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Proc Natl Acad Sci U S A. Jul 7, 2009; 106(27): 11034–11039.
Published online Jun 19, 2009. doi:  10.1073/pnas.0902463106
PMCID: PMC2708782
Biochemistry

Structure of a serine protease poised to resynthesize a peptide bond

Abstract

The serine proteases are among the most thoroughly studied enzymes, and numerous crystal structures representing the enzyme–substrate complex and intermediates in the hydrolysis reactions have been reported. Some aspects of the catalytic mechanism remain controversial, however, especially the role of conformational changes in the reaction. We describe here a high-resolution (1.46 Å) crystal structure of a complex formed between a cleaved form of bovine pancreatic trypsin inhibitor (BPTI) and a catalytically inactive trypsin variant with the BPTI cleavage site ideally positioned in the active site for resynthesis of the peptide bond. This structure defines the positions of the newly generated amino and carboxyl groups following the 2 steps in the hydrolytic reaction. Comparison of this structure with those representing other intermediates in the reaction demonstrates that the residues of the catalytic triad are positioned to promote each step of both the forward and reverse reaction with remarkably little motion and with conservation of hydrogen-bonding interactions. The results also provide insights into the mechanism by which inhibitors like BPTI normally resist hydrolysis when bound to their target proteases.

Keywords: trypsin, bovine pancreatic trypsin inhibitor, enzyme mechanisms

Serine proteases are found throughout all 3 domains of cellular life and function in a wide range of physiological processes, including digestion, protein maturation and turnover, hemostasis, and immune responses (1). Approximately 0.6% of human protein-encoding genes are predicted to specify serine proteases, and this family is even more prevalent in other organisms, notably the arthropods (2, 3). A large body of biochemical and structural data have established a 2-step mechanism for hydrolysis of peptide bonds by this class of proteases (4), as shown in Scheme 1.

Scheme 1.
Two-step mechanism of serine protease action.

The first step of the reaction is a nucleophilic attack by the catalytic serine residue (Ser-195 in trypsin and other members of the chymotrypsin, or S1, family) on the carbonyl carbon atom of the residue labeled P1, generating a covalent acyl-enzyme intermediate and a new peptide amino terminus, on the P1′ residue. A second nucleophilic attack, by a water molecule, leads to hydrolysis of the acyl-enzyme, releasing the new carboxyl group and restoring the catalytic Ser residue to its initial state. In addition to the Ser residue, the mechanism of most, but not all, serine proteases depends on the side chains of a His and an Asp residue (His-57 and Asp-102 in the chymotrypsin family) (5). The His side chain serves as a base for activating the nucleophilic species and as an acid that transfers a proton to the leaving group in each step of the reaction. The Asp side chain is generally believed to stabilize the charge on the protonated His residue, but the details of the interaction between these 2 residues are not fully agreed on (6, 7).

Crystallographic studies of serine proteases with bound substrates and inhibitors have provided detailed structural information about the enzyme–substrate complex (8, 9), the acyl-enzyme (1013), and the high-energy tetrahedral intermediates that separate these species (1417). Crystal structures with bound P1 products have also been reported (1820), but at relatively low resolution or in forms that appear to be mixtures of the carboxyl, tetrahedral, and acyl-enzyme species. As far as we are aware, no structure of a bound P1 product has been reported, probably because this product usually diffuses away rapidly. Precisely locating the newly generated termini in an appropriate complex would provide insights into both steps of the mechanism, because the P1′ amino nitrogen atom is the leaving group for the first nucleophilic substitution, and the position of the P1 carboxyl group defines the endpoint in the trajectory of the deacylation step.

Although rebinding of the hydrolysis products is disfavored for most substrates, an important exception was discovered by Finkenstadt and Laskowski (21), who showed that a natural serine protease inhibitor, soybean trypsin inhibitor (SBTI), was slowly hydrolyzed when bound to trypsin and that the peptide bond could be resynthesized. Since then, it has been found that SBTI is one of a very large number of inhibitors, described as “standard mechanism” or “Laskowski mechanism” inhibitors, that act by binding tightly to the active sites of their targets as a substrate would, but resist hydrolysis for many hours or longer (2225). Because the products of hydrolysis remain physically associated, the resynthesis of these inhibitors is an intramolecular reaction and is, therefore, much more favorable than the equivalent intermolecular reaction at modest reactant concentrations. Despite the prevalence of Laskowski inhibitors (25), the mechanisms by which they resist proteolysis remain poorly understood (2629).

