Structural Determinants of Factor IX(a) Binding in Nitrophorin 2, a Lipocalin Inhibitor of the Intrinsic Coagulation Pathway

Abstract

Nitrophorin 2 (NP2) is a salivary lipocalin from Rhodnius prolixus that binds with coagulation factors IX (fIX) and IXa (fIXa). Binding of NP2 with fIXa results in potent inhibition of the intrinsic factor Xase complex. A panel of site-directed surface mutants of NP2 was generated to locate determinants of high affinity fIX(a) binding. The locations of the mutations were based on comparisons with the related, but less potent, inhibitor nitrophorin 3 (NP3). Three point mutants (K21A, K92A, and V94A) were found that clearly reduced the inhibitory potency as measured by the activity of a reconstituted factor Xase system. Binding of NP2 with fIXa and fIX as measured by surface plasmon resonance and isothermal titration calorimetry was reduced in a similar manner. Of the three mutants, two (K92A and V94A) were located on the loop connecting ␤-strands E and F of the lipocalin ␤-barrel. The largest changes were seen with the K92A mutation, which lies at the apex of the loop, with a smaller effect being seen with mutation of Val 94 . Combination of four E-F loop mutations (K92A, A93K, V94A, E97A) in a single mutant reduced the inhibitory potency and binding to levels similar to those seen with NP3 without affecting heme or histamine binding. The intrinsic factor Xase complex (fXase)1 is an essential component of the mammalian blood coagulation cascade consisting of factors IXa (fIXa), VIIIa (fVIIIa), and X (fX) assembled on an anionic phospholipid surface (1). Disruption of this reaction results in two of the most important human bleeding disorders, hemophilias A (fVIII deficiency) and B (fIX deficiency) (1). Nitrophorin 2 (NP2) is a salivary anticoagulant protein from the blood-feeding insect Rhodnius prolixus that targets the intrinsic fXase complex (2, 3). It is a hyperbolic mixed-type inhibitor that acts by interfering with assembly of the proteolytic complex and/or phospholipid binding (4). NP2 does not inhibit the amidolytic activity of fIXa but rather blocks fXa formation in reactions lacking either phospholipid vesicles or fVIIIa (4). Factor X activation is not inhibited in the absence of fVIIIa and phospholipid suggesting that interactions between fX and fIXa are not directly influenced by NP2, while those between fIXa and fVIIIa/phospholipid are (4). Nevertheless, direct binding of NP2 with fIX and fIXa has been shown using surface plasmon resonance (SPR), while in the same study, no interaction of NP2 with fVIII was seen (5). Binding with fIX also results in inhibition of fIXa generation by both fXIa and the tissue factor-fVIIa complex (5). Removal of the Gla (␥-carboxyglutamic acid-rich) domain of fIX was found to eliminate the SPR interaction, suggesting that the NP2 binding site is located at or near this region, a primary point of interaction between fIXa and the phospholipid surface (5) and fVIIIa (6).NP2 is a member of the nitrophorin (NP) group, consisting of four related salivary lipocalins in R. prolixus that are remarkable in taking on numerous functions related to blood feeding (7-10). NPs are heme...

Antihemostatic Molecules from Saliva of Blood-Feeding Arthropods

“…This interaction occurs through the Gla domain, as fIXa lack-ing this domain does not bind NP2 [49] . The interaction is complex, with two-phase kinetics possibly indicative of a conformational change involving domain swapping [50] . Mutation analysis has shown that the interaction involves a series of resides at the apex of the EF loop [50] .…”

Examination of the Ligand-Binding and Enzymatic Properties of a Bilin-Binding Protein from the Poisonous Caterpillar Lonomia obliqua

“…The recombinant protein was refolded by dilution into a large excess of 20 mM Tris (pH 8.5), 0.4 M arginine HCl and then concentrated by ultrafiltration. These refolding methods have been successfully used in numerous studies on insect lipocalin structure and function [9], [10].…”

“…The observed heats were converted to enthalpies and fit to a single-site binding model using the Microcal-Origin software package. The nitrophorin 2 (NP2) used in ITC experiments was produced in E. coli using methods described previously [9], [13], and purified by gel filtration chromatography on Sephacryl S-100.…”

Structure and mechanism in salivary proteins from blood-feeding arthropods

“…Comparison of the NP4 and NP2 crystal structures shows that the surfaces of the two proteins are significantly different in shape, due mainly to an extended C-terminal region in NP4 (Andersen and Montfort, 2000). Site directed mutagenesis of the NP2 sequence, guided by comparisons between NP2, NP3 and NP4, identified an area of the protein surface formed mainly by residues of the EF loop that is critical to high affinity binding of NP2 with factors IX and IXa (Gudderra, et al, 2005). …”