Crystal structure of tick tyrosylprotein sulfotransferase reveals the activation mechanism of the tick anticoagulant protein madanin

Ticks pose a substantial public health risk as they transmit various pathogens. This concern is related to the adept blood-sucking strategy of ticks, underscored by the action of the anticoagulant, madanin, which is known to exhibit an approximately 1000-fold increase in anticoagulant activity following sulfation of its two tyrosine residues, Tyr51 and Tyr54. Despite this knowledge, the molecular mechanism underlying sulfation by tick tyrosylprotein sulfotransferase (TPST) remains unclear. In this study, we successfully prepared tick TPST as a soluble recombinant enzyme. We clarified the method by which this enzyme proficiently sulfates tyrosine residues in madanin. Biochemical analysis using a substrate peptide based on madanin and tick TPST, along with the analysis of the crystal structure of the complex and docking simulations, revealed a sequential sulfation process. Initial sulfation at the Tyr51 site augments binding, thereby facilitating efficient sulfation at Tyr54. Beyond direct biochemical implications, these findings considerably improve our understanding of tick blood-sucking strategies. Furthermore, combined with the utility of modified tick TPST, our findings may lead to the development of novel anticoagulants, promising avenues for thrombotic disease intervention and advancements in the field of public health. Ticks pose a substantial public health risk as they transmit various pathogens. This concern is related to the adept blood-sucking strategy of ticks, underscored by the action of the anticoagulant, madanin, which is known to exhibit an approximately 1000-fold increase in anticoagulant activity following sulfation of its two tyrosine residues, Tyr51 and Tyr54. Despite this knowledge, the molecular mechanism underlying sulfation by tick tyrosylprotein sulfotransferase (TPST) remains unclear. In this study, we successfully prepared tick TPST as a soluble recombinant enzyme. We clarified the method by which this enzyme proficiently sulfates tyrosine residues in madanin. Biochemical analysis using a substrate peptide based on madanin and tick TPST, along with the analysis of the crystal structure of the complex and docking simulations, revealed a sequential sulfation process. Initial sulfation at the Tyr51 site augments binding, thereby facilitating efficient sulfation at Tyr54. Beyond direct biochemical implications, these findings considerably improve our understanding of tick blood-sucking strategies. Furthermore, combined with the utility of modified tick TPST, our findings may lead to the development of novel anticoagulants, promising avenues for thrombotic disease intervention and advancements in the field of public health. Despite their small size, ticks pose a major public health concern. They can transmit various pathogens to their hosts, some of which can cause major infectious diseases (1Jongejan F. Uilenberg G. The global importance of ticks.Parasitology. 2004; 129 Suppl: S3-S14Crossref PubMed Scopus (1410) Google Scholar, 2Walker A.R. Eradication and control of livestock ticks: biological, economic and social perspectives.Parasitology. 2011; 138: 945-959Crossref PubMed Scopus (44) Google Scholar, 3Fang L.-Q. Liu K. Li X.-L. Liang S. Yang Y. Yao H.-W. et al.Emerging tick-borne infections in mainland China: an increasing public health threat.Lancet Infect. Dis. 2015; 15: 1467-1479Abstract Full Text Full Text PDF PubMed Google Scholar). For example, Borrelia species, which cause Lyme disease and severe fever with thrombocytopenia syndrome virus, a type of flavivirus, are transmitted from ticks to humans (3Fang L.-Q. Liu K. Li X.-L. Liang S. Yang Y. Yao H.-W. et al.Emerging tick-borne infections in mainland China: an increasing public health threat.Lancet Infect. Dis. 2015; 15: 1467-1479Abstract Full Text Full Text PDF PubMed Google Scholar, 4Yu X.-J. Liang M.-F. Zhang S.-Y. Liu Y. Li J.-D. Sun Y.-L. et al.Fever with thrombocytopenia associated with a novel Bunyavirus in China.N. Engl. J. Med. 2011; 364: 1523-1532Crossref PubMed Scopus (1258) Google Scholar). These diseases are a global public health issue, and blood-sucking by ticks, followed by transmission of pathogens, is a core event in their etiopathogenesis. One strategy for efficient blood-sucking involves the use of madanin, an anticoagulant found in tick saliva (5Iwanaga S. Okada M. Isawa H. Morita A. Yuda M. Chinzei Y. Identification and characterization of novel salivary thrombin inhibitors from the ixodidae tick, Haemaphysalis longicornis.Eur. J. Biochem. 