To gain structural insights into the mechanisms of both serine proteases and their inhibitors, we have determined the crystal structure of the complex formed between the cleaved form of a Laskowski-mechanism inhibitor, bovine pancreatic trypsin inhibitor (BPTI), and a trypsin variant with the catalytic Ser residue (Ser-195) replaced with Ala, rat anionic trypsin S195A. BPTI is one of the most extensively studied protease inhibitors and also one of the most effective: The dissociation constant for the complex formed with bovine trypsin is ≈10−13 M, and the half-time for hydrolysis has been estimated to be several years (30). A ribbon representation of the structure of intact BPTI bound to rat anionic trypsin is shown in Fig. 1A. As described below, the structure of the complex with the cleaved inhibitor highlights the degree to which each step in the catalytic mechanism is accommodated with minimum structural rearrangement of the enzyme and substrate. The results also support a model in which the Laskowski-mechanism inhibitors act by preventing hydrolysis of the acyl-enzyme intermediate and promoting reformation of the peptide bond.

Fig. 1.
Structure of the BPTI–trypsin complex and resynthesis of the scissile bond of cleaved BPTI by rat trypsin. (A) Ribbon diagram representation of the complex formed between rat trypsin and the intact form of BPTI, drawn from the atomic coordinates ...

Results and Discussion

Structure of Cleaved BPTI Bound to Trypsin.

BPTI with the reactive-site peptide bond (between Lys-15 and Ala-16) cleaved was prepared by using a modification of the procedure in ref. 31, in which 1 of the 3 disulfide bonds of the native protein was selectively reduced, thereby increasing the hydrolysis rate by ≈3,000-fold. After treatment with trypsin, the cleaved inhibitor was isolated and the disulfide bond reformed to generate the species designated here as BPTI*. As illustrated in Fig. 1B, the peptide bond is efficiently resynthesized when BPTI* is incubated with rat trypsin, as has been shown with other proteases (32, 33). The complex of BPTI* and S195A rat trypsin readily formed P3221 crystals that were isomorphous with those formed by complexes of the wild-type enzyme and intact BPTI (34, 35). X-ray diffraction data were collected by using cryopreserved crystals of the BPTI and BPTI* complexes, with resolution limits of 1.49 and 1.46 Å, respectively. Structures of the complexes were determined by the method of molecular replacement, using the structure of the complex of rat trypsin and intact BPTI determined at 1.8-Å resolution (35). Refinement yielded R values of 18.7% and 17.5% for complexes of trypsin with BPTI and BPTI*, respectively. Refinement and data collection statistics are provided in SI Table 1.

The structure of the active-site regions of the enzyme and inhibitor are shown in Fig. 2, for both the intact inhibitor bound to wild-type trypsin (A) and the cleaved inhibitor bound to S195A trypsin (B). As observed for other enzyme-bound Laskowski inhibitors (29), the scissile amide group of the intact inhibitor displayed planar geometry, and the carbonyl carbon was ideally positioned for attack by the Oγ atom of Ser-195 (shown in red on the surface representation of the enzyme in Fig. 2). The electron density map calculated for the complex containing cleaved BPTI showed the newly generated amino and carboxyl groups in well-defined positions in the enzyme active site. Hydrolysis of the peptide bond was accommodated by a small change in the conformation of Lys-15 and a displacement of ≈1 Å in the position of the nitrogen atom of Ala-16.

Fig. 2.
Structures of the intact and cleaved trypsin-binding regions in complexes of rat trypsin with BPTI and BPTI*. (A and B) The enzyme active site is shown in a surface representation, and the primary binding residues of the inhibitors are represented as ...

The electron density maps in the region of Cys-38 of the cleaved inhibitor indicated the presence of 2 side-chain conformations, one nearly identical to that seen in the intact inhibitor, and the other differing by rotation of the χ1 dihedral angle by −100°, thereby changing the chirality of the Cys-14–Cys-38 disulfide bond. The occupancy of the altered conformation was estimated to be ≈20%. This disulfide isomerization has been detected at a very low level (≈5%) by NMR spectroscopy in free intact BPTI (36, 37) and in a crystal structure of a BPTI mutant with 3 amino acid replacements in the trypsin-binding loop (38, 39). The alternate isomer is accommodated within the constraints of the complex with essentially no perturbation of Cys-14 or the backbone of either Cys residue. Beyond the active-site region, the structures of the enzyme and inhibitor were essentially identical in the 2 complexes.

