An Intermolecular π-Stacking Interaction Drives Conformational Changes Necessary to β-Barrel Formation in a Pore-Forming Toxin

Abstract

The crystal structures of the soluble monomers of the pore-forming cholesterol-dependent cytolysins (CDCs) contain two α-helical bundles that flank a twisted core β-sheet. This protein fold is the hallmark of the CDCs, as well as of the membrane attack complex/perforin immune defense proteins and the stonefish toxins. To form the β-barrel pore, a core β-sheet is flattened to align the membrane-spanning β-hairpins. Concomitantly with this conformational change, the two α-helical bundles that flank the core β-sheet break their restraining contacts and refold into two membrane-spanning β-hairpins of the β-barrel pore. The studies herein show that in the monomer structure of the archetype CDC perfringolysin O (PFO), a conserved Met-Met-Phe triad simultaneously contributes to maintaining the twist in this core β-sheet, as well as restricting the α-helical–to–β-strand transition necessary to form one of two membrane-spanning β-hairpins. A previously identified intermolecular π-stacking interaction is now shown to disrupt the interactions mediated by this conserved triad. This is required to establish the subsequent intermolecular electrostatic interaction, which has previously been shown to drive the final conformational changes necessary to form the β-barrel pore. Hence, these studies show that the intermolecular π-stacking and electrostatic interactions work in tandem to flatten the core β-sheet and initiate the α-helical–to–β-strand transitions to form the β-barrel pore. IMPORTANCE A unique feature of the CDC/MACPF/SNTX (cholesterol-dependent cytolysin/membrane attack complex perforin/stonefish toxin) superfamily of pore-forming toxins is that the β-strands that comprise the β-barrel pore are derived from a pair of α-helical bundles. These studies reveal the molecular basis by which the formation of intermolecular interactions within the prepore complex drive the disruption of intramolecular interactions within each monomer of the prepore to trigger the α-helical–to–β-strand transition and formation of the β-barrel pore.

A Key Motif in the Cholesterol-Dependent Cytolysins Reveals a Large Family of Related Proteins

“…The largest changes in T m were observed for G274A (−9°C) and Y231A:Y273A (−12°C). The increased activity and lower T m s for the mutants of Y231 and Y273 are similar to what we have observed for mutations that destabilize D3, which makes it easier for D3 to unfold and form the β-barrel pore ( 8 , 9 , 18 , 19 ). In contrast, alanine-substituted G274 results in an unstable protein with variable activity, which appears to be the result of large side-chain-dependent repulsive forces caused by the alanine sidechain, specifically with the backbones of Y273 and R275 ( Table S1 ).…”

“…The sequential formation of intermolecular π-stacking and electrostatic interactions drive the flattening of the core β-sheet ( 18 ) by bringing β-strands 1 and 4 of two adjacent monomers into alignment within the prepore complex to form the upper wall of the β-barrel pore ( 13 , 17 ). Loss of either intermolecular interaction traps PFO in a prepore complex.…”

“…These studies show the F/Y-F/Y-X n -YGR motif forms critical interactions necessary for the function of the αβ loop. These interactions impact the subsequent formation of the key intermolecular π-stacking and electrostatic interactions, which drive the prepore-to-pore transition ( 18 , 19 ). Once these two intermolecular interactions are initiated, the prepore is irrevocably committed to its transition to the pore.…”

“…This transition shortens this loop by about 9 Å, thereby placing additional stress on the K336-E183 electrostatic interaction, which breaks the final contacts between D3 and domains 1 and 2 to allow the formation of the β-barrel pore. Hence, the αβ loop must undergo specific structural transitions in the prepore to facilitate those intermolecular interactions ( 18 , 19 ) that drive the prepore-to-pore transition.…”

“…D3 is in a metastable state; it must remain stable in the soluble monomer but undergoes significant secondary and tertiary structural changes to form the β-barrel pore, whereas D1 and D4 remain largely unchanged ( 8 , 9 , 11 , 13 ). We have shown that specific intramolecular interactions stabilize the structure of D3 in the monomer, which are broken by the formation of the intermolecular electrostatic and π-stacking interactions ( 18 , 20 ). The interactions mediated by residues Y231 and Y273 contribute to stabilizing D3, such that weakening them decreases the T m and increases the rate of pore formation, whereas replacing G274 with side-chain-containing residues greatly reduces the T m , which results in an unstable protein.…”