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Logo of jbcThe Journal of Biological Chemistry
 
J Biol Chem. 2016 February 26; 291(9): 4671–4683.
Published online 2015 December 22. doi:  10.1074/jbc.M115.698613
PMCID: PMC4813490

Molecular Basis of Enhanced Activity in Factor VIIa-Trypsin Variants Conveys Insights into Tissue Factor-mediated Allosteric Regulation of Factor VIIa Activity*An external file that holds a picture, illustration, etc.
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Abstract

The complex of coagulation factor VIIa (FVIIa), a trypsin-like serine protease, and membrane-bound tissue factor (TF) initiates blood coagulation upon vascular injury. Binding of TF to FVIIa promotes allosteric conformational changes in the FVIIa protease domain and improves its catalytic properties. Extensive studies have revealed two putative pathways for this allosteric communication. Here we provide further details of this allosteric communication by investigating FVIIa loop swap variants containing the 170 loop of trypsin that display TF-independent enhanced activity. Using x-ray crystallography, we show that the introduced 170 loop from trypsin directly interacts with the FVIIa active site, stabilizing segment 215–217 and activation loop 3, leading to enhanced activity. Molecular dynamics simulations and novel fluorescence quenching studies support that segment 215–217 conformation is pivotal to the enhanced activity of the FVIIa variants. We speculate that the allosteric regulation of FVIIa activity by TF binding follows a similar path in conjunction with protease domain N terminus insertion, suggesting a more complete molecular basis of TF-mediated allosteric enhancement of FVIIa activity.

Keywords: allosteric regulation, coagulation factor, molecular dynamics, serine protease, x-ray crystallography

Introduction

Allosteric mechanisms play a vital role in the timely initiation and progression of the blood coagulation cascade. At the site of injury, membrane-bound tissue factor (TF)4 interacts with zymogen coagulation factor VII (FVII) and its active form (FVIIa). The FVIIa·TF complex initiates the coagulation cascade by activating FIX and FX, leading to thrombin generation and eventually wound healing (1). Conversion of FVII to FVIIa involves proteolytic cleavage at the Arg15-Ile16 peptide bond,5 producing a disulfide-linked two-chain molecule with a light chain consisting of a phospholipid-interactive γ-carboxyglutamic acid domain, two epidermal growth factor (EGF)-like domains, and a heavy chain trypsin-like protease domain (2) (Fig. 1A). In trypsin, the N terminus formed upon activation spontaneously enters the activation pocket to form a salt bridge with residue Asp194. This key interaction leads to optimal alignment and architecture of the oxyanion hole and primary specificity pocket (S1), resulting in a mature active site for substrate binding and catalysis (3). In FVIIa, the newly formed N terminus fails to completely insert into the activation pocket (4), leading to a non-optimal configuration of the catalytic machinery, rendering FVIIa “zymogen-like” with inferior catalytic efficiency. TF binding allosterically corrects these defects in the catalytic domain by stabilizing the 170 loop (amino acids 170–178) in conjunction with activation loops 1–3 (AL1–3) and promotes N terminus insertion (Fig. 1B) (5, 6). This transforms FVIIa into its catalytically competent form and increases amidolytic activity by 40-fold (7). Furthermore, TF ensures optimal orientation and positioning of the FVIIa catalytic domain above the membrane surface and generates exosites for incoming macromolecular substrates, thereby enhancing the proteolytic activity by ~105-fold (8, 9).

FIGURE 1.
Overview of the FVIIa-WT·sTF complex and variant nomenclature. A, full view of the FVIIa-WT·sTF (Protein Data Bank code 1dan) complex with the FVIIa protease domain in gray, light chain in blue, sTF in orange, Ca2+ ions in yellow, and ...

Previous studies have provided details of TF-binding regions in the FVIIa light chain and on structural changes in the protease domain upon cofactor binding. However, the full extent of TF-mediated structural changes has yet to be fully elucidated, and key components of the allosteric pathways remain elusive (10). To further understand the molecular basis of TF-mediated allosteric regulation of FVIIa activity, we considered FVIIa variants displaying superior catalytic efficiency in the absence of TF, variants modified in the vicinity of either the 170 loop (11) or the activation pocket (7), or a variant with the 170 loop replaced by that of trypsin (FVIIa-YT) (12). Interestingly, FVIIa-YT displays a similar extent of N terminus insertion as FVIIa wild type (WT) in the absence of TF; however, FVIIa-YT displays improved catalytic efficiency. Recent studies with thrombin, another trypsin-like serine protease, reveal a highly plastic protease fold for the apo form of thrombin that undergoes structural transitions upon cofactor binding (13,19). In particular, segment 215–217 was shown to be vital for substrate access and cofactor-mediated allosteric regulation, which may also be the case in TF-mediated allosteric regulation of FVIIa activity (20, 21).

We hypothesized that the improved catalytic efficiency of FVIIa-YT is due to stabilization of the 215–217 segment with Tyr172 playing an important role. To test our hypothesis, we investigated the FVIIa-YT variant and two variants of FVIIa-YT where this tyrosine is either replaced by serine (FVIIa-ST) or phenylalanine (FVIIa-FT) (Fig. 1D). Data presented here confirm that Tyr172 stabilizes the 215–217 segment and AL3 in the absence of TF, maturing the primary specificity pocket and enhancing catalytic efficiency independently of complete protease domain N terminus insertion. Molecular dynamics (MD) simulations supported the experimental results and suggested the direct involvement of Trp215 in TF-mediated allosteric changes in the protease domain, further corroborated by fluorescence quenching studies and solvent-accessible surface area (SASA) calculations. Based on these observations, we hypothesize that stabilization of segment 215–217 in the open conformation is a key step in TF-mediated allosteric regulation of FVIIa.

Experimental Procedures

Materials

H-d-Phe-Phe-Arg chloromethyl ketone TFA salt (FFR) was from Bachem (Basel, Switzerland). d-Ile-Pro-Arg p-nitroanilide (S-2288) was from Chromogenix (Mölndal, Sweden). All other chemicals were from Sigma-Aldrich and of analytical grade or the highest quality commercially available. Recombinant wild-type human FVIIa was prepared as described previously (11). Recombinant human soluble tissue factor 1–219 (sTF) was prepared as described (22) with the modification of using the reductase-deficient Escherichia coli strain BL21 Origami (Novagen, Germany). Factor Xa was purchased from Molecular Innovations.

