A Molecular Mechanism for Lys49-Phospholipase A2 Activity Based on Ligand-induced Conformational Change

Abstract

Agkistrodon contortrix laticinctus myotoxin is a Lys49 -phospholipase A 2 (EC 3.1.1.4) isolated from the venom of the serpent A. contortrix laticinctus (broad-banded copperhead). We present here three monomeric crystal structures of the myotoxin, obtained under different crystallization conditions. The three forms present notable structural differences and reveal that the presence of a ligand in the active site (naturally presumed to be a fatty acid) induces the exposure of a hydrophobic surface (the hydrophobic knuckle) toward the C terminus. The knuckle in A. contortrix laticinctus myotoxin involves the side chains of Phe 121 and Phe 124 and is a consequence of the formation of a canonical structure for the main chain within the region of residues 118 -125. Comparison with other Lys49 -phospholipase A 2 myotoxins shows that although the knuckle is a generic structural motif common to all members of the family, it is not readily recognizable by simple sequence analyses. An activation mechanism is proposed that relates fatty acid retention at the active site to conformational changes within the C-terminal region, a part of the molecule that has long been associated with Ca 2؉ -independent membrane damaging activity and myotoxicity. This provides, for the first time, a direct structural connection between the phospholipase "active site" and the C-terminal "myotoxic site," justifying the otherwise enigmatic conservation of the residues of the former in supposedly catalytically inactive molecules.

Cellular pathology induced by snake venom phospholipase A2 myotoxins and neurotoxins: common aspects of their mechanisms of action

“…In addition, the dimeric character of these toxins may enhance their membrane-damaging effects by increasing binding affinity owing to the divalent interaction, as opposed to the monovalent binding of monomers; the latter ones are still capable of exerting myotoxicity and cytotoxicity, though with a lower potency than the dimers [22,95]. An FA molecule bound to the inactive catalytic site has been observed in the crystal structure of some of these myotoxins, and proposed to induce the exposure of a hydrophobic knuckle involving the side chains of hydrophobic residues that may contribute to bilayer disorganization [96]. This hypothesis might explain the strict conservation of the active site in these enzymatically-inactive molecules [96].…”

“…An FA molecule bound to the inactive catalytic site has been observed in the crystal structure of some of these myotoxins, and proposed to induce the exposure of a hydrophobic knuckle involving the side chains of hydrophobic residues that may contribute to bilayer disorganization [96]. This hypothesis might explain the strict conservation of the active site in these enzymatically-inactive molecules [96]. It might also explain the puzzling observation that alkylation of the His48 of the catalytic centre of Lys49 myotoxins reduces their toxicity [97,98], as this chemical modification might interfere with the binding of the FA molecule, thus precluding the conformational change that exposes the hydrophobic knuckle.…”

Insights into the structure and function of fungal β‐mannosidases from glycoside hydrolase family 2 based on multiple crystal structures of the Trichoderma harzianum enzyme

“…This may achieved by using a combi-nation of techniques [12], by an ensemble refinement of crystallographic data [13,14], or by solving multiple crystal structures of the same target, especially in different crystal forms (e.g. [15] and [16]). …”

Bothrops snake myotoxins induce a large efflux of ATP and potassium with spreading of cell damage and pain

“…Asp49 PLA 2 myotoxins depend on their enzymatic activity to induce skeletal muscle fiber damage (6). In contrast, the catalytically inactive Lys49 PLA 2 homologs use, as major determinant of toxicity, a C-terminal region (residues 115-129), which presents a variable combination of cationic and hydrophobic/aromatic residues and forms membrane pores (11)(12)(13). Both types of myotoxins cause a large influx of Ca 2+ in muscle cells, which triggers a cascade of events such as loss of mitochondrial function, widespread proteolysis, myofibrillar hypercontraction, and additional degenerative events that still await a detailed description (6,14).…”