Kinetic characterization of the N-terminally derivatized peptides
The synthesis of the derivatized peptide substrates has been described (Gagnon et al. 2002). The kinetic parameters KM and kcat of guinea pig liver TGase were determined for a series of CBz-derivatized Gln-Xaa peptides as well as for the Boc-derivatized Gln-Gly dipeptide ( Table 1). The seven CBz-peptides assayed gave similar KM and kcat values, whereas the Boc-dipeptide did not appear to act as a donor substrate, showing no reactivity at concentrations up to 40 mM, the limit of its solubility. The similar KM and kcat values obtained for all the CBz-derivatized Gln-Xaa peptides indicate that the identity of the C-terminal amino acid does not significantly affect productive binding or turnover. Thus, these substrates result in a similar catalytic efficiency. Because amino acids having side chains with widely differing chemical and structural attributes were assayed and because we observed that the nature of these side chains does not affect substrate recognition or efficiency, we can conclude that substrate binding does not depend on the nature of the C-terminal amino acid. However, the nature of the N-terminal functional group, Boc or CBz, has an important effect on substrate recognition by the enzyme. Namely, Boc-Gln-Gly does not serve as a donor substrate, whereas Folk and Cole (1966) observed that CBz-Gln does act as a substrate of tissue type TGase, albeit a poor one, although Gln-Gly does not. Our kinetic data confirm that the presence of the CBz group contributes importantly to recognition of dipeptide substrates by tissue TGase, presumably by allowing better binding to the enzyme.
Modeling of substrate binding
To obtain more information regarding the mode of binding of acyl-donor substrates on tissue TGase, we attempted to model substrate binding using the coordinates of the crystal structure of tissue TGase from red sea bream that were obtained at 2.5 Å resolution. This structure, although lacking the activating calcium ion, was chosen over the crystal structure of human tissue TGase because the latter was cocrystallized with GDP and is therefore in the inactive state (Liu et al. 2002). The percentage of identity of the catalytic domains of red sea bream TGase with guinea pig liver TGase is >55%, and the percentage of homology is ∼70%. Furthermore, all of the active site residues (W236, C272, H300, W329, H332, and Y515 of red sea bream tissue TGase; Fig. 1) are conserved in all sequenced tissue TGases, justifying the use of the coordinates of fish transglutaminase for our modeling in the absence of coordinates for the guinea pig liver TGase. As a first step toward modeling the acyl-donor substrate binding on tissue TGase, we created all of the substrates given in Table 1 in silico with the BIOPOLYMER module of InsightII, and their structures were energy-minimized. All adopted a “Y”-shaped extended conformation, with the vertical line representing the Gln side chain and the two arms pointing upwards being the peptide backbone, with the CBz or Boc group on one side and on the other, the C-terminal amino acid. These conformations were used as starting structures for automated and manual docking at the active site of tissue TGase.
The crystal structure of red sea bream TGase presents a shallow, narrow cleft running diagonally on its surface, passing over the active site (Fig. 2). We identified this cleft as the possible binding site of the small peptide substrate's backbone and N-terminal functional group, as it permitted maximal Gln side chain insertion into the active site, bringing the γ-carboxamide group into close proximity with the catalytic triad. Visual inspection suggests that it would be difficult for it to be located in any significantly different manner because of steric clashes.