Factor VII Mutagenesis and Protein Expression

Human wild-type FVII cDNA was cloned into a QMCF vector (Icosagen AS, Estonia), and all variants were generated using a PCR-based site-directed mutagenesis method with KOD Xtreme Hot Start DNA polymerase (Novagen) or a QuikChange Lightning XL (Agilent) kit according to the manufacturers' instructions. Introduction of the desired mutations was verified by DNA sequencing of the entire FVII cDNA region (MWG Biotech, Germany). QMCF Technology, a semistable episomal mammalian expression system obtained from Icosagen AS (Estonia), was used for expression of the FVII variants in a QMCF CHO cell line (CHO-EBNALT85), and cells were cultivated according to the manufacturer's instructions. During a period of 3–4 weeks, the transfected cell cultures were expanded to 2–10 liters, and the media were harvested by centrifugation and 0.22-μm filtration.

Protein Purification and Verification

For all FVII variants, expression medium was adjusted to pH 6.0, CaCl2 was added to 5 mm, and benzamidine HCl was added to a final concentration of 10 mm. All purification steps were performed using an ÄKTA Explorer system (GE Healthcare) and consisted of an immunoaffinity purification step (γ-carboxyglutamic acid domain-specific) performed as described (23) except at pH 6.0 using a histidine buffer and eluting with 20 mm EDTA. This was followed by a concentration and purification step using a prepacked 6-ml ResourceQTM (GE Healthcare) column at pH 6.0, eluting with a step gradient to 100 mm NaCl, 60 mm CaCl2 in 10 mm histidine. Activation of FVII variants was performed by passing the protein solution through a custom packed Tricon column (GE Healthcare) with factor Xa coupled to Sepharose 4B FF CNBr (GE Healthcare). Protein identity was verified using intact electrospray ionization-TOF mass spectrometry, and purity was shown to be >95% by Novex 4–12% SDS-PAGE (Life Technologies). The amount of active protein was determined by active site titration using FFR and measuring residual S-2288 activity (24).

Functional Evaluation of FVIIa 170 Loop Variants

All functional assays were carried out in 50 mm Hepes, pH 7.4, 0.1 m NaCl, 5 mm CaCl2, 0.01% Tween 20 (assay buffer) and monitored at 405 nm in a microplate reader (SpectraMax 340, Molecular Devices Corp., Sunnyvale, CA) using a Nunc F96-well plate (non-treated clear) with a 200-μl assay volume at 25 °C. sTF binding studies using S-2288 were performed essentially as described (12) using 0–3 μm sTF. Kinetic parameters of S-2288 hydrolysis were determined for the FVIIa variants with 0–12.5 mm S-2288, and the Ki for inhibition by pABA was determined in a competitive activity assay using 1 mm S-2288 as described (11). Carbamylation of the N-terminal Ile16 was investigated by incubating in 0.2 m KNCO (Sigma-Aldrich) and measuring residual activity at 1 mm S-2288 as described (11). All functional studies were performed in the absence or presence of 3 μm sTF. Data analysis and curve fitting were performed using GraphPad Prism 6.0.

FVIIa Variant Preparation, Crystallization, and Data Collection

Preparation of samples for x-ray crystallography was performed by inhibiting FVIIa-YT, -ST, and -FT with FFR and adding sTF in a 1:1 molar relationship. Protein integrity was verified using SDS-PAGE. Diffraction quality crystals were obtained using the hanging drop method at 22 °C with two different conditions: for FVIIa-YT and -FT, 0.1 m sodium citrate buffer at pH 5.1, 16.6% PEG 3350 (Hampton Research), 12.5% 1-propanol, and for FVIIa-ST, 0.1 m cacodylate buffer at pH 5.6, 12% PEG 8000 (Hampton Research). All diffraction data were collected at MaxLab IV beam-line I911-3 (25). Data were integrated and scaled using the XDS package (26). Molecular replacement was performed with the Phenix.Phaser software (27) and a FVIIa-WT·sTF-FFR complex as a search model. The subsequent refinement and model building were performed using iterative cycles of Phenix.Refine (28) in the Phenix program package (29), utilizing MolProbity (30) and TLS (31), followed by computer graphic model corrections by Coot software (32). The three generated structures were deposited in the Protein Data Bank as FVIIa-YT (Protein Data Bank code 4z6a), FVIIa-ST (Protein Data Bank code 4zmA), and FVIIa-FT (Protein Data Bank code 4ylq).

Acrylamide Tryptophan Quenching

Fluorescence intensity measurements were performed on a Cary Eclipse spectrofluorometer (Agilent Technologies) equipped with a four-cell magnetic stirrer sample holder and Peltier element using a set of four 10 × 10-mm QS quartz cuvettes (Hellma Analytics, Germany). 150 nm FVIIa variant in assay buffer kept at 15, 25, or 37 °C was titrated with 0–0.5 m acrylamide using a volume replacement approach by preparing two identical solutions for the titration where one was spiked with 5.63 m acrylamide (Bio-Rad). Data were collected with excitation at 295 nm (5-nm slit width) and emission at 330 nm (20-nm slit width); the integration time was 0.5 s. The collected data were baseline-corrected, and inner filtering effects were addressed by a correction method for a right-angled fluorescence setup (33) with the correction factor being Fcorr = Fobs × 100.125 × [acrylamide] using a ϵ295 of 0.25 m−1 cm−1 for acrylamide and a path length of fluorescence measurement of 0.5 cm. Stern-Volmer plots were generated, and the data were analyzed using a dynamic collision quenching model (34) in GraphPad Prism 6.0.

equation image

The observed correlation between the determined Stern-Volmer quenching constant (Ksv) values and the calculated SASA values was evaluated using a Pearson correlation approach in GraphPad Prism 6.0 to determine whether it could be explained by random sampling (α = 0.01).

