3g8h
Crystal structure of phospholipase A2 ammodytoxin C from vipera ammodytes ammodytesCrystal structure of phospholipase A2 ammodytoxin C from vipera ammodytes ammodytes
Structural highlights
FunctionPA2BC_VIPAA Snake venom phospholipase A2 (PLA2) that acts as a presynaptic neurotoxin, an inhibitor of blood coagulation, and has been found to bind with high affinity to intracellular proteins. The response of indirectly stimulated neuromuscular preparations to ammodytoxin (Atx) is triphasic. The first phase, the transient inhibition of the acetylcholine (ACh) release, starts soon after the addition of Atx and lasts for several minutes. This phase is probably independent of Atx enzymatic activity. The effect may be due to the specific binding of the toxin to presynaptic receptors. These receptors, called N-type receptors, are still unidentified. It is noteworthy that a neuronal isoform of the M-type PLA2 receptor (R180) has been identified as a high-affinity receptor for Atx in neuronal plasma membranes. It was demonstrated however that this receptor is not essential for expression of neurotoxicity by Atx. The second phase corresponds to an augmentation of neurotransmitter release. A peak is reached 10-20 min after exposure of the preparation to Atx and is followed by a gradual reduction. In this phase, the enzymatic activity of Atx of the mammalian is not significant. It is speculated that the increased release of neurotransmitter in this phase is induced by the interference of Atx with voltage-gated potassium channels. Measurements of ionic showed however that voltage-gated potassium channels are not affected by Atx. The third phase of the response of neuromuscular preparations to Atx, which corresponds to a complete and irreversible paralysis, is clearly dependent on the hydrolytic activity of the toxin. In addition to its presynaptic neurotoxicity, Atx shows an anticoagulant activity by binding with high affinity to activated coagulation factor X (F10) thus inhibiting the formation of the prothrombinase complex (FX/FV) and its activity (IC(50) is 240 nM). Surprisingly, Atx was discovered to bind intracellular proteins such as calmodulin (CaM), 14-3-3 proteins gamma (YWHAG) and epsilon (YWHAE), as well as R25, a mitochondrial integral membrane protein found in cerebral cortex. These findings raised a doubt about the dogma of the exclusively extracellular action of PLA2s, defended by the potential instability of these molecules in the reducing environment of the eukaryotic cytosol coupled with their possible inability to act as enzymes in this cellular compartment, due to too low concentration of calcium ions. This hypothesis was challenged efficiently by demonstrating the internalization of AtxA into a culture cells, but still remains to be directly demonstrated in vivo (By similarity). PLA2 catalyzes the calcium-dependent hydrolysis of the 2-acyl groups in 3-sn-phosphoglycerides.[1] [2] [3] Evolutionary Conservation Check, as determined by ConSurfDB. You may read the explanation of the method and the full data available from ConSurf. Publication Abstract from PubMedAmmodytoxin A (AtxA) and its natural isoform AtxC from the venom of Vipera ammodytes ammodytes belong to group IIA-secreted phospholipases A(2) which catalyze the hydrolysis of glycerophospholipids and exhibit strong neurotoxic and anticoagulant effects. The two isoforms, which differ in sequence by only two amino acid residues (Phe124>Ile and Lys128>Glu), display significant differences in toxicity and anticoagulant properties and act on protein targets including neurotoxic proteic receptors and coagulation factor Xa with significantly different strengths of binding. In order to characterize the structural basis of these functional differences, we have determined the crystal structures of the two isoforms. Comparison of the structures shows that the mutation Lys128>Glu in AtxC could perturb interactions with FXa, resulting in lower anticoagulant activity, since the side chain of Glu128 is partly buried, making a stabilizing hydrogen bond with the main-chain nitrogen atom of residue Thr35. This interaction leads to a displacement of the main polypeptide chain at positions 127 and 128 (identified by mutagenesis as important for interaction with FXa), and a different orientation of the side chain of unmutated Lys127. The mutation Phe124>Ile in AtxC induces no significant conformational changes, suggesting that the differences in toxicity of the two isoforms are due essentially to differences in surface complementarity in the interaction of the toxin with the neurotoxic protein receptor. The crystal structures also reveal a novel dimeric quaternary association involving significant hydrophobic interactions between the N-terminal alpha-helices of two molecules of ammodytoxin related by crystallographic symmetry. Interactions at the dimer interface include important contributions from Met7, which is unique to ammodytoxin. Equilibrium sedimentation experiments are consistent with the crystallographic model. Competition experiments using SPR technology show complete inhibition of AtxA binding to FXa by calmodulin (CaM). The crystal structure shows that the C-terminal region, important for binding to FXa and CaM, is fully exposed and accessible for interaction with proteic receptors in both the monomeric and dimeric forms of ammodytoxin described here. Comparative structural studies of two natural isoforms of ammodytoxin, phospholipases A2 from Vipera ammodytes ammodytes which differ in neurotoxicity and anticoagulant activity.,Saul FA, Prijatelj-Znidarsic P, Vulliez-le Normand B, Villette B, Raynal B, Pungercar J, Krizaj I, Faure G J Struct Biol. 2010 Mar;169(3):360-9. Epub 2009 Oct 24. PMID:19857576[4] From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine. See AlsoReferences
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