High-Resolution Reconstruction of the Serine–Protease Mechanism.

Together with previously determined crystal structures of enzyme–inhibitor complexes and acyl-enzyme intermediates, the structure of BPTI* bound to trypsin contributes to a detailed structural description of the steps making up the entire serine protease mechanism. Fig. 2C shows a superposition of the catalytic residues of 4 structures: the BPTI–trypsin complex (carbon atoms colored green), the BPTI*–S195A trypsin complex (orange carbon atoms), bovine trypsin bound to a tetrahedral transition-state analog (purple carbon atoms) (16), and an acyl-enzyme intermediate formed by bovine trypsin and a peptide-nitroanilide substrate (gray carbon atoms) (13). The close superposition of the catalytic residues in the 4 structures suggests that the reaction proceeds with minimal structural changes in the active site.

A reconstruction of the peptide hydrolysis reaction is illustrated in Fig. 3, using the 4 superimposed structures described above. For clarity, only the side chains of Ser-195 and His-57 are shown, along with the scissile peptide unit or the boronate transition-state analog. To illustrate the geometry of potential hydrogen bonds involving the Nε2 atom of His-57, a hydrogen atom bound to this site was added to each model by using standard geometry. As noted previously, the structure of the BPTI–trypsin complex, shown in Fig. 3A, displays all of the features expected of a productive enzyme–substrate complex, with the Ser-195 Oγ atom positioned to attack the carbonyl carbon of the substrate (indicated by the arrow), and the Nε2 atom of His-57 positioned to activate the Ser oxygen by accepting its proton.

Fig. 3.
Structural reconstruction of the catalytic mechanism for peptide hydrolysis by serine proteases. (A) The enzyme–substrate complex, drawn from atomic coordinates of the structure of the BPTI–trypsin complex. (B) The tetrahedral intermediate ...

Formation of the acyl-enzyme intermediate requires displacement of the Nα atom of the P1′ residue and transfer of a proton to this atom. This reaction is presumed to take place via a tetrahedral intermediate, with a structure and energy closely approximating that of the transition state. Superposition of the BPTI–trypsin complex and the complex with the transition-state analog indicates that the tetrahedral intermediate in this reaction is formed by small motions (<1 Å) of the P1 Nα atom, the carbonyl carbon and the Ser-195 Oγ atom, as illustrated in Fig. 3B. A model of the resulting acyl-enzyme (Fig. 3C) was constructed by superimposing the structures of the BPTI*–trypsin complex and that of the acyl-enzyme intermediate. Fig. 3C shows that the superimposed position of the Nα atom is within 3 Å of the carbonyl carbon of the acyl intermediate and is also positioned to exchange a proton with His-57. Thus, the P1′ residue of the cleaved inhibitor lies in just the position expected for the product of the first step in the catalytic reaction and is poised to reverse this step by attacking the acyl-enzyme carbonyl.

The location of the water molecule necessary to hydrolyze the acyl-enzyme intermediate in the second step of the reaction has been the subject of considerable debate (40, 41), but the most plausible candidate appears to be the solvent molecule (S-25) identified by Radisky et al. (13) in their structures of productive acyl-enzyme complexes, and in crystal structures of acyl-enzymes formed with porcine pancreatic elastase (12, 42) and Streptomyces griseus proteinase A (11). The position of this water molecule, represented by a red sphere in Fig. 3D, places it within 3 Å of both the acyl carbon atom and the Nε2 atom of the His-57 side chain. The water molecule overlaps the superimposed position of the α-amino nitrogen atom of Ala-16 in the BPTI* structure (Fig. 3C), implying that the P1 residue must be significantly displaced from the position seen in the BPTI* complex for the water molecule to gain access to the active site and attack the acyl intermediate.