Molecular Dynamics Simulations of FVIIa Variants

For MD simulations, the FVIIa 170 loop variants were constructed using the x-ray crystallographic structure of FVIIa-YT as a template. For the ST and FT variants, Tyr172 was mutated to Ser and Phe, respectively, while preserving the side chain rotamer of the template. FVIIa-WT was based on a representative structure of benzamidine-inhibited FVIIa (Protein Data Bank code 1kli (35)). The complex of sTF 1–213 and FVIIa was constructed starting from Protein Data Bank code 1dan as described and graciously provided by Ohkubo et al. (8). In all structures, the co-crystallized inhibitor was removed. 100-ns conventional MD simulations of the FVIIa-WT·sTF complex, FVIIa-WT, and the three FVIIa variants with the trypsin 170 loops were performed using the NAMD 2.7 software package (36) with the CHARMM27 force field (37) and the TIP3P water model (38). An integration time step of 1.0 fs was used for the velocity Verlet algorithm. Simulations were carried out at constant pressure (P = 1 atm) and constant temperature (T = 310 K) controlled by the Langevin thermostat (damping coefficient, 5/ps) and the Nosé-Hoover Langevin piston barostat (39, 40), respectively. Throughout, anisotropic pressure coupling was applied for the barostat using a piston damping coefficient of 5/ps, a piston period of 100 fs, and piston decay of 50 fs. Long range electrostatic forces were calculated using the particle mesh Ewald method (41) using a grid spacing of ~1 Å and a fourth order spline for interpolation. Electrostatic forces were updated every 4th fs. van der Waals interactions were cut off at 12 Å in combination with a switching function beginning at 10 Å. Periodic boundary conditions was applied in x-, y-, and z-directions. The potential energy in all systems was initially minimized using 500 steps of the conjugated gradient method.

SASA

The SASA was calculated for all tryptophans in the simulated FVIIa variants during the entire time course at a probe radius of 1.4 Å using the standard implementation (measure command) in VMD (42). The same calculations were made for the crystal structures with FFR present. For graphical comparison of SASA values for Trp215 between variants, the data were smoothened using the Savistsky and Golay method in GraphPad Prism 6.0 with a window size of 10 and a second degree polynomial.

Results

The 170 Loop Is Linked to Cofactor Binding and Amidolytic Activity

Previous studies have shown that the conformation of the 170 loop is affected by the binding of TF to FVIIa (2, 6). In agreement, impairment of cofactor interaction was observed for the three FVIIa variants as assessed by the effect of sTF on FVIIa amidolytic activity (S-2288) (Fig. 2, A–D, and Table 1). FVIIa-YT displayed an 84-fold compromise in its ability to bind to sTF but could be fully stimulated by the addition of saturating sTF, reaching a higher final amidolytic activity than FVIIa-WT·sTF (Fig. 2, A and D). This indicates that the engineered 170 loop in FVIIa-YT selectively affects sTF binding but is still able to mediate allosteric communication to the FVIIa active site upon sTF binding. From the kinetics of S-2288 hydrolysis, we found that the increased FVIIa-YT activity at saturating sTF concentration was entirely due to an increased kcat value with no change in the Km value compared with the FVIIa-WT·sTF complex (Fig. 2D and Table 1). In addition, we reproduced the higher activity of FVIIa-YT without sTF with an increase in kcat/Km of 8.3-fold over FVIIa-WT (12). The importance of Tyr172 was evident as the removal of a single hydroxyl group (FVIIa-FT) resulted in a markedly decreased potentiation of activity and a loss in sTF affinity (Fig. 2, A and B, and Table 1) with an accompanying reduction of the intrinsic activity to half that of FVIIa-WT (Fig. 2C and Table 1). The FVIIa-ST variant showed a partial rescue of sTF affinity (Table 1), maintaining a 4-fold higher intrinsic activity compared with FVIIa-WT, whereas the activity level at saturating sTF concentration was significantly reduced (Fig. 2A and Table 1). Independent surface plasmon resonance analysis confirmed sTF affinity values for the three FVIIa variants obtained by the amidolytic activity strategy (data not shown).

FIGURE 2.
Cofactor binding and amidolytic activity of FVIIa variants. A, initial velocity (Vint) of 1 mm S-2288 hydrolysis by 15 nm FVIIa-WT (○) in black, FVIIa-YT ([open diamond]) in orange, FVIIa-ST (□) in cyan, and FVIIa-FT ([open triangle]) in green as ...
TABLE 1
Functional investigation of 170 loop variants

Inhibitor Binding Reveals Changes in S1 Pocket Maturation

To further investigate the effect of the engineered 170 loops on the active site, we probed the S1 pocket by binding of pABA, a small molecule inhibitor known to occupy the S1 pocket and oxyanion hole (35). Consistent with an immature S1 pocket, FVIIa-WT was poorly inhibited by pABA in the absence of sTF (Ki 1485 μm; Fig. 3A and Table 1). The Ki values for FVIIa-YT and FVIIa-ST were significantly decreased in agreement with their increased amidolytic activity and a more mature S1 pocket (Table 1). FVIIa-FT exhibited an intermediate Ki value. Binding of sTF to FVIIa-WT is known to mature the active site (23) and resulted in a ~30-fold decreased Ki value (49.3 μm). At saturating sTF concentrations, FVIIa-WT, FVIIa-YT, FVIIa-ST, and FVIIa-FT all reached similar Ki values (Fig. 3A and Table 1) as anticipated for FVIIa-YT and FVIIa-ST but not for FVIIa-FT due to its much lower activity toward S-2288. This suggests that FVIIa-FT has a mature S1 pocket in the presence of sTF but possibly impaired substrate binding in the S2-S3 pockets.

FIGURE 3.
Functional characterization of FVIIa variants. A, titration of 10–100 nm FVIIa-WT (○) in black, FVIIa-YT ([open diamond]) in orange, FVIIa-ST (□) in cyan, and FVIIa-FT ([open triangle]) in green with 0–30 mm pABA inhibitor normalized ...

Mutagenesis of the 170 Loop Affects N Terminus Protection

A functional marker for FVIIa “zymogenicity” is the extent of N terminus insertion, which can be perturbed by TF binding or by mutagenesis in FVIIa (7, 12). The carbamylation assay (Fig. 3B and Table 1) correlates Ile16 (N terminus) solvent exposure to residual activity and has been successfully used to assess the extent of protease domain N terminus insertion (4). In the absence of sTF, FVIIa-WT, FVIIa-YT, and FVIIa-ST showed similar levels of N terminus protection, whereas the protection level was slightly decreased in FVIIa-FT (Fig. 3B). Addition of sTF had a pronounced effect on N terminus insertion with FVIIa-WT showing complete protection, whereas FVIIa-YT achieves very little protection, suggesting that the improved amidolytic activity observed for this variant is independent of complete N terminus insertion. Interestingly, the FVIIa-ST N terminus is protected when compared with FVIIa-YT, correlating with the increased sTF affinity, whereas FVIIa-FT showed the lowest level of protection gained from sTF addition in agreement with a poor catalytic activity and sTF affinity.

