Evidences of ternary complex formation in Trypanosoma cruzi trans

TcTS substrate-induced binding site

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Evidences of ternary complex formation in Trypanosoma cruzi trans-sialidase catalysis

Isadora A. Oliveira1, Arlan S. Gonçalves2, Jorge L. Neves3, Mark von Itzstein4, and Adriane R.

Todeschini1

1Laboratório de Glicobiologia Estrutural e Funcional, Instituto de Biofísica Carlos Chagas Filho,

Universidade Federal do Rio de Janeiro, Rio de Janeiro, RJ 21941-902, Brazil.

2Instituto Federal do Espírito Santo, Campus Vila Velha, Vila Velha, ES 29106-010, Brazil.

3Departamento de Química Fundamental, Universidade Federal de Pernambuco, Recife, PE 50740-560, Brazil.

4Griffith University, Institute for Glycomics, Gold Coast Campus, Gold Coast, Queensland 4222,

Australia.

*Running title: TcTS substrate-induced binding site

To whom correspondence should be addressed: Adriane R. Todeschini, Laboratório de Glicobiologia Estrutural e Funcional, Instituto de Biofísica Carlos Chagas Filho, Universidade Federal do Rio de Janeiro. Av. Carlos Chagas Filho, 373, bloco D, sala 3, Cidade Universitária, Rio de Janeiro, Tel.: (55 21) 2562-6543; Fax: (55 21) 2280-8193; E-mail: [email protected]. Keywords: trans-sialidase; Enzyme mechanisms; Molecular dynamics; NMR; flexibility. _____________________________________________________________________________________ Background: Trypanosoma cruzi trans-sialidase (TcTS) is a potential target for Chagas disease chemotherapy. Results: The binding of a sialoside donor opens a second cavity that supports acceptor substrate binding. Conclusion: The cooperative binding of both substrates to TcTS is required for transfer reaction. Significance: This is the first time that the acceptor substrate binding site is shown in TcTS. ABSTRACT

Trypanosoma cruzi trans-sialidase (TcTS) is a key target protein for Chagas disease chemotherapy. In this study, we investigated the implications of active site flexibility on the biochemical mechanism of TcTS. Molecular dynamics studies revealed remarkable plasticity in the TcTS catalytic site, demonstrating, for the first time, how donor substrate engagement with the enzyme induces an acceptor binding site in the catalytic pocket

that was not previously captured in crystal structures. Furthermore, NMR data showed cooperative binding between donor and acceptor substrates, supporting theoretical results. In summary, our data put forward a coherent dynamic framework to understand how a glycosidase evolved its highly efficient trans-glycosidase activity.

Nearly 1% of the genome of any organism encodes glycoside hydrolases5 (GH) (1). Many of these genes have been classified and grouped into families according to their sequence similarities. Proteins from the same GH family can share the biochemical mode of action and even key residues for catalysis. Although it has been established that almost all of the classical mechanisms undergo acid/base catalysis with carboxyl-containing residues as the most frequent catalytic nucleophiles, three of the 130 GH families use a tyrosine for this role instead. This differential aspect was first described for the Trypanosoma cruzi trans-sialidase (TcTS) (2). TcTS is a

http://www.jbc.org/cgi/doi/10.1074/jbc.M112.399303The latest version is at JBC Papers in Press. Published on November 5, 2013 as Manuscript M112.399303

Copyright 2013 by The American Society for Biochemistry and Molecular Biology, Inc.

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retaining GH (3) member of the family number 33 (GH33) (4) that preferentially transfers sialic acid units to terminal β-galactopyranoside (β-Galp)-containing

molecules and synthesizes α2-3-linkages rather than acts as a hydrolase (5). TcTS activity has been involved in a vast myriad of functions in the biology of T. cruzi (6,7,8,9) and in the pathology of Chagas disease, a life-threatening illness that affects 10 million people in 21 endemic countries (10). In an effort to decipher the mechanism of TcTS catalysis, Watts et al. (11) demonstrated that fluor-based sialyl inactivators formed a covalent intermediate with the enzyme. The occurrence of such an intermediate was further observed in several enzymes of the GH33 family (12,13). Based on structural (11) and kinetic studies (14), Y342 is considered to be the catalytic nucleophile residue of TcTS (11), while D59 is proposed to act as a general acid/base catalyst in a double-displacement reaction (14) that follows a classical ping-pong mechanism (15), in which the sialosyl-aglycone may leave the active site to allow entry of an acceptor substrate. According to this hypothesis, however, a water molecule could attack the sialosyl-enzyme intermediate before the acceptor substrate reaches the binding site, which would result in hydrolysis rather than in an efficient sugar transfer. Thus, a mechanism involving a ternary complex would support the high rates for the transfer reaction, wherein the acceptor moiety is located in the acceptor binding site before the sialic acid transfer, which would displace water molecules from the catalytic pocket. Such a model would require additional conformational rearrangements in TcTS, since the crystal structure of this enzyme shows a notably narrow acceptor binding site for accommodating both the sialoside donor and β-Galp acceptor substrates simultaneously (16). Numerous works presenting evidences of plasticity in the catalytic cleft of TcTS have arisen. Molecular dynamics (MD) simulations have shown that two hydrophobic residues, Y119 and W312, play important roles in TcTS flexibility by controlling the entry of substrates into the catalytic pocket (17,18). Classical (19) and hybrid quantum mechanics/MD (20) simulations combined with mutagenesis studies (19,21) have identified other key residues that probably contribute to the

