Insilco analysis of an interaction between an endogenous peptide fragment of NUR77 receptor from human cells and USAG1 protein - may induce teeth regeneration

doi:10.21203/rs.3.rs-4193367/v1

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Insilco analysis of an interaction between an endogenous peptide fragment of NUR77 receptor from human cells and USAG1 protein - may induce teeth regeneration

https://doi.org/10.21203/rs.3.rs-4193367/v1

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BMP-7 and WNT signaling are responsible for the development of teeth. USAG-1 (uterine sensitization associated gene-1) inhibits bone morphogenetic protein-7 protein (BMP-7) as well as WNT receptors leading to suppression of tooth development and growth. The inhibition of USAG-1 using an antibody has been shown to induce tooth regeneration in mice. Hence, USAG-1 is a drug target that is promising enough for teeth generation. In this paper, for the first time, it has been proposed that an endogenous peptide that is part of the NUR-77 receptor in human cells may inhibit USAG-1 protein to induce tooth regeneration when injected locally. The peptide FSRSLHSL of the NUR-77 receptor interacts with the usag-1 protein through enough hydrogen bonds as shown by the docking studies. BCL-2 expression determines the mineralogical composition of enamel crystals of tooth structure. The peptide FSRSLHSL doesn’t convert the character of BCL-2 to proapoptotic although the addition of an extra amino acid “L” to derive FSRSLHSLL leads to an orthogonal behavior.

Bioinformatics

USAG1

Endogenous peptide

NUR-77

BCL-2

Teeth regeneration Simulation

It is important to find the essential molecules that help in the development of teeth. The bone morphogenetic protein (BMP) and Wnt molecules regulate the morphogenesis of a single tooth demonstrating the importance of molecular signals that influence organogenesis. The morphogenesis of more teeth requires BMP signalling[1], while the growth of extra teeth relies on Wnt signalling. However, it remains uncertain whether BMP or Wnt signalling is essential for tooth counting. Teeth agenesis is a phenomenon wherein the total number of teeth deviates from the normal count either by being more or less in numbers.[2–5] The total number of teeth in an individual might vary from person to person. The prevalence of congenital increases or decreases is estimated to be in the range of 0.1 to 3.6 %. Supenumerary teeth, often called teeth agenesis, are dental structures that deviate from the normal number of teeth in an individual, either by being more or less in numbers.

Bone Morphogenetic Protein- 7, a homodimer 35-kDa protein, plays an important role in the development of an embryo and the associated events. USAG-1(uterine sensitization associated gene-1), expressed predominantly in the kidneys, inhibits BMP-7’s bioactivity which is crucial for tooth development.[6–12] By way of knocking out the gene Usag-1, tooth regeneration has been shown. Hence, inhibition of Usag-1 may provide a way for teeth regeneration.

In this study, through docking and simulation studies for the first time, it is shown that an endogenous 8 amino acid peptide derived from human cell-based Nur-77 receptor may serve as an agent, injected locally,[13, 14] to modify the behavior of Usag-1 and help in tooth development.

Software and Modules

The atomic level interactions and stability of the USAG1 protein complex between peptides were built and examined by using Schrodinger's bioluminent, BIOVIA Discovery studio, and Desmond simulation software.[15, 16] In order to investigate the molecular mechanisms underlying the interaction between peptides and the simulated USAG1 protein, the Homology module, the protein preparation module, the Grid generation module, the Site map prediction module, the Ligprep module, and the glide docking module were utilised.

Preparation and Validation of Homology modeling protein

The homology model of the USAG1 protein was fabricated in the three-step process. First, the fasta sequence of the protein was downloaded from the UniProt public domain and given as an input to search the RCSB database for the matching templates. Secondly, the BLAST search was performed to find suitable templates with the similarity index. Further, the template sequence and USAG1 sequence were aligned by the align sequence module. Finally, the model was constructed by a knowledge-based modeling method with the parameters of minimizing all non-template residues, preserving rotomers, optimizing side chains and limiting the insertion 1o 10 residues[17–19]. The constructed model was analyzed for 20 parameters and the Ramachandran plot. Further loops are refined by the refine loops protocol and the corrected 3D model was subjected to interaction studies.

Protein and ligand preparation protocol

The energy-stabilised peptide FSRSLHSL was used to investigate its interaction with the USAG1-modelled protein.[20] The pre-processed protein was optimized using the methods detailed.

GRID generation for analysis of the binding pocket

Since the modelled protein lacks a crystallized molecule, a standard grid with a 4Ǻ crop site that maps at the closest site point on the generated protein USAG1 was found using the site map procedure. An appropriate grid was designed for docking the peptide based on the site map locations.

Peptide docking

The peptide docking protocol was set up with a loading binding grid file and the centroid of the grid was picked to bind the peptide. The dimension was automatically fixed based on the surface of the peptide. Further, the peptide file was loaded and cis-amide bonds were used to generate the different conformation of the FSRSLHSL peptide[21]. The more accurate MM-GBSA scoring method was used to find the docking score and the binding energy values.

System Builder and Dynamic Simulation Set up

Using the Desmond software's system builder and dynamic simulation procedure, the best-docked pose of the FSRSLHSL peptide stability was investigated. First, the complex was solvated using the previously established SPC solvent model. The buffer box size was calculated at a distance of 10.0 A, and the boundary condition was set up as an orthorhombic box shape. Additionally, the complex protein's surface occupation is used to minimise the final volume, and the system was neutralised by adding sodium and chloride ions. The procedure for molecular dynamic simulation was used for the solvated model. The trajectory recording interval was set to 100 ps with a 1.2 energy gap, and the simulation time was set at 100 ns. A total of 1000 frames are produced at a temperature of 300 k and pressure of 1.01325 bar in the NPT ensemble class. Prior to simulation, the model was relaxed.[19, 22, 23] The analysis of dynamic interactions for the 100 ns simulations was done by plotting the relevant graphs involving pressure, temperature and volume.

Preparation and Validation of Homology modeling protein

The BLAST run found two matching templates with a similarity index of 42.37% and the 3D coordinates are copied from the template. The homology model developed the model structure (Fig. 1) for the USAG1 protein and the quality of model was not satisfactory due to the steric clashes’ amino acids in the loop region. The loop refinement module alters the conformation of loop region and rectifies the clashes and all the 20 features of protein reliability report were satisfied (Fig. 2b). Further, it is confirmed by Ramachandran plot with all the amino acids are in the allowed region (Fig. 2b).

The modeled protein energy was minimized to local minima to make stable environment and the site map protocol identified the cavity where the drug molecule possible to bind. Based on the binding spot deified by the site map a grid was generated at the radius of 11Å (Fig. 3a). Protein and ligand preparation protocol. Glide algorithm positioned the FSRSLHSL peptide (Fig. 3a) with proper orientation inside the binding grid. The Fig. 3c illustrate the binding of best conformation with the USAG1 protein. The interaction between the active side amino acid and the sidechain fragments of peptide was analyzed submitted for the further studies. The overall dock score and MMGBSA score was found to be -7.566 kcal/mol and − 56.52 kcal/mol respectively.

The interaction analysis of the peptide and residues of protein USAG1 showed that the peptide formed 5 different types of networks. Majorly, hydrogen bonds formed between Trp 113- C = O, Lys 121-C = O, Glu103-NH₂, Ser 128-Imidazole NH, Trp131-OH and Glu174-OH. Additionally, Arg 126 formed a salt bridge and three positively charged amino acids (Lys 121, Arg132 and Arg 170) make the peptide fit well in the cavity of protein. Hydrophobic amino acids aid both the end of a peptide to bind on the surface of USAG1 (Fig. 4).

To perform the stability simulation studies, initially, water molecules were added to the complex system. The system builder module added 79484 water molecules and Na⁺ and Cl⁻ added to neutralize the system (Fig. 5).

The USAG1 protein and USAG1- FSRSLHSL complex were shown in Fig. 6, it proved that the 49 amino acids involved in stabilizing the complex during the simulation. The dimerization amino acid cys104 interaction not formed during the docking also formed and supported the binding of peptides (Fig. 6).

Protein-ligand contacts and heat map illustrate the type of interaction and the stable bonds forming amino acids during 100ns simulation. Gly199, Ser128, Trp131 and Asn173 interactions are stable throughout the simulation. Other amino acid interactions support the peptide to make proper configuration fit in the binding pocket. Also, the H-bonds and hydrophobic interaction play a vital role in the stability of the complex.

The dimerization amino acid Cys 104 initially supports the molecule to go inside the cavity and Trp 131 and Glu 174 formed 62% stronger bonds and other amino acids form the bonds strength range of 31–51%.

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The authors declare no competing interests.

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Version 1

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Insilco analysis of an interaction between an endogenous peptide fragment of NUR77 receptor from human cells and USAG1 protein - may induce teeth regeneration

Status:

Version 1

Abstract

Figures

Introduction

Materials and methods

Software and Modules

