scholarly journals The Role of Factor XIa (FXIa) Catalytic Domain Exosite Residues in Substrate Catalysis and Inhibition by the Kunitz Protease Inhibitor Domain of Protease Nexin 2

2011 ◽  
Vol 286 (36) ◽  
pp. 31904-31914 ◽  
Author(s):  
Ya-Chi Su ◽  
Tara N. Miller ◽  
Duraiswamy Navaneetham ◽  
Robert T. Schoonmaker ◽  
Dipali Sinha ◽  
...  
Keyword(s):  
Protease Inhibitor ◽  
Catalytic Domain ◽  
Kunitz Protease Inhibitor ◽  
Factor Xia ◽  
Protease Nexin ◽  
Kunitz Protease Inhibitor Domain

scholarly journals Structural and Mutational Analyses of the Molecular Interactions between the Catalytic Domain of Factor XIa and the Kunitz Protease Inhibitor Domain of Protease Nexin 2

2005 ◽  
Vol 280 (43) ◽  
pp. 36165-36175 ◽  
Author(s):  
Duraiswamy Navaneetham ◽  
Lei Jin ◽  
Pramod Pandey ◽  
James E. Strickler ◽  
Robert E. Babine ◽  
...  
Keyword(s):  
Protease Inhibitor ◽  
Catalytic Domain ◽  
Point Mutations ◽  
Protein Data Bank Code ◽  
Mutant Form ◽  
Wild Type ◽  
Site Mutation ◽  
Kunitz Protease Inhibitor ◽  
Factor Xia ◽  
Protease Nexin

Factor XIa (FXIa) is a serine protease important for initiating the intrinsic pathway of blood coagulation. Protease nexin 2 (PN2) is a Kunitz-type protease inhibitor secreted by activated platelets and a physiologically important inhibitor of FXIa. Inhibition of FXIa by PN2 requires interactions between the two proteins that are confined to the catalytic domain of the enzyme and the Kunitz protease inhibitor (KPI) domain of PN2. Recombinant PN2KPI and a mutant form of the FXI catalytic domain (FXIac) were expressed in yeast, purified to homogeneity, co-crystallized, and the structure of the complex was solved at 2.6 Å (Protein Data Bank code 1ZJD). In this complex, PN2KPI has a characteristic, disulfide-stabilized double loop structure that fits into the FXIac active site. To determine the contributions of residues within PN2KPI to its inhibitory activity, selected point mutations in PN2KPI loop 1 11TGPCRAMISR20 and loop 2 34FYGGC38 were tested for their ability to inhibit FXIa. The P1 site mutation R15A completely abolished its ability to inhibit FXIa. IC50 values for the wild type protein and the remaining mutants were as follows: PN2KPI WT, 1.28 nm; P13A, 5.92 nm; M17A, 1.62 nm; S19A, 1.86 nm; R20A, 5.67 nm; F34A, 9.85 nm. The IC50 values for the M17A and S19A mutants were not significantly different from those obtained with wild type PN2KPI. These functional studies and activated partial thromboplastin time analysis validate predictions made from the PN2KPI-FXIac co-crystal structure and implicate PN2KPI residues, in descending order of importance, Arg15, Phe34, Pro13, and Arg20 in FXIa inhibition by PN2KPI.

Structural and Mutational Analysis of Canonical Loop Residues In Protease Nexin 2 Involved In Factor XIa and Trypsin Inhibition.