2003; 270: 1926-1934Crossref PubMed Scopus (88) Google Scholar). Madanin and its analogous blood coagulation inhibitory proteins are prevalent among closely related species (6Thompson R.E. Liu X. Ripoll-Rozada J. Alonso-García N. Parker B.L. Pereira P.J.B. et al.Tyrosine sulfation modulates activity of tick-derived thrombin inhibitors.Nat. Chem. 2017; 9: 909-917Crossref PubMed Google Scholar). Such proteins characteristically comprise tyrosine residues primed for sulfation, a modification considered to underlie their shared anticoagulant activity (Fig. S1). Specifically, madanin possesses a unique structure containing two tyrosine residues (Tyr51 and Tyr54) whose sulfation amplifies its anticoagulant activity (Fig. 1B) (6Thompson R.E. Liu X. Ripoll-Rozada J. Alonso-García N. Parker B.L. Pereira P.J.B. et al.Tyrosine sulfation modulates activity of tick-derived thrombin inhibitors.Nat. Chem. 2017; 9: 909-917Crossref PubMed Google Scholar). The sulfated madanin strongly inhibits the production of thrombin, a key process in the host's blood coagulation cascade, enabling ticks to safely consume blood. Similarly, hirudin, a blood coagulation inhibitory protein found in leeches, undergoes sulfation at its tyrosine residues (7Stewart V. Ronald P.C. Sulfotyrosine residues: interaction specificity determinants for extracellular protein–protein interactions.J. Biol. Chem. 2022; 298102232Abstract Full Text Full Text PDF Scopus (7) Google Scholar, 8Maraganore J.M. Chao B. Joseph M.L. Jablonski J. Ramachandran K.L. Anticoagulant activity of synthetic hirudin peptides.J. Biol. Chem. 1989; 264: 8692-8698Abstract Full Text PDF PubMed Google Scholar, 9Skrzypczak-Jankun E. Carperos V.E. Ravichandran K.G. Tulinsky A. Westbrook M. Maraganore J.M. Structure of the hirugen and hirulog 1 complexes of α-thrombin.J. Mol. Biol. 1991; 221: 1379-1393Crossref PubMed Scopus (270) Google Scholar). Notably, the anticoagulant drug bivalirudin has been formulated based on the structure and function of hirudin (10Mehran R. Lansky A.J. Witzenbichler B. Guagliumi G. Peruga J.Z. Brodie B.R. et al.Bivalirudin in patients undergoing primary angioplasty for acute myocardial infarction (HORIZONS-AMI): 1-year results of a randomised controlled trial.Lancet. 2009; 374: 1149-1159Abstract Full Text Full Text PDF PubMed Scopus (371) Google Scholar). Tick tyrosylprotein sulfotransferase (TPST) is postulated to function as a type II membrane protein localized predominantly within the Golgi apparatus and is the enzyme responsible for tyrosine sulfation, where it catalyzes the transfer of a sulfate group from 3′-phosphoadenosine-5′-phosphosulfate (PAPS) to the tyrosine residues of madanin (Fig. 1A) (6Thompson R.E. Liu X. Ripoll-Rozada J. Alonso-García N. Parker B.L. Pereira P.J.B. et al.Tyrosine sulfation modulates activity of tick-derived thrombin inhibitors.Nat. Chem. 2017; 9: 909-917Crossref PubMed Google Scholar, 7Stewart V. Ronald P.C. Sulfotyrosine residues: interaction specificity determinants for extracellular protein–protein interactions.J. Biol. Chem. 2022; 298102232Abstract Full Text Full Text PDF Scopus (7) Google Scholar). This biochemical modification leads to approximately 1000-fold enhancement in the interaction between madanin and thrombin. Despite the importance of this process, the molecular mechanism underlying sulfation remains elusive, primarily because of the lack of structural information on tick TPST. In the present study, we successfully prepared tick TPST as a soluble recombinant enzyme and investigated the method by which it proficiently sulfates the tyrosine residues within madanin. To further our understanding, we determined the crystal structure of the complex between tick TPST and madanin, aiming to shed light on the detailed molecular mechanism underlying this interaction. Our investigations spanned specific interactions between tick TPST and madanin, the catalytic mechanism of tick TPST, and the identification of the pivotal residues involved in the sulfation process. Overall, our research might serve