Quantitative Analysis of Bacterial Toxin Affinity and Specificity for Glycolipid Receptors by Surface Plasmon Resonance

195

235

Equinatoxin II (EqtII) belongs to a unique family of 20-kDa pore-forming toxins from sea anemones. These toxins preferentially bind to membranes containing sphingomyelin and create cation-selective pores by oligomerization of 3-4 monomers. In this work we have studied the binding of EqtII to lipid membranes by the use of lipid monolayers and surface plasmon resonance (SPR). The binding is a two-step process, separately mediated by two regions of the molecule. An exposed aromatic cluster involving tryptophans 112 and 116 mediates the initial attachment that is prerequisite for the next step. Steric shielding of the aromatic cluster or mutation of Trp-112 and -116 to phenylalanine significantly reduces the toxin-lipid interaction. The second step is promoted by the N-terminal amphiphilic helix, which translocates into the lipid phase. The two steps were distinguished by the use of a double cysteine mutant having the N-terminal helix fixed to the protein core by a disulfide bond. The kinetics of membrane binding derived from the SPR experiments could be fitted to a two-stage binding model. Finally, by using membraneembedded quenchers, we showed that EqtII does not insert deeply in the membrane. The first step of the EqtII binding is reminiscent of the binding of the evolutionarily distant cholesterol-dependant cytolysins, which share a similar structural motif in the membrane attachment domain.Targeting and attachment of proteins to membranes is one of the key steps in many cellular processes (1-3). Protein-membrane interactions have been studied intensively in recent years with many different examples of proteins and membranes. These interactions can be promoted at the lipid-water interface by lipid anchors, electrostatic forces or surface-exposed aromatic and aliphatic residues (1, 2, 4). Compared with protein-protein interactions, details of protein-membrane interactions are poorly defined. Some of the best characterized examples are a phospholipase C pleckstrin homology domain specific for phosphatidylinositol trisphosphate (5) and small protein kinase-C-conserved (C2) domains specific for zwitterionic, particularly phosphatidylcholine membranes (6).Another group of proteins interacting with lipid membranes are pore-forming toxins (PFT) 1 (7-10), which bind to membranes before eliciting their toxic effects via the formation of transmembrane pores. The most studied PFT are bacterial since this group includes important virulence factors. Few examples of eukaryotic PFT have been well characterized, exceptions being the actinoporins, cytolysins found exclusively in sea anemones (10, 11). Members of this family have properties distinct from other PFT: they are composed of 175-179 amino acids, contain no cysteine residues, have pIϾ9.5, and show a preference for sphingomyelin (SM)-containing membranes. Actinoporins act on cellular and model lipid membranes by forming cation-selective pores with a hydrodynamic diameter of ϳ2 nm. The mechanism of pore formation involves at least two steps: binding of the water soluble m...

Section: Resultsmentioning

confidence: 99%

Two-step Membrane Binding by Equinatoxin II, a Pore-forming Toxin from the Sea Anemone, Involves an Exposed Aromatic Cluster and a Flexible Helix

Hong¹,

Gutiérrez-Aguirre²,

Barlič³

et al. 2002

195

235

“…However, the Gal␤3GalNAc disaccharide moiety is necessary for binding (10,11). Recent studies using surface plasmon resonance to study binding of purified H C fragment have extended these findings and demonstrated a preference for GD1b over GT1b and negligible binding to GQ1b gangliosides (12).…”

mentioning

confidence: 75%

The Structures of the HC Fragment of Tetanus Toxin with Carbohydrate Subunit Complexes Provide Insight into Ganglioside Binding

Emsley

Fotinou

Black

et al. 2000

137

136

Tetanus toxin (TeNT) 1 and the botulinum toxins (BoNTs) are members of the family of clostridial neurotoxins, produced by Clostridium tetani and Clostridium botulinum, respectively. These protein toxins are structurally and functionally related, each being synthesized as a 150-kDa single polypeptide, which is processed to give a 50-kDa amino-terminal L chain, disulfide-bonded to a 100-kDa carboxyl-terminal H chain. These toxins cause paralysis by inhibiting release of neurotransmitter from presynaptic nerve terminals. The differences in clinical symptoms that tetanus and botulinum toxins exhibit are due to the distinct sites of action of the toxins. TeNT undergoes retrograde transport from the neuromuscular junction to the central nervous system and targets inhibitory neurons within the spinal cord causing a spastic paralysis. In contrast, BoNTs do not undergo retrograde transport and target peripheral sensory neurons resulting in flaccid paralysis.

“…Study of macromolecule-ligand interaction by SPR method depends solely on the mass changes during the reaction (18,19). Additionally, in SPR the ability to form model membrane assemblies, monolayers, or bilayers incorporating the biologic receptors which mimic the natural environment offer additional opportunities to study, in molecular terms, the surfaceassociated phenomena.…”

Section: Resultsmentioning

confidence: 99%

Surface Plasmon Resonance Studies Resolve the Enigmatic Endotoxin Neutralizing Activity of Polymyxin B

Thomas¹,

Surolia²,

Surolia³

1999

Polymyxin B (PMB), a cyclic cationic peptide antibiotic, despite its severe side effects continues to occupy a premiere position for treating endotoxicosis. Its mode of neutralization of endotoxin has remained elusive for the last three decades. Several synthetic peptide mimics of PMB, capable of binding endotoxin, have been made. However, the binding ability alone appears to be a deceptive indicator of endotoxin neutralizing activity as molecules with similar binding propensities could either sequester or opsonize the toxin. Hence identification of additional physical parameters which describe adequately the outcome of PMB-endotoxin interaction become imperative. Surface plasmon resonance (SPR) studies reported here show that several mimics of PMB despite exhibiting lipopolysaccharide binding affinities comparable with it but, unlike it, do not sequester the endotoxin. These studies thus provide a striking illustration of the difference in the behavior of PMB, vis a vis its mimics toward the endotoxin lamellae, and define further, in chemical terms, mechanism of the action of PMB and allow us to posit that the design of molecules as effective antidotes for sepsis should incorporate the ability to sequester endotoxin specifically.Release of miniscule (nanomolar) quantities of lipopolysaccharide (LPS), 1 the major structural component of the Gramnegative bacterial outer membrane, in systemic bacterial infections frequently leads to a relatively common but often fatal constellation of symptoms termed as the endotoxic shock (1, 2). The treatment for endotoxic shock which is characterized by deranged hemodynamics, coagulation abnormalities, and multiple system organ failure, continues to remain nonspecific and supportive because of the absence of specific interventional strategies (3). However, the mechanisms by which endotoxin (LPS) acts on the target cells are increasingly being understood, which in turn has led to the development of several experimental approaches for treating endotoxicosis. These include sequestration of LPS by peptides or anti-LPS antibodies (4 -7), use of its antagonistic homologs that prevent its binding to the target cells (8), or the molecules that abrogate signaling to the pathways leading to the production of inflammatory cytokines such as tumor necrosis factor, interleukin-1, etc. (9). Despite these many interventional modalities and the severe side effects associated with the use of polymyxin B (PMB), a cyclic cationic peptide antibiotic, for the treatment of endotoxicosis, it continues to occupy the premiere position in our armamentarium for combating endotoxicosis (3, 4). Only recently have its mode of interaction with LPS and the structural features involved therein been elucidated (6, 10 -12). These and earlier studies have led to the proposal that the asymmetric distribution of the basic and nonpolar groups in polymyxin B impart to it an amphiphilicity that is both necessary and sufficient for its LPS neutralizing activity. That this is indeed the case was proven by a subsequent st...