Interaction of Group IA Phospholipase A2 with Metal Ions and Phospholipid Vesicles Probed with Deuterium Exchange Mass Spectrometry

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Abstract
Deuterium exchange mass spectrometric evaluation of the cobra venom (Naja naja naja) group IA phospholipase A2 (GIA PLA2) was carried out in the presence of metal ions Ca2+ and Ba2+ and phospholipid vesicles. Novel conditions for digesting highly disulfide bonded proteins and a methodology for studying protein−lipid interactions using deuterium exchange have been developed. The enzyme exhibits unexpectedly slow rates of exchange in the two large α-helices of residues 43−53 and 89−101, which suggests that these α-helices are highly rigidified by the four disulfide bonds in this region. The binding of Ca2+ or Ba2+ ions decreased the deuterium exchange rates for five regions of the protein (residues 24−27, 29−40, 43−53, 103−110, and 111−114). The magnitude of the changes was the same for both ions with the exception of regions of residues 24−27 and 103−110 which showed greater changes for Ca2+. The crystal structure of the N. naja naja GIA PLA2 contains a single Ca2+ bound in the catalytic site, but the crystal structures of related PLA2s contain a second Ca2+ binding site. The deuterium exchange studies reported here clearly show that in solution the GIA PLA2 does in fact bind two Ca2+ ions. With dimyristoylphosphatidylcholine (DMPC) phospholipid vesicles with 100 μM Ca2+ present at 0 °C, significant areas on the i-face of the enzyme showed decreases in the rate of exchange. These areas included regions of residues 3−8, 18−21, and 56−64 which include Tyr-3, Trp-61, Tyr-63, and Phe-64 proposed to penetrate the membrane surface. These regions also contained Phe-5 and Trp-19, proposed to bind the fatty acyl tails of substrate.

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