as a pivotal basis for the development of next-generation anticoagulants, providing a promising avenue to address pressing public health concerns. We engineered and expressed a soluble active domain of the tick TPST, excluding its anticipated membrane-binding and stem regions, as a recombinant protein in Escherichia coli. To evaluate the sulfotransferase activity of tick TPST, we designed and chemically synthesized five distinct peptides based on the vicinity of two tyrosine residues in madanin following sulfation (Fig. 1B). The sulfotransferase activity of Y51F54-peptide was significantly higher than that of F51Y54-peptide in tick TPST, as determined using activity assays and KM, kcat, and kcat/KM analyses (Fig. 1, C, D, and E). The results indicated that Y51F54-peptide has 25 times higher specific activity, one-ninth KM, and 7 times higher kcat/KM than F51Y54-peptide, making it more susceptible to sulfation. Both peptides have similar kcat values, suggesting that the substrate-binding affinity differences primarily influence the sulfation susceptibility of Y51F54-peptide. Next, we prepared N-terminal fluorescein-peptide substrates (Fig. 1B). We measured the binding affinity of these two types of peptides (Y51F54- and F51Y54-peptides) in the presence of 3′-phosphoadenosine-5′-phosphate (PAP) using fluorescence polarization. The results revealed that the binding affinity to Y51F54-peptide (Kd = 4.04 ± 0.42 μM) was approximately ten times stronger than that to F51Y54-peptide (Kd = 40.76 ± 4.74 μM) (Fig. 1F). Differences in the degree of sulfation and binding activities were observed between Y51F54- and F51Y54-peptides; we postulated that the tyrosine residue at the Tyr51 site is more readily sulfated by tick TPST than that at the Tyr54 site. Subsequently, we analyzed the time course of sulfation for an extended peptide harboring the tyrosine residues Y51 and Y54 (Y51Y54-peptide) (Fig. 1G). Assuming that sulfation occurs at both sites of this peptide and represents the sum of two simple first-order reactions (model_1), we obtained a very good fitting curve with two significantly different rate constants (blue curve, R2 = 0.97). Conversely, the assumption that both sites undergo sulfation at the same rate constant (model_2) yielded an inadequate fitting curve (purple curve, R2 = 0.69). Similarly, the assumption that sulfation at only one site (model_3) did not produce an appropriate fitting curve (orange curve, R2 = 0.74). These observations suggest that the peptide undergoes sulfation at both tyrosine residues and that this occurs with significantly different efficiencies. To investigate the influence on the second tyrosine residue following the sulfation of one of the residues, we synthesized two peptide variants, each with one of the tyrosine residues already subjected to sulfation modification. The binding affinities of the sulfated peptides (Y51Ys54-and Ys51Y54-peptides; Ys represent sulfated tyrosine residue) were Kd = 1.5 ± 0.2 μM and 20.4 ± 1.0 μM, respectively (Fig. 1F). The peptides with either Y51 or Y54 sulfated demonstrated stronger binding than the peptides where the corresponding positions had Phe. These results suggest that the sulfation modification of the first tyrosine residue may promote the sulfation of the second tyrosine residue. Integrating these results with all biochemical experiments conducted thus far, we strongly suggest that the order of sulfation modification of the two tyrosine residues typically begins with Y51 and that the sulfation of Y51 promotes the subsequent sulfation of Y54. Furthermore, the ability of the prepared tick TPST to bind to the peptides was maintained even after overnight incubation at 20 °C, suggesting that the enzyme is likely stable (Fig. S2: F51Y54-peptide∗). To investigate the structural basis for the sulfation mechanism of the two tyrosine residues in madanin by tick TPST, we determined the crystal structure of tick TPST. The crystal structure of the core domain of tick TPST complexed with PAP and the F51Y54-peptide was resolved at a resolution of 1.55 Å (Fig. 2, A and B). The data collection and refinement statistics are summarized in Table S1. The asymmetric unit of the tick TPST-PAP-F51Y54-peptide crystal contains two tick TPST molecules that assemble into a dimer. The root-mean-square deviation (r.m.sd) values for the