Figure 1.
The molecular structures of PFO and ILY.
Shown in A is a ribbon representation of the crystal structure of PFO [7]. The domain 3 β5 strand and associated α-helix (α1) that swing away from β4 are highlighted in red. The locations of Asn-197 (at the D2–D3 interface) and Val-322 (buried under the loop formed by α1β5) are shown in space-filled atoms. The twin α-helical bundles in D3 (cyan) extend into the twin transmembrane β-strands (TMHs). The conserved undecapeptide is shown in blue in D4. In panel B an enlarged view of the conserved undecapeptide loop and the CRM containing loop L1 of PFO is shown. In Panel C the analogous structures are shown for ILY as are shown in panel B for PFO.All structures were derived from the crystal structures of PFO and ILY [6], [7]). In panel D we show the structural changes in PFO as it makes the transition from the bound monomer state to the membrane embedded oligomer. The membrane embedded monomer structure is based on the 3D reconstruction of the pneumolysin pore fitted with the PFO crystal structure [57]. D3 breaks its contacts with D2 and swings out in order to extend the α-helical bundles (in cyan) into the twin TMHs. This transition also repositions Asn-197 from the D2–D3 interface to a solvent exposed position within the lumen of the membrane pore. Prior to or simultaneously with the disruption of the D2–D3 interface the α1β5 loop (red) swings away from β4 thus exposing the edge β4 (as well as exposing Val-322 to the solvent), which can then pair with β1 of a second monomer. Upon transition to the pore the oligomeric complex undergoes a 40 Å vertical collapse to insert the β-barrel pore into the bilayer [23]. After β5 breaks contact with β4 the location of α1β5 loop is not known, its position in the model is for illustrative purposes only. All structures were generated using VMD [58].
Table 1.
Cytolytic activity of PFO derivatives with mutations in the Arg-468 residue.
Figure 2.
Binding and oligomerization of PFO and PFOR468A.
(A) Binding of PFOβ4β5 and PFOR468A•β4β5 to human RBCs (4×106/ml in a final volume of 0.5 ml) was measured by flow cytometry. The disulfide locked β4β5 versions of each protein [25] were used to prevent the lysis of the RBCs during flow cytometry. (B). Oligomerization of PFO and PFOR468A(both toxins were maintained at 440 nM)on human RBCs (concentrations ranged from 2.5×107/ml to 2.5×108/ml in a final volume of 40 µl) was determined using SDS-agarose gel electrophoresis (SDS-AGE) and the proteins were detected with anti-PFO antibody after transfer to nitrocellulose paper. The analyses are representative of at least 3 experiments.
Figure 3.
Disruption of the β4β5 interface of PFO and PFOR468A.
A cysteine was substituted for Val-322, located in the D3 β4 strand. Each derivative was labeled with NBD and incubated in the presence (dashed line) and absence (solid line) of human erythrocyte ghost membranes. The fluorescence emission intensity of NBD was measured from 500–600 nm. The data are representative of 3 experiments.
Figure 4.
FRET-detected monomer association of PFO and PFOR468A.
A cysteine was substituted for Asp-30, located in domain 1 and the derivatives were labeled with Alexa Fluor 488 (donor, D) or Alexa Fluor 568 (acceptor, A).A 4∶1 molar ratio of A-labeled PFOR468A (dashed line) or unlabeled PFOR468A (U; solid line) to D-labeled toxin was incubated in the presence of human erythrocyte ghost membranes and fluorescence emission intensity of D was measured.
Figure 5.
Disruption of the D2/D3 interface in PFO and PFOR468A.
(A) A cysteine was substituted for TMH1 residue Asn-197, which is located within the D2/D3 interface. Each derivative was labeled with NBD and incubated in the presence (dashed line) and absence (solid line) of human erythrocyte ghost membranes. If TMH1 breaks its contact with D2 then Asn-197 moves from a buried, nonpolar location at the interface with D2 to the lumen of the pore. An NBD positioned at this location will therefore undergo a nonpolar to polar transition, which results in the quenching of the fluorescence emission. (B) Unlabeled native PFO or PFOR468A were mixed at a 4∶1 molar ratio with PFON197C-NBD and PFOR468A•N197C-NBD derivatives. The fluorescence emission intensity of NBD was measured from 500 to 600 nm. These data are representative of 3 experiments.
Figure 6.
TMH insertion in PFO and PFOR468A.
A cysteine was substituted for Ala-215 in PFO and PFOR468A, which is located in TMH1.The sidechain of Ala-215 is in an aqueous environment in the soluble monomer, but enters the membrane upon formation of the membrane spanning β-barrel [27]. Therefore an NBD probe positioned at this site undergoes a polar to nonpolar transition that is detected by an increase in the fluorescence emission of the probe.(A) Each NBD-labeled derivative was incubated in the presence (dashed line) and absence (solid line) of human erythrocyte ghost membranes. (B) Unlabeled native PFO or PFOR468A were mixed in a 4∶1 molar ratio with PFOA215C-NBD and PFOR468A•A215C-NBD derivatives. The fluorescence emission for all experiments wasrelative to the maximum emission change observed for NBD-labeled PFO. The fluorescence emission intensity of NBD was measured from 500–600 nm. The data are representative of 3 experiments.
Figure 7.
ILY binding and pore formation on cholesterol-rich liposomes.
(A) Binding of PFO, ILY and ILYDM to cholesterol-rich POPC liposomes was measured by SPR. The data is representative of 3 experiments. (B) Pore formation on liposomes was measured as the emission intensity of CF increased upon dilution as pores are formed in the liposomes. The change in the emission intensity of CF over time in an untreated sample was subtracted from the experimental data. The data are representative of at least 3 analyses. ILYDM contains glycine substitutions for the ILY CRM residues Thr-517 and Leu-518, which knocks out CRM-dependent binding to cholesterol-rich membranes [22].(C) To measure the insertion of the β-barrel pore a cysteine was substitutedand modified with NBDfor TMH1 residue Ala-215 of PFO or its analog, His-242 in ILY. Each derivative was incubated in the presence (dashed line) and absence (solid line) of cholesterol-rich liposomes. As the soluble monomer binds to and forms a pore in the membrane the NBD probe positioned in TMH1 makes the transition from a polar environment in the soluble monomer (solid line) to the nonpolar environment of the membrane (dashed line), which is reflected by an increase in the NBD fluorescence emission [27].
Figure 8.
The β4–β5 interaction in cholesterol bound ILY.
Cysteines were substituted for Val-322 in β4 of PFO or the analogous Val-349 in β4 of ILY and modified with NBD. Upon membrane binding β5 rotates away from β4, thus, the probe makes a transition from a nonpolar environment in the soluble monomer (solid line) to a polar environment in the membrane oligomer (dashed line), which results in the quenching of the NBD fluorescence [25]. The data are representative of 3 experiments.
Table 2.
PFO and its derivatives used herein.