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

Shan Qin,Abhay H Pande,Kathleen N Nemec,Xiaomei He,Suren A Tatulian

doi:10.1074/jbc.m506789200

Abstract

The phospholipase A(2) (PLA(2)) enzymes are activated by binding to phospholipid membranes. Although the N-terminal alpha-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 membrane-bound 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.

Highlights

phospholipase A2 (PLA2) gain their full activity only when they bind to phospholipid micelles or membranes, an effect known as interfacial activation (4 – 6)
Experimental evidence accumulated to date suggests that the N-terminal ␣-helix of group I/II PLA2s is a crucial structural domain necessary for a productive mode membrane binding and activity of the enzymes [13], conformational changes do occur in these enzymes during interfacial activation involving the N-terminal helix, and these conformational changes are group-specific
We present our findings on the effects of substitution of the N-terminal helix of human group IB PLA2 by that of human group IIA PLA2 on the enzyme activity, membrane binding strength, membrane-induced conformational changes, and the precise mode of membrane binding

Summary

EXPERIMENTAL PROCEDURES

Materials—The lipids 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) and 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoglycerol (POPG) were purchased from Avanti Polar Lipids (Alabaster, AL). Adsorption of cationic protein or peptide molecules to the membrane reduces the negative surface charge of the membrane, which results in a protein (or peptide) dose-dependent deprotonation of fluorescein and an increase in the fluorescence intensity This effect was used to measure binding isotherms for PLA2s and their N-terminal peptides. (When the N 3 C directionality of helices is considered, the interhelical angle would be 173.8o, but in terms of infrared order parameters, parallel and antiparallel helices are equivalent.) Polarized ATRFTIR spectroscopy was used to measure the amide I bands of the membrane-bound, segmentally 13C-labeled chimeric N10IIA/IB PLA2 at parallel and perpendicular orientations of the infrared light. This allows determination of all nine angles between the protein and membrane coordinate systems, using the laws of direction cosines, which in turn allows transformation of the protein atom coordinates from the system ⌺p to the system ⌺m

RESULTS AND DISCUSSION

TABLE ONE

Molecule pI