alignment of the protomers (using 328 Cαs) were 0.199 Å, indicating that the protomers of tick TPST in the dimer are structurally almost identical (Fig. 2A). The catalytic domain of tick TPST comprises a single α/β motif with a five-stranded parallel β-sheet, flanked on both sides by α-helices (Fig. 2B). The 5′-phosphosulfate-binding (5′-PSB) motif (11Kakuta Y. Pedersen L.G. Pedersen L.C. Negishi M. Conserved structural motifs in the sulfotransferase family.Trends Biochem. Sci. 1998; 23: 129-130Abstract Full Text Full Text PDF PubMed Scopus (154) Google Scholar, 12Negishi M. Pedersen L.G. Petrotchenko E. Shevtsov S. Gorokhov A. Kakuta Y. et al.Structure and function of sulfotransferases.Arch. Biochem. Biophys. 2001; 390: 149-157Crossref PubMed Scopus (287) Google Scholar), which is contained within a strand-loop-helix consisting of β4 and α1, is central to this structural motif. Moreover, β7 and α6 are key elements that include the 3′-phosphate-binding (3′-PB) motif. The 5′-PSB and 3′-PB motifs are conserved among all members of the sulfotransferase family (11Kakuta Y. Pedersen L.G. Pedersen L.C. Negishi M. Conserved structural motifs in the sulfotransferase family.Trends Biochem. Sci. 1998; 23: 129-130Abstract Full Text Full Text PDF PubMed Scopus (154) Google Scholar, 13Teramoto T. Fujikawa Y. Kawaguchi Y. Kurogi K. Soejima M. Adachi R. et al.Crystal structure of human tyrosylprotein sulfotransferase-2 reveals the mechanism of protein tyrosine sulfation reaction.Nat. Commun. 2013; 4: 1572Crossref PubMed Scopus (52) Google Scholar, 14Shi R. Lamb S.S. Bhat S. Sulea T. Wright G.D. Matte A. et al.Crystal structure of StaL, a glycopeptide antibiotic sulfotransferase from Streptomyces toyocaensis.J. Biol. Chem. 2007; 282: 13073-13086Abstract Full Text Full Text PDF PubMed Scopus (20) Google Scholar, 15Moon A.F. Edavettal S.C. Krahn J.M. Munoz E.M. Negishi M. Linhardt R.J. et al.Structural analysis of the sulfotransferase (3-O-sulfotransferase isoform 3) involved in the biosynthesis of an entry receptor for herpes simplex virus 1.J. Biol. Chem. 2004; 279: 45185-45193Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar, 16Kakuta Y. Pedersen L.G. Carter C.W. Negishi M. Pedersen L.C. Crystal structure of estrogen sulphotransferase.Nat. Struct. Biol. 1997; 4: 904-908Crossref PubMed Scopus (239) Google Scholar). Its dimerization is attributed to the protruding regions from the α-helices 2, 3, and 4. This protruding region comprises a major portion of the dimeric interface. Moreover, the elongated α-helix 15 at the C-terminus contributes to the formation of the dimer interface (Figs. 2A, S3, and S4B). The crystal structure of tick TPST exhibits similarities with that of human TPST1 and TPST2 (Fig. S4A) (13Teramoto T. Fujikawa Y. Kawaguchi Y. Kurogi K. Soejima M. Adachi R. et al.Crystal structure of human tyrosylprotein sulfotransferase-2 reveals the mechanism of protein tyrosine sulfation reaction.Nat. Commun. 2013; 4: 1572Crossref PubMed Scopus (52) Google Scholar, 17Teramoto T. Sakakibara Y. Liu M.-C. Suiko M. Kimura M. Kakuta Y. Snapshot of a Michaelis complex in a sulfuryl transfer reaction: crystal structure of a mouse sulfotransferase, mSULT1D1, complexed with donor substrate and accepter substrate.Biochem. Biophys. Res. Commun. 2009; 383: 83-87Crossref PubMed Scopus (19) Google Scholar). A considerable structural divergence between the tick and human TPSTs is the extended length of the C-terminal alpha helix (α15) in tick TPST (Figs. 2A, S3, and S4B). This helix forms interactions with another protomer during dimer formation. Additionally, a unique characteristic is the β-sheet configuration in the N-terminal. The electron density in this region appears dispersed with several indistinct segments, indicating that its structural integrity might be compromised. However, the primary beta-strand at the N-terminus (β1) collaborates with the β2-β3 strands of another protomer to establish a β-sheet comprising three strands (Figs. 2A, S3, and S4C). This arrangement potentially acts as a supplementary element for the stabilization of the dimeric assembly. Owing to these structural variations, the dimeric interface of tick TPST (2657 Å2) is more expansive than its counterpart in human TPST2 (1535 Å2). Such differences may underlie the enhanced stability of the tick TPST dimer, possibly