On the Mechanism of Pore Formation by Melittin

Abstract

The mechanism of pore formation of lytic peptides, such as melittin from bee venom, is thought to involve binding to the membrane surface, followed by insertion at threshold levels of bound peptide. We show that in membranes composed of zwitterionic lipids, i.e. phosphatidylcholine, melittin not only forms pores but also inhibits pore formation. We propose that these two modes of action are the result of two competing reactions: direct insertion into the membrane and binding parallel to the membrane surface. The direct insertion of melittin leads to pore formation, whereas the parallel conformation is inactive and prevents other melittin molecules from inserting, hence preventing pore formation.Lytic or antimicrobial peptides are small proteins (12-50 amino acids) that affect cells by disrupting the barrier function of lipid membranes (1). There is much interest in these peptides because of their potential pharmaceutical applications, e.g. as cancer drugs and antibiotics (2). Because of their small size and high stability, they can be obtained in large quantities. Of all lytic peptides, the 26-residue melittin is the best studied to date (for review see Ref.3). Melittin is the major constituent of the venom of the European honeybee Apis mellifera. Melittin has been reported to have anticancer effects and is already being used to treat pain and arthritis in Asia (4).Melittin can bind within milliseconds to lipid membranes and adopts an amphipatic ␣-helical conformation, oriented either parallel or perpendicular to the plane of the membrane. The perpendicular conformation is embedded in the membrane and is needed for pore formation, whereas the parallel conformation is inactive (5-10). These observations led to the proposal of a two-step model for pore formation, where melittin at low concentrations binds parallel to the membrane (Fig. 1A, step 1) and at higher concentrations shifts toward the perpendicular orientation (step 2), causing pore formation (10 -12). The transition from parallel to perpendicular is still poorly understood, especially because the cationic (5ϩ at neutral pH) melittin interacts strongly with the lipid headgroups (partitioning coefficient of 10 4 -10 5 M Ϫ1 for phosphatidylcholine (PC) 2 (12-17)), and the energy of the transition must be very high (3,11). Based on the high affinity of melittin for PC headgroups, it has been widely accepted that the concentration of melittin needed for pore formation is not dependent on the absolute melittin concentration but rather on the ratio of melittin to lipid molecules. However, this important implication has never been directly tested by varying the lipid concentration and determining the fractions of bound and free melittin. In this study we analyzed the lipid concentration dependence of melittin action and studied the reversibility of melittin binding, thereby resolving pore formation from binding events. MATERIALS AND METHODSMelittin was purchased from Genscript (Piscataway, NJ), and lipids were from Avanti Polar Lipids (Albaster, AL). Liposomes ...

FLO11 Gene Is Involved in the Interaction of Flor Strains of Saccharomyces cerevisiae with a Biofilm-Promoting Synthetic Hexapeptide

“…The flo11 mutant showed a higher tolerance to melittin than the A9 strain and its parental 3238-32 strain. Melittin must diffuse through the outer layers of the cell to reach the lipid cell membranes, where it is active through a cytolytic mechanism (36). Yeast CW is usually composed of approximately 2% chitin.…”

“…Melittin was originally isolated from the venom of the European honeybee Apis mellifera. It folds into an amphipathic ␣-helix that gives the intrinsic capacity to interact with cell membranes, leading to pore formation and nonspecific cytolysis and cell death (35,36).…”

Role of the inflammasome in defense against venoms

“…A large class of cytotoxic venom components consists of proteins with direct cytolytic activities that are dependent on the ability of these compounds to bind and disrupt cellular membranes either through direct pore formation or cleavage of membrane phospholipids (5)(6)(7). This class of toxins includes various cytolytic cationic peptides, such as melittin in bee venom, scorpion toxins in scorpion venom, lycotoxins in wolf spider venom (7,8), direct lytic factor in Indian Cobra venom, and Crotamine in South American rattlesnake venom (9,10).…”

Selective Membrane Disruption: Mode of Action of C16G2, a Specifically Targeted Antimicrobial Peptide

“…In particular, because C16G2 is novel, in that its antimicrobial killing region is conjoined to a non-AMP peptide region of equal size, it may well have a different mode of action compared with "classical" linear cationic AMPs. In this study, we investigated the mode of action of C16G2 and its ability to specifically target and disrupt the membrane of S. mutans in comparison to the widespectrum AMP melittin B (3,17,25,38), which was utilized as a positive control for expected membrane disruption activity.…”