After a second tetrahedral intermediate (Fig. 3E), the hydrolysis reaction attains the final product species, represented in Fig. 3F, where the carboxyl group of the P1 residue in the BPTI*–trypsin complex is shown superimposed on the Ser-195 and His-57 side chains of the intact BPTI–trypsin structure. This superposition places the Ser Oγ atom 2.7 Å from the carboxyl carbon, exactly the distance observed between the Ser oxygen and the scissile carbonyl carbon in the complex with intact BPTI. The vector connecting the Ser Oγ atom and the carbonyl carbon is also nearly perpendicular to the plane of the carboxyl group. Thus, the carboxyl group appears to be ideally positioned for a nucleophilic attack to initiate the resynthesis reaction, implying that its position represents the final point in the trajectory of the hydrolysis reaction.

The conserved position of the His-57 side chain in structures representing different stages of the catalytic mechanism is particularly striking. The serine–protease mechanism requires that His-57 act as a base in activating each of the nucleophilic-attack steps, as an acid in transferring a proton to each leaving group. This dual role might, in principle, be accommodated either by a fixed strategic placement of the side chain, as originally proposed by Wang (43) or a movement of this side chain, as in the “histidine ring-flip” mechanism proposed by Ash et al. (44). Comparison of the structures shown in Fig. 3 indicates that the requirements of the His side chain can be met with little or no reconfiguration. Each of the nucleophilic atoms is positioned to form a hydrogen bond with the His side chain, and the model of the tetrahedral intermediates places the Nε2 atom of His-57 in a position midway between the nucleophile and leaving group atoms, poised to accept or donate the hydrogen in either direction. In all of the structures, the inferred hydrogen bonds deviate significantly from linearity, but the deviations are all quite similar, with angles ranging from 130° to 150°. Although the positions of hydrogen atoms on the nucleophilic atoms are more difficult to infer than that of the protonated His Nε2 atom, similarly bent hydrogen bonds are expected when the Nε2 atom is the acceptor rather than donor.

The nonlinearity of the His–Ser hydrogen bond has been noted previously, and it has been suggested that a weaker hydrogen bond might facilitate the proton transfer steps (5), although the relationship between hydrogen-bond geometry and proton-exchange rates does not appear to have been analyzed quantitatively in this system. A qualitative argument can be made, however, that the very similar geometries for the different His-57 hydrogen bonds modeled in Fig. 3 may contribute to an overall rate enhancement by minimizing the depths of the energy wells corresponding to the intermediates in the reaction. Rotation of the His side chain from the position observed in the crystal structures could likely enhance the interaction with the Ser-195 Oγ atom, for instance, but would almost certainly weaken the hydrogen bonds with the Nα leaving group and the hydrolytic water molecule. As a consequence, the enzyme–substrate complex would be stabilized at the expense of disfavoring formation of the subsequent intermediates. Such is likely not the case, as strongly indicated by the nearly equivalent atom positions seen in the structures representing different stages of the reaction. The arrangement of symmetrical and equally bent hydrogen bonds between His-57 and the nucleophile and leaving group was presciently suggested by Wang in 1970 (43), but appears not to have been discussed since.

Another distinctive and conserved feature of the serine proteases is the “oxyanion hole,” a grouping of amide nitrogen atoms positioned to stabilize the negative charge formed on the carbonyl oxygen atom in the tetrahedral intermediates. In members of the chymotrypsin family, this role is played by the backbone amide groups of Gly-193 and Ser-195, as illustrated in Fig. 4. In the structures of tetrahedral transition-state analogs, each of the amide groups is positioned to form a hydrogen bond with the oxyanion (Fig. 4A). These hydrogen bonds are also observed in the structures of complexes with inhibitors such as BPTI and in the acyl-enzyme intermediate, even though the plane of the carbonyl group is rotated significantly in these structures. In the complex with cleaved BPTI, however, the pattern of hydrogen bonds is subtly changed, with the P1 carboxyl group rotated so that each of its oxygen atoms is positioned to form a hydrogen bond with one of the backbone amide groups, but not the other (Fig. 4B). This arrangement is thus able to satisfy the hydrogen-bonding potential of the backbone amide groups while interacting with the negative charge distributed over the 2 carboxyl oxygen atoms. In this respect as well, the structure of the BPTI*–trypsin complex suggests that the serine proteases have evolved to form stable interactions with the species representing each step in the mechanism, with minimal structural rearrangements.