augmenting its enzymatic resilience. To elucidate the catalytic reaction mechanism of tick TPST, we meticulously investigated the structure around the active site, focusing on the regions binding to PAP and the madanin peptide (Fig. 2F). In the context of its natural substrate, PAPS, the 5′ phosphate group of PAP, where the sulfate group is attached, was found to interact with Arg74. Additionally, the oxygen atom of the Tyr side chain in the madanin peptide, which receives the sulfate group, exhibited interactions with Glu95. Notably, Lys154, carrying a positive charge, was positioned near the region expected to be involved in sulfate transfer. The sulfotransferase activity of tick TPST with Ala mutations in each of these three residues was significantly reduced or below the detectable limit (Fig. 2G). Considering this spatial arrangement and the anticipated patterns of chemical interactions, we postulate the following: Arg74 acts as a general acid catalyst, facilitating the dissociation of the sulfate group from PAPS. Glu95 functions as a general base catalyst, enhancing the nucleophilic attack capabilities of the acceptor oxygen in the tyrosine residue of the substrate peptide. Lys154 plays a pivotal role in stabilizing the transition state by neutralizing the negative charge of the transferring sulfate group during the transition. Based on these insights, we established that each of these residues made critical contributions to the sulfate transfer reaction. Based on the binding state of PAP, we constructed a docking model for PAPS, subsequently generating a putative ES complex (Fig. S5) (17Teramoto T. Sakakibara Y. Liu M.-C. Suiko M. Kimura M. Kakuta Y. Snapshot of a Michaelis complex in a sulfuryl transfer reaction: crystal structure of a mouse sulfotransferase, mSULT1D1, complexed with donor substrate and accepter substrate.Biochem. Biophys. Res. Commun. 2009; 383: 83-87Crossref PubMed Scopus (19) Google Scholar, 18Kakuta Y. Petrotchenko E.V. Pedersen L.C. Negishi M. The sulfuryl transfer mechanism. Crystal structure of a vanadate complex of estrogen sulfotransferase and mutational analysis.J. Biol. Chem. 1998; 273: 27325-27330Abstract Full Text Full Text PDF PubMed Scopus (123) Google Scholar). We have described the proposed steps of the sulfotransferase reaction in Figure 2H. The electron flow depicted in this representation suggests that the sulfotransfer reaction is catalyzed in such a manner. Remarkably, our predictions align closely with the crystal structure observations of human TPST2, where residues Arg78, Glu99, and Lys158, including their conformations, exhibit striking structural similarities (Fig. S4D). Furthermore, it has been reported that the sulfotransferase activity of human TPST2 is substantially reduced in Ala mutants of the corresponding three residues (13Teramoto T. Fujikawa Y. Kawaguchi Y. Kurogi K. Soejima M. Adachi R. et al.Crystal structure of human tyrosylprotein sulfotransferase-2 reveals the mechanism of protein tyrosine sulfation reaction.Nat. Commun. 2013; 4: 1572Crossref PubMed Scopus (52) Google Scholar), which aligns well with the results of mutation experiments in tick TPST. Theoretical studies of the sulfotransferase reaction mechanism for human TPST2 have also reported results that fundamentally support our proposed mechanism (19Marforio T.D. Giacinto P. Bottoni A. Calvaresi M. Computational evidence for the catalytic mechanism of tyrosylprotein sulfotransferases: a density functional theory investigation.Biochemistry. 2015; 54: 4404-4410Crossref PubMed Scopus (11) Google Scholar, 20Šmak P. Tvaroška I. Koča J. The catalytic reaction mechanism of tyrosylprotein sulfotransferase-1.Phys. Chem. Chem. Phys. 2021; 23: 23850-23860Crossref PubMed Google Scholar). These concordances further strengthen our propositions. Such acid–base concerted sulfate transfer reactions are commonly observed in sulfate-transfer enzymes, suggesting an SN2-like in-line displacement mechanism (12Negishi M. Pedersen L.G. Petrotchenko E. Shevtsov S. Gorokhov A. Kakuta Y. et al.Structure and function of sulfotransferases.Arch. Biochem. Biophys. 2001; 390: 149-157Crossref PubMed Scopus (287) Google Scholar). The conservation of these residues among TPSTs and their broad conservation in sulfotransferases (Figs. S3 and S4D) support this speculation (13Teramoto T. Fujikawa Y. Kawaguchi Y. Kurogi K. Soejima M. Adachi R. et al.Crystal structure of human tyrosylprotein sulfotransferase-2 reveals the mechanism of protein tyrosine sulfation reaction.Nat. Commun. 