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 1147-1147
Author(s):  
Duraiswamy Navaneetham ◽  
Dipali Sinha ◽  
Peter N. Walsh
Keyword(s):  
Inhibitory Activity ◽  
Significant Contribution ◽  
Catalytic Domain ◽  
Hydrophobic Interactions ◽  
Conflicts Of Interest ◽  
Side Chain ◽  
Trypsin Inhibition ◽  
Factor Xia ◽  
Protease Nexin ◽  
Loop 2

Abstract Abstract 1147 Factor XIa (FXIa) activates FIX and is regulated by platelet-secreted protease nexin 2 (PN2) that contains a Kunitz-type protease inhibitor (KPI) domain. Trypsin is regulated by basic pancreatic trypsin inhibitor (BPTI). The primary and tertiary structures of trypsin and the catalytic domain of FXIa are highly homologous, and KPI and BPTI are nearly identical structurally. We have previously identified two loop structures (loops 1 and 2) in the KPI domain of PN2 that interact with residues in the FXIa catalytic domain. Based on the structure of the FXIa/KPI complex crystal structure, residues within loops 1 and 2 were mutated for experiments examining the inhibition of FXIa and trypsin. Results show that the loop-1-region P1 site residue Arg15 of PN2KPI plays a major role in FXIa inhibition by protruding into the S1 specificity pocket of FXIa. Ala mutation at this site renders PN2KPI non-inhibitory for both FXIa and trypsin. BPTI has Lys15 at the P1 site. BPTI inhibits both FXIa and trypsin significantly less effectively than PN2KPI. PN2KPI-R15K lost FXIa inhibitory activity, whereas BPTI-K15R substantially gained affinity for FXIa. Like FXIa, trypsin preferred BPTI-K15R showing a significant enhancement in affinity. Thus, a major determinant of the inhibitory activity of PN2KPI and BPTI against FXIa and trypsin is the P1 residue, with Arg being preferred over Lys for both inhibitors and both proteases. In addition, loop 1 residues Pro13 and Arg20 make important contributions to both FXIa and trypsin inhibition as demonstrated by significantly elevated Ki values for Ala mutations (P13A, and R20A) at these sites. In contrast, Ser19 makes no significant contribution to inhibition of either FXIa or trypsin whereas Met17 makes a significant contribution to the inhibition of trypsin, but not FXIa. In loop 2, only Phe34 is identified as a residue making significant contributions to the inhibition of both FXIa and trypsin, since the PN2KPI-F34A mutant displayed reduced inhibitory activity for both FXIa (6-fold) and for trypsin (3-fold). To rationalize these findings, we examined the crystal structures of the FXIa(catalytic domain)/PN2KPI complex, and the trypsin/BPTI complex. Structurally, the PN2KPI loop-1, P1-site residue Arg15 makes a complex primary interaction with Asp189 of both FXIa and trypsin. Disruption of this site by R15A mutation renders PN2KPI non-inhibitory because it preempts salt bridge interactions from two nitrogen atoms of the guanidinium group of Arg15 with Asp189 and Gly218 in FXIa. In addition, the Arg15 carbonyl oxygen forms hydrogen bonds with main-chain nitrogen atoms of one of the catalytic triad residues, Ser195, and with Gly193. The other important interaction in FXIa/PN2KPI or trypsin/BPTI is hydrophobic, between PN2KPI-Phe34 and FXIa-Tyr143 and between BPTI-Val34 and trypsin-Tyr151. This intermolecular interaction is further strengthened by an intramolecular interaction in which the side chain of Phe34 packs closely with the side chain of Met17 within PN2KPI, altogether forming a strong hydrophobic patch in FXIa-PN2KPI and trypsin-PN2KPI. PN2KPI-F34A disrupts both inter- and intramolecular hydrophobic interactions, leading to discernable reductions in affinity for both FXIa and trypsin. Despite occupying extreme positions in the autolysis loops, (143YRKLRDKI151 in FXIa and 143NTKSSGTSY151 in trypsin), Tyr143/151 residues still orient themselves in close proximity to Phe34. Thus, loop-1 residues of PN2KPI establish complex ionic interactions that play a major role, which is supplemented by the loop-2 residue, Phe34 (in PN2KPI) or Val34 (in BPTI), which establish hydrophobic interactions with residues in FXIa and trypsin leading to very high-affinity enzyme-inhibitor complexes. Disclosures: No relevant conflicts of interest to declare.

