The sea anemone actinoporin (Arg‐Gly‐Asp) conserved motif is involved in maintaining the competent oligomerization state of these pore‐forming toxins

Abstract

Sea anemone actinoporins constitute an optimum model to investigate mechanisms of membrane pore formation. All actinoporins of known structure show a general fold of a b-sandwich motif flanked by two a-helices. The crucial structure for pore formation seems to be the helix located at the N-terminal end. The role of several other protein regions in membrane attachment is also well established. However, not much is known about the protein residues involved in the oligomerization required for pore formation. Previous detailed analysis of the soluble three-dimensional structures of different wild-type and mutant actinoporins from Stychodactyla helianthus suggested residues which could be involved in this oligomerization. One of these stretches contains a conserved sequence compatible with an integrin-binding RGD motif. The results presented now deal with mutants affecting this motif in the well-characterized actinoporin sticholysin II. Small modifications along this three-residue sequence had profound effects on its solubility. Just a single methyl group yielded an RAD mutant version with a highly diminished haemolytic activity and altered oligomerization behaviour. The results obtained are discussed in terms of a key role for the RGD motif in maintaining the actinoporins' pore-competent state of protein oligomerization. Structured digital abstractStnII and StnII bind by molecular sieving (1, 2)

Evolution of the Cytolytic Pore-Forming Proteins (Actinoporins) in Sea Anemones

“…The RGD domain (R144, G145, and D146) forms a bend in the protein between two β–strands and was hypothesized to maintain correct oligomerization in order to form a functional pore, but may also play an important role in directing protein attachment to some integrin-like receptors [28]. Many of the actinoporin candidates in clade 2A are instead equipped with KGD (R144K) residues, and the actinoporin candidates in clade 2M are equipped with EGD (R144E) residues or have a deletion in this region (Figure 2).…”

“…Several residues have been manipulated to identify functionally important regions within the protein [25], revealing an aromatic-rich region that forms the phosphocholine (POC) binding site, with a single amino acid residue (W112 in Equinatoxin II (EqII)) taking on a key role in initiating sphingomyelin recognition and pore formation [11,26,27]. Although events leading to oligomerization remain uncertain, both the RGD domain (R144, G145, and D146 in EqII) and a single valine residue (V60 in EqII) are thought to direct protein attachment and play a key role in this process [25,28]. Ultimately, a key hydrophobic arginine (R31 in EqII) and other hydrophobic residues in the α-helix at the N-terminal region are involved in cell membrane penetration and the formation of the ion conductive pathway [29,30,31,32,33,34], forming a selective pore from four monomers [12,35,36], although oligomerizations involving eight or nine peptides have also been proposed [37,38].…”

Synergistic Action of Actinoporin Isoforms from the Same Sea Anemone Species Assembled into Functionally Active Heteropores

“…These amino acid analyses were performed on a Biochrom 20 automatic analyzer (GE Healthcare). All protein batches used were also previously characterized in terms of recording their far-UV circular dichroism (CD) spectra on a Jasco 715 spectropolarimeter, also as described (20,21,56,57).…”

“…Protein Binding to Lipid Vesicles-Binding was measured using isothermal titration calorimetry (ITC) as described before (21,30,61), using a VP-ITC calorimeter (MicroCal). Briefly, protein solutions at 1.5-10.0 M concentration were titrated by injection of 10-or 20-l aliquots of lipid suspensions (phospholipid concentration, 0.85-5.00 mM).…”

A RNA-seq approach to identify putative toxins from acrorhagi in aggressive and non-aggressive Anthopleura elegantissima polyps

“…Although classified into four paralogous groups, all cytolysins form pores in the cellular membrane, creating an ionic imbalance that results in cytolysis [ 18 , 28 - 31 ]. Unlike other classes of toxins discussed here, cytolysins do not have disulfide bonds, relying instead on several amino acid residues for proper folding [ 18 , 32 ]. In term of function, cytolysins are ideal candidate agents for the localized necrosis observed in the victim of an intraspecific aggressive encounter, however, they cannot form pores in cnidarian cells because cnidarians lack the target lipid sphingomyelin in their cell membranes [ 18 , 33 , 34 ].…”