2013; 4: 1572Crossref PubMed Scopus (52) Google Scholar, 21Tanaka S. Nishiyori T. Kojo H. Otsubo R. Tsuruta M. Kurogi K. et al.Structural basis for the broad substrate specificity of the human tyrosylprotein sulfotransferase-1.Sci. Rep. 2017; 7: 8776Crossref PubMed Scopus (19) Google Scholar). In the complex of tick TPST-PAP-F51Y54-peptide, a strong interaction was evidenced by the definition of six (F−3, D−2, E−1, Y0, E+1, and E+2) of the eleven residues comprising the F51Y54-peptide within the electron density map (Fig. 2E). Subsite numbers ranging from −3 to +2 were identified to account for binding to each side chain of the substrate peptide, with the tyrosine residue accepting SO3− defined as subsite 0. The interaction pattern of the bound F51Y54-peptide with tick TPST, adopting an L-shaped conformation, was found to be fundamentally consistent between the two protomers, bending nearly perpendicular at position −1 (Fig. 2, C and D). Intriguingly, F51Y54-peptide, with a clear electron density observable from positions −3 to +2 (Fig. 2E), interacted with tick TPST across almost the entire range, indicating that all side chains within these positions interact to some extent with tick TPST (Fig. 2, C and D). This pattern indicates an efficient process of sulfation of madanin's Tyr54 residue by tick TPST. Notably, both main and side chains of the Phe residue at position −3 in F51Y54-peptide (corresponding to Tyr51 in madanin: Fig. 1B) interacted with a separate protomer that formed a dimer (Fig. 2C), suggesting that this dimerization significantly influenced the substrate-binding capability of tick TPST. These observations fundamentally align with those of the crystal structures of human TPST1 and TPST2 C4 peptide complexes, illustrating a similar mode of substrate recognition across TPSTs from humans to ticks (Fig. S4) (13Teramoto T. Fujikawa Y. Kawaguchi Y. Kurogi K. Soejima M. Adachi R. et al.Crystal structure of human tyrosylprotein sulfotransferase-2 reveals the mechanism of protein tyrosine sulfation reaction.Nat. Commun. 2013; 4: 1572Crossref PubMed Scopus (52) Google Scholar, 21Tanaka S. Nishiyori T. Kojo H. Otsubo R. Tsuruta M. Kurogi K. et al.Structural basis for the broad substrate specificity of the human tyrosylprotein sulfotransferase-1.Sci. Rep. 2017; 7: 8776Crossref PubMed Scopus (19) Google Scholar). Based on the findings of our biochemical experiments, we ascertained that the sulfotransferase activity of tick TPST and its binding affinity for the substrate peptide are higher for Y51F54-peptide than for F51Y54-peptide (Fig. 1, C–F). Furthermore, it has been shown that the sulfation of one tyrosine residue enhances the binding affinity to the peptide (Fig. 1F). We validated these observations using docking simulations and energy calculations with the crystal structure of tick TPST. First, we created docking models for the scenarios where Tyr51 (Fig. 3A) and Tyr54 (Fig. 3D) of madanin are sulfated and estimated the binding energy for each structure. The docking modeling results showed that the binding energy for the scenario where Tyr51 of madanin is sulfated was −526 kcal/mol, whereas that for Tyr54 was −503 kcal/mol. These findings corroborate with the biochemical experimental data, underscoring the reliability of these docking models. These data indicate that sulfation likely occurred at Tyr51 before Tyr54. In further docking modeling, the binding energy when Tyr51 is at the active site with Tyr54 being sulfated was −547 kcal/mol (Fig. 3B) and that when Tyr54 is at the active site with Tyr51 being sulfated was −561 kcal/mol (Fig. 3E). This finding corroborates with the biochemical experimental data, suggesting that once a tyrosine residue is sulfated, it becomes more conducive for sulfation of the second tyrosine residue (Fig. 3, C and F). Furthermore, the most likely scenario is that Tyr51 undergoes sulfation first (Fig. 3A), followed by Tyr54 (Fig. 3E), indicating an efficient sulfation process at both sites. When Tyr51 is initially sulfated, within the binding mode where Tyr54 is sulfated, its sulfate group interacts with Arg101, Gln108, and Trp109, enhancing its binding (Fig. 3E). This can strengthen the binding of madanin with sulfated Tyr51 to tick TPST. The residue corresponding to Arg101 and Trp109 are conserved in both human TPST1 and TPST2 (Fig. S3), suggesting that the mechanism for recognizing the sulfated tyrosine residue at −3 position of the substrate protein might be shared across human TPST1 and TPST2 (13Teramoto T. Fujikawa Y. Kawaguchi Y. Kurogi K. Soejima M. Adachi R. et al.Crystal structure of human tyrosylprotein sulfotransferase-2 reveals the mechanism of protein tyrosine sulfation reaction.Nat. Commun. 