P1-231: The Kunitz protease inhibitor domain controls homodimerization of the amyloid precursor protein

2010 ◽  
Vol 6 ◽  
pp. S241-S241
Author(s):  
Naouel Ben Khalifa ◽  
Jean-Christophe Renauld ◽  
Pierre J. Courtoy ◽  
Donatienne Tyteca ◽  
Jean-François Collet ◽  
...  
Keyword(s):  
Protease Inhibitor ◽  
Amyloid Precursor Protein ◽  
Precursor Protein ◽  
Kunitz Protease Inhibitor ◽  
Kunitz Protease Inhibitor Domain

The Role of the P1 Residue of Protease Nexin 2 and Basic Pancreatic Trypsin Inhibitor in the Mechanism of Factor XIa Inhibition.

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 2015-2015
Author(s):  
Duraiswamy Navaneetham ◽  
Dipali Sinha ◽  
Peter N. Walsh
Keyword(s):  
Steady State ◽  
Protease Inhibitor ◽  
Trypsin Inhibitor ◽  
Tight Binding ◽  
Basic Pancreatic Trypsin Inhibitor ◽  
Factor Xia ◽  
Pancreatic Trypsin Inhibitor ◽  
Kunitz Type ◽  
Protease Nexin ◽  
Kunitz Type Protease Inhibitor

Abstract Factor XIa (FXIa), a plasma serine protease that activates FIX, is regulated by protease nexin 2 (PN2), a Kunitz-type protease inhibitor (KPI) secreted on platelet activation. The Kunitz protease inhibitor domain of protease nexin 2 (PN2KPI) is highly homologous (45% primary sequence identity) to basic pancreatic trypsin inhibitor (BPTI, another Kunitz-type protease inhibitor) and their backbone tertiary structures are nearly identical (J Biol Chem280: 36165, 2005; J Mol Biol230: 919, 2005). However, PN2KPI (Ki 1 nM) is 600-fold more potent as a FXIa inhibitor than bovine basic pancreatic trypsin inhibitor (BPTI; Ki 630 nM). The present study is aimed at examining why these two structurally similar inhibitors are so different in their affinities and specificities in FXIa inhibition and analyzing the mechanisms of FXIa inhibition by these two inhibitors. Reasoning that the P1 residue (Arg15 in PN2KPI and Lys15 in BPTI) might play a crucial role in determining the affinity and specificity for FXIa we expressed BPTI-K15R (mimicking PN2KPI at the P1 site) and PN2KPI-R15K (mimicking BPTI at the P1 site) and examined their inhibitory properties. BPTI-K15R was found to inhibit FXIa with a Ki (10 nM) ~60-fold tighter than BPTI (Ki 630 nM), whereas PN2KPI-R15K inhibited FXIa with a Ki (32 nM) that was 32- fold impaired compared with PN2KPI (Ki 1 nM). Progress curves of peptidyl fluorogenic substrate (Boc-Glu-Ala-Arg-AMC) hydrolysis by FXIa in the presence of all the inhibitors except BPTI clearly demonstrated higher initial rates that continuously decreased until reaching a steady state, typical of slow inhibition. Progress curves obtained using BPTI, on the other hand, were found to be linear at all concentrations of the inhibitor. Analysis using steady state kinetics clearly demonstrated that BPTI is a competitive inhibitor in FXIa hydrolysis of the peptidyl chromogenic substrate, pyro-Glu-Pro-Arg-pNA. Analysis of progress curves using kinetic equations for slow, tight-binding inhibitors confirmed that the formation of the FXIa:PN2KPI complex occurs in a single step that is slow, whereas formation of FXIa:PN2KPI-R15K, FXIa:BPTI and FXIa:BPTI-K15R complexes do not conform to the same kinetic mechanism. Thus PN2KPI and BPTI inhibit FXIa by distinctly different mechanisms and the Arg at the P1 site of PN2KPI confers specificity and high affinity for FXIa inhibition and in part determines the mechanism of PN2KPI as a slow, tight binding inhibitor.

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