The crystal structure of nitrophorin 2 - A trifunctional antihemostatic protein from the saliva of Rhodnius prolixus

Nitrophorin 2 (NP2) (also known as prolixin-S) is a salivary protein that t ransports nitric oxide, binds histamine, and acts as an anticoagulant durin g blood feeding by the insect Rhodnius prolixus. The 2.0-Angstrom crystal s tructure of NP2 reveals an eight-stranded antiparallel beta-barrel containi ng a ferric heme coordinated through His(57), Similar to the structures of NP1 and NP4. All four Rhodnius nitrophorins transport NO and sequester hist amine through heme binding, but only NP2 acts as an anticoagulant, Here, we demonstrate that recombinant NP2, but not recombinant NP1 or NP4, is a pot ent anticoagulant; recombinant NP3 also displays minor activity. Comparison of the nitrophorin structures suggests that a surface region near the C te rminus and the loops between beta strands B-C and E-F is responsible for th e anticoagulant activity. NP2 also displays larger NO association rates and smaller dissociation rates than NP1 and NP4, which may result from a more open and more hydrophobic distal pocket, allowing more rapid solvent reorga nization on ligand binding. The NP2 protein core differs from NP1 and NP4 i n that buried Glu(53), which allows for larger NO release rates when deprot onated, hydrogen bonds to invariant Tyr(81). Surprisingly, this tyrosine li es on the protein surface in NP1 and NP4.