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We performed the Structural modeling of catalytic domain of TgCPC1 with server I-TASSER, the template selected for homology modeling was Dipeptidyl ...
Structural Modeling of Toxoplasma gondii TGME49_289620 Proteinase Mateo Murillo León, Diego Mauricio Moncada Giraldo, Diego Alejandro Molina, Aylan Farid Arenas, and Jorge Enrique Gómez Grupo de Estudio en Parasitología Molecular (GEPAMOL) [email protected]

Abstract. Cysteine proteinases play key roles in host-parasite interactions, including host invasion, parasite differentiation, and intracellular survival. Toxoplasma gondii expresses five cysteine proteases, including one cathepsin L‑like (TgCPL), one cathepsin B‑like (TgCPB) and three cathepsin C‑like (TgCPC1, 2 and 3) proteases. We performed the Structural modeling of catalytic domain of TgCPC1 with server I-TASSER, the template selected for homology modeling was Dipeptidyl peptidase I (Cathepsin C) (PDB code: 1JQP). The C-Score of structural modeling of catalytic domain was -0.5; in the L-domain there are nine ɑ-helices and two β-strands and in the right domain there are four β-strands and two ɑ-helices. Cys-440, His-651 and Asn-676, form the cysteine protease catalytic triad in the active site. Adjacent to active site there is a Tyr441 this residue may be involved in the binding of the N terminus of the peptide substrate. A tyrosine residue (Tyr 578) that binds a chloride ion in the crystal structures of rat and human Cathepsin C is also conserved. Keywords: cathepsin, structural modeling, Toxoplasma gondii, protease.

1

Introduction

Peptidases play a critical role in protein catabolism by hydrolysis of peptide bonds in the polypeptides. Peptidases are classified into the seven categories based on the principal catalytic residue in the active site: Aspartic, Cysteine, Glutamic, Serine, Threonine, Metallo and Mixed, each of which can be further divided into clans and families [1]. Cathepsin peptidases belong to the C1 family clan CA of “Papain-like” cysteine peptidases are widely distributed in eukaryotic organisms. During catalysis, a basic amino acid in the catalytic triad, usually histidine, de-protonates the cysteine thiol group, which attacks the carbonyl carbon group in the substrate for hydrolysis [2]. Cysteine proteinases play key roles in host-parasite interactions, including host invasion, parasite differentiation, and intracellular survival [3] Toxoplasma gondii expresses five members of the C1 family of cysteine proteases, including one cathepsin L‑like (TgCPL), one cathepsin B‑like (TgCPB) and three cathepsin C‑like (TgCPC1, 2 and 3) proteases. T. gondii cathepsins Cs are exopeptidases L.F. Castillo et al. (eds.), Advances in Computational Biology, Advances in Intelligent Systems and Computing 232, DOI: 10.1007/978-3-319-01568-2_43, © Springer International Publishing Switzerland 2014

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and remove dipeptides of unblocked N-terminal substrates of proteins or peptides, whereas cathepsin B and L are endopeptidases[4]. CPC1 was the most highly expressed cathepsin mRNA in tachyzoites (by real-time PCR). T. gondii cathepsins Cs are required for peptide degradation in the parasiphorous vacuole of transgenic tachyzoites was completely inhibited CPC1 was the most highly expressed cathepsin mRNA in tachyzoites (by real-time PCR). T. gondii cathepsins Cs are required for peptide degradation in the parasiphorous vacuole of transgenic tachyzoites was completely inhibited by the cathepsin C inhibitor [4]. Our aim was to determinate the tridimensional structure of catalytic region of TgCPC1 (TGME49_289620) by structural homology.

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Materials and Methods

The Sequence of the TGME49_289620 protein from T.gondii was obtained from Genbank with the accession number: EEB01247. Secondary structure prediction was performed by web-based program namely Psipred [5] and the tertiary structure with the server I-Tasser [6]. The template selected for homology modeling was Dipeptidyl peptidase I (Cathepsin C) of crystallographic model deduced by Olsen et al., 2001. (PDB code: 1JQP). The functional regions were determined with PROSITE [7], SMART [8], SOSUI [9] and NCBI CD [10]. FATCAT [11] was used for alignment of TgCPC1 with TgCPCL (PDB code: 3F75). The structure was evaluated for its backbone conformation and stereochemical properties by PROCHECK Ramachandran Plot [12]. Visualization was performed with Chimera 1.8 [13].

