Calcium-Driven Folding of RTX Domain β-Rolls Ratchets Translocation of RTX Proteins through Type I Secretion Ducts

Abstract

Calcium-binding RTX proteins are equipped with C-terminal secretion signals and translocate from the Ca(2+)-depleted cytosol of Gram-negative bacteria directly into the Ca(2+)-rich external milieu, passing through the "channel-tunnel" ducts of type I secretion systems (T1SSs). Using Bordetella pertussis adenylate cyclase toxin, we solved the structure of an essential C-terminal assembly that caps the RTX domains of RTX family leukotoxins. This is shown to scaffold directional Ca(2+)-dependent folding of the carboxy-proximal RTX repeat blocks into β-rolls. The resulting intramolecular Brownian ratchets then prevent backsliding of translocating RTX proteins in the T1SS conduits and thereby accelerate excretion of very large RTX leukotoxins from bacterial cells by a vectorial "push-ratchet" mechanism. Successive Ca(2+)-dependent and cosecretional acquisition of a functional RTX toxin structure in the course of T1SS-mediated translocation, through RTX domain folding from the C-terminal cap toward the N terminus, sets a paradigm that opens for design of virulence inhibitors of major pathogens.

Kinetics of DNA uptake during transformation provide evidence for a translocation ratchet mechanism

“…Chemical asymmetries can bias the Brownian walk of the chain. Experimental examples for such asymmetries include compaction of DNA during DNA injection into host cells by the Agrobacterium tumefaciens type 4 secretion system (9) and calcium-induced folding of proteins exported by the type 1 secretion system of Bordetella pertussis (10). A conceptually simple mechanism for ratcheting would be the existence of molecules (chaperones) binding to the polymer only on one side of the membrane; they hinder backward diffusion and thus bias the polymer translocation.…”

Bacterial Translocation Ratchets: Shared Physical Principles with Different Molecular Implementations

“…[82] Only the early steps of substrate translocation depend on the ABC transporter. [78,81] Once the C-terminus of the cargo protein reaches the extracellular space, Ca 2þ -dependent protein folding can initiate the intramolecular ratchet [5] (see next paragraph), suggesting that the process of "threading" depends on ATP Single-molecule experiments show that chaperone ComE accelerates DNA uptake; comparison of force-dependent translocation kinetics between experiment and theory provides strong evidence for translocation ratchet mechanism. [3,35] T4SS Ejection of DNA into eukarytic target cells…”

“…[83] The translocation of this protein class across the cell envelope of Gram negative bacteria is powered by Ca 2þ -dependent protein folding. [5] The part of the protein facilitating T1SS-mediated transport is the C-terminal repeat-in-toxin (RTX) domain. [84] A non-cleavable C-terminal signal sequence mediates the initiation of transport by inserting the C-terminus in the T1SS duct.…”

“…[94] The folding of the Cterminal RTX segment then triggers a translocation ratchet mechanism via segment-by-segment folding of the entire RTX domain. [5] This cooperativity results in an acceleration of the ratcheting process that allows for translocation of the Nterminal effector domain. [5] Interestingly, the application of external antibodies against RTX could substitute external Ca 2þ with regard to functional secretion by the T1SS, thereby creating an artificial chaperone-assisted translocation ratchet that is similar to the examples above discussing DNA translocation.…”

“…Structural data together with functional analysis indicate that an intramolecular ratchet drives RTX secretion by Ca 2þ specific folding without the requirement for chaperones. [5] 2. DNA Uptake During Transformation and the Type II Secretion/Type IV Pilus System Bacterial transformation is the uptake and inheritable integration of DNA from the environment.…”