Tyr172 Directly Stabilizes Segment 215–217 and AL3

To investigate the conformation of the 170 loop from trypsin in FVIIa, we determined the x-ray crystal structure of the three variants with sTF and the irreversible active site inhibitor FFR (2). The three FVIIa variants crystallized in identical space groups with highly similar unit cell dimensions, allowing meaningful structural comparison. Data collection and refinement statistics are summarized in Table 2. Comparison of the protease domain of FVIIa-WT (Protein Data Bank code 1dan (2)) and the three variants revealed the same structural topology (Cα root mean square deviation of the protease domain (241 residues) of 0.27–0.39 Å) outside the immediate surroundings of the 170 loop and AL2–3 (residues 184–193 and 220–225) (Fig. 4, A–D). The FVIIa-YT map (2FoFc) lacked electron density for the Ca2+-dependent γ-carboxyglutamic acid domain presumably due to the presence of citrate in the crystallization condition and the cryoprotectant for this variant.

TABLE 2
Data collection and refinement statistics of x-ray crystallography
FIGURE 4.
Structural analysis reveals important role of Tyr172. A, comparison of FVIIa-WT (Protein Data Bank code 1dan; dark blue) and FVIIa-YT (orange) showing the insertion of Tyr172, which displaces HOH1. Water molecules found in the region of AL2–3 ...

From the FVIIa-YT·sTF complex structure, it was evident that the trypsin 170 loop interacts with the active site region. This was illustrated by hydrogen bonding from the hydroxyl group of Tyr172 to the backbone nitrogen of Gln217 (3.1 Å) and the backbone carbonyl of Phe225 (2.5 Å) (Fig. 4, A and B). In addition, Tyr172 may further stabilize the 215–217 segment by favorable electrostatic aromatic interaction with Trp215 (closest ring carbons, 3.7–3.9 Å). In addition, the Tyr172 side chain displaces a water molecule (HOH1) present in the FVIIa-WT structure, which may play a stabilizing role in the FVIIa-WT·sTF complex (Fig. 4B), supporting the increased amidolytic activity observed for the FVIIa-YT·sTF complex. It was also observed that a smaller serine at position 171, compared with glutamine in FVIIa-WT, seemed to enable the shorter 170 loop from trypsin to interact with the AL3 backbone (Fig. 5A).

FIGURE 5.
Structural effects of 170 loop substitution. A, alignment of the 170 loop from FVIIa-WT and FVIIa-YT showing removal of the possible Gln171 clash in FVIIa-YT by replacement to a serine at position 171. B, alignment of the 170 loop main chain of FVIIa-WT, ...

The removal of the benzene and shortening of the hydroxyl group placement in FVIIa-ST resulted in a loss of electron density for residues Ser172 and Pro173 in the 170 loop (Fig. 4C). This may result from competition of the shortened and possibly more mobile loop with a cacodylate ion found in both the FVIIa-ST and FVIIa-WT structures (Fig. 4C). The structural data obtained for FVIIa-FT revealed 170 loop and AL2–3 conformations very similar to that observed for FVIIa-YT with Phe172 occupying the same position as Tyr172. The N terminus for the three variants was inserted in the activation pocket as in the FVIIa-WT·sTF crystal structure, possibly due to the presence of sTF and active site inhibitor. In general, shortening of the 170 loop seemed to affect the structural integrity of the TF-binding helix (residues 165–169) where the final turn of the helix was skewed for all three variants (Fig. 5B) with FVIIa-YT and -FT [var phi]/ψ angles of Asp167 and Cys168 outside the typical α-helix regions (Fig. 5C). This correlated with the observed loss of sTF affinity and lower extent of N terminus insertion for these variants, whereas FVIIa-ST with [var phi]/ψ angles closer to those of FVIIa-WT exhibited improved sTF affinity and a higher extent of N terminus insertion in the TF-bound complex. From these data, it seems that the orientation of the 170 loop and thus the structural integrity of TF-binding helix alter the extent to which the N terminus (Ile16) can be inserted into the activation pocket as a consequence of sTF binding.

Molecular Dynamics Simulations Track Trp215 Movement

To allow for an unbiased observation of the dynamic behavior and a detailed understanding of the effects of Tyr172 on the protease domain, we performed 100 ns classical MD simulations for FVIIa-WT and the three variants without active site inhibitor and sTF. In addition, we also performed 100-ns classical MD simulations for FVIIa-WT in complex with sTF without active site inhibitor.

Our simulations show that AL1–3, including segment 215–217 that harbors Trp215 (supplemental Movies S1–S5), are highly flexible and undergo significant structural changes. The rearrangements are most pronounced in FVIIa-WT where Trp215 not only releases from the aryl binding pocket (S3-S4) but the S1 pocket collapses entirely as indicated by the short distance between Trp215 and Ser195 (Fig. 6, A–C). This suggests that the FVIIa molecule undergoes a transition where the 215–217 segment moves from an open conformation through an intermediate to a fully collapsed state where especially Trp215 occludes the substrate-binding region (Fig. 6, A–D). In the absence of sTF, this conformational transition from an open to a collapsed population is absent in FVIIa-YT but is reintroduced in the FVIIa-ST and -FT variants (Fig. 6D). Intriguingly, the 215–217 segment is stabilized in the FVIIa-WT·sTF complex in a manner similar to that observed for FVIIa-YT (Fig. 6D). Additionally, it was observed that FVIIa-FT displayed a significant collapse of the TF-binding helix in good agreement with the relatively low sTF affinity and that Trp215 is released from the S3 pocket into a position where it can interfere with substrate binding to S2/S3 sites and conceivably cause the low amidolytic activity. Furthermore, it seems that this mechanism is independent of N terminus insertion as the salt bridge between the amino group of Ile16 and Asp194 was present for the duration of all simulations.

FIGURE 6.
Trp215 location in relation to the active site. Representative conformations of segment 215–217 measured as Trp215 relative distances to the catalytic triad are shown. A, open; B, intermediate; C, collapsed. Each state is depicted on the scatter ...

Fluorescence Quenching Shows Changes to Tryptophan Accessibility

To correlate the MD simulations with an experimentally measurable quantity, we calculated SASA values for all tryptophans in the FVIIa protease domain throughout the simulation time course (Fig. 7, A–C). The SASA values were compared with results from a fluorescence quenching assay reporting on tryptophan solvent exposure by monitoring the loss of overall tryptophan fluorescence intensity upon addition of acrylamide (Fig. 7D) (34). According to the SASA calculations, three of the eight tryptophan residues (Trp61, Trp207, and Trp215) were partially or fully exposed (Fig. 7B). This agreed well with the fluorescence quenching data where a significant level of quenching, or exposed tryptophans, was observed in FVIIa-WT (Fig. 7D and Table 3). The observed linearity of the quenching data allowed for the assumption of a collision quenching mechanism to predict the Ksv (43). In accordance with a collision quenching mechanism, increased quenching was observed with increasing temperatures (data not shown) (34).

