Role of the Tryptophan Residues in the Specific Interaction of the Sea Anemone Stichodactyla helianthus’s Actinoporin Sticholysin II with Biological Membranes

Abstract

Actinoporins are pore-forming toxins from sea anemones. Upon interaction with sphingomyelin-containing bilayers, they become integral oligomeric membrane structures that form a pore. Sticholysin II from Stichodactyla helianthus contains five tryptophans located at strategic positions; its role has now been studied using different mutants. Results show that W43 and W115 play a determinant role in maintaining the high thermostability of the protein, while W146 provides specific interactions for protomer-protomer assembly. W110 and W114 sustain the hydrophobic effect, which is one of the major driving forces for membrane binding in the presence of Chol. However, in its absence, additional interactions with sphingomyelin are required. These conclusions were confirmed with two sphingomyelin analogues, one of which had impaired hydrogen bonding properties. The results obtained support actinoporins' Trp residues playing a major role in membrane recognition and binding, but their residues have an only minor influence on the diffusion and oligomerization steps needed to assemble a functional pore.

Stichodactyla helianthus' de novo transcriptome assembly: Discovery of a new actinoporin isoform

“…Within this context, Leu-113 in StnIII, corresponding to Trp-111 in StnI and Trp-110 in StnII stands out because this residue belongs to the so called cluster of aromatic residues, responsible for driving membrane binding and involved in maintaining the required hydrophobicity to achieve that goal. In agreement with this observation, StnII Trp-110 has been recently described as penetrating the hydrophobic membrane core (Garcia-Linares et al, 2016b). Accordingly, substitution by another highly hydrophobic amino acid, as is the case with Leu-113, is expected to have only minor influence on StnIII membrane interaction ability.…”

“…Most of these potential actinoporins showed a universally conserved motif present in all previously characterized actinoporins: "Y/FDYNWYSNWW" (Garcia-Linares et al, 2016a). Residues in bold are part of a cluster of aromatic residues involved in membrane binding (Garcia-Linares et al, 2016b;Garcia-Ortega et al, 2011;Malovrh et al, 2000), while the underlined tyrosine belongs to the sphingomyelinspecific phosphorylcholine binding site (Mancheno et al, 2003). These are two of the most significant structural signatures regarding actinoporin activity.…”

Biophysical and biochemical strategies to understand membrane binding and pore formation by sticholysins, pore-forming proteins from a sea anemone

“…Replacement of Trp 111 by Cys caused an eightfold decrease in the hemolytic activity of rStI due to a loss in its ability to interact with membranes (Penton et al 2011). These results are in agreement with those of García-Linares et al (2016), who concluded that the Trp residues of actinoporins play a major role in membrane recognition and binding but they have only minor influence on the diffusion and oligomerization steps needed to assemble a functional pore. The Pro 80 residue is located in one of the conformational loops of StI (Pardo-Cea et al 2011) and is strictly conserved in actinoporins (Hervis et al 2014).…”

Multigene Family of Pore-Forming Toxins from Sea Anemone Heteractis crispa

“…Subgroup ID3 includes actinoporins D4, E5, E8, E11, and Hct-S23 with substitutions Gln17 and 172 being found in two other subgroups, ID2 and ID4. Subgroup ID4 is distinguished by the presence of actinoporins with Phe112 in POC site, its role in membrane binding is still being discussed [ 48 , 52 ].…”

“…To clarify the role of amino acid residues during membrane binding, wild-type actinoporins, as well as recombinant and mutant ones that are produced in Escherichia coli have been used. [ 11 , 29 , 35 , 36 , 37 , 39 , 42 , 43 , 44 , 45 , 46 , 47 , 48 , 49 , 50 , 51 , 52 , 53 ]. In general, the process of pore formation by actinoporins involves its binding to a sphingomyelin of cytoplasmic membranes through the aromatic POC site, transition of a N-terminal α-helical region (1–25 aa) to the lipid-water interface, oligomerization of 3–4, 8, or 9 monomers within the membrane interface, and the insertion of the N-terminal region into membrane hydrophobic core resulted in the creation of the functionally active protein-lipid pore [ 4 ].…”