Free Energy Landscape and Rate Estimation of the Aromatic Ring Flips in Basic Pancreatic Trypsin Inhibitor Using Metadynamics

Aromatic side-chains (phenylalanine and tyrosine) of a protein flip by 180° around the Cβ-Cγ axis (χ2 dihedral of side-chain) producing two symmetry-equivalent states. The ring-flip dynamics act as an NMR probe to understand local conformational fluctuations. Ring-flips are categorized as slow (ms onwards) or fast (ns to near ms) based on timescales accessible to NMR experiments. In this study, we investigated the ability of the infrequent metadynamics approach to discriminate between slow and fast ring-flips for eight individual aromatic side-chains (F4, Y10, Y21, F22, Y23, F33, Y35, F45) of basic pancreatic trypsin inhibitor (BPTI). Well-tempered metadynamics simulations were performed to observe ring-flipping free energy surfaces for all eight aromatic residues. The results indicate that χ2 as a standalone collective variable (CV) is not sufficient to classify fast and slow ring-flips. Most of the residues needed χ1 (N−Cχα) as a complementary CV, indicating the importance of librational motions in ring-flips. Multiple pathways and mechanisms were observed for residues F4, Y10, and F22. Recrossing events are observed for residues F22 and F33, indicating a possible role of friction effects in the ring-flipping. The results demonstrate the successful application of the metadynamics based approach to estimate ring-flip rates of aromatic residues in BPTI and identify certain limitations of the approach.

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

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.

Low Molecular Weight Heparin Modulates the Binding and Reactivity of Factor Xa towards Basic Pancreatic Trypsin Inhibitor: A Surface Plasmon Resonance and Kinetics Study.

Previous studies by us have shown that the blood coagulation factor IXa is modulated by low molecular weight heparin (LMWH), resulting in greater reactivity of this protease with basic pancreatic trypsin inhibitor (BPTI). Since thrombin, factor Xa and factor VIIa share a high degree of homology with factor IXa and all are capable of binding heparin, we examined the potential ability of LMWH to modulate the reactivity of these coagulation proteases towards BPTI. Recombinant BPTI containing a His6-tag on the amino terminal end was constructed, expressed in bacteria and purified by nickel-chelating affinity chromatography (HisTrap). His6-BPTI had identical inhibitory activity as wild-type BPTI towards both trypsin and factor IXa. Surface plasmon resonance (Biacore 3000) was used to examine the binding and reacitvity of His6-BPTI to various coagulation proteases under different conditions. Initial biacore studies (using factor IXa) suggested that the carboxymethyl dextran moeity of CM5 biacore chips can paritally mimic heparin binding to the coagulation proteases, resulting in all sensed interactions being largely a reflection of the heparin-bound enzyme conformer. Thus, a hydrophobic HPA chip that lacks the carboxymethyl dextran matrix was used to prepare self-assembling monolayer surfaces of 100 mol% 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) or 90 mol% DOPC and 10 mol% of the nickel-chelating phospholipid 1,2 dioleoyl-sn-glycero-3-{[N-(5-amino-1-carboxypentyl)iminodiacetic acid]succinyl} (DOGS-NTA). His6-BPTI was efficiently, specifically and reproducibly captured only onto the surface containing DOGS-NTA. This binding was also dependent on the presence of nickel, and the generated BPTI-phospholipid surface was found to bind trypsin in a BPTI-dependent fashion. Efficient regeneration of the surface was accomplished between cycles by sequential injections of 10 mM EDTA and 10 mM NaOH (to completely remove the enzyme-BPTI complexes) followed by re-loading of the surface with nickel and re-capture of BPTI. Subsequent injections of thrombin, factor IXa, factor Xa, factor VIIa (both in the absence and presence of saturating levels of soluble tissue factor) over the BPTI-phospholipid surface was examined in the absence and presence of 10 μM of the LMWH enoxaparin (15 oligosaccharide units). As previously observed, the binding of factor IXa to BPTI was greatly enhanced by the presence of enoxaparin. Interestingly, the binding of factor Xa to the BPTI surface was also substantially enhanced by enoxaparin, while that of thrombin was only moderately (though consistently) enhanced and that of factor VIIa remained unaffected. Solution-based inhibition studies with factor Xa and BPTI or the isolated second Kunitz-type inhibitor of tissue factor pathway inhibitor (TFPI-K2) confirmed a 2- to 3-fold enhancing effect of enoxaparin on factor Xa reacitivity towards BPTI, with no effect apparent towards TFPI-K2. The enhancing effect of LMWH towards BPTI was also observed with high levels of fondaparinux (100 μM). Although the high levels of fondaparinux required are likely not clinically relevant and presumably reflect the poorer binding of fondaparinux (5 oligosaccharide units) to factor Xa compared with enoxaparin, these results underscore the ability of LMWH to modulate factor Xa reactivity upon occupation of the heparin-binding exosite and may have implications for the rational drug design of specific factor Xa inhibitors.