Our goal is to design the peptides of tandem-repeated sequence (PTRSs) to imitate C-type lectin domain, family 5, member A (CLEC5A) docking with dengue virus. In order to ensure the PTRs and the envelope protein (E protein) of dengue virus have an interaction, all the structure of PTRSs and proteins and their interactions were modeled using Rosetta. First, we utilized RosettaCM (Comparative modeling with Rosetta) to generate the structure of E protein from a local strain (PL046) based on the crystal structure (PDB: 1OAN). Second, we used the ab initio method to predict the PTRSs structures purely based on their sequences. Then, we utilized the clustering method to cluster the results and find the most probable structure of the PTRS. Finally, we verified the interactions between these predicted PTRSs and the E protein based on the global protein-protein docking. Figure 1 shows the flow of our simulation.
Figure 1. The flow of our simulation procedures
RosettaCM (Comparative modeling with Rosetta)
Purpose: To generate the structure of E protein (PL046)
- Create a threaded model
- Target sequence in a fasta file
- Sequence alignment file in the Grishin format
- Template PDB file
Output: A PDB file of threaded template.
Commands: partial_thread.static.linuxgccrelease @flag_threading
- Target sequence in a fasta file
- RosettaScripts xml file:
- The weight files
- Target sequence fragments files: 3-residue fragments and 9-residue fragments
- The PDB file of threaded template from step 1
Output: PDB files after hybridization.
Commands: rosetta_scripts.static.linuxgccrelease @flag_hybridize
-chemical:exclude_patches LowerDNA UpperDNA Cterm_amidation SpecialRotamer VirtualBB ShoveBB VirtualDNAPhosphate VirtualNTerm CTermConnect sc_orbitals pro_hydroxylated_case1 pro_hydroxylated_case2 ser_phosphorylated thr_phosphorylated tyr_phosphorylated tyr_sulfated lys_dimethylated lys_monomethylated lys_trimethylated lys_acetylated glu_carboxylated cys_acetylated tyr_diiodinated N_acetylated C_methylamidated MethylatedProteinCterm
- Disulfide bond
- PDB files generated from step 2
Output: About 10,000 results. (The lowest Rosetta score of the structure is shown in Figure 2)
Commands: relax.static.linuxgccrelease @flag_relax
Figure 2. The homology structure of PL046 E protein in cyan based on the deposited structure (PDB: 1OAN) in magenta
Purpose: To generate the structure of the PTRSs
- Protein sequence in fasta format
- Fragment library: 3-residue fragments and 9-residue fragments
- Psipred-ss2 (optional)
The last two inputs were generated from Robetta Fragment
Output: About 20,000 results. (Two of the representative structures are shown in Figure 3)
Commands: AbinitioRelax.static.linuxgccrelease @flags
We did not find the "funnel shape" from the plot of "Rosetta energy score vs RMSD" for the best 1,000 structures, which suggests there might be no single stable conformation.
Figure 3. Two of the representative structures of PTRS
Purpose: To cluster the results and find the most probable structure
- Rescore the thousands of results
- Energy cut-off
- Extract the results
Input: The ab initio results.
Output: The clustering scores.
score_jd2.static.linuxgccrelease -in:file:s 110_7/*.pdb -out:file:silent scored_silent110_7.out -out:file:silent_struct_type binary -in:file:fullatom
Input: The clustering scores.
Output: The results passing the energy cut-off.
python make_sub_silent_file_percentile.py scored_silent110_7.out ecut110_7.out -1 0.10
Input: The results passing the energy cut-off.
Output: The structures sorted by clustering. (We chose the lowest score to simulate protein and protein docking.)
Commands: cluster.static.linuxgccrelease @flag_cluster
Protein-Protein Docking (global docking)
Purpose: To find the interaction between PTRSs and E Protein
Input: The structures of ligand (PTRS or CLEC5A) and receptor (E protein) in the same input file.
Output: About 10,000 results. (The most 100 frequent docking sites are shown in Figure 4)
Commands: docking_protocol.static.linuxgccrelease @flag_global_docking
Figure 4. The best 100 results (based on the Rosetta score) of PTRS-1 or PTRS-2 docking to PL046 E protein (in red). The space above the plane (in grey) indicates the external surface of the virions, where the interactions occur.
The weakness of our design is that the PTRSs from the gold nanoparticles and the ones from the glass fiber compete for the same binding sites on the E protein. Moreover, if the E proteins on the virus particles are fully covered by the gold nanoparticles, there are no sites available to interact the PTRSs from the glass fiber.
Repulsion between gold nanoparticles
To assess this potential problem, we used DLVO theory to calculate the repulsion between gold nanoparticles to estimate the number of gold nanoparticles that would bind to a virus particle. The structure of dengue virus is icosahedral, and there are three E proteins on each surface. The distances between the potential binding sites can be obtained from the structure in protein data bank (1K4R). DLVO theory can be described as Equation 1.
Wtotal(D) = Wa(D) + Wr(D) = -AR/12D + 2πεε0RΨδ2exp(-κD) Equation 1.
Wtotal(D): total energy
Wa(D): van der Waals interaction energy
Wr(D): electrostatic interaction energy
A: Hamaker constant = 2.5×10-19 J
R: radius = 1.3x10-8 m (based on the size of gold nanoparticles)
C: center to center distance
ε: permittivity of vacuum = 8.854x10-12 F/m
ε0: dielectric of solution = 80.4 (at 20°C)
Ψδ2: Stern layer potential (zeta potential) = 1.794 mV
κ-1: diffuse layer thickness = 7.95x10-10 m
We took several representative positions on adjoining faces of the icosahedron to calculate the interactions between the gold nanoparticles based on DLVO theory. We found the total energies are all positive (Table 1), and these total energies are also larger than the typical biological interactions (~0.5 kcal/mol or 3.49 x1021 J). The results suggest that there will always be free faces on the virus particles to interact with the PTRS-2 conjugated on the test line.
Table 1. The representative energies of the interaction between the gold nanoparticles.
|Attractive energy (10-21J)||-3.3||-4.6||-7.2||-4.5|
|Repulsive energy (10-21J)||51.0||51.3||51.6||51.3|
|Total energy (10-21J)||47.7||46.7||44.4||46.8|
|Attractive energy (10-21J)||-7.8||-7.5||-6.7||-7.8|
|Repulsive energy (10-21J)||51.7||51.7||51.6||51.7|
|Total energy (10-21J)||43.9||44.2||44.9||43.9|
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