FIGURE 7.
Evaluation of tryptophan surface accessibility. A, conformations of Trp215 in FVIIa-WT during MD simulations with van der Waals surface area in red in an open (0 ns), intermediate (73 ns), and collapsed conformations with collapsed S1 pocket (99 ns). ...
TABLE 3
Tryptophan surface accessibility

Consistent with the distance plots (Fig. 6D), Trp215 exhibited varying SASA values over the time course of the simulations for FVIIa-WT, -ST, and -FT, whereas those of FVIIa-YT and FVIIa-WT·sTF showed lower and more stable levels (Fig. 7C and Table 3). In agreement with these observations, FVIIa-YT showed a significant decrease in quenching at 15 °C, whereas FVIIa-FT was only moderately protected, and FVIIa-ST showed a total quenching increase compared with FVIIa-WT (Fig. 7D and Table 3). The SASA values for Trp215 correlated well (Pearson r value of 0.99 and p < 0.01) with the Ksv values for the examined variants compared with Trp61/Trp207 where the relationship between the measured and experimental data was less pronounced (Fig. 7, E–G). A control experiment with FFR added to all variants gave the expected normalization of quenching values to that of inhibited FVIIa-WT. This was in good agreement with the calculated SASA values, reflecting complete shielding of Trp215, which should result in a significant decrease in overall quenching due to the large contribution from this residue to the total tryptophan surface-accessible area (~33%). These findings support that the acrylamide quenching is highly sensitive to the conformation of Trp215 even with the background signal from the remaining tryptophan residues.

Discussion

The TF-mediated allosteric regulation of FVIIa activity has been investigated for several decades, generating a wealth of experimental evidence for two distinct allosteric pathways (Fig. 1B). In the current model, the two pathways have a suggested common origin at the FVIIa-TF interface where especially the insertion of Met164 from FVIIa into a pocket of TF has proven essential for the propagation of the allosteric signal to the FVIIa active site (10, 23). From Met164, the two pathways branch out with pathway I moving through the TF-binding helix to tether it and the 170 loop to the protease domain through Arg173 and Gly223 (24). Pathway II propagates through Leu163 and Phe225 to stabilize AL3, which in turn influences Ser185 in AL2, allowing N terminus insertion, which stabilizes a Val17 to Ala221a interaction and the Cys191-Cys220 disulfide pair (44). Here we attempt to elaborate on these two pathways and propose a more complete molecular basis of TF-mediated allosteric enhancement of FVIIa activity.

The crystal structure of FVIIa-YT in complex with sTF revealed that Tyr172, as in trypsin (45), is inserted into a cavity in the FVIIa protease domain, forming key hydrogen bonds with Gln217 and Phe225 and favorable electrostatic interaction with Trp215. These key interactions, missing in the FVIIa-ST and -FT crystal structures, result in stabilization of segment 215–217 and AL3 in FVIIa-YT. Both Gln217 and Phe225 have been shown to be components of the two consensus TF-mediated allosteric pathways in FVIIa (5, 10, 46) that were recently suggested to encompass Trp215 (20). In addition, the hydroxyl group of Tyr172 displaces a water molecule (HOH1; Fig. 4, A and B) found in the FVIIa-WT·sTF complex, which may result in a more stable hydrogen bond network, leading to the observed higher activity for the FVIIa-YT·sTF complex. It is quite likely that the introduced 170 loop from trypsin stabilizes allosteric pathway I via Tyr172 interactions in the absence of sTF, resulting in the increased amidolytic activity without complete N terminus insertion. In the presence of sTF, the inability of the FVIIa-YT protease domain N terminus to completely insert in the activation pocket may stem from the strain imposed by Tyr172 on the 170 loop by making direct contacts with AL2 and AL3. Although these interactions stabilize the 215–217 segment and AL3, leading to improved activity, they may interfere with allosteric pathway II and have a deleterious effect on the neighboring AL1. AL1 and AL2 along with other key interactions accommodate the N terminus. Previous studies have shown the critical role of Tyr172 in engineering substrate specificity (47) and Na+ mimicry (48) in trypsin, warranting future studies probing the influence of Tyr172 on the conformation of segment 215–217 and the intrinsic activity of trypsin.

It has previously been reported that a five-residue truncation of the FVIIa 170 loop to the length found in trypsin resulted in a 3-fold decrease in amidolytic activity (12). The FVIIa-ST variant investigated here has an identical 170 loop length but showed a 4-fold increase in amidolytic activity. From the crystal structures of FVIIa-ST and -YT (Fig. 5A), it was evident that the additional changes relative to FVIIa-WT, specifically Gln171 to serine, removed a clash with AL2–3, allowing for the increased activity. This in turn suggests that shortening of the 170 loop in FVIIa-WT should result in gained activity if Gln171 was concomitantly mutated to a non-clashing residue (e.g. Ser or Ala). A similar effect was not observed for FVIIa-FT as the activity was decreased significantly compared with that of FVIIa-WT. It is possible that Phe172 may help stabilize the S1 pocket by locating itself into the cavity normally found in FVIIa but is unable to stabilize the 215–217 segment, resulting in the observed decrease in amidolytic activity in combination with an increase in S1 pocket maturity. This effect became even more pronounced in the presence of sTF with the cofactor still able to mature the S1 pocket to FVIIa-WT levels through the proposed pathway II (10) but unable to potentiate amidolytic activity. The unfavorable conformation of the TF-binding helix may explain this as it is likely to result in the low extent of N terminus insertion, which without the effects of Tyr172 results in a significant destabilization of the whole activation pocket and the active site (5). In conclusion, despite incomplete N terminus insertion, a combination of several factors appears to contribute to the activity gain of FVIIa-YT, including 170 loop shortening, removal of a Gln171 clash, direct stabilization of 215–217 segment and AL3 through Tyr172, and the displacement of a water molecule, which may enhance an allosteric pathway from Leu163 to the 215–217 segment.

