The Crystal Structure of Nitrophorin 2: A TRIFUNCTIONAL ANTIHEMOSTATIC PROTEIN FROM THE SALIVA OF RHODNIUS PROLIXUS

John F Andersen,William R Montfort

doi:10.1074/jbc.m002857200

Abstract

Nitrophorin 2 (NP2) (also known as prolixin-S) is a salivary protein that transports nitric oxide, binds histamine, and acts as an anticoagulant during blood feeding by the insect Rhodnius prolixus. The 2.0-A crystal structure of NP2 reveals an eight-stranded antiparallel beta-barrel containing a ferric heme coordinated through His(57), similar to the structures of NP1 and NP4. All four Rhodnius nitrophorins transport NO and sequester histamine through heme binding, but only NP2 acts as an anticoagulant. Here, we demonstrate that recombinant NP2, but not recombinant NP1 or NP4, is a potent anticoagulant; recombinant NP3 also displays minor activity. Comparison of the nitrophorin structures suggests that a surface region near the C terminus and the loops between beta strands B-C and E-F is responsible for the 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 reorganization on ligand binding. The NP2 protein core differs from NP1 and NP4 in that buried Glu(53), which allows for larger NO release rates when deprotonated, hydrogen bonds to invariant Tyr(81). Surprisingly, this tyrosine lies on the protein surface in NP1 and NP4.

Highlights

Nitrophorin 2 (NP2) is a salivary protein that transports nitric oxide, binds histamine, and acts as an anticoagulant during blood feeding by the insect Rhodnius prolixus
The NP2 protein core differs from NP1 and NP4 in that buried Glu53, which allows for larger NO release rates when deprotonated, hydrogen bonds to invariant Tyr81
For expression in Escherichia coli, methionine was added to the N terminus of the protein, and this residue was found to be well ordered in the crystal and stabilized through interaction with an adjacent NP2 molecule

Summary

A TRIFUNCTIONAL ANTIHEMOSTATIC PROTEIN FROM THE SALIVA OF RHODNIUS PROLIXUS*

Nitrophorin 2 (NP2) ( known as prolixin-S) is a salivary protein that transports nitric oxide, binds histamine, and acts as an anticoagulant during blood feeding by the insect Rhodnius prolixus. The NPs are all Fe(III) heme proteins, but the sequence groups differ in both their NO binding and anticoagulation properties. NP4 binding to NO (but not to cyanide or ammonia) induces a large conformational change in the protein that results in burial of the NO ligand in the distal pocket and a substantial increase in NO binding affinity [12]. We have determined the crystal structure of recombinant NP2 and compared the anticoagulation activity of this protein with recombinant samples of NP1–NP4 This reveals significant differences between NP2 and both NP1 and NP4, including the surprising use of invariant amino acid residues in completely new ways

EXPERIMENTAL PROCEDURES

RESULTS

DISCUSSION