Crystal Structure of Human Group X Secreted Phospholipase A2

Abstract

The crystal structure of human group X (hGX) secreted phospholipase A 2 (sPLA 2 ) has been solved to a resolution of 1.97 Å. As expected the protein fold is similar to previously reported sPLA 2 structures. The active site architecture, including the positions of the catalytic residues and the first and second shell water around the Ca 2؉ cofactor, are highly conserved and remarkably similar to the group IB and group IIA enzymes. Differences are seen in the structures following the (1-12)-Nterminal helix and at the C terminus. These regions are proposed to interact with the substrate membrane surface. The opening to the active site slot is considerably larger in hGX than in human group IIA sPLA 2 . Furthermore, the electrostatic surface potential of the hGX interfacial-binding surface does not resemble that of the human group IIA sPLA 2 ; the former is highly neutral, whereas the latter is highly cationic. The cationic residues on this face of group IB and IIA enzymes have been implicated in membrane binding and in k cat * allostery. In contrast, hGX does not show activation by the anionic charge at the lipid interface when acting on phospholipid vesicles or short-chain phospholipid micelles. Together, the crystal structure and kinetic results of hGX supports the conclusion that it is as active on zwitterionic as on anionic interfaces, and thus it is predicted to target the zwitterionic membrane surfaces of mammalian cells.

Interfacial Kinetic and Binding Properties of the Complete Set of Human and Mouse Groups I, II, V, X, and XII Secreted Phospholipases A2

“…Based on overlaying the sequences of the mouse and human sPLA 2 s onto the known three-dimensional x-ray structure of hGIIA, the following enzymes are predicted to contain one or more tryptophans on their membrane binding surface: hGIB, hGIID, hGV, mGIB, mGIIC, mGIID, mGV, mGX (hGXII has a distinct primary structure making it difficult to locate the putative membrane binding surface). The recently determined structure of hGX shows a single tryptophan on the putative membrane binding surface (40). With the exception of mGIB, all of these sPLA 2 s hydrolyze PC vesicles without a discernible lag.…”

Kinetic Evaluation of Cell Membrane Hydrolysis during Apoptosis by Human Isoforms of Secretory Phospholipase A2

“…The following recombinant human sPLA 2 isoforms were prepared as described previously: hGIIA (27,28), hGV (29), and hGX (19). The hGV enzyme was generously provided by Dr. Wonhwa Cho (University of Illinois, Chicago).…”

“…The relationship of each step to membrane behavior varies among the different isoforms (17)(18)(19)(20). A prominent example is the degree to which initial adsorption (step 1) depends on the presence of negative charge at the membrane surface (18,21,22).…”

“…For hGV, the presence of a tryptophan residue at the interfacial binding surface of the enzyme diminishes this requirement and allows some adsorption to a zwitterionic interface (17,24). In the case of the hGX enzyme, there appears to be a smaller or perhaps no requirement for an anionic interface (18,19). For snake venom sPLA 2 from Agkistrodon piscivorus piscivorus, either or both steps may be limiting depending on the physical state of the membrane (15).…”

Secretory Phospholipase A2

“…21,30) The crystal structure reveals that the opening to the active site of sPLA 2 -X is considerably larger than that of sPLA 2 -IIA and that the electrostatic surface potential of the sPLA 2 -X interfacial-binding surface is charge neutral, which allows sPLA 2 -X to be as active on zwitterionic as on anionic membranes. 38) Since sPLA 2 -X shows no affinity for HSPG, it does not act on cells via the HSPG-shuttling pathway. 30) sPLA 2 -IIF and -III exhibit unique arachidonate-releasing properties, where the regions characteristic of these enzymes play regulatory roles.…”