The MD simulations allowed tracking of segment 215–217 movement in the three FVIIa 170 loop variants and FVIIa-WT. A clear picture emerged of FVIIa-YT being able to retain the open active conformation, whereas FVIIa-ST, FVIIa-FT, and FVIIa-WT collapsed into inactive closed states with Trp215 occluding the active site. Addition of sTF to FVIIa-WT stabilized segment 215–217 in the active open conformation, very similar to that of FVIIa-YT. This is in agreement with recent hydrogen-deuterium exchange mass spectrometry (20) where an increase in Trp215 backbone amide protection was seen upon sTF addition. The combined approach of SASA calculation and in-solution quenching used here allowed an elaboration on these observations. The approach showed a lower degree of quenching for the more active variants correlating with the SASA calculations, supporting the suggested activity-regulating mechanism observed in the MD simulations for the 170 loop swap variants. From these observations, we speculate that the final mechanism in the conversion of FVIIa into its catalytically competent state involves segment 215–217 moving from a collapsed to a more open conformation upon TF binding, allowing substrate access to the active site. The two allosteric pathways in FVIIa (10) may work in unison to allow for the stabilization of segment 215–217 in an open conformation because both stabilization of the 170 loop and AL1–3 through insertion of the N terminus are needed to maintain FVIIa in a fully catalytically competent state. The exact distribution of the suggested open and collapsed states in FVIIa, their relation to the N terminus insertion event, and whether this distribution is affected by binding of TF remain to be explored.

The ability of the trypsin 170 loop to stabilize the 215–217 segment in FVIIa prompted an investigation of the conformation surrounding the 170 loop and the active site in other trypsin-like proteases (Fig. 8). An interesting pattern emerges from the analyzed crystal structures with FVIIa and trypsin located at opposite ends of the spectrum with regard to reported activity. Three of the proteases involved in blood coagulation, factor IXa (49), factor Xa (50), and thrombin (51), all show possible stabilization of segment 215–217 via a conserved water hydrogen bond network between the Glu217 carboxyl group and the 170 loop. This mode of stabilization, however, may be more transient than the Tyr172-mediated mechanism observed in trypsin. Ethylene glycol improves factor IXa activity ~20-fold (52) and may mimic the role of Tyr172. In factor Xa, the presence of three consecutive serines (171–173) may mitigate the mobility of the water network (48), leading to higher intrinsic activity of this protease (53). In thrombin, recent work has shown that the apo form of thrombin is highly flexible (17) and exists in an open/collapsed equilibrium controlled by the position of the 215–217 segment (16, 18, 54) where the addition of Na+ shifts the equilibrium in favor of the open form. We speculate that in the apo form the water network in thrombin facilitates a 170 loop-mediated stabilization of segment 215–217. In chymotrypsin, Trp172 makes a weak electrostatic interaction with the backbone of Pro225 and may stabilize the 215–217 segment by an edge-face stacking interaction with Trp215 (55). From the structures reviewed here, we speculate that stabilization of the 215–217 segment in an open conformation through 170 loop interactions could be a recurring theme in trypsin-like proteases (56) and that the structural mechanism behind this has diverged through evolution. This may accommodate the development of allosteric regulatory control as decreased intrinsic activity creates the need for cofactors to achieve full activity. Such intricate mechanisms in conjunction with the generation of new exosites due to cofactor binding allow for the necessary control of protease activity in the complex enzymatic cascades of blood coagulation.

FIGURE 8.
Overview of 170 loop-mediated active site stabilization in trypsin-like serine proteases. A, alignment of the selected proteases with residues corresponding to Tyr172, Trp215, Asp217, and Pro225 in trypsin shown in red. Structural overviews of plausible ...

Author Contributions

A. B. S. designed the research, conducted experiments, analyzed the results, and wrote and revised the manuscript. J. J. M. designed the research, conducted the molecular dynamics simulations, and wrote the manuscript. L. A. S. and A. A. P. supported the experiments and analyzed results. H. Ø., M. T. O., and O. H. O. designed the research, reviewed the results, and revised the manuscript. P. S. G. designed the research, reviewed the results, and wrote and revised the manuscript.

Supplementary Material

Supplemental Data:

Acknowledgments

We thank Michael P. Petersen and Anette S. Petersen for excellent technical assistance. We also thank Hanne Grøn, Grant E. Blouse, and Egon Persson for valuable scientific discussions and comments.

*A. B. S., L. A. S., A. A. P., H. Ø., O. H. O., and P. S. G. are employees of Novo Nordisk A/S.

The atomic coordinates and structure factors (codes 4z6a, 4zma, and 4ylq) have been deposited in the Protein Data Bank (http://wwpdb.org/).

An external file that holds a picture, illustration, etc.
Object name is sbox.jpgThis article contains supplemental Movies S1–S5.

5Chymotrypsin numbering used throughout the article.

4The abbreviations used are:

TF
tissue factor
sTF
soluble tissue factor 1–219
FVIIa
activated coagulation factor VIIa
FVII
zymogen coagulation factor VII
AL1–3
activation loops 1–3
MD
molecular dynamics
SASA
solvent-accessible surface area
FFR
H-d-Phe-Phe-Arg chloromethyl ketone TFA salt
S-2288
d-Ile-Pro-Arg-p-nitroanilide
pABA
p-aminobenzamidine
Ksv
Stern-Volmer quenching constant.

