Molecular modeling and docking characterization of Dectin-1 (PAMP) receptor of Bubalus bubalis

Experimental and Molecular Pathology 92 (2012) 7–12

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Molecular modeling and docking characterization of Dectin-1 (PAMP) receptor of Bubalus bubalis Brijesh S. Yadav a,⁎, Vijay Tripathi c, Ajeet Kumar a, Md. Faheem Khan d, Abhijit Barate e, Ajay Kumar b, Bhaskar Sharma a a

Division of Biochemistry, Indian Veterinary Research Institute, Izatnagar 243122, India Division of Animal Biotechnology, Indian Veterinary Research Institute, Izatnagar 243122, India Centre of Bioinformatics IIDS, University of Allahabad, Allahabad 211002, India d Department of Plant Science, M.J.P. Rohilkhand University, Bareilly 243122, India e Laboratory of Infectious Diseases & Vaccine Development, Kangwon National University, South Korea b c

a r t i c l e

i n f o

Article history: Received 11 September 2011 and in revised form 14 September 2011 Available online 8 October 2011 Keywords: Dectin-1 receptor β-glucan Homology modeling Docking

a b s t r a c t Dectin-1, is a type II transmembrane receptor protein which contains a single extracellular CTLD (C-type lectin domain), stalk, transmembrane domain and an ITAM (immunoreceptor tyrosine-based activation motifs) in its cytoplasmic tail. Dectin-1 has the ability to recognize fungal β-glucans, which are carbohydrate PAMPs found predominantly in fungal cell walls. The recognition of fungal β-glucans by Dectin-1 helps in a variety of cellular responses, like host protection, such as fungal uptake and killing, and the production of inflammatory cytokines and chemokines. In this study we predicted the 3D (three dimensional) structure of Dectin-1 receptor based on homology modeling using MODELLER 9v8 software. The TMHMM server was used for the prediction of transmembrane helices. DALI, PROFUNC, Q-Site Finder, PINTS servers and PASS software used for the prediction of functional sites in the modeled Dectin-1 receptor. The docking investigation of Dectin-1 receptor with β-glucan suggests that ASP150, ASP113, GLY106, and GLU196 amino acids are the catalytic residues which form a shallow groove in the protein surface and bind to ligand β-glucan. We hope that this work will help in in-silico screening, structure-based design, and in understanding the structural basis of ligand binding to the Dectin-1 receptor. © 2011 Elsevier Inc. All rights reserved.

Introduction The rate of new discovered protein sequences is very high, whereas, the rate of detailed structural information about proteins by X-ray diffraction or nuclear magnetic resonance spectroscopy (NMR) is far less. Thus, there is an essential need for theoretical methods to predict protein structure from sequences. Presently, many protein 3D structures are predicted by homology modeling. This method involves aligning a desired protein to one or more nonredundant homologous sequences of a template protein and evaluating the alignment using a scoring matrix. Differing side chains can be added from a side chain library according to known side chain preferences, followed by extensive energy minimization to eliminate steric clashes (Schwede et al., 2000). The predicted threedimensional (3 D) structure of proteins using the homology modeling method is imprecise. Considering the uncertainties involved in

⁎ Corresponding author. E-mail address: [email protected] (B.S. Yadav). 0014-4800/$ – see front matter © 2011 Elsevier Inc. All rights reserved. doi:10.1016/j.yexmp.2011.09.018

homology modeling, it is imperative that the initial 3D model be verified to assess its structural integrity and biological relevance before it is used in structure-based drug design projects (Marti et al., 2003). Refining of homology models is usually based either on energy minimization, limited conformational sampling using molecular dynamics (MD) in conjunction with a detailed force field, or more extensive sampling using simplified force fields (Schonbrun et al., 2002). Many endogenous and exogenous ligands for Dectin-1 have been reported, but the receptors mainly recognize a ligand known as fungal β-glucans. These are carbohydrate PAMPs predominantly present in fungal cell walls. They consist primarily of (1–3)-β-D-linked polymer backbones with (1–6)-β-linked side chains of varying length and distribution (Romani, 2004, Tsoni and Brown, 2008). Dectin-1 receptor has three parts: a single extracellular C-type lectin-like domain (CTLD), a transmembrane region, and a cytoplasmic tail that contains a single tyrosine-based activation motif. Its alternative splicing generates two major Dectin-1 isoforms and a number of minor isoforms. The two major isoforms bind to β-glucan, but differ in the presence or absence of a stalk region and their ability to bind and induce