plasticity of the binding site. Moreover, experimental evidences for such plasticity arose from the observation that the non-functional variant of the trans-sialidase (TSY342H) (22) undergoes conformational changes upon sialoside binding, suggesting the opening of a second binding site that accommodates a β-Galp moiety (6). Active site rearrangement following the binding of sialoside was further proposed for the active TcTS (23). Results of TSY342H incubated with α2-6-sialyllactose in the presence of lacto-N-tetraose show that incorrect fitting of sialoside into the binding site of TSY342H does not trigger β-Galp binding, which corroborates this hypothesis (6). Additionally, surface plasmon resonance results show that lactose (Lac) binds to the inactive mutant TSD59N in the presence of α2-3-sialyllactose (3-SL) (24). The sequential entry of both substrates, leading to the formation of a ternary complex would support the catalytic efficiency of the sialic acid transfer reaction, but would contradict the crystallographic data (24). Herein, we investigated the implications of TcTS active site flexibility on its biochemical mechanism by analyzing the protein-binding dynamics of both donor and acceptor substrates on three TcTS variants – the enzymatically active form (wtTS) and two inactive mutants, TSY342H and TSD247A – using MD simulations and NMR spectroscopy studies. We demonstrated for the first time that upon 3-SL binding, wtTS and TSY342H, but not TSD247A, undergo conformational changes, opening a second cleft in the catalytic site and allowing the acceptor substrate to bind. The simulations carried out with the Lac molecule bound to the second site of wtTS and TSY342H showed a stable ternary complex with the acceptor substrate in position for a subsequent transfer reaction, which sheds light on the catalytic mechanism. Our results show remarkable structural plasticity upon engagement of acceptor substrate in the TcTS active site, a property that must be considered in future studies on the catalytic mechanism of the enzyme and in the design of new selective inhibitors. EXPERIMENTAL PROCEDURES Expression and purification of His-tagged fusion proteins – Expression and purification of wtTS,


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TSY342H, and TSD247A proteins were carried out according to Carvalho et al. (19). Briefly, a freshly transformed bacterial colony with the His-tagged protein insert was selected and inoculated in 100 ml Luria Bertani medium. Twenty milliliters from this pre-inoculum was transferred to 4 l of Terrific Broth medium and grown at 37 °C until an optical density at 600 nm of 0.8 was reached. Protein expression was induced by addition of 0.12 mM isopropyl β-D-thiogalactoside (IPTG) and the cells were incubated overnight at 28 °C. The bacterial cells were harvested by centrifugation and washed with saline solution. The pellet was lysed with 20 mM Tris-HCl pH 8.0, 0.5 M NaCl, 2% Triton X-100, 1 mM EDTA, and protease inhibitors (5 mg/ml trypsin inhibitor, 0.1 μM iodoacetamide, 1 mM PMSF, 2 μg/ml leupeptin, 30 μM E64) and sonicated. The lysed cells were cleared by centrifugation (32,816 x g, 15 min at 4 °C), and the supernatant was loaded into three 5 ml HiTrap IMAC HP columns (GE Healthcare, USA) that were charged with Ni+2 ions. Next, trans-sialidases were eluted using a stepwise imidazole gradient of up to 250 mM. Protein fractions were dialyzed against Tris-HCl buffer, loaded into a MonoS column, and eluted using a linear gradient of NaCl. Sample purity was assessed by SDS-PAGE. Proteins were concentrated using Amicon Ultra 50K NMWL (Millipore, USA) as needed. TcTS activity measurements – Sialidase activity was determined by measuring the fluorescence of 4-methyl-umbelliferone (MUone) released by the hydrolysis of 4-methyl-umbelliferyl- N-acetylneuraminic acid (MUNANA) (Sigma, St Louis, MO, USA), as described by Ribeirao et al. (25). The hydrolysis reaction was carried out in 100 µl of 20 mM phosphate buffer pH 7.4 containing variable concentrations of MUNANA (0.05, 0.1, 0.15, 0.2, 0.3, 0.4, 0.5, 1.0, 2.0, 3.0, and 4.0 mM) and TcTS proteins as follows: wtTS 5 µg/ml, TSY342H 1 mg/ml, and TSD247A 1 mg/ml. The fluorescence emission of MUone at 450 nm was monitored for 1 h at 37 °C under 365 nm excitation in a Spectramax M5e instrument (Molecular Devices, CA, USA). Kinetic parameters were determined by direct fit of the rate versus substrate concentration data to the Michaelis-Menten equation using GraphPad Prism

version 5 (GraphPad Software, San Diego, CA, USA). Specific trans-sialidase activity was measured by following the transfer of sialic acid from 3-SL (Carbosynth, Berkshire, UK) to acetyl N-[1-14C] lactosamine ([14C]LacNAc, American Radiolabeled Chemicals, St. Louis, MO, USA), as described by Ribeirao et al. (25). The reactions were performed in 50 µl of 20 mM phosphate buffer pH 7.4, 0.5 mg/ml BSA (Sigma, St Louis, MO, USA), 5 mM 3-SL, 2.5 mM N-acetyl-lactosamine (LacNAc, Sigma, St Louis, MO, USA), and 0.52 nmol (10.5 μM) of [14C]LacNAc (50 mCi/mmol, American Radiolabeled Compounds). The reactions were started by adding the enzymes wtTS, TSY342H, and TSD247A at 5 µg/ml, 2 mg/ml, and 1 mg/ml, respectively. The reaction volumes were incubated for 10 min at room temperature and stopped by the addition of 1 ml of deionized water. The sialylated [14C]LacNAc was purified by chromatography in 0.5 ml of Dowex 1x4 (Sigma, St Louis, MO, USA) in acetate form, equilibrated with 20 ml of deionized water. Unreacted [14C]LacNAc was washed out by 20 ml of deionized water. Bound sialylated [14C]LacNAc was eluted with 3 ml of 1 M ammonium acetate and quantified by liquid scintillation. The specific activities of each enzyme were determined as the rate of reaction product by time and protein concentration. The significance was assessed by one-way analysis of variance (ANOVA) using GraphPad Prism 5. System setup for molecular dynamics – The structure of wtTS was modeled according to previous work (19) because there are nine missing residues (DPAASSSER) between amino acids A399 and G409 in the 1MS3 structure (24). The selected model was validated by a Ramachandran plot obtained through the PROCHECK server (26). Mutations on TSY342H and TSD247A were made using Swiss-PDB-Viewer (27) on the modeled wtTS to generate the TSY342H and TSD247A variants. The coordinates of ligands 3-SL and Lac were extracted from Protein Data Bank (PDB) code 1S0I. The structure geometries of both oligosaccharides were optimized using a Monte Carlo algorithm in GHEMICAL (28). The lower energy structures of the ligands were


