Structural determinants of factor IX(a) binding in nitrophorin 2, a lipocalin inhibitor of the intrinsic coagulation pathway.
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 beta-strands E and F of the lipocalin beta-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.
Gudderra
Nanda P
NP
Laboratory of Malaria and Vector Research NIAID, National Institutes of Health, Bethesda, Maryland 20892, USA.
Ribeiro
José M C
JM
Andersen
John F
JF
D000595:Amino Acid Sequence; D000818:Animals; D002151:Calorimetry; D002352:Carrier Proteins / Q000737:chemistry*; D002462:Cell Membrane / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D005164:Factor IX / Q000737:chemistry; D015949:Factor IXa / Q000737:chemistry*; D006418:Heme / Q000737:chemistry; D006420:Hemeproteins / Q000737:chemistry*; D006632:Histamine / Q000737:chemistry; D006801:Humans; D020128:Inhibitory Concentration 50; D007700:Kinetics; D054835:Lipocalin 1; D008956:Models, Chemical; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D017354:Point Mutation; D011485:Protein Binding; D012239:Rhodnius / Q000378:metabolism*; D012471:Salivary Proteins and Peptides / Q000737:chemistry*; D017386:Sequence Homology, Amino Acid; D020349:Surface Plasmon Resonance; D013997:Time Factors
D002352:Carrier Proteins; D006420:Hemeproteins; C518443:LCN1 protein, human; D054835:Lipocalin 1; D012471:Salivary Proteins and Peptides; C090560:nitrophorin; D006418:Heme; D006632:Histamine; D005164:Factor IX; D015949:Factor IXa