Fig. 4.
Hydrogen bonding in the oxyanion hole of trypsin bound to a transition-state analog (A; PDB ID code 1BTZ) and cleaved BPTI (B; PDB ID code 3FP7). Amide backbone hydrogen atoms for Gly-193 and Ser-195 were added by using standard geometry. The boron atom ...

Implications for Inhibitor Mechanisms.

The structure of the BPTI*–trypsin complex also provides insights into the mechanism by which BPTI and related inhibitors resist hydrolysis. From the structures of intact inhibitors bound to proteases, it has widely been thought that the rigidity of the enzyme–inhibitor complexes effectively blocks the catalytic mechanism before the formation of the tetrahedral intermediate or acyl-enzyme. An alternative, or supplementary, model suggests that the acyl-enzyme intermediate forms readily, but that the peptide bond is more rapidly reformed, so that the intact form seen in crystal structures predominates (2629). This model is supported by experiments directly demonstrating formation of an acyl-enzyme intermediate by subtilisin and chymotrypsin inhibitor II (29) and analysis of the pH dependence of inhibitor hydrolysis rates (45).

As illustrated in Fig. 3C, the P1′ Nα atom in the cleaved inhibitor is, in fact, held in a position favorable for attack on the acyl carbonyl atom to reform the intact inhibitor. In addition, the P1 residue (Ala-16) appears to block entry of the water molecule that is required for the second step in the hydrolysis reaction. Because the position of Ala-16 is stabilized by interactions with both the enzyme and the inhibitor, it is likely that extensive disruption of one or both structures is required for the P1′ amine to be replaced by the water molecule. Thus, the the stability of the complex favors reformation of the peptide bond and disfavors hydrolysis of the acyl-enzyme intermediate. If hydrolysis does proceed, the position of the newly generated carboxyl group would also promote reversal of the reaction, as demonstrated experimentally (Fig. 1B and refs. 21, 32, and 33).

In may also be significant that the Laskowski mechanism has been found almost exclusively in inhibitors of serine proteases. Rawlings et al. (25) have identified 13 clans of Laskowski serine protease inhibitors with distinct 3D folds, indicating that the mechanism has evolved many times. At present, however, there are very few examples of inhibition of other protease types by a mechanism in which a peptide segment binds in a substrate-like manner and resists hydrolysis (25). The best-characterized example appears to be Streptomyces metalloproteinase inhibitor (SMPI) (46), but this inhibitor is much more susceptible to hydrolysis than most of the serine protease inhibitors, with a turnover time of <10 min when bound to thermolysin (46). These observations suggest that the 2-step mechanism of the serine proteases may be a key factor in the inhibition mechanism, perhaps because binding of the P1 amino group and the hydrolytic water molecule are mutually exclusive. In contrast, proteases in which the hydrolytic water directly attacks the peptide carbonyl group are likely to be less susceptible to this inhibition mechanism. However, there is no obvious reason that cysteine or threonine proteases, which act via an acyl-enzyme intermediate, could not be inhibited in this way, and such inhibitors may eventually be found.

Conclusions

The structure of the BPTI*–trypsin complex, together with structures representing other steps in the serine-protease reaction mechanism, suggests that both the forward and reverse reactions proceed with a remarkable economy of motion. A subtle rotation of the plane of the scissile bond allows the carbonyl carbon to be attacked from alternate directions in the 2 steps of the reaction, and the location of the catalytic His residue allows it to act as a proton acceptor or donor to the nucleophile or leaving group, respectively, with little or no change in position. Furthermore, the His side chain and the amide groups of the oxyanion hole appear to be positioned to provide comparable stabilization of each of the catalytic intermediates and transition states. This arrangement may help minimize energetic differences among the intermediates, thereby increasing the overall catalytic efficiency of the enzyme (47). The Laskowski-mechanism inhibitors offer an instructive counter example of this principle, in which the very high stability of the enzyme–substrate complex effectively traps the reaction in a deep local minimum.

Materials and Methods

Protein Samples.

Bovine cationic trypsin and wild-type BPTI (aprotinin) were obtained from Sigma and Roche Diagnostics, respectively. Trypsin was further purified by affinity chromatography using immobilized SBTI. Wild-type and S195A rat anionic trypsin were prepared by using a yeast expression system and purified by ion-exchange and affinity chromatography, as described by Hedstrom and colleagues (48, 49).

Preparation of Cleaved BPTI.