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semi-empirically optimized using the RM1 Hamiltonian (29) in the MOPAC 2009 software (30). Charges were calculated by GAMES-US, utilizing the base 6-31G* and the RESP.pl perl script (http://q4md-forcefieldtools.org/RED/). Finally, the molecules were parameterized in the OPLS/AA force field (31),(32) with MKTOP software (33). Molecular dynamics simulations – Five systems were prepared: wtTS with 3-SL, wtTS with 3-SL and Lac, TSD247A with 3-SL, TSY342H with 3-SL, and TSY342H with 3-SL and Lac. The coordinates of systems with 3-SL were obtained by overlapping the constructed model and the crystal structure from PDB code 1S0I in which the TcTS mutant D59A was co-crystallized with 3-SL. For systems containing Lac, the coordinates of this molecule were obtained using molecular docking (as described in the next section). All systems were analyzed with the PROPKA server (34) in order to find the local protonation state of all residues at pH 7.4. The MD simulations were run by the GROMACS 4.0.3 computational package (35,36) using OPLS/AA force field. Systems were simulated with NPT ensemble to keep the temperature and pressure constants at 310 K and 1 bar, respectively. Proteins with or without the ligands were immersed in dodecahedrical boxes with periodic boundary conditions and filled by TIP3P water molecules and chloride anions for neutralization of the systems’ total charge. All systems were energy-minimized using a steepest-descent algorithm with and without position restraint, followed by conjugate gradient and quasi-Newton methods until a force of less than 41.84 kJ mol-1 nm-1 was reached. The systems were equilibrated for 500 ps with position-restrained MD for heavy atoms, which was followed by 20 ns of free MD. We also used particle mesh Ewald (PME) method, 6-12 Lennard-Jones potential with a 1 nm cutoff and the LINCS algorithm in the MD steps. Molecular Docking – Lac was docked on the protein+3-SL complex. For the protein, Gasteiger charges were calculated using AutoDock Tools (37), and for 3-SL, RESP charges were calculated using the RESP.pl script downloaded from the

R.E.D. III server (http://q4md-forcefieldtools.org/RED/). Coordinates with respective charges were added to the docking input containing the coordinates of the protein. We utilized a grid of 26 nm x 36 nm x 36 nm (XYZ), which was centered at the active site. One hundred possible conformations were calculated by AutoDock Vina (38), and the best ones were selected by considering the docking energy, the number of conformers in each cluster, and the distance from and location of key residues for interaction in the pocket. Diffusion Ordered Spectroscopy (DOSY) – 3-SL, methyl-S-(5-acetamide-3,5-dideoxy-d-glycero-β-d-galacto-2-nonulopyranosil-ate-(2,3)-β-galactopyranosidic acid (NeuSGal), methyl 4-O-β-D-galactopyranosyl-β-D-glucopyranoside (Me-Lac), lacto-N-neotetraose (LacNTet), and BSA were dissolved in deuterated PBS pH 7.5 (not corrected for isotope effects). Solutions of wtTS, TSY342H, and TSD247A in 20 mM Tris-HCl were exchanged with deuterated PBS using four cycles of concentration with Amicon Ultra 50K NMWL (Millipore, USA) and diluted in deuterated PBS. One hundred microliters of 0.1 mM protein solution (TcTSs or BSA) were titrated with the sialoside (0.2, 0.4, 0.6, 0.8, 1.0, 1.2, 1.6, and 2.0 mM) or LacNTet (0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, and 0.9 mM) as negative control. The solution containing 0.1 mM protein plus sialoside was further titrated with Me-Lac (0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, and 0.9 mM) in Shigemi tubes. NMR spectra were obtained at a probe temperature of 293 K on a Bruker DMX 600 MHz equipped with a 5 mm self-shielded gradient triple resonance probe. All DOSY experiments were performed with a stimulated echo using bipolar gradient pulses for diffusion, one spoil gradient, and the 3-9-19 pulse sequence with gradients for water suppression, according to the Bruker pulse program stebpgp1s19. The gradient duration was set to δ = 10 ms (protein-only experiments) and 20 ms (protein-carbohydrate complexes experiments), and the diffusion time was set to Δ = 40 ms. Gradient strength was linearly varied in 16 steps from 0.6 G/cm to 6.7 G/cm. For each of the 16 gradient amplitudes, eight transients of 192 complex data points were acquired to a 10.0 ppm spectral width. The pulse repetition delay between


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each scan was 3 s. The data acquisition and analysis were performed using spectrometer software Topspin 3.2 (Bruker Corporation) and GraphPad Prism 5. Isotopic protein labeling – A freshly transformed bacterial colony with the His-tagged wtTS insert was grown at 37 °C in 15N-2H-enriched M9 minimal media to obtain 15N-2H-labeled protein samples, according to Tugarinog et al. (39). Protein expression was induced with 1.0 mM IPTG for 12 h. After harvesting, cells were resuspended in buffer containing 50 mM Tris-HCl and 0.5 mM NaCl pH 8.0, and disrupted by sonication. Following centrifugation at 5000 x g for 10 min, isotopically labeled protein was purified and concentrated as described for unlabeled TcTSs.

The 2H-15N-labeled wtTS sample was dissolved in 20 mM phosphate buffer pH 7.5, prepared with 2H2O, providing a final protein concentration of approximately 200 µM. NMR spectra of isotopically labeled samples with or without 2 mM NeuSGal, or with 2 mM NeuSGal and 2 mM Me-Lac were recorded at 303 K on a Bruker DMX 600 MHz spectrometer equipped with cryo-probe 5 mm CPTCI 1H. All 1H-15N heteronuclear single quantum coherence (HSQC) spectra were recorded by setting up a resolution of 1024 × 128 points and spectral window of 10.822 kHz and 3.040 kHz for 1H and 15N, respectively, according to the trosyetf3gpsi pulse sequence. All NMR data were processed and analyzed using Topspin 3.2. RESULTS Characterization of enzyme kinetic parameters – To assess the extent of enzyme activity loss due to the mutations, we tested the enzyme variants for both hydrolysis and trans-sialidase activities. Sialidase activity was assayed by continuous fluorimetric detection of MUone, the product of hydrolysis of MUNANA (25). TSD247A and TSY342H showed higher Km than wtTS (Table 1), suggesting that the mutant proteins have lower sialic acid affinity than wtTS. The turnover constants and catalytic efficiencies of mutant proteins were found to be more than 300 times lower than wtTS (Table 1), supporting their residual hydrolytic activity.