3

Results

The TGME49_289620 are encoded in chromosome IX, this protein consist of 730 amino acids. The C-Score of structural modeling of catalytic domain was -0.5; the Catalytic domain consists of a L- domain and a R-Domain. The Left (L) domain (Residues: Leu 412 - Asp635) and the Right (R) domain (Residues: Leu 636- Met 730). There are nine ɑ-helices and two β-strands in the L-domain and in the right domain there are four β-strands and two ɑ-helices. Cys-440, His-651 and Asn-676, form the cysteine protease catalytic triad in the active site. Adjacent to active site there is a Tyr441 this residue may be involved in the binding of the N terminus of the peptide substrate. A tyrosine residue (Tyr 578) that binds a chloride ion in the crystal structures of rat and human Cathepsin C is also conserved. In our model the Asp1, which is a key residue that is involved in docking the substrate via interaction with the amino group and is conserved is all cysteine proteases, is far to the active site, but the Asp386 was found in this position and the propeptide occupied the deep active site cleft. Results of FACAT showed that TgCPC1 and TgCPCL are significantly similar (P-value =3.41e-09), and they has 216 equivalent positions with an RMSD of 2.11. In this alignment we found that CPC1 have a stabilizing disulfide Bond (Cys-437 to Cys-508) that is highly conserved among other papain-like cysteine.

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Fig. 1. Structure of TgCPC1 in complex with its propeptide. Stereoview looking into the active site cleft with the left (L) domain on the left, the right (R) domain on the right, and the propetide on the top. N- and C-terminal residues of each polypeptide are labeled. The catalytic triad (Cys440, His651, and Asn676) with side chains shown as sticks, disulfide bonds is also shown as sticks.

Fig. 2. View of catalytic triad. Cys440, His651 and Asn676, form the cysteine protease catalytic triad in the active site. Asp386 of the propeptide occupied the deep active site cleft.

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Discussion

The amino acid sequences of the catalytic regions are similar to the mature papain family cathepsins, including cathepsins B and L from T. gondii. Several residues known to be important to activity are conserved in TgCPC: Cys, His and Asn, which form the cysteine protease catalytic triad in the active site [14].

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The catalytic domain is significantly similar to CPCL that consist of two domains divided by the deep active site cleft; the left (L) domain is primarily ɑ-helical, whereas the right (R) domain contains a β-barrel-like motif that is decorated by a few short ɑ-helices [15]. In our model the Asp 1 was absent in the catalytic site, as well as results of TgCPCL crystallography structure. The TgCPL contains the three stabilizing disulfide bonds that are highly conserved among other papain-like cysteine [15] in our model we found only one conserved bond. A tyrosine residue that binds a chloride ion in the crystal structures of rat and human Cathepsin C is also conserved (Tyr 578). Adjacent to the active site cysteine, a distinctive residue may be involved in the binding of the N terminus of the peptide substrate. This tyrosine motif appears to be unique in the papain family proteases, in which tryptophan usually occupies the position adjacent to the active site residue. This Cathepsin contain a tyrosine based motif, YXXϕ, where X is any amino acid and ϕ is an amino acid with a bulky hydrophobic side chain, and/or an amino acid cluster dileucine motif located in their citoplasmatic tails know to participle in endosomal/lysosomal protein sorting in higher eukaryotes [16]. maturase for roptry proteins involved in modulation of the host cell [16]. TgCPL is predicted to be a type II membrane protein, it has an enzymatic activity with a low pH optimum and it occupies a membrane-bound structure in the apical region of extracellular parasites [17]. Studies found that Cathepsin C may play a role in cell growth, differentiation and protease activation [3].In other parasites was found that cysteine proteases may alter the adaptive immune response from a Th1 response to a Th2 response: The best studied protease from T. cruzi is a cathepsin L-like cysteine protease called cruzipain which induces a Th2 cytokine profile in spleenic cells. This result points to a major role for cruzipain in the direction of the immune response towards a Th2 profile, which is beneficial for survival of the parasite in the host [18]. Recent studies show that TgCPB and TgCPL vaccines elicited strong humoral and cellular immune responses in mice, both of which were Th-1 cell mediated. In addition, all of the vaccines protected the mice against infection with virulent T. gondii RH tachyzoites, with the multi-gene vaccine (pTgCPB/TgCPL) providing the highest level of protection [19].

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Conclusion

TgCPC1 from T.gondii showed that Cys-440, His-651 and Asn-676, give the chemical environment for the cysteine protease catalytic triad activity, and the Asp386 on the propeptide is near to active site at 3.9Å of distance to active Cys440 that was blocking the deep active site cleft.

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