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Table 1 Target and template protein information. Target

Dectin-1 protein (Bubalus bubalis)

Dectin1 2BPE Dectin-1 2BPE Dectin-1 2BPE Dectin-1 2BPE

Template PDB ID

Protein

Sequence identity

2BPE 1YPQ 1YXK

Murine Dectin-1 (Mus musculus) Human oxidized low density lipoprotein 42% (Homo sapiens) Human lectin-like oxidized low-density lipoprotein (H. sapiens)

59% 42%

MEYQSSVENLDEDGYTQLDFSSRNITRRSIVSEKGLCAASSHWRLIAVTLGILCSVMLVV 60 ————————————————————————————————————————————————— TVVLSTSGVFSSSCSPNWITHEDSCYLFSTLLDSWDGSKRQCFQLGSNLLKIDSSKELEF 120 ————MGGFSQSCLPNWIMHGKSCYLFSFSGNSWYGSKRHCSQLGAHLLKIDNSKEFEF 54 * **.** **** * .****** :** ****:* ***::*****.***:** IARQVSSQPDHSFWIGLSRRRTEEPWLWEDGSTLLSNLFQIRSTVTKKDSSHNCAWIHVS 180 IESQTSSHRINAFWIGLSRNQSEGPWFWEDGSAFFPNSFQVRNAVPQESLLHNCVWIHGS 114 * *.**: ::*******.::* **:*****:::.* **:*.:*.::. ***.*** * DIYDQLCSAHSYSICEKKLSV——— 201 EVYNQICNTSSYSICEKELKHHHHHH 140 ::*:*:*.: *******:*.

cellular responses (Heinsbroek et al., 2006). Dectin-1 is a dendritic cell-specific receptor, now known to be expressed by many other cell types such as monocytes, macrophages, neutrophils, and a subset of T cells (Taylor et al., 2002). The levels of Dectin-1 expression can be significantly influenced by various factors such as steroids, some cytokines, and microbial stimuli. For example, IL-4, IL-13, and GM-CSF (granulocyte–macrophage colony-stimulating factor) cause significant up regulation of Dectin-1 expression. On the other hand, IL-10, LPS, and dexamethasone trigger down regulation of Dectin-1 expression (Willment and Brown, 2008). Phylogenetic analysis of Dectin-1 sequences shows that the Dectin-1 of buffalo and cattle are closely related and their structural and functional properties are also very similar to each other (Barate et al., 2010). The recognition of fungal β-glucans by Dectin-1 results in a variety of cellular responses, some of which are host protective such as fungal uptake and killing, and the production of inflammatory cytokines and chemokines. Interestingly, Dectin-1 also induces the production of IL-10, an anti-inflammatory cytokine whose role during fungal infection is unclear. The presence of IL-10 has traditionally been thought of as disadvantageous to the host during fungal infection. However, recent investigations have led to the suggestion that the inhibitory action of IL-10 on leukocyte activation may be important for limiting host injury during severe inflammation (Romani and Puccetti, 2006). The present study was designed to predict the 3D structure of Dectin-1 receptor based on homology modeling and docking of the Dectin-1 receptor with β-glucan.

Materials and method Sequence alignment and molecular modeling of Dectin-1 The Dectin-1 protein sequence of Bubalus bubalis (NCBI accession number: ABR24825) was retrieved from NCBI. Homology modeling of Dectin-1 of B. bubalis was performed in the following steps: template selection from the Protein Data Bank (Berman et al., 2002) (PDB), sequence-template alignment, model building, model refinement and validation (Table 1). The Protein-BLAST algorithm against the Protein Data Bank was used to carry out the sequence homology search. The sequence and 3D structure of template protein were extracted from the PDB database. The Blast program was run with BLOSUM62 as a scoring matrix, word size 3, gap penalty of 11 and gap extension penalty of 1. High resolution crystal structures of homologous protein as a template were considered for homology modeling. The crystal structure of murine Dectin-1 (PDB ID: 2BPD.B) (known as the beta-glucan receptor in humans) with a 58.91% sequence identity was considered as the best hit. The murine Dectin-1 crystal structure was used as a template to generate a comparative 3 D model of Dectin-1 of B. bubalis (Fig. 3) by MODELLER 9v8 (Eswar et al., 2006). MODELLER generated several preliminary models which were ranked based on their DOPE scores (Table 2). Some models with the lowest DOPE score were selected and the stereochemical properties of each model were assessed by PROCHECK (Laskowski et al., 1993). PROCHECK analysis of the model was performed to determine whether the residues were falling in the most favored region in the

Fig. 1. Sequence alignment of Dectin-1 of Bubalus bubalis and 2BPE.B (Template).