The sialic acid transfer was assessed as described by Ribeirao et al. (25). TSY342H and TSD247A were close to inactivity, with specific activity 13,000 and 240 times lower than wtTS, respectively (Fig. 1). Using this method, we did not observe detectable reaction product in a continuous manner for TSY342H and TSD247A, in contrast to the high activity displayed by wtTS. In order to comprehend the molecular basis of the loss of activity of the mutated enzymes, we performed MD simulations of wtTS, TSY342H, and TSD247A complexed to 3-SL. Y342H mutation affects the structure of protein-carbohydrate complex – Given that histidine is a residue frequently found in reaction mechanisms, functioning either as a nucleophile (40) or as a basic catalyst (41,42), the inactivation of enzymatic activity due to the Y342H mutation was unexpected, although extensively reported (43-46). Throughout the MD simulation, H342 was observed to internalize into the active site, losing interaction with polar residues, such as R35, E230, and S285 (Fig. 2A) and approaching nonpolar residues like L36 and A179 (Fig. 2B). The novel interaction network of H342 prevented it from binding to sialic acid of 3-SL, the distance from which increased from 5 up to 8 Å during the simulation (Fig. 2C). The inner position of H342 placed it apart from D59, with the distance reaching 11 Å at the end of the simulation (Fig. 2D). Such a gap would impair both TcTS activities, since the supposed nucleophile could not reach the donor substrate. Besides, the position adopted by H342 also contributed to its reduced solvation in contrast to that observed for Y342 within the wtTS+3-SL complex. Internalization of the Y342 residue on the TSY342H+3-SL complex had an impact on the desolvation of the whole catalytic cleft, as demonstrated by its accessible surface area (Fig. 2E), and specially on D59, which can be observed in the water radial distribution function (RDF) relative to this residue (Fig. 2F). The difficulty of D59 in accessing the solvent hampered its ability to act as an acid/basic catalyst, preventing this residue from activating water molecules to attack the sialyl-enzyme intermediate, which provides an explanation for the inactivity of this mutated protein.


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How a silent residue becomes meaningful: influence of D247A mutation in protein-carbohydrate dynamics – Enzyme kinetics data raised the question of how the D247A mutation can be important enough to promote such a loss of catalytic activities observed in TSD247A. In wtTS, it was observed that D247 interacted directly with the glycerol motif of 3-SL, particularly with the hydroxyl from C9 of sialic acid. The distance between these two groups was stable at approximately 2.2 Å (Fig. 3A) and favored hydrogen bond formation (Fig. 3B). The D247A mutation, however, disrupted these interactions (Fig. 3A), since it altered both the side chain length and the charge of the residue. Therefore, in TSD247A, the sialic acid glycerol moiety was separate from A247, which resulted in the complete absence of hydrogen bonds between them (Fig. 3B). Another series of remarkable residues influenced by the D247A mutation is the arginine triad, which is composed of R35, R245, and R314. These residues are responsible for fixing the sialoside carboxyl group in the proper position for catalysis (24). Analysis of the wtTS and TSD247A MD simulations showed important differences in the network of interactions (Figs. 3C and 3D). In the wtTS+3-SL system, the carboxyl motif of 3-SL was close to R245 (Fig. 3C), while it shifted towards R35 in the complex containing TSD247A (Fig. 3D), which suggests that the 3-SL was not correctly positioned for catalysis. The D247A mutation also disrupted the hydrogen bond formed between the guanidine motif of R245 and the D247 carboxyl group observed in wtTS (Figs. 3E and 3F). Meanwhile, loss of the charge of D247 favored hydrogen bond formation between R245 and another nearby acid residue, E230 (Figs. 3G and 3H). This feature may reduce the basic character of E230, which, in turn, could reduce the nucleophilic character of Y342. We also observed that the D247A mutation altered the solvation of important residues in the catalytic cleft. In particular, the RDF of solvent relative to D59 suggests that this residue was less solvated in the complex containing the mutant TSD247A than in wtTS+3-SL (data not shown). Such limited contact of this aspartate with water molecules would prevent their activation, a

required step for the occurrence of the hydrolysis reaction. Moreover, the D247A mutation impaired the interaction between Y248 and W312 observed in the wtTS+3-SL simulation (Figs. 4A and 4B). In the TSD247A complex, Y248 moved 5 Å away from W312 (Fig. 4C), which increases W312 motility, as shown by its root mean square deviation (RMSD, Fig. 4D). In TSD247A+3-SL MD, Y248 shifted away from the catalytic site, whereas in the wtTS+3-SL system, the interaction between Y248 and W312 moved this last residue away from Y119 (Fig. 4E) and caused Y119 to be closer to 3-SL (Fig. 4B). Surprises of protein-carbohydrate interaction: a new cavity revealed – W312 movement also contributed to the formation of a second cavity in the wtTS+3-SL complex, which was not present in the TSD247A+3-SL system. By the trajectory analysis of the MD simulations, we identified pronounced structural differences among wtTS, TSY342H, TSD247A, and the crystal structure code 1S0I. Many residues from the wtTS and TSY342H complexes were shifted away from 3-SL, which favored the opening of a second cavity adjacent to the donor substrate (Figs. 5A and 5B, respectively) not observed in crystal structure (Fig. 5C). The Y119 movement is in agreement with previous works (14,24). Furthermore, the D59 residue of wtTS and TSY342H underwent a great conformational change, being shifted 7 Å away from the 3-SL glycosidic bond (Fig. 5E). The new position of D59 in wtTS+3-SL and TSY342H+3-SL restrained its role as a basic catalyst for both enzyme reactions. Interestingly, the additional cleft was not observed in the TSD247A MD (Fig. 5D), where D59 was close to the donor substrate (Fig. 5E). The second cleft of wtTS was previously suggested (25,47), since binding of the acceptor substrate was only achieved when the donor substrate was present (23,24). Such an argument is strengthened by the observation that the inactive mutant TSD247A did not display the second cleft during the MD simulations, which suggests that binding of the acceptor substrate is imperative for the trans-sialidase reaction to take place.