2013; 4: 1572Crossref PubMed Scopus (52) Google Scholar, 21Tanaka S. Nishiyori T. Kojo H. Otsubo R. Tsuruta M. Kurogi K. et al.Structural basis for the broad substrate specificity of the human tyrosylprotein sulfotransferase-1.Sci. Rep. 2017; 7: 8776Crossref PubMed Scopus (19) Google Scholar). Indeed, the possibility of recognizing sulfated tyrosine at the −3 position has been discussed by studies on human TPSTs (13Teramoto T. Fujikawa Y. Kawaguchi Y. Kurogi K. Soejima M. Adachi R. et al.Crystal structure of human tyrosylprotein sulfotransferase-2 reveals the mechanism of protein tyrosine sulfation reaction.Nat. Commun. 2013; 4: 1572Crossref PubMed Scopus (52) Google Scholar, 21Tanaka S. Nishiyori T. Kojo H. Otsubo R. Tsuruta M. Kurogi K. et al.Structural basis for the broad substrate specificity of the human tyrosylprotein sulfotransferase-1.Sci. Rep. 2017; 7: 8776Crossref PubMed Scopus (19) Google Scholar). Our findings suggest a sequential sulfation process in which the sulfation of one tyrosine residue potentially enhances the sulfation of the next. Notably, our data indicate that initial sulfation likely occurs at the Tyr51 site, amplifying the binding capacity and enabling efficient sulfation at the subsequent Tyr54 site. Furthermore, we developed a docking model using Alphafold2 (22Jumper J. Evans R. Pritzel A. Green T. Figurnov M. Ronneberger O. et al.Highly accurate protein structure prediction with AlphaFold.Nature. 2021; 596: 583-589Crossref PubMed Scopus (13826) Google Scholar), projecting the interaction between the full-length madanin protein (79 residues) and tick TPST, as opposed to partial peptides of the madanin protein (Fig. 3G). This model suggests that the two active sites formed by the dimerization of tick TPST could concurrently accommodate two madanin molecules without steric hindrance. This finding implies that the dimerization of tick TPST may facilitate efficient sulfation of the second tyrosine residue in madanin, following the sulfation of the first. In summary, our biochemical findings using the madanin peptide coupled with docking simulations offer crucial insights into the efficient sulfation mechanism of the two unique tyrosine residues in madanin by tick TPST. The molecular mechanism revealed in this study, which involves sulfation-mediated activation of the madanin as a proteinaceous anticoagulant agent, shares a notable similarity with the mechanism in humans, whereby the primary sulfation by the glycosaminoglycan sulfotransferase on heparin and heparan sulfate, both anticoagulant molecules, streamlines the subsequent sulfation process (7Stewart V. Ronald P.C. Sulfotyrosine residues: interaction specificity determinants for extracellular protein–protein interactions.J. Biol. Chem. 2022; 298102232Abstract Full Text Full Text PDF Scopus (7) Google Scholar, 15Moon A.F. Edavettal S.C. Krahn J.M. Munoz E.M. Negishi M. Linhardt R.J. et al.Structural analysis of the sulfotransferase (3-O-sulfotransferase isoform 3) involved in the biosynthesis of an entry receptor for herpes simplex virus 1.J. Biol. Chem. 2004; 279: 45185-45193Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar). Although these hypotheses require additional experimental validation, they provide a crucial perspective for understanding madanin's sulfation. Our elucidation of the mechanism by which tick TPST activates madanin sheds light on the intricacies of tick blood-sucking strategies and opens avenues for developing proactive measures for the management of tick populations and curbing tick-associated diseases. Furthermore, combined with the utility of the adjusted tick TPST, this may lead to the development of novel anticoagulants, promising avenues for thrombotic disease intervention, and advancements in the field of public health.