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180

Table 2 Comparative study of DOPE score of five models predicted through MODELLER and overall quality factor determination through ERRAT.

135 90

Psi (degrees)

9

45 0 -45

S.N.

Model predicted through MODELLER

DOPE score KJmol−1

Overall quality factor ERRAT

1. 2. 3. 4. 5.

PBP.99990001 PBP.99990002 PBP.99990003 PBP.99990004 PBP.99990005

− 6450.298 − 6524.533 − 6248.761 − 6183.876 − 6217.563

62.89 72.39 65.64 60.48 65.25

-90 -135

-180

-135

-90

-45

0

45

90

135

180

Phi (degrees) Fig. 2. Ramachandran map of Dectin-1 protein of Bubalus bubalis.

Ramachandran plot (Table 3). The model with the least number of residues in the disallowed region was selected for further studies. Protein structure validation The PROCHEK server was used for the validation of modeled Dectin-1 protein structure. The quality of the models was evaluated with respect to energy and stereochemical geometry. The ProSA (Wiederstein and Sippl, 2007) Web server was employed to evaluate energy and verify the 3D server to evaluate the local compatibility of the model related to good protein structure (Fig. 6). Prediction of transmembrane helices The TMHMM server was used to predict the transmembrane helices in Dectin-1 of B. bubalis. Protein functional sites PINTS, Q-Site Finder (Laurie and Jackson, 2005) PROFUNC (Laskowski et al., 2005)servers and PASS software were used to predict the structure based protein functional site. Molecular docking The AutoDock 4.0 (Morris et al., 2009) program was used for the molecular docking analysis of Dectin-1 protein with β 1, 3 glucan.

The two dimensional structure of β 1, 3 glucan substrate was taken from the pubchem (Wang et al., 2009) server of NCBI and converted into the 3D coordinate via the CORINA server. The β 1, 3 glucan substrate was docked against modeled protein via mentioned docking software. The Lamarckian genetic algorithm was used in AutoDock to perform the automated molecular dockings. Default parameters were used except number of runs. The docking of β 1, 3 glucan with Dectin-1 protein was performed in two steps. In the preliminary step of docking identification of the potential binding sites on the Dectin-1 protein of B. bubalis was performed and in the second step, the whole surface of the protein was covered with very large grid maps, created by AutoGrid. The X, Y, Z dimensions of the grid were set to 72 Å with grid points separated by 0.375 Å. Results and discussion Sequence analysis and homology model analysis The Dectin-1protein of B. bubalis (NCBI protein accession number: ABR24825) is 201 amino acids long and shows structural similarity with the crystal structure of murine Dectin-1 protein of Mus musculus (PDB ID: 2BPD.B). Murine Dectin-1 protein was selected as the template on the basis of lowest e-value (5e-43), highest resolution (2.25 Å) and maximum identity (59%). An alignment of Dectin-1 protein of B. bubalis with the template 3D structure of murine Dectin-1 protein of M. musculus (PDB ID: 2BPD.B) shown in Fig. 1. MODELLER 9v8 was used to generate the homology model of Dectin-1 protein of B. bubalis according to the crystal structure of 2BPE.B. A total five models were generated and their discrete optimized potential energy (DOPE) was calculated using model-single.top script. The model no. 2 (PBP.B99990002.pdb) having maximum score was considered as the best model of Dectin-1 protein of B. bubalis. PyMOL (DeLano, 2002) software was used to visualize the model and find out the maximum numbers of helices, turns and sheets in the protein. Protein structure analysis PROCHECK, VERIFY3D and ERRAT programs were used for the predicted model validation. PROCHECK analysis of the modeled protein showed that 72.39% of the residues were found in allowed regions of the Ramachandran plot (Fig. 2). Among the 129 residues, 104 residues were found in the most favored region, 14 in additional allowed

Table 3 Ramachandran plot calculations for 3D model of Dectin-1 protein.

Fig. 3. 3D structure of Dectin-1 protein of Bubalus bubalis.

