Evidence for the Regulatory Role of the N-terminal Helix of Secretory Phospholipase A2 from Studies on Native and Chimeric Proteins

Abstract

The phospholipase A 2 (PLA 2 ) enzymes are activated by binding to phospholipid membranes. Although the N-terminal ␣-helix of group I/II PLA 2 s plays an important role in the productive mode membrane binding of the enzymes, its role in the structural aspects of membrane-induced activation of PLA 2 s is not well understood. In order to elucidate membrane-induced conformational changes in the N-terminal helix and in the rest of the PLA 2 , we have created semisynthetic human group IB PLA 2 in which the N-terminal decapeptide is joined with the 13 C-labeled fragment, as well as a chimeric protein containing the N-terminal decapeptide from human group IIA PLA 2 joined with a 13 C-labeled fragment of group IB PLA 2 . Infrared spectral resolution of the unlabeled and 13 C-labeled segments suggests that the N-terminal helix of membranebound IB PLA 2 has a more rigid structure than the other helices. On the other hand, the overall structure of the chimeric PLA 2 is more rigid than that of the IB PLA 2 , but the N-terminal helix is more flexible. A combination of homology modeling and polarized infrared spectroscopy provides the structure of membrane-bound chimeric PLA 2 , which demonstrates remarkable similarity but also distinct differences compared with that of IB PLA 2 . Correlation is delineated between structural and membrane binding properties of PLA 2 s and their N-terminal helices. Altogether, the data provide evidence that the N-terminal helix of group I/II PLA 2 s acts as a regulatory domain that mediates interfacial activation of these enzymes.Enzymes of the phospholipase A 2 (PLA 2 ) 2 family hydrolyze the sn-2 ester bond of diacylglycerophospholipids and thus initiate the biosynthesis of lipid-derived mediators, such as eicosanoids and platelet-activating factor, which are potent mediators of inflammation, allergy, and tumorigenesis (1-3). These enzymes are divided into several groups and subgroups based on their amino acid sequences, tissue distribution, and functional features (1, 2).PLA 2 s gain their full activity only when they bind to phospholipid micelles or membranes, an effect known as interfacial activation (4 -6). The molecular mechanism of interfacial activation has been the subject of debate over several decades. Initially, similarities of crystal structures of PLA 2 s with and without bound substrate mimics argued against conformational changes in the enzymes during interfacial activation (4). Later, the interfacially activated form of group IB PLA 2 was modeled by anion-mediated dimers (7), which revealed significant conformational changes compared with "inactive" monomeric forms, mainly involving the loop that has the functionally important Tyr 69 , and the presence of an assisting water molecule (8). Several spectroscopic studies also identified conformational changes in group IB and IIA PLA 2 s upon binding to phospholipid micelles or membranes. Fluorescence studies indicated that the N-terminal helix of porcine group IB PLA 2 becomes rigid upon enzyme-substrate complex formation at the m...

Structural basis for the recruitment and activation of the Legionella phospholipase VipD by the host GTPase Rab5

“…Deletion of N18 reduced the PLA 1 activity of VipD 19-564 but did not interfere with allosteric activation of the catalytic site. We hypothesize that N18 bearing a short amphipathic α-helix may facilitate the correct positioning of the PLA 1 domain toward the lipid layer, promote substrate diffusion from the lipid-water interface into the catalytic site, or a combination of both effects as has been previously described for secretory PLA 2 enzymes (32,33). Interestingly, the N-terminal tail of VpdA (residues 11-54) and the N-terminal segment of P. aeruginosa ExoU (residues 57-96) showed structural similarity to the equivalent region of VipD despite lacking sequence homology (Fig.…”

Structural analysis of proteins by isotope-edited FTIR spectroscopy

“…The ligated sample was diluted 10-fold in the denaturing buffer, and 5 mM Tris (2-carboxyethyl) phosphine hydrochloride was added to reduce disulfides between internal cysteines of PLA 2 and thiophenol and/or benzyl mercaptan [16]. The protein was refolded by dialysis and purified using ion exchange and size exclusion column chromatography, as described [12,13,19]. The semisynthetic PLA 2 was identified by immunoblotting and had nearly the same specific phospholipid hydrolyzing activity as the recombinant PLA 2 .…”

“…The N-terminal decapeptide of human pancreatic PLA 2 containing a C-terminal thioester group (Ala-Val-Trp-Gln-Phe-Arg-Lys-Met-Ile-Lys-COSCH 2 COOH) was synthesized by SynPep Corp. (Dublin, CA, USA). The fragment of PLA 2 Cys 11 -Ser 126 , named ΔN10, was expressed in Escherichia coli in an M9 minimal medium containing 0.2% 13 C 6 -D-glucose as a sole metabolic source of carbon so as to produce a uniformly 13 C-labeled recombinant protein.…”

“…PLA 2 enzymes undergo a substantial increase in their phospholipid hydrolyzing activity upon binding to cellular membranes [4,9], yet the structural aspects of the interfacial activation remain uncharacterized. The N-terminal α-helix of certain secreted PLA 2 s, including pancreatic PLA 2 , has been shown to play an important regulatory role in the enzyme function [12,13,19]. Here, peptide ligation was used to produce a semisynthetic human pancreatic PLA 2 in which the N-terminal helix was unlabeled while the rest of the protein was 13 C-labeled.…”

“…The N-terminal α-helix of certain secreted PLA 2 s, including pancreatic PLA 2 , has been shown to play an important regulatory role in the enzyme function [12,13,19]. Here, peptide ligation was used to produce a semisynthetic human pancreatic PLA 2 in which the N-terminal helix was unlabeled while the rest of the protein was 13 C-labeled. The segmentally isotope labeled PLA 2 was studied by direct transmission and ATR-FTIR spectroscopy to identify the structural dynamics of the labeled and unlabeled parts of the protein as well as to determine the precise membrane binding mode of PLA 2 .…”