References

1. Davie E. W., Fujikawa K., and Kisiel W. (1991) The coagulation cascade: initiation, maintenance, and regulation. Biochemistry 30, 10363–10370 [PubMed]
2. Banner D. W., D'Arcy A., Chène C., Winkler F. K., Guha A., Konigsberg W. H., Nemerson Y., and Kirchhofer D. (1996) The crystal structure of the complex of blood coagulation factor VIIa with soluble tissue factor. Nature 380, 41–46 [PubMed]
3. Huber R., and Bode W. (1978) Structural basis of the activation and action of trypsin. Acc. Chem. Res. 266, 114–122
4. Higashi S., Nishimura H., Aita K., and Iwanaga S. (1994) Identification of regions of bovine factor VII essential for binding to tissue factor. J. Biol. Chem. 269, 18891–18898 [PubMed]
5. Rand K. D., Andersen M. D., Olsen O. H., Jørgensen T. J., Ostergaard H., Jensen O. N., Stennicke H. R., and Persson E. (2008) The origins of enhanced activity in factor VIIa analogs and the interplay between key allosteric sites revealed by hydrogen exchange mass spectrometry. J. Biol. Chem. 283, 13378–13387 [PubMed]
6. Pike A. C., Brzozowski A. M., Roberts S. M., Olsen O. H., and Persson E. (1999) Structure of human factor VIIa and its implications for the triggering of blood coagulation. Proc. Natl. Acad. Sci. U.S.A. 96, 8925–8930 [PubMed]
7. Persson E., Kjalke M., and Olsen O. H. (2001) Rational design of coagulation factor VIIa variants with substantially increased intrinsic activity. Proc. Natl. Acad. Sci. U.S.A. 98, 13583–13588 [PubMed]
8. Ohkubo Y. Z., Morrissey J. H., and Tajkhorshid E. (2010) Dynamical view of membrane binding and complex formation of human factor VIIa and tissue factor. J. Thromb. Haemost. 8, 1044–1053 [PMC free article] [PubMed]
9. McCallum C. D., Hapak R. C., Neuenschwander P. F., Morrissey J. H., and Johnson A. E. (1996) The location of the active site of blood coagulation factor VIIa above the membrane surface and its reorientation upon association with tissue factor: a fluorescence energy transfer study. J. Biol. Chem. 271, 28168–28175 [PubMed]
10. Persson E., and Olsen O. H. (2011) Allosteric activation of coagulation factor VIIa. Front. Biosci. 16, 3156–3163 [PubMed]
11. Persson E., Bak H., Østergaard A., and Olsen O. H. (2004) Augmented intrinsic activity of Factor VIIa by replacement of residues 305, 314, 337 and 374: evidence of two unique mutational mechanisms of activity enhancement. Biochem. J. 379, 497–503 [PubMed]
12. Soejima K., Mizuguchi J., Yuguchi M., Nakagaki T., Higashi S., and Iwanaga S. (2001) Factor VIIa modified in the 170 loop shows enhanced catalytic activity but does not change the zymogen-like property. J. Biol. Chem. 276, 17229–17235 [PubMed]
13. Gandhi P. S., Chen Z., Mathews F. S., and Di Cera E. (2008) Structural identification of the pathway of long-range communication in an allosteric enzyme. Proc. Natl. Acad. Sci. U.S.A. 105, 1832–1837 [PubMed]
14. Pozzi N., Vogt A. D., Gohara D. W., and Di Cera E. (2012) Conformational selection in trypsin-like proteases. Curr. Opin. Struct. Biol. 22, 421–431 [PMC free article] [PubMed]
15. Bah A., Garvey L. C., Ge J., and Di Cera E. (2006) Rapid kinetics of Na+ binding to thrombin. J. Biol. Chem. 281, 40049–40056 [PubMed]
16. Niu W., Chen Z., Gandhi P. S., Vogt A. D., Pozzi N., Pelc L. A., Zapata F., and Di Cera E. (2011) Crystallographic and kinetic evidence of allostery in a trypsin-like protease. Biochemistry 50, 6301–6307 [PMC free article] [PubMed]
17. Lechtenberg B. C., Johnson D. J., Freund S. M., and Huntington J. A. (2010) NMR resonance assignments of thrombin reveal the conformational and dynamic effects of ligation. Proc. Natl. Acad. Sci. U.S.A. 107, 14087–14092 [PubMed]
18. Vogt A. D., Chakraborty P., and Di Cera E. (2015) Kinetic dissection of the pre-existing conformational equilibrium in the trypsin fold. J. Biol. Chem. 290, 22435–22445 [PMC free article] [PubMed]
19. Huntington J. A., and Esmon C. T. (2003) The molecular basis of thrombin allostery revealed by a 1.8 Å structure of the “slow” form. Structure 11, 469–479 [PubMed]
20. Song H., Olsen O. H., Persson E., and Rand K. D. (2014) Sites involved in intra- and interdomain allostery associated with the activation of factor VIIa pinpointed by hydrogen-deuterium exchange and electron transfer dissociation mass spectrometry. J. Biol. Chem. 289, 35388–35396 [PMC free article] [PubMed]
21. Madsen J. J., Persson E., and Olsen O. H. (2015) Tissue factor activates allosteric networks in factor VIIa through structural and dynamic changes. J. Thromb. Haemost. 13, 262–267 [PubMed]
22. Freskgård P. O., Olsen O. H., and Persson E. (1996) Structural changes in factor VIIa induced by Ca2+ and tissue factor studied using circular dichroism spectroscopy. Protein Sci. 5, 1531–1540 [PubMed]
23. Persson E., Nielsen L. S., and Olsen O. H. (2001) Substitution of aspartic acid for methionine-306 in factor VIIa abolishes the allosteric linkage between the active site and the binding interface with tissue factor. Biochemistry 40, 3251–3256 [PubMed]
24. Persson E., and Olsen O. H. (2009) Activation loop 3 and the 170 loop interact in the active conformation of coagulation factor VIIa. FEBS J. 276, 3099–3109 [PubMed]
25. Ursby T., Unge J., Appio R., Logan D. T., Fredslund F., Svensson C., Larsson K., Labrador A., and Thunnissen M. M. (2013) The macromolecular crystallography beamline I911-3 at the MAX IV laboratory. J. Synchrotron Radiat. 20, 648–653 [PMC free article] [PubMed]
26. Kabsch W. (2010) XDS. Acta Crystallogr. D Biol. Crystallogr. 66, 125–132 [PMC free article] [PubMed]
27. McCoy A. J., Grosse-Kunstleve R. W., Adams P. D., Winn M. D., Storoni L. C., and Read R. J. (2007) Phaser crystallographic software. J. Appl. Crystallogr. 40, 658–674 [PubMed]
28. Afonine P. V., Grosse-Kunstleve R. W., Echols N., Headd J. J., Moriarty N. W., Mustyakimov M., Terwilliger T. C., Urzhumtsev A., Zwart P. H., and Adams P. D. (2012) Towards automated crystallographic structure refinement with phenix.refine. Acta Crystallogr. D Biol. Crystallogr. 68, 352–367 [PMC free article] [PubMed]