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Does the second site in wtTS and TSY342H support acceptor substrate binding? – The second cleft observed in wtTS and TSY342H complexed with 3-SL was 8 Å x 12 Å x 13 Å, which should be large and deep enough to lodge the acceptor substrate. To address this hypothesis, we docked a Lac molecule into the new site of wtTS+3-SL and TSY342H+3-SL systems and performed MD simulations on the ternary complex. During the simulation, we perceived that the Lac remained stable in the second cleft, which favored stabilization of 3-SL in a more likely position for catalysis, as observed from its RMSD in the ternary complex compared with the binary complex (Fig. 6A). Also, we observed that the RMSD of 3-SL was larger and more unstable in the binary system than in the ternary system (2 Å on average). The same pattern was shown by the binary and ternary complexes formed by TSY342H and its ligands; however, 3-SL achieved notable stabilization just later in the MD simulation time (Fig. 6B). Other residues were in a more favorable situation for the transfer reaction in the ternary system than in the binary one, such as D59. The carboxyl group of D59 moved toward the Lac 3-OH, which favored its proton abstraction. Lac binding also reduced D59 solvation in the wtTS ternary complex (Fig. 6C), which suggests that entry of the acceptor substrate displaced water molecules from the catalytic site and supported the high rates of transfer reaction exhibited by wtTS. It is noteworthy that W312 had a remarkable shift during MD of the wtTS ternary complex. Initially close to the sialoside galactosyl ring (Fig. 7A), W312 disrupted the interaction with donor substrate and moved toward the acceptor substrate (Fig. 7B). These changes were evident in the analysis of distances of W312 Cζ2 and β-Galp C1 from 3-SL, and of W312 and D-glucopyranoside (Glc) C4 from Lac (Fig. 7C). Besides, the movement of W312 resulted in a radical alteration in the dihedral angle formed by carbons α and β (Fig. 7D). Experimental binding of TcTSs to donor and acceptor substrates by NMR – The results obtained so far suggest that sialoside binding induces a conformational change in the architecture of the wtTS active site in order to

enable the binding of acceptor substrate. The same cooperative binding profile was observed for TSY342H. For the inactive variant TSD247A, however, the opening of a second cavity was not observed. Since this protein has all catalytic residues intact, its inactivity may be due to the inability of the acceptor substrate to bind to the catalytic cleft in the presence of sialoside. To support the MD findings, we performed binding experiments using DOSY. DOSY-NMR experiments provide the observed diffusion constants (Dobs) of the ligands, which represent a sum among its molar fractions bound to the protein or not. By the conversion of the molar fractions to molar concentrations, it is possible to calculate the dissociation constants, Kds, as a measure of the binding affinity of the protein-ligand complex (48). Since TSD247A and TSY342H showed only residual sialidase activity, even a hydrolysable donor substrate such as 3-SL would be adequate for our binding studies. Nevertheless, binding studies with wtTS required a donor substrate resistant to enzymatic hydrolysis. It is known that some sialidases are not able to cleave thiosialosides (49). Thus, we explored the resistance of the thiosialoside NeuSGal (Fig. 8A) to wtTS hydrolysis by monitoring the reaction using one-dimensional (1D) 1H NMR. After 24 h of incubation (Figs. 8B and 8C), the resonances corresponding to H3eq and H3ax signals of the α- and β-anomers of free sialic acid at 2.72 and 1.62 ppm and 2.21 and 1.84 ppm, respectively (3), were not detectable. Neither was the signal corresponding to H1 of free methyl-galactoside at 4.4 ppm. These data demonstrated that NeuSGal was a suitable substrate for DOSY assays with wtTS. Our data showed a regular binding profile for the wtTS+NeuSGal complex, without ligand saturation even at the higher dose tested (Fig. 9A). NeuSGal displayed a Kd of 0.456 mM for wtTS binding (Table 2) and notably, Me-Lac was able to bind to wtTS only in the presence of NeuSGal, with a Kd of 0.015 mM (Table 2). During Me-Lac titration, Dobs values from sialoside did not change significantly, suggesting that there was no displacement of sialoside from the active site by Me-Lac. The same binding pattern was observed for the complexes formed among TSY342H and its ligands (Fig. 9B), with Kds values of 0.234 mM


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and 0.052 mM for 3-SL and Me-Lac (Table 2), respectively. When bound to TSD247A, 3-SL displayed a Kd of 0.107 mM (Table 2), lower than that observed when complexed to TSY342H. However, in contrast to TSY342H and wtTS complexes, Me-Lac did not bind considerably to TSD247A in the presence of sialoside (Fig. 9C). In the presence of BSA, used as negative control, Me-Lac presented a Dobs of 2.82x10-10 m²/s, similar to that for the titration of Me-Lac to the binary TSD247A complex. These results support the MD data showing that lactoside does not bind to the TSD247A+3-SL complex. Together, DOSY and MD simulation data show a differential cooperativity among ligands and TcTS variants, supporting the existence of a ternary complex during wtTS catalysis. How wtTS notices oligosaccharides – To investigate whether wtTS was able to alter its active site conformations upon ligand binding, we use bidimensional (2D) 1H-15N HSQC spectra in NMR. Such a strategy is capable of identifying subtle conformational changes of proteins upon ligand binding (50,51), even without chemical shift assignments (52,53). By monitoring the conformational changes of wtTS upon NeuSGal binding, we observed that several chemical shifts of amide groups arising from wtTS were perturbed in the presence of NeuSGal. When Me-Lac was added to the mixture, significant spectral changes were observed (Fig. 10A). Moreover, additional chemical shift perturbations were perceived compared with wtTS in the presence of sialoside only. The addition of Me-Lac altered the chemical shifts of many amide groups already perturbed by sialoside binding in all regions of 2D spectrum (Fig. 10B and 10C), notably in the tryptophan side chain region (Fig. 10D), suggesting that lactoside binds close to the sialoside binding site. DISCUSSION