Ramachandran plot statistics

Modeled protein

Template

% Amino acid % Amino acid % Amino acid % Amino acid RMS Z-score Bond angle Bond length

86.7 9.4 0.3 0.3

89.1 10.0 0.9 0.0

in most favored regions in additional allowed regions in generously allowed regions in disallowed regions

1.035 0.743

0.778 0.584

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3

0.9 0.8 0.7 0.6 0.5 0.4 0.3 0.2 0.1 0 -0.1

window size 10 window size 40

50

100

150

200

250

300

350

Knowledge-based energy

3D-ID Averaged score

10

400

Fig. 4. Verify 3D score profile.

DALI, PROFUNC, Q-Site Finder, PINTS servers and PASS software were used for the prediction of functional sites in the modeled Dectin-1 protein of B. bubalis. The DALI server showed a very close match with 2BPE (chain B) (Z-score - 28.7, RMSD - 0.1). These servers detected the following putative functional site residues in the modeled protein: SER-84, GLY-106, SER-107, ASN-108, LEU-109, LEU110, LYS-111, ASP-113, GLY-136, TRP- 148, GLU-149 and ASP-150 with significant matches. Molecular docking analysis Compound β 1, 3 glucan has been shown to be a potent inhibitor of Dectin-1 protein. Molecular docking of the modeled Dectin-1 protein was performed with β 1, 3 glucan. We have performed rigid

10

0

Z-score

Z-score

X-ray NMR

0

-5

-5

-10

-10

-15

-15

Number of residues

800

199

Identification of functional sites

5

600

-2

server, modeled proteins have 1 sheet, 3 beta hairpins, 2 beta bulges, 9 strands, 2 helices, 12 beta turns and 2 gamma turns. We have also predicted the transmembrane region in modeled protein using TMHMM server. This server identifies the transmembrane region from amino acid 43 to 65, inside region from 66 to 201 amino acid and 1 to 42 amino acids begins in the outside region.

5

400

-1

Fig. 6. Energy plot for the predicted Dectin-1 protein by ProSA.

X-ray NMR

200

0

Sequence position

10

0

1

-3 71

region, no residue in generously allowed region and 2 residues in disallowed region. The statistical score of the Ramachandran plot shows that 86.7% are in the most favored region, 11.7% in additional allowed region, 0.0% in generously allowed region and 1.7% in disallowed region. The above results indicate that the protein model is reliable. Verify3D score profile was used to access the quality of the model. Fig. 4 shows the Verify3D profile of the modeled protein, residues having an averaged 3D-1D score greater than zero should be considered reliable. The computability score for all the residues in the modeled protein are above zero. The Z score of the protein is representing the overall quality and measures the deviation of the total energy of the protein structure. The Z score of the protein is displayed in this plot with a dark black point (Fig. 5). Groups of structures determined from different sources (NMR, X-ray) are distinguished by different colors (NMR in dark blue and X-ray in light blue). In this plot the Z score value of the obtained model (PBP.99990002) of Dectin-1 of B. bubalis (−4.97) is located within the space of protein related to NMR. This value is very close to the value of the template (−5.51) which suggests that the obtained model is reliable and very close to experimentally determined structure. The Z-Score of modeled protein is within the acceptable range (−10 to 10, negative Z-scores are good and depend on the length of the protein). PROFUNC and STRIDE servers were used to predict the secondary structure of modeled Dectin-1, recognize helix, sheet and coil regions (Fig. 7). STRIDE server reveals 58 (28.86%) residues were in α helices and 48 (23.88%) residues were in β sheets, 81 (41.30%) residues were in coils and 14 (6.97) residues were in β turns. According to PROFUNC

-20

2

1000

-20

0

200

400

600

800

1000

Number of residues

Fig. 5. Z-Score Plot shows that z score value of proteins (dark blue region represents protein determined by NMR and light blue represents protein determined by X ray) The two black dots represent Z-score of the template and model protein.

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Fig. 7. Secondary structure alignment of modeled Dectin-1 protein of Bubalus bubalis.

