Structural and Functional Effects of Tryptophans Inserted into the Membrane-binding and Substrate-binding Sites of Human Group IIA Phospholipase A₂

Abstract

Phospholipase A(2) (PLA(2)) enzymes become activated by binding to biological membranes and hydrolyze phospholipids to free fatty acids and lyso-phospholipids, the precursors of inflammatory mediators. To understand the functional significance of amino acid residues at key positions, we have studied the effects of the substitution of Val(3) (membrane binding surface) and Phe(5) (substrate binding pocket) of human group IIA PLA(2) by tryptophan on the structure and function of the enzyme. Despite the close proximity of the sites of mutations, the V3W mutation results in substantial enhancement of the enzyme activity, whereas the F5W mutant demonstrates significantly suppressed activity. A structural analysis of all three proteins free in buffer and bound to membranes indicates that large differences in activities result from distinct conformational changes in PLA(2)s upon membrane binding. Although PLA(2) and the V3W mutant demonstrate a decrease in helical content and an increase in helix flexibility, the F5W mutant experiences partial distortion of the alpha-helical structure presumably resulting from the tendency of Trp(5) to insert into the membrane. Furthermore, whereas the PLA(2) and the V3W mutant bind to the membrane at similar and apparently productive-mode orientation, the F5W mutant binds to membranes with a distinctly different orientation. It is suggested that both the stimulatory effect of the V3W mutation and the inhibitory effect of the F5W mutation result from the high affinity of Trp for the membrane-water interface. Although Trp(3) at the membrane binding face of PLA(2) facilitates the proper membrane binding of the enzyme, Trp(5) in the internal substrate binding site causes partial unwinding of the N-terminal helix in order to interact with the membrane.

Assessment of cytotoxicity, apoptosis and DNA damages in Colo320 colorectal cancer cells selected for oxaliplatin resistance

“…For example, the bioactive synthetic peptide p 115-129 of B. asper myotoxin II becomes roughly one order of magnitude more potent in all its membrane damaging effects if its cluster of three tyrosines is substituted by tryptophans, 43 a bulky and highly hydrophobic amino acid known to have a critical role in the interaction of proteins with membranes because of its tendency to insert. [44][45][46] Moreover, the related synthetic peptide pEM-2 47 , a variant of p 115-129 , was shown to insert into phospholipid monolayers and bilayers. 48 Another suggestion along this line derives from crystallographic studies of ACL myotoxin (Lys49 PLA 2 ), which revealed a conformational shift that exposes the buried side chains of two phenylalanine residues within its C-terminal region upon occupation of its (in)active site by a ligand.…”

Phospholipase A2 Biochemistry

“…It has been shown that a mutant form of the GIB enzyme with the pancreatic loop from 62–66 removed has increases in activity against zwitterionic substrate, and a decrease in activity against negatively charged substrate [63]. Recent studies using a GIIA enzyme with a Trp residue mutated into the interfacial binding region dramatically increased zwitterionic phospholipid hydrolysis [64], as well as penetration [65, 66]. The only other sPLA 2 enzymes to have high affinity for zwitterionic vesicles are the GV, and GX enzymes [67–69] which also share the characteristic of having Trp residues in the interfacial binding region.…”

Interaction of the Dengue Virus Fusion Peptide with Membranes Assessed by NMR: The Essential Role of the Envelope Protein Trp101 for Membrane Fusion

“…28 These properties of tryptophan residues make them appropriate for stabilizing the interaction of integral membrane proteins with lipid bilayers; 31 in some cases, an "aromatic belt" is present. 28 They may also make crucial contributions to the conformational stability of peptides and proteins, 32 making them key players in protein/peptide-membrane interactions. Tryptophan residues serve as anchors at interfaces and have a significant effect on conformation, which, in turn, is crucial for the adaptation and stabilization of peptides and transmembrane protein segments present in lipid bilayers.…”