29. Adams P. D., Afonine P. V., Bunkóczi G., Chen V. B., Davis I. W., Echols N., Headd J. J., Hung L.-W., Kapral G. J., Grosse-Kunstleve R. W., McCoy A. J., Moriarty N. W., Oeffner R., Read R. J., Richardson D. C., Richardson J. S., Terwilliger T. C., and Zwart P. H. (2010) PHENIX: a comprehensive Python-based system for macromolecular structure solution. Acta Crystallogr. D Biol. Crystallogr. 66, 213–221 [PMC free article] [PubMed]
30. Davis I. W., Leaver-Fay A., Chen V. B., Block J. N., Kapral G. J., Wang X., Murray L. W., Arendall W. B. 3rd, Snoeyink J., Richardson J. S., and Richardson D. C. (2007) MolProbity: all-atom contacts and structure validation for proteins and nucleic acids. Nucleic Acids Res. 35, W375–W383 [PMC free article] [PubMed]
31. Winn M. D., Murshudov G. N., and Papiz M. Z. (2003) Macromolecular TLS refinement in REFMAC at moderate resolutions. Methods Enzymol. 374, 300–321 [PubMed]
32. Emsley P., Lohkamp B., Scott W. G., and Cowtan K. (2010) Features and development of Coot. Acta Crystallogr. D Biol. Crystallogr. 66, 486–501 [PMC free article] [PubMed]
33. Lutz H., and Luisi P. L. (1983) Correction for inner filter effects in fluorescence spectroscopy. Helv. Chim. Acta 66, 1929–1935
34. Lakowicz J. (2007) Principles of Fluorescence Spectroscopy, 3rd Ed., pp. 277–293, Springer, New York
35. Sichler K., Banner D. W., D'Arcy A., Hopfner K. P., Huber R., Bode W., Kresse G.-B., Kopetzki E., and Brandstetter H. (2002) Crystal structures of uninhibited factor VIIa link its cofactor and substrate-assisted activation to specific interactions. J. Mol. Biol. 322, 591–603 [PubMed]
36. Phillips J. C., Braun R., Wang W., Gumbart J., Tajkhorshid E., Villa E., Chipot C., Skeel R. D., Kalé L., and Schulten K. (2005) Scalable molecular dynamics with NAMD. J. Comput. Chem. 26, 1781–1802 [PMC free article] [PubMed]
37. MacKerell A. D., Bashford D., Bellott M., Dunbrack R. L., Evanseck J. D., Field M. J., Fischer S., Gao J., Guo H., Ha S., Joseph-McCarthy D.,Kuchnir L., Kuczera K., Lau F. T., Mattos C., Michnick S., Ngo T., Nguyen D. T., Prodhom B., Reiher W. E., Roux B., Schlenkrich M., Smith J. C., Stote R., Straub J., Watanabe M., Wiórkiewicz-Kuczera J., Yin D., and Karplus M. (1998) All-atom empirical potential for molecular modeling and dynamics studies of proteins. J. Phys. Chem. B 102, 3586–3616 [PubMed]
38. Jorgensen W. L., Chandrasekhar J., Madura J. D., Impey R. W., and Klein M. L. (1983) Comparison of simple potential functions for simulating liquid water. J. Chem. Phys. 79, 926
39. Martyna G. J., Tobias D. J., and Klein M. L. (1994) Constant pressure molecular dynamics algorithms. J. Chem. Phys. 101, 4177
40. Feller S. E., Zhang Y., Pastor R. W., and Brooks B. R. (1995) Constant pressure molecular dynamics simulation: the Langevin piston method. J. Chem. Phys. 103, 4613
41. Essmann U., Perera L., Berkowitz M. L., Darden T., Lee H., and Pedersen L. G. (1995) A smooth particle mesh Ewald method. J. Chem. Phys. 103, 8577–8593
42. Humphrey W., Dalke A., and Schulten K. (1996) VMD: visual molecular dynamics. J. Mol. Graph. 14, 33–38 [PubMed]
43. Eftink M. R., and Ghiron C. A. (1981) Fluorescence quenching studies with proteins. Anal. Biochem. 114, 199–227 [PubMed]
44. Olsen O. H., Rand K. D., Østergaard H., and Persson E. (2007) A combined structural dynamics approach identifies a putative switch in factor VIIa employed by tissue factor to initiate blood coagulation. Protein Sci. 16, 671–682 [PubMed]
45. Gaboriaud C., Serre L., Guy-Crotte O., Forest E., and Fontecilla-Camps J. C. (1996) Crystal structure of human trypsin 1: unexpected phosphorylation of Tyr151. J. Mol. Biol. 259, 995–1010 [PubMed]
46. Petrovan R. J., and Ruf W. (2000) Role of residue Phe225 in the cofactor-mediated, allosteric regulation of the serine protease coagulation factor VIIa. Biochemistry 39, 14457–14463 [PubMed]
47. Hedstrom L., Perona J. J., and Rutter W. J. (1994) Converting trypsin to chymotrypsin: residue 172 is a substrate specificity determinant. Biochemistry 33, 8757–8763 [PubMed]
48. Page M. J., Bleackley M. R., Wong S., MacGillivray R. T., and Di Cera E. (2006) Conversion of trypsin into a Na+-activated enzyme. Biochemistry 45, 2987–2993 [PubMed]
49. Zögg T., and Brandstetter H. (2009) Structural basis of the cofactor- and substrate-assisted activation of human coagulation factor IXa. Structure 17, 1669–1678 [PubMed]
50. Salonen L. M., Bucher C., Banner D. W., Haap W., Mary J. L., Benz J., Kuster O., Seiler P., Schweizer W. B., and Diederich F. (2009) Cation-π interactions at the active site of factor Xa: dramatic enhancement upon stepwise N-alkylation of ammonium ions. Angew. Chem. Int. Ed. Engl. 48, 811–814 [PubMed]
51. Pineda A. O., Carrell C. J., Bush L. A., Prasad S., Caccia S., Chen Z.-W., Mathews F. S., and Di Cera E. (2004) Molecular dissection of Na+ binding to thrombin. J. Biol. Chem. 279, 31842–31853 [PubMed]
52. Sturzebecher J., Kopetzki E., Bode W., and Hopfner K. P. (1997) Dramatic enhancement of the catalytic activity of coagulation factor IXa by alcohols. FEBS Lett. 412, 295–300 [PubMed]
53. Hopfner K. P., Brandstetter H., Karcher A., Kopetzki E., Huber R., Engh R. A., and Bode W. (1997) Converting blood coagulation factor IXa into factor Xa: dramatic increase in amidolytic activity identifies important active site determinants. EMBO J. 16, 6626–6635 [PubMed]
54. Carter W. J., Myles T., Gibbs C. S., Leung L. L., and Huntington J. A. (2004) Crystal structure of anticoagulant thrombin variant E217K provides insights into thrombin allostery. J. Biol. Chem. 279, 26387–26394 [PubMed]
55. Cohen G. H., Silverton E. W., and Davies D. R. (1981) Refined crystal structure of gamma-chymotrypsin at 1.9 Å resolution. Comparison with other pancreatic serine proteases. J. Mol. Biol. 148, 449–479 [PubMed]
56. Gohara D. W., and Di Cera E. (2011) Allostery in trypsin-like proteases suggests new therapeutic strategies. Trends Biotechnol. 29, 577–585 [PMC free article] [PubMed]

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