Structure-function relationship from vesicle to cytosol

Pore formation

At first glance, the primary sequence of PA does not look like that of a membrane-spanning protein. A hydrophobicity plot lacking any patterns which are common to possible membrane-spanning domains. The structures of other multimeric membrane proteins (such as diphtheria toxin) provide the answer to how PA manages to span the membrane. It is thought that PA acts like these multimeric membrane proteins that form β-barrels made from stretchs of both polar and non-polar amino acids from each monomer.^[10]

Greek-key motif.

The formation of the β-barrel pore is facilitated with a drop in pH. To form the barrel when the pH drops, PA63 domain 2 must undergo the greatest conformation change. Upon examination of the structure of domain 2 (Fig. 7), one can see that this domain contains a Greek-key motif (the gold portion in Fig. 7). A general schematic of a Greek-key motif is shown in Fig. 8. Attached to the Greek-key in domain 2 is a large disordered loop. The necessity of this loop in pore formation is shown through using mutagenesis and proteolysis of the loop with chymotrypsin. Additional electrophysiological measurements of cysteine substitutions place the amino acids of this loop inside the lumen of the membrane inserted pore. The disordered loop in domain 2 also has a pattern of alternating hydrophobic and hydrophilic amino acids, which is a pattern conserved in the membrane-spanning portions of porins. The only problem is that the loop is not large enough to span a membrane in a β-barrel. This membrane insertion could only occur with additional conformational changes. A large conformational change takes place where the Greek-key motif unfolds, forming a β-hairpin that projects downward into the membrane and forms a β-barrel with the other 6 monomers of the complex (figures 9a and 9b). The final pore has a diameter of 12 Å (1.2 nm), which fits the theoretical value of this model.^[10]

This model would require large conformational changes in domain 2 along with the breaking of many hydrogen bonds as the Greek-key motif peels away from the center of the domain. Petosa et al. proposed a model of how this occurs.^[10] Insertion of the PA Greek key motifs into the membrane occurs when the heptamer is acidified. On artificial bilayers, this occurs when the pH is dropped from 7.4 to 6.5, suggesting that the trigger for insertion involves a titration of histidines. This indeed fits the sequence of PA since domain 2 contains a number of histidines (shown as asterisks in figure 9a). Three histidine residues are found in the disordered loop, one of which lies with a Greek-key histidine within a cluster of polar amino acids. This cluster (including the two histidines, three arginines and one glutamate) is embedded at the top of the Greek-key motif, so it is easy to see that the protonation of these histidines would disrupt the cluster. Furthermore, another histidine is located at the base of the Greek-key motif along with a number of hydrophobic residues (on the green segment in figures 7 and 9a). At pH 7.4 this segment is ordered, but when the crystals are grown at pH 6.0, it becomes disordered. This order to disorder transition is the initial step of PA membrane insertion.

PA is endocytosed as a soluble heptamer attached to its receptors, with LF or EF attached to the heptamer as cargo. The first step after endocytosis is the acidification of the endocytotic vesicle. The acidification plays two roles in the lifespan of the toxin. First, it helps to relax the tight grip of the CMG2 or TEM8 receptor on PA, facilitating the pore formation (the different receptors allow for insertion at a slightly different pH).^[12] Second, the drop in pH causes a disordered loop and a Greek-key motif in the PA domain 2 to fold out of the heptamer pre-pore and insert through the wall of the acidic vesicle, leading to pore formation (Figures 7–9).

Santelli et al. explained more about the process after they determined the crystal structure of the PA/CMG2 complex.^[12] The structure of this complex shows the binding of CMG2 by both domain 2 and 4 of PA. This interaction demonstrates less freedom to unfold the Greek key. Further analysis shows that seven of the nine histidines in PA are on the domain 2/domain 4 interface. Protonation of these histidines causes the domains to separate enough to allow the Greek-key to flop out and help form the β-hairpin involved in insertion. In addition, when PA binds to CMG2, insertion no longer occurs at a pH of 6.5, as it does when inserted into an artificial membrane. Instead it requires a pH of 5.0 for insertion in natural cells. This difference was explained to be the result of the pocket next to the MIDAS motif in CMG2. This pocket contains a histidine buried at the bottom where domain 2 attaches. This histidine is protonated at a lower pH and adds greater stability to PA. This added stability keeps the Greek-key from being able to move until more acidic conditions are met. These histidines all work in conjunction to keep the heptamer from inserting prematurely before endocytosis occurs.

Santelli and colleagues (Fig. 10) also built a hypothetical structure of the membrane-inserted PA/CMG2 structure. This model shows that the β-barrel is about 70 Å (7 nm) long, 30 Å (3 nm) of which span the membrane and the 40 Å (4 nm) gap is actually filled in with the rest of the extracellular portion of the CMG2 receptor (~100 residues). CMG2 provides additional support to the pore.

Protein translocation[edit]

Diagram of protein translocation.

Several recent studies demonstrate how the PA63 pore allows the EF and LF into the cytoplasm when its lumen is so small. The lumen on the PA63 pore is only 15 Å (1.5 nm) across, which is much smaller than the diameter of LF or EF. Translocation occurs through a series of events which begin in the endosome as it acidifies. LF and EF are pH sensitive, and as the pH drops, their structures lose stability. Below a pH of 6.0 (the pH in an endosome), both LF and EF become disordered molten globules. When a molecule is in this conformation, the N-terminus is freed and drawn into the pore by the proton gradient and positive transmembrane potential. A ring of seven phenylalanines at the mouth endosome side of the pore (phenylalanine clamp) assists in the unfolding of LF or EF by interacting with the hydrophobic residues found in LF or EF. The proton gradient then begins to lace the protein though the pore. The lacing mechanism is driven by the gradient, but requires the phenylalanine clamp for a ratcheting motion. The first 250 residues of EF and LF have an irregular alternating sequence of basic, acidic, and hydrophobic residues. The interplay between the phenylalanine clamp and the protonation state cause a ratcheting effect that drives the protein though until enough has crossed into the cytoplasm to drag the rest through the pore as the N-terminus refolds (Fig. 11).