proteins, consistent with the translocation hypothesis, but the function of these domains in CDI remains to be determined.…”

Colicin Biology

“…Alternatively, the C-terminal domain might translocate through the OM bilayer by conformational changes induced by its interaction with the phospholipids (468,469,470) or at the interface between the porin trimer and the LPS leaflet. It has also been proposed that for group A colicins, interactions between the colicin N-terminal domain and the Tol machinery would displace Tol-Tol interactions (such as the TolB-Pal complex) (417) and then induce an OM defect and the subsequent entry of the cytotoxic domain (147). Nevertheless, the Tol proteins are not involved in the translocation of pore-forming C-terminal domains or in their insertion into the inner membrane (188).…”

“…When bound to its immunity protein, the affinity of colicin E9 for TolB decreases to ϳ14 M (248). A comparison of dissociation constants between TolB and the two partners, the translocation domain of colicin E9 (ColE9-T) and Pal (0.9 M versus 0.03 M), is not consistent with the displacement of the TolB-Pal interaction by the colicin E9 N-terminal domain during transit (417). However, identical measurements in the presence of Ca 2ϩ give similar affinities for TolB-ColE9-T and TolB-Pal complexes (0.08 M versus 0.09 M) (417) ( Table 3).…”

“…Here again, truncation studies have defined the ␤-propeller of TolB as the colicin binding site (72). Therefore, this domain is responsible for the interaction of TolB with both the Pal lipoprotein and the colicin N-terminal domain (550,658), and it has been demonstrated recently that the formation of the colicin E9 N-terminal domain-TolB complex displaces the TolB-Pal interaction (417) (Table 3), a result in agreement with earlier in vivo experiments demonstrating that colicin N activity does not require the TolB subunit (192). Using surface plasmon resonance and ITC, the dissociation constant for the colicin E9 (devoid of immunity)-TolB interaction was measured at ϳ1 M (248), an affinity that is 10-fold higher than that of the colicin A-TolB interaction measured by ITC (228) ( Table 3).…”

“…Once BtuB binds colicin E9, OmpF (which is a trimer consisting of three ␤-barrels with three independent pores) is recruited to interact with a natively unstructured segment of the colicin's translocation domain, forming a BtuB-OmpF-colicin translocon (275). This portion of the translocation domain then enters the cell through an OmpF pore to recruit TolB (417). The colicin's cytotoxic domain (conferring nuclease activity), now in close proximity to an OmpF trimer, probably enters the periplasm through another OmpF pore (698).…”