To test this hypothetical binding mode, we performed automated docking experiments using AutoDock 3.0 software (Morris et al. 1998). Preliminary docking experiments were performed using the PDB file 1G0D. We observed that the CBz-Gln-Gly and Boc-Gln-Gly substrates never entered the active site during the 50 runs performed. Indeed, the active site of the enzyme is visibly blocked by a Trp residue, W329 (Fig. 1) which is conserved in all tissue TGases as well as in Factor XIII (Yee et al. 1994), epidermal (Ahvazi et al. 2002), and keratinocyte TGases. This apparently results from the structure having been resolved in the absence of substrate. Because automated docking is performed without movement of the protein atoms, it is unlikely to succeed in identifying a binding mode for the substrate Gln side chain inside the active site. Thus, we manually increased the accessibility of the active site of the structure of red sea bream TGase by introducing torsions in the Cα–Cβ (−83.13°) and Cβ–Cγ (+48.52°) bonds of residue W329. These torsions opened the active site cavity while minimizing steric clashes between W329 and the rest of the protein. With this starting structure, the AutoDock procedure resulted in several structures with the substrate Gln entering the active site (Table 2). This indicates that W329 may prevent entry to the active site, acting as a ‘gate’ that could regulate TGase activity. Of the 50 runs performed with CBz-Gln-Gly, only four generated a structure where the substrate's Gln side chain had entered the active site (Table 2). This low number of hits is expected because the substrate was allowed a high degree of conformational flexibility. Three of the four bound structures positioned the CBz group at position 0; the last one positioned the CBz at position 1 (Fig. 2). With the Boc-Gln-Gly substrate, only six of the 50 runs generated a structure where the Gln side chain had entered the active site. Of these six structures, two had the Boc at position 2, three at position 0, and one at position 1. It should be noted that these positions are not precisely defined but represent restricted zones within which positioning was observed. These AutoDock results allowed us to establish three potential binding sites at the surface of the enzyme for the N-terminal functional group. Position 0, which is in the diagonal cleft, appears to be favored for binding. This suggests that positioning of the N-terminal functional group in the cleft is favorable to productive binding of the Gln side chain inside the active site cavity. The low number of runs that resulted in a structure where Gln was bound in the active site reflects the generally weak affinity of tissue TGase for these dipeptide substrates. This in turn is manifested in their millimolar KM values. We also observed that after minimization of these 10 structures using InsightII, 75% (3/4) of the CBz-Gln-Gly structures formed an H-bond with conserved residue Y515 of the active site, whereas only 16% (1/6) of the Boc-Gln-Gly structures had formed this H-bond ( Table 2).
To further test the hypothesis that the binding site of the N-terminal functional group is in the cleft defined by positions 0 and 2 and to identify the preferred position for the N-terminal functional group in the cleft, we manually docked the acyl-donor substrate at four different positions on the enzyme. First, the reacting Gln side chain was positioned inside the active site respecting the conditions described in the Materials and Methods section, while the N-terminal functional group was positioned in one of the four different positions on the surface of the enzyme (Fig. 2). These positions place the N-terminal functional group alternatively in the cleft (positions 0 and 2), in a small cavity perpendicular to the cleft (position 1), or directly on top of residue W236 (position 3). Steric clashes were more important in position 3, which was chosen as a negative control for the molecular modeling procedure. Then, steric clashes between the enzyme and the substrate were energy-minimized while the enzyme/substrate distance was constrained, as described in Materials and Methods. This caused the active site to ‘open up’ as residue W329 was displaced, indicating that this residue is very mobile, because it readily moves away from the active site after merely a minimization.
A 10-picosecond molecular dynamics simulation was then performed on the generated enzyme-substrate complexes to explore the immediate conformational space. One hundred conformers were thus generated. The conformer with the lowest energy was further energy-minimized, and the resulting structure was analyzed. This resulting structure with each substrate was always of lower energy than the structure generated before the molecular dynamics simulation. The backbone of the enzyme was maintained fixed during Trial 1 of manual docking (Table 3) to determine the most plausible binding site for the N-terminal functional group in order for the small CBz-Gln-Gly substrate to adopt a conformation that best fit the enzyme's structure and not the opposite. This experiment was also performed two more times with varying conditions, including independent manual substrate positioning, a 25 Å or 40 Å layer of water covering the active site, and with no constraint on the enzyme backbone, which generated similar results (Table 3). In all three independent trials with CBz-Gln-Gly, the enzyme-substrate interaction energy was the lowest and was consistently negative when the CBz was positioned in the upper left part of the cleft (position 0; Fig. 2), indicating that it is the only one of the four positions tested that is energetically favorable and reproducible for binding the CBz group for a small peptide substrate. Thus, it appears that the preferred position for docking the CBz group on the surface of the enzyme is located in the upper left corner of the diagonal cleft (position 0). This position also allows better insertion of the reacting Gln side chain into the active site based on the anti-conformation of the Gln side chain in the active site compared to the other three positions (data not shown). Manual docking experiments therefore gave us similar results to those obtained with automated docking, while providing additional insights into the preferred position of the N-terminal functional group. The remainder of the docking experiments were performed with manual docking of the substrates on tissue TGase.
Following the docking studies on the putative positioning of the CBz group using CBz-Gln-Gly, binding of the CBz-Gln-Xaa substrates was modeled with the N-terminal functional group in position 0. The Boc-Gln-Gly substrate was modeled as a control to ensure that the modeling results are consistent with its lack of reactivity as a substrate (Table 1). For each test, the substrate was manually positioned in an independent manner. During the calculations, only the residues including atoms within 20 Å of the catalytic Sγ atom of C272, the water molecules, and the substrate were mobile. Constraints were applied as described in Materials and Methods to retain the substrate Gln proximal to the catalytic residues, thus increasing the likelihood of modeling productive enzyme/substrate complexes. After the minimization/molecular dynamics methodology was performed, the interaction energy between the enzyme and each substrate was analyzed. For the CBz-Gln-Gly substrate, analyzed in 13 tests, we found that the interaction energy (ΔG) between ligand and protein was negative, thus favorable, in all but one case ( Table 4). In the single case where the interaction energy was positive, the electrostatic component of the interaction energy between enzyme and substrate was very high (+158.9 kcal/mole). In this case, the carboxylate of CBz-Gln-Gly was found to be in closer proximity than usual to a conserved, negatively charged residue of red sea bream TGase, Glu360, which could result in such an unfavorable electrostatic energy. Nonetheless, the average of the 13 data sets suggests that there is a favorable interaction between the enzyme and the CBz-Gln-Gly substrate. The average van der Waals energy between the enzyme and the CBz-Gln-Gly ligand was −53 ± 5 kcal/mole. While examining the interactions between the enzyme and CBz-Gln-Gly, we found that each of the 13 structures generated showed an H-bond between the Oη proton of Y515 and the Oδ of Gln from CBz-Gln-Gly. Also, 77% of the structures (10/13) displayed an H-bond between Nε of H332 and Hε of Gln side chain. The proximity and orientation displayed in this H-bond pairing resembles the interaction that takes place during catalysis when the acid/base catalyst, H332, protonates the ammonia leaving group. The high prevalence of these H-bonds suggests that they are important in binding the glutaminyl residue at the active site.
For the CBz-Gln-Xaa substrates (Xaa = Ala, Val, Leu, Phe, Ser, or Gly-Gly), we obtained similar results. Interaction energies between the enzyme and the substrates were negative and thus favorable for all substrates, independently of the side chain of the C-terminal amino acid. Also, all of these molecules formed the same H-bond between the proton of Oη of Y515 and the Oδ of Gln, as observed for CBz-Gln-Gly, whereas 86% (6/7) of them formed an H-bond between Nε of H332 and Hε of the Gln side chain. These results indicate that the nature of the second amino acid does not significantly affect efficiency of binding to tissue TGase, which correlates well with the kinetic data from Table 1.
To evaluate the contribution of the N-terminal functional group to substrate binding, we compared the binding of Boc-Gln-Gly and CBz-Gln-Gly to tissue TGase. The Boc-Gln-Gly peptide was manually docked similarly to the CBz-Gln-Gly peptide with the N-terminal functional group at position 0 or position 2. The results at position 2 were similar to those for CBz-Gln-Gly at position 2; that is, they were of considerably higher energy and were not pursued (data not shown). The binding of Boc-Gln-Gly at position 0 did not generate reproducible results as CBz-Gln-Xaa did. Indeed, some results generated poor interaction energy between enzyme and substrate, whereas others gave as good an interaction as the CBz peptides. Of the 13 trials, 38% (5/13) generated a positive interaction energy, indicating unfavorable binding. Furthermore, the H-bond between the Boc-Gln-Gly substrate and Y515 was found in only 69% (9/13) of the trials, whereas the H-bond with H332 was found in 62% of the trials (8/13), which is significantly less than for the CBz-derivatized peptides. In addition, the average van der Waals energy is −44 ± 4 kcal/mole, which is more than 10 kcal/mole higher than for the average of all CBz-derivatized peptides (CBz-Gln-Gly and CBz-Gln-Xaa). Taken together, these results suggest that the presence of the Boc group does not allow for binding that is as constant and reproducible as for the CBz-Gln-Xaa substrates. It thus appears that the Boc group is deleterious to proper binding, because it does not allow a constant productive insertion of Gln side chain into the active site.
Modeling of the tetrahedral and acyl-enzyme intermediate
To minimize any potential bias arising from the manual docking undertaken during the modeling study and to investigate more thoroughly the importance of the H-bond network with Y515 and H332 identified for the binding of the CBz-peptides, we created in silico the tetrahedral and acyl-enzyme intermediates for the reaction of tissue TGase with CBz-Gln-Gly. These intermediates have a covalent bond between the Cδ atom of Gln from the donor substrate and Sγ of the catalytic Cys residue, thus eliminating the requirement for manual docking of the substrate. The acyl-enzyme and tetrahedral intermediates were constructed respecting the χ1–χ2 and χ2–χ3 plots dihedral angles (Janin et al. 1978) for the substrate Gln and C272. Two starting structures for the acyl-enzyme intermediate were constructed, one with the CBz group at position 0 and another at position 2. These dihedral angles still respected the χ1–χ2 and χ2–χ3 plots at the end of the simulation, indicating that the generated structure was coherent with regards to the conformation of the two residues involved in the covalent bond between enzyme and substrate (data not shown). After the simulation, a lower energy for the modeled system was obtained for the acyl-enzyme intermediate with the CBz group at position 0 compared to position 2 (−4513 kcal/mole versus −4461 kcal/mole). This is consistent with the hypothesis that position 0 represents the actual CBz binding site because it is energetically more favorable.
While analyzing the covalent structures generated, we observed that the acyl portion of the molecule adopted a conformation very similar to the model of TGase with the noncovalently docked substrate. Indeed, the RMSD of the Gln side chains of CBz-Gln-Gly in the Michaelis complex and the acyl portion of the acyl-enzyme and tetrahedral intermediates are very low (Table 5), indicating near-identical positioning of the Gln side chain inside the active site for these three modeled structures (Fig. 3A–C). Also, the dihedral angles χ1 and χ2 are nearly identical among the three structures, which demonstrates that the conformation of the Gln side chain in the Michaelis complex is the same as in the tetrahedral and acyl-enzyme intermediates. This suggests that the insertion of the Gln side chain of the docked substrate is coherent and thus there was no bias in positioning during manual docking. The H-bond between the reactive Gln γ-carboxamide group and the enzyme Y515 residue was present in the acyl-enzyme intermediate. The H-bond between Nε of H332 and Hε of Gln side chain was not present because the Gln side chain in the acylenzyme intermediate no longer carries the NH2 group.
There are two H-bonds in the tetrahedral intermediate structure (Fig. 3B) that are similar to the H-bonds between CBz-Gln-Gly and the enzyme in the Michaelis complex. The first H-bond occurs between the oxyanion generated by nucleophilic attack and the hydroxyl function of Y515. The second occurs between Nε of Gln and the acidic proton of protonated H332. This is consistent with the known mechanism of the enzyme, where the leaving group NH2 is protonated by the imidazolium group of H332. The H-bond formed between the oxyanion and Y515 could significantly stabilize the tetrahedral intermediate. These results, along with the docking study of CBz- or Boc-derivatized peptides, support the importance of these H-bonds.
Structural analysis of CBz-peptide binding
The 13 structures generated by molecular dynamics following manual docking of CBz-Gln-Gly peptides and the seven structures generated with the CBz-Gln-Xaa substrates were analyzed as a group. The Gln side chain enters the active site almost perpendicularly with respect to the center of mass of the enzyme. Figure 3D illustrates one of the trial structures, chosen because it was typical and representative. The conformation of the substrate Gln side chain is the low-energy anti-conformation. The χ1 and χ2 dihedral angles of the substrate Gln are near 180°, which gives a trans-conformation to the side chain that is common for amino acids. Three aromatic residues, W236, W329, and H300 that appear to form a tunnel leading to the catalytic residues, surround the side chain. As previously discussed, residue W329 is displaced by the insertion of the substrate Gln side chain into the active site so as to open the active site that it blocks in the unbound state. This displacement places the W329 side chain in a position very similar to that achieved manually as part of the automated docking experiments (Cα − Cβ = −95.53° and Cβ − Cγ = +82.4°). The roof of the tunnel is formed by a loop of residues P356–G369 (Fig. 1). The two methylene groups of the Gln side chain of the substrate are in contact with the indole group of residue W236. This maximizes the hydrophobic interaction between the indole group of W236 and the methylene groups of the substrate Gln side chain during the course of the simulation—as evidenced by the concerted movement of these two groups during the dynamics simulation. The substrate γ-carboxamide, as mentioned earlier, adopts a conformation allowing it to form H-bonds with residues Y515 and H332. In this conformation, the nucleophilic attack by the thiolate can only be achieved from the top or the bottom of the substrate γ-carboxamide, in keeping with an addition-displacement mechanism. If the attack occurs from the top, the oxyanion generated could form a stabilizing H-bond with the hydroxyl group of Y515. Such a bond is observed in the model of the tetrahedral intermediate.