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J. Biol. Chem., Vol. 280, Issue 26, 25022-25028, July 1, 2005

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

Nanda P. Gudderra, José M. C. Ribeiro, and John F. Andersen

From the Laboratory of Malaria and Vector Research NIAID, National Institutes of Health, Bethesda, Maryland 20892

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 Val⁹⁴. 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.

Received for publication, April 21, 2005

^* The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

To whom correspondence should be addressed: NIH/NIAID, Twinbrook 3 Bldg., 12735 Twinbrook Parkway, Bethesda, MD 20892. Tel.: 301-402-5871; Fax: 301-402-2201; E-mail: jandersen{at}niaid.nih.gov.

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