The 14-38 disulfide bond of BPTI was selectively reduced by incubating the native protein (at a concentration of ≈ 0.2 mM) with 4 mM DTT at 25 °C in a solution also containing 0.1 M Tris·Cl (pH 8.7), 1 mM EDTA, and 0.2 M KCl. After 1 min, the reaction was quenched by acidification with 0.025 volumes of 6 M HCl, and the selectively reduced protein was purified by RP-HPLC using a Vydac C18 column eluted with a gradient of acetonitrile in 0.1% trifluoroacetic acid. The partially reduced BPTI was then incubated with 1 molar equivalent of bovine trypsin at 25 °C in the presence of O.15 M Na-citrate (pH 3.4) and 2 mM EDTA. The reaction mixture was maintained under a nitrogen atmosphere, and progress of the hydrolysis reaction was monitored by HPLC, as illustrated in Fig. 1B. The hydrolysis reaction was typically complete after 5 days, at which time cleaved BPTI was separated from trypsin by RP-HPLC. To reform the 14-38 disulfide by reaction with molecular oxygen, the purified protein was incubated at pH 8 and room temperature for ≈48 h. The identity of the cleaved BPTI (BPTI*) was confirmed by matrix-assisted laser desorption mass spectrometry.

Crystallography.

Samples for crystallization were prepared by mixing trypsin and BPTI or BPTI* in a molar ratio of 1:1.2 and a total protein concentration of 10 mg/mL. The protein solutions also contained 10 mM CaCl2. Crystals were grown at room temperature by using the hanging-drop vapor diffusion technique. Drops contained 2 μL of the protein mixture plus 2 μL of well solution composed of 0.1 M Tris·Cl (pH 7.5–8.5), 0.2 M Li2SO4, and 16–32% polyethylene glycol-4000.

Crystals were soaked briefly in reservoir solution plus 20% (vol/vol) ethylene glycol as cryoprotectant before freezing in liquid propane at 100 K. During data collection, crystals were maintained at 100 K in a stream of evaporated nitrogen (Oxford Cryosystems). Data were collected with use of a CCD detector and a rotating anode FR591 generator (Nonius Delft).

Structures of the complexes of BPTI or BPTI* bound to rat trypsin were determined in the P3221 space group by molecular replacement using the atomic coordinates for Protein Data Bank (PDB) ID code 3TGI. The structures were refined by iterative cycles of torsion angle-simulated annealing, geometry and atom positional minimization, and restrained individual B-factor refinement using maximum-likelihood target functions implemented in the computer program CNS (50). Each cycle of refinement was interleaved with manual adjustment and rebuilding by using the program O (51) while inspecting σA-weighted 2|Fo| − |Fc|, 3|Fo| − 2|Fc|, and |Fo| − |Fc| difference maps and composite simulated-annealing omit maps. As phases improved, peaks in the |Fo| − |Fc| difference maps with >4.8-σ intensity and with appropriate geometry and disposition of H-bonding donors or acceptors were assigned to solvent molecules. Where indicated by residual positive and negative electron density peaks, certain residues were modeled with 2 or 3 alternate conformations. Diagrams representing the structures were drawn by using the computer program PyMOL (DeLano Scientific), and the align function of this program was used to superimpose the atomic coordinates of PDB ID codes 1BTZ, 2AGE, 3FP6, and 3FP7.

Supplementary Material

Supporting Information:

Acknowledgments.

We thank Dr. Lizbeth Hedstrom (Brandeis University, Waltham, MA) for sharing the yeast strains and protocols used to produce recombinant rat trypsin; Mr. Kigen Curtice for assistance in the protein preparations; Dr. W. Miachel Hanson (University of Utah Mass Spectrometry and Proteomics Core Facility) for mass spectrometry analyses; and Dr. Hedstrom and Dr. Evette Radisky for helpful discussions and comments on the manuscript. This work was supported by National Institutes of Health Grants GM42494 (to D.P.G.) and GM067994 (to M.P.H.).

Footnotes

The authors declare no conflict of interest.

This article is a PNAS Direct Submission.

Data deposition: The atomic coordinates have been deposited in the Protein Data Bank, www.pdb.org (PDB ID codes 3FP6 and 3FP7).

This article contains supporting information online at www.pnas.org/cgi/content/full/0902463106/DCSupplemental.

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