Table 4 Docking energy. S.N

Inhibition constant Ki (μm)

Torsonal energy (Kcal/mol)

Inter molecular energy (Kcal/mol)

RMSD(Å)

Ligand efficiency

Hydrogen bond

Binding energy

1 2 3 4 5 6 7 8 9 10

386.34 387.91 49.99 100.50 385.96 104.61 387.93 386.83 2.96 121.56

+ 5.37 + 5.37 + 5.37 + 5.37 + 5.37 + 5.37 + 5.37 + 5.37 + 5.37 + 5.37

-10.03 − 10.02 − 7.14 − 8.95 − 10.03 − 6.71 − 10.02 − 10.02 − 8.92 − 6.62

46.47 46.5 41.81 49.32 46.49 39.77 46.47 46.49 49.55 39.12

− 0.14 − 0.14 − 0.05 − 0.11 − 0.14 − 0.04 − 0.14 − 0.14 − 0.11 − 0.04

0 1 4 2 1 4 1 3 4 1

− 4.66 − 4.65 − 1.77 − 3.58 − 4.66 − 1.34 − 4.6 − 3.58 − 1.25 − 4.65

docking, where the target and ligand molecules were kept in a nonflexible state (not changing the bond length, bond angle or torsional angle). All amide bonds were set rigidly and all ligand bonds were allowed to move freely. The AUTOGRID program assigned the precalculated grid map to each atom type present in the ligand molecule. We studied the interaction of β 1, 3 glucan with Dectin-1 protein on the basis of docking several energies i.e. free energy binding energy (ΔGb), inhibition constant, intermolecular energy, torsional energy, RMS and internal energy are given in Table 4. The best binding energy of β 1, 3 glucan with Dectin-1 protein is −1.25 Kcal/mol. The highest binding energy correlates with the highest binding efficiency in protein and drug interactions. The following amino acid residues of Dectin-1 protein played an active role in the interaction with β 1, 3 glucan compounds: GLY106, ASP113, SER138, ARG139, THR142, GLU143, GLU144, TRP148, ASP150, GLY151, LEU155, PHE159, ILE161, ASN173 and GLU196. The β 1, 3 glucan was bound with these active amino acids of Dectin-1 protein with 1, 2, 3 and 4 hydrogen bonds. The negative and low value of binding energy as well as maximum hydrogen

Residues

PHE159:O TRP148:OGLU144:OEGLY151:OSER138:HG ILE161:OLEU155:O PHE159:O ARG139:OGLU143:OE1ASN173:HDETHR142:HN PHE159:O ILE161:OTRP148:HE1LEU155:O ASP150:OD2ASP113:OD2GLY106:OGLU196:OE2 ASP152:O

bonding showed strong and most favorable binding between protein and ligand molecules. Of 10 docked confirmations, the 9th one is the best confirmation because β 1, 3 glucan binds with four hydrogen bonds and has the highest binding energy (− 1.25 Kcal/mol) and the highest RMSD value. These amino acid residues were involved in this confirmation: ASP150:OD2, ASP113:OD2, GLY106: O, GLU196:OE2.

TMHMM posterior probabilities for 1.2

probability

1 0.8 0.6 0.4 0.2 0

20

40

60

transmembrane

80

100 inside

120

140

160

180

200

outside

Fig. 8. Transmembrane region of Dectin-1 protein.

Fig. 9. Interaction of β 1, 3 glucan with Dectin-1 protein.

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The number of hydrogen bonds formed and catalytic sites involved in the protein–ligand complex are shown in Fig. 9.

Conclusion Dectin-1 is a lectin receptor for β glucan that is important for innate macrophage recognition of fungi and contributes to phagocytosis and the induction of inflammatory cytokines. A homology model of Dectin-1 receptor of B. bubalis built and validated using the Ramachandran plot. ERRAT and PROCHECK software were used to develop a reliable model for structure based drug design. Validation by software showed that the homology model score is similar to the crystal structure of the template. The TMHMM server identified the transmembrane region from amino acids 43 to 65, inside region from 66 to 201 amino acids and outside region from 1 to 42 amino acids (Fig. 8). Functional site analysis predicted the extracellular region of Dectin-1 receptor showed similarity with the catalytic site of the template. This docking study of the Dectin-1 receptor showed that of 10 docked confirmations, the 9th one was the best confirmation because β 1, 3 glucan binds with four hydrogen bonds, with the highest binding energy (− 1.25 Kcal/mol) and highest RMSD value. It is our hope that this study will facilitate understanding of the structural and functional basis of ligand binding to the Dectin-1 receptor.

Conflict of interest The authors declare that there are no conflicts of interest.

Acknowledgments We are grateful to Director, Indian Veterinary Research Institute and Indian Council of Agriculture Reseach (ICAR) for supporting this investigation.

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