Once restricted to Latin America, Chagas disease, which is caused by the parasite T. cruzi, is a worldwide challenge that has now spread to North America, Europe, and the western Pacific region (54). Despite this disease’s health and economic costs, its chemotherapeutic treatment is still notably unsatisfactory (55), and designing

new drugs is an urgent matter. The key roles of TcTS in the pathogenesis combined with its absence in mammalian hosts have suggested TcTS as a promising target for preventing the invasion of host cells by the parasite. Both the trans-sialidase and sialidase reactions of TcTS have interested researchers all over the world, since it has been proven that T. cruzi incorporates sialic acid through a mechanism different from mammalian sialyltransferases (56), which catalyzes the sialylation with CMP-activated sialic acid substrates. However, almost 30 years of research has not been sufficient to unveil this enzyme’s reaction mechanism completely. In this study, NMR spectroscopy and molecular modeling were applied to the trans-sialidase theme to elucidate the molecular basis of its reaction mechanism. The analysis of MD simulations has shown that upon 3-SL binding, wtTS and TSY342H undergo conformational changes that open a second cleft in the catalytic site adjacent to the sialoside binding cleft. Such a cavity was not observed in TSD247A, which kept the sialoside closely bound to the active site. These data are in agreement with the DOSY results that showed that Me-Lac was not able to bind to TSD247A, even in the presence of 3-SL. Docking of Lac into wtTS+3-SL and TSY342H+3-SL systems was further followed by MD. From the solvated MD analysis of the ternary system, we perceived that Lac remained stable in the second cleft by placing the D59 carboxyl group in the proper position for proton abstraction of the 3-OH in Lac, which favored the transfer reaction. Moreover, lactose binding reduced solvent exposure of the catalytic site. These results do not support the suggested role of D59 as an acid catalyst because in the ternary system, this residue is too distant from the sialoside to protonate its aglycone. Such speculation is strengthened by the low theoretical pKa presented by D59, which was already suggested by other groups (57,58). Whereas Amaya et al. (14) have argued that the D59 pKa tends to be higher due to its proximity to Y119, we observed that these two residues were only in close proximity when the acceptor substrate was present in the cleft. This finding strongly suggests that D59 acts as a basic catalyst only. In contrast, the D96 residue has a


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theoretical pKa high enough to act as an acid catalyst. This residue is highly conserved among viral and bacterial sialidases and acts as the acid/base catalyst in these enzymes (13,59). D96 was also considered to be a potential catalyst in the work of Buschiazzo et al. (16) and was cited together with Y342 and E230 as the conserved polar residues of the catalytic triad. However, D96 interacted with the glycerol portion of 2,3-didehydro-2-deoxy-N-acetylneuraminic acid (Neu5NAc2en) in the crystal structure of TcTS (24), while in a crystal structure released in 2004, a TcTS mutated at D59A and soaked with 3-SL revealed an interaction between the D96 and the N-acetyl moiety of the sialoside (14). Despite the position of D96, the TcTS crystals did not favor its role as an acid catalyst and there is still no consensus on its role. One hypothesis is that before the nucleophilic attack, the sialoside changes its ring conformation and adopts a configuration favorable for interaction with D96, a position that was never captured in crystals. The residue W312 orchestrates the conformational change in the wtTS catalytic site after the binding of 3-SL. Other works have already attributed a role to this tryptophan as a gateway that provides the opening/closure motion of the TcTS binding cleft (17,19). When the acceptor substrate is bound, however, the W312 moves far away from the sialoside toward the acceptor, which can place it in the best position for catalysis following its ejection after the reaction takes place, and corroborates the lever/shovel function reported by Mitchell et al. (18). The interaction observed in MD between β-Galp rings from both substrates is in line with the experimental results of Todeschini et al. (60), which showed transference of saturation among the protein and the H1, H3, and H4 from the donor β-Galp and the H1 and H3 protons from the acceptor β-Galp using Saturation Transfer Difference (STD)-Total Correlation Spectroscopy (TOCSY), same protons observed in this study to interact with W312. This ability of the tryptophans to interact closely with the Gal rings has been widely observed (61-64). Such an interaction is suggested to be stacked CH/π, based on quantum calculations (65). Taken together, these data contribute to unveiling the role of W312 in wtTS

catalysis, whose importance was observed previously in the inactive TSW312A mutant (21).

Comparison of the structure of wtTS with the structure of TSY342H clarified the internalization of H342 into the active site, toward hydrophobic residues. The new chemical environment experienced by this residue hampers the access of water molecules to the sialoside, impairing the hydrolysis reaction. This result provides an explanation for the reduction of the hydrolytic activity of TSY342H and is consistent with the residual sialidase activity demonstrated here and by other groups (66). The great distance between H342 and E230 impedes proton abstraction from the imidazole ring (H342). Thus, although histidine would be a better nucleophile than tyrosine, since it would have a negative ionic form stabilized by resonance, such a form becomes unlikely to occur as there are no basic residues close by that can act as a base. These data help to understand how the Y342H mutation can affect the catalytic activity of wtTS.

The effect of the D247A mutation on wtTS activity was demonstrated previously (19) and the enzyme kinetics were detailed here. From our MD data, the role of the D247 residue for the active site architecture can be dimensioned. This residue establishes hydrogen bonds with D247 and E230 while holding R245 apart from E230. Hydrogen bond formation between R245 and E230 would reduce the acidic character of this glutamate and hamper proton abstraction from Y342, which is a crucial step of the trans-sialidase reaction. Furthermore, the D247 residue itself is located in a narrow cleft, establishing hydrogen bonds with the 3-SL glycerol motif and assisting the orientation of sialoside into position for catalysis. The glycerol portion of sialoside was observed to be a relevant group for ligand binding, since STD-NMR studies using sialoside derivatives showed that the sequential removal of hydroxyl groups reduced the binding of sialoside probes to wtTS (67). Buchini and coworkers (68) have observed that the cleft where the glycerol motif is located is larger and more hydrophobic than the equivalent from human cytoplasmic neuraminidase. These researchers have also demonstrated that the incorporation of bulky and hydrophobic substituents into C9 of sialic acid analogs, such as benzoyl and umbelliferyl


