Dataset Information

Nuclear Magnetic Resonance-Based Structural Characterization and Backbone Dynamics of Recombinant Bee Venom Melittin.

ABSTRACT: In recent years, there has been a resurgence of interest in melittin and its variants as their therapeutic potential has become increasingly evident. Melittin is a 26-residue peptide and a toxic component of honey bee venom. The versatility of melittin in interacting with various biological substrates, such as membranes, glycosaminoglycans, and a variety of proteins, has inspired a slew of studies that aim to improve our understanding of the structural basis of such interactions. However, these studies have largely focused on melittin solutions at high concentrations (>1 mM), even though melittin is generally effective at lower (micromolar) concentrations. Here we present high-resolution nuclear magnetic resonance studies in the lower-concentration regime using a novel method to produce isotope-labeled (15N and 13C) recombinant melittin. We provide residue-specific structural characterization of melittin in dilute aqueous solution and in 2,2,2-trifluoroethanol/water mixtures, which mimic melittin structure-function and interactions in aqueous and membrane-like environments, respectively. We find that the cis-trans isomerization of Pro14 is key to changes in the secondary structure of melittin. Thus, this study provides residue-specific structural information about melittin in the free state and in a model of the substrate-bound state. These results, taken together with published work from other laboratories, reveal the peptide's structural versatility that resembles that of intrinsically disordered proteins and peptides.

SUBMITTER: Ramirez L

PROVIDER: S-EPMC6333091 | biostudies-literature | 2018 May

REPOSITORIES: biostudies-literature

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Nuclear Magnetic Resonance-Based Structural Characterization and Backbone Dynamics of Recombinant Bee Venom Melittin.

Ramirez Lisa L Shekhtman Alexander A Pande Jayanti J

Biochemistry 20180430 19

In recent years, there has been a resurgence of interest in melittin and its variants as their therapeutic potential has become increasingly evident. Melittin is a 26-residue peptide and a toxic component of honey bee venom. The versatility of melittin in interacting with various biological substrates, such as membranes, glycosaminoglycans, and a variety of proteins, has inspired a slew of studies that aim to improve our understanding of the structural basis of such interactions. However, these ...[more]

PMID: 29668274

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Project description:Melittin is a basic 26-amino-acid polypeptide that constitutes 40-60% of dry honeybee (Apis mellifera) venom. Although much is known about its strong surface activity on lipid membranes, less is known about its pain-producing effects in the nervous system. In this review, we provide lines of accumulating evidence to support the hypothesis that melittin is the major pain-producing substance of bee venom. At the psychophysical and behavioral levels, subcutaneous injection of melittin causes tonic pain sensation and pain-related behaviors in both humans and animals. At the cellular level, melittin activates primary nociceptor cells through direct and indirect effects. On one hand, melittin can selectively open thermal nociceptor transient receptor potential vanilloid receptor channels via phospholipase A2-lipoxygenase/cyclooxygenase metabolites, leading to depolarization of primary nociceptor cells. On the other hand, algogens and inflammatory/pro-inflammatory mediators released from the tissue matrix by melittin's pore-forming effects can activate primary nociceptor cells through both ligand-gated receptor channels and the G-protein-coupled receptor-mediated opening of transient receptor potential canonical channels. Moreover, subcutaneous melittin up-regulates Nav1.8 and Nav1.9 subunits, resulting in the enhancement of tetrodotoxin-resistant Na(+) currents and the generation of long-term action potential firing. These nociceptive responses in the periphery finally activate and sensitize the spinal dorsal horn pain-signaling neurons, resulting in spontaneous nociceptive paw flinches and pain hypersensitivity to thermal and mechanical stimuli. Taken together, it is concluded that melittin is the major pain-producing substance of bee venom, by which peripheral persistent pain and hyperalgesia (or allodynia), primary nociceptive neuronal sensitization, and CNS synaptic plasticity (or metaplasticity) can be readily induced and the molecular and cellular mechanisms underlying naturally-occurring venomous biotoxins can be experimentally unraveled.

Project description:Protein kinases are ubiquitous enzymes with critical roles in cellular processes and pathology. As a result, researchers have studied their activity and regulatory mechanisms extensively. Thousands of X-ray structures give snapshots of the architectures of protein kinases in various states of activation and ligand binding. However, the extent of and manner by which protein motions and conformational dynamics underlie the function and regulation of these important enzymes is not well understood. Nuclear magnetic resonance (NMR) methods provide complementary information about protein conformation and dynamics in solution. However, until recently, the large size of these enzymes prevented researchers from using these methods with kinases. Developments in transverse relaxation-optimized spectroscopy (TROSY)-based techniques and more efficient isotope labeling strategies are now allowing researchers to carry out NMR studies on full-length protein kinases. In this Account, we describe recent insights into the role of dynamics in protein kinase regulation and catalysis that have been gained from NMR measurements of chemical shift changes and line broadening, residual dipolar couplings, and relaxation. These findings show strong associations between protein motion and events that control kinase activity. Dynamic and conformational changes occurring at ligand binding sites and other regulatory domains of these proteins propagate to conserved kinase core regions that mediate catalytic function. NMR measurements of slow time scale (microsecond to millisecond) motions also reveal that kinases carry out global exchange processes that synchronize multiple residues and allosteric interconversion between conformational states. Activating covalent modifications or ligand binding to form the Michaelis complex can induce these global processes. Inhibitors can also exploit the exchange properties of kinases by using conformational selection to form dynamically quenched states. These investigations have revealed that kinases are highly dynamic enzymes, whose regulation by interdomain interactions, ligand binding, and covalent modifications involve changes in motion and conformational equilibrium in a manner that can be correlated with function. Thus, NMR provides a unique window into the role of protein dynamics in kinase regulation and catalysis with important implications for drug design.

Project description:The ability of the peptides melittin, [Ala-14]melittin (P14A) and whole bee venom to lyse red blood cells (RBC) and to cause shape transformation, binding, partitioning and changes in volume of the cells during haemolysis, as well as the action of the bivalent cations Zn2+ and Ca2+, chlorpromazine, albumin and plasma on the peptide-induced haemolysis of RBC in high ionic-strength solution, have been investigated. The protective effect of all inhibitors depends on whether they have been added to the media before or after the cells. When added before the cells they reduced significantly the rate of peptide-induced haemolysis and shape transformation. The effect was maximal when agents acted simultaneously after introduction of the cells into the media containing both inhibitors and peptides. Incubation of the cells in isotonic solution before the addition of peptides enhanced 2-3-fold the RBC susceptibility (i.e. rate of haemolysis) to lytic action of the same amount of peptides, and increased the order of the haemolytic reaction, although the power law coefficient did not exceed a value of 2 for all peptides, suggesting that haemolysis is attributable to the monomeric or dimeric forms of the peptides. Partition coefficients were of the order of approximately 10(6) M-1, and P14A possessed a value 3-fold larger compared with melittin and bee venom, which correlated with its enhanced haemolytic activity. The protective action of inhibitors against peptide-induced haemolysis has been explained on the basis of their ability to compete with peptide binding at an early stage of peptide-membrane interaction, and not as a result of inhibition of a pre-existing peptide-induced pore. Whereas melittin increased the volume of RBC during haemolysis, P14A, melittin in the presence of phospholipase A2 or bee venom, reduced the volume in a concentration-dependent manner. The present data reveal the significant role of the initial stage of peptide-membrane interaction and peptide structure in the mechanism of haemolysis. These data are not consistent with a lipid-based mechanism of peptide-induced haemolysis, indicating that the mode of peptide-protein interaction is an important and decisive step in the haemolytic mechanism. It should be noted that data (in the form of three additional Tables) on the ability of inhibitors to protect cells from haemolysis when inhibitor and peptide act simultaneously are available. They are reported in Supplementary Publication SUP 50178, which has been deposited at the British Library Document Supply Centre, Boston Spa, Wetherby, West Yorkshire LS23 7BQ, U.K., from whom copies can be obtained on the terms indicated in Biochem. J. (1996) 313, 9.

Dataset Information

Nuclear Magnetic Resonance-Based Structural Characterization and Backbone Dynamics of Recombinant Bee Venom Melittin.

Publications

Nuclear Magnetic Resonance-Based Structural Characterization and Backbone Dynamics of Recombinant Bee Venom Melittin.

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