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increases their specificity for TcTS against a human neuraminidase. These data are in line with ours, and highlight the importance of D247 for the structure of wtTS and its interaction with ligands, which suggests that this residue has a direct role in trans-sialidase specificity and should be considered for the design of new drugs targeted to this enzyme. Using DOSY-NMR spectroscopy, we demonstrated that TSY342H and wtTS, but not TSD247A, interacted with Me-Lac only after sialoside binding, which supports our MD data and previous work (6). Furthermore, the conformational switch triggered by sialic acid donor binding in wtTS was indeed confirmed by the differences in chemical shifts observed in 1H-15N HSQC spectra. Surface plasmon resonance results obtained by Buschiazzo et al. (24) showed that Lac bound to an inactive TSD59N mutated enzyme in the presence of 3-SL, which is consistent with our theory. However, the authors were unable to show the existence of two distinct binding sites when monoclinic crystals of the active wtTS were soaked in Lac and Neu5NAc2en, which indicated that the Lac binding site was too narrow to accommodate the Lac moiety of the donor and acceptor substrates simultaneously. The results of our work demonstrate that additional conformation rearrangements occur in the TSY342H and wtTS such that they can bind the acceptor substrate. In previous results using inactive TSY342H incubated with α2,6-sialyllactose in the presence of LacNTet, we showed that incorrect fitting of sialoside into the binding site of wtTS did not trigger β-Galp binding, which corroborates our double induced site hypothesis. These data suggest that the interaction of Neu5NAc2en with TcTS demonstrated by Buschiazzo et al. (24) is not sufficient to induce the required conformational rearrangement to accommodate acceptor and donor substrates simultaneously. Conformational fluctuations at the catalytic cleft of TcTS have previously been suggested by our group and others (17-20,69). Indeed, MD simulations have led to a better understanding of the importance of protein

flexibility in the catalytic mechanism of several enzymes (70) and were recently employed together with NMR to demonstrate the opening of two sialoside binding sites in influenza virus neuraminidase (71). The structural data of TcTS reported in this study reveal a remarkable alteration in the wtTS catalytic site, showing for the first time an acceptor substrate binding site in TcTS, which has been suggested to exist by many groups but has not been captured in crystal structures to date. Our data support the formation of a ternary complex during the trans-sialidase reaction since acceptor binding was shown to displace water molecules from the active site by adjusting the position of the sialyl donor in the cleft to favor the transfer. These data support the prevalence of trans-glycosylation over hydrolysis. Our data suggest that trans-sialylation by TcTS follows neither a classical sequential nor a ping-pong mechanism, but a hybrid of these two systems in which the enzyme intermediate is formed after the ternary complex (72). Many examples of enzymes that do not fall into the strict classification of sequential or ping-pong mechanisms but lie somewhere in between these two systems have been reported (73,74). Furthermore, a hybrid ping-pong sequential mechanism fits all of the data published to date (11,15,25,72,75).

In conclusion, this work showed, for the first time, the acceptor substrate binding site in TcTS, which has been suggested to exist by many groups but has never had its structure revealed. Our experimental and structural results strongly support the formation of a ternary complex for the trans-sialidase reaction of this enzyme. Taken together, our results put forward a coherent dynamic framework to understand how glycosidase evolved to become a highly efficient trans-glycosidase and suggest that the structure of wtTS with two cavities would be a better model for rational drug design addressed to trans-sialidase.


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Acknowledgements – We thank Professor Fabio Almeida for his essential help with ideas for the DOSY experiments and Centro Nacional de Ressonância Magnética Nuclear (Rio de Janeiro, Brazil) for the use of their NMR facilities. FOOTNOTES *This work was supported by grants from Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq), Fundação Carlos Chagas Filho de Amparo à Pesquisa do Estado do Rio de Janeiro (FAPERJ), Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES), and the National Institute for Science and Technology in Vaccines (INCT-V). ¹To whom correspondence should be addressed: Laboratório de Glicobiologia Estrutural e Funcional, Instituto de Biofísica Carlos Chagas Filho, Universidade Federal do Rio de Janeiro. Av. Carlos Chagas Filho, 373, bloco D, sala 3, Cidade Universitária, Rio de Janeiro, Tel.: (55 21) 2562-6543; Fax: (55 21) 2280-8193; E-mail: [email protected]. 2Instituto Federal do Espírito Santo, Campus Vila Velha, Vila Velha, ES 29106-010, Brazil.

3Departamento de Química Fundamental, Universidade Federal de Pernambuco, Recife, PE 50740-560, Brazil. 4Griffith University, Institute for Glycomics, Gold Coast Campus, Gold Coast, Queensland 4222, Australia. 5The abbreviations used are: [14C]LacNAc, acetyl N-[1-14C] lactosamine; 1D, one-dimensional; 2D, bidimensional; 3-SL, α2,3-sialyllactose; ANOVA, analysis of variance; Dobs, observed diffusion constants; DOSY, Diffusion Ordered Spectroscopy; GH, glycoside hydrolase; Glc, D-glucopyranoside; HSQC, heteronuclear single quantum coherence; IPTG, isopropyl β-D-thiogalactoside; Kd, dissociation constant; Lac, lactose; LacNAc, N-acetyl-lactosamine; LacNTet, lacto-N-neotetratose; MD, molecular dynamics; Me-Lac, methyl 4-O-β-D-galactopyranosyl-β-D-glucopyranoside; MUNANA, 2’-(4-methyl-umbelliferyl)-α-D-N-neuraminic acid; MUone, 4-methyl-umbelliferone; Neu5NAc2en, 2,3-didehydro-2-deoxy-N-acetylneuraminic acid; NeuSGal, methyl-S-(5-acetamide-3,5-dideoxy- d-glycero-β-d-galacto-2-nonulo-pyranosilate-(2,3)-β-galactopyranosidic acid; PDB, Protein Data Bank;


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PME, particle mesh Ewald; RDF, radial distribution function; RMSD, root mean square deviation; SAS, solvent accessible surface; STD, Saturation Transfer Difference; TcTS, Trypanosoma cruzi trans-sialidase; TOCSY, Total Correlation Spectroscopy; TSD247A, TcTS D247A mutated; TSY342H, TcTS Y342H mutated; wtTS, wild type trans-sialidase; β-Galp, β-galactopyranoside. FIGURE LEGENDS FIGURE 1. trans-Sialidase activity of TcTS variants. Sialic acid transfer reaction was performed according to Ribeirao et al. (25). The sialylated [14C]LacNAc was quantified after 10 min incubation of donor and acceptor substrates with wtTS (blue bar), TSY342H (red bar), and TSD247A (green bar). The formed product was normalized by protein amount. FIGURE 2. Alterations in hydrophobicity in the catalytic cleft of TSY342H. Initial (A) and final (B) frames of MD simulations of TSY342H+3-SL system. 3-SL and H342 (center of images) are represented by sticks colored in cyan; the residues of the cleft at 3 Å away from H342 are represented in lines and balls, colored according to polar character (nonpolar residues in white, polar in green, negatively charged in red, and positively charged in blue). Distances of imidazole ring from H342 to 3-SL (C) or to D59 (D) throughout the simulation time. E. Solvent accessible surface (SAS) of residues from the active site during MD. F. Radial distribution function of water molecules relative to D59 from wtTS and TSY342H binary systems. Data relative to the wtTS+3-SL system are represented by blue lines; those relative to the TSY342H+3-SL system are represented by red lines. FIGURE 3. The D247A mutation alters the interaction among sialoside and residues of the catalytic cleft. Distance (A) and number of hydrogen bonds (B) between the glycerol portion of sialoside and carboxyl group of D247 (wtTS, blue) or methyl group of A247 (TSD247A, green) during MD simulation time course of wtTS and TSD247A binary systems. The net of interactions formed among the arginine triad (R35, R245, R314, sticks colored in green), D/A247 (sticks colored in magenta), E230 (sticks colored in yellow), and 3-SL (sticks colored in cyan) on final frames of MD simulations of binary complexes with wtTS (C) and TSD247A (D). Distance (E) and hydrogen bond formation (F) between guanidine group of R245 and carboxyl group of D247 (wtTS, blue) or methyl group of A247 (TSD247A, green). Distance (G) and hydrogen bond formation (H) between guanidine group of R245 and carboxyl group of E230 on binary systems with wtTS (blue) and TSD247A (green). FIGURE 4. Differential flexibility of W312 caused by D247A mutation. Final frames of MD simulations of binary systems composed of 3-SL and wtTS (A) and TSD247A (B). C. Distance between hydroxyl group from Y248 and indole ring of W312 during the course of simulation. D. Root mean square deviation (RMSD) of W312 relative to its initial position during MD. E. Distance between hydroxyl group from Y119 and indole ring of W312 throughout the simulation. Data related to binary system wtTS+3-SL are represented by blue lines; data related to the TSD247A+3-SL system are represented by green lines. FIGURE 5. Structural alterations caused by mutations: opening of a second cleft. A. Final frame of MD simulation of binary complex wtTS+3-SL. B. Final frame of MD simulation of binary complex TSY342H+3-SL. C. Crystal structure of TSD59A with 3-SL from PDB ID 1S0I. D. Final frame of MD simulation of binary complex TSD247A+3-SL. The proteins are represented in van der Waals spheres colored in gray and the 3-SL molecules are represented by sticks colored in cyan. Residues from the catalytic cleft are represented by mesh colored as follows: catalytic triad (E230, Y/H342, D/A59) in yellow, Y119 and W312 in orange, D/A247 in magenta, and Y248 in red. E. Distance between C2 of sialic acid from 3-SL and carboxyl group of D59 during MD simulation time course of wtTS (blue), TSY342H (red) and TSD247A (red) binary systems.


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FIGURE 6. Acceptor substrate stabilizes 3-SL into the active site. Root mean square deviation (RMSD) of 3-SL relative to protein coordinates on binary (light lines) and ternary (dark lines) complexes formed by 3-SL, lactose and wtTS (A, blue lines) or TSY342H (B, red lines) during MD. C, Radial distribution function of water molecules relative to D59 from wtTS on binary (light blue line) and ternary (dark blue line) systems. FIGURE 7. Lactose alters the position of W312. Frames of 3.5 ns (A) and 20 ns (B) from MD simulation of the wtTS+3-SL+Lac ternary complex. Proteins are represented in cartoon and the residues and oligosaccharides are represented by sticks colored as follows: 3-SL in cyan, Lac in green, W312 and Y119 in orange. C. Distances of Cζ2 from W312 to C1 of Gal from 3-SL (gray line) and to C4 of Glc from Lac (black line). D. Variation of dihedral angle formed between Cα and Cβ of W312 throughout MD simulation. FIGURE 8. Wild type TcTS does not hydrolyze NeuSGal. A, Chemical structure of NeuSGal. 1H NMR spectra of NeuSGal (5 mM) before (B) and after 24 h incubation (C) with 0.2 mM wtTS. Both spectra were acquired in PBS/2H2O pH 7.4 at 25 ºC. There are not differences in chemical shifts of sialic acid between two spectra. FIGURE 9. DOSY-NMR reveals differential interaction modes of substrates with TcTS variants. Observed diffusion coefficients (Dobs) upon sialoside (black circles) and Me-Lac (gray squares) titrations in wtTS (A), TSY342H (B), and TSD247A (C). Distinct sialosides were used (NeuSGal for wtTS binding experiments and 3-SL for TSY342H and TSD247A) to avoid enzyme reaction. Dashed line indicates the Dobs of free Me-Lac. FIGURE 10. Wild type TcTS (wtTS) undergoes conformational changes triggered by the binding of donor and acceptor substrates. A. Overlap of 1H-15N HSQC spectra of wtTS in the absence (blue) or in the presence of NeuSGal (green) and NeuSGal and Me-Lac (red). B, C and D. Detail of regions of the spectra indicated in A.


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Table 1


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Figure 10


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TodeschiniIsadora A. Oliveira, Arlan S. Gonçalves, Jorge L. Neves, Mark von Itzstein and Adriane R.

-sialidase catalysisTrypanosoma cruzi transEvidences of ternary complex formation in

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Evidences of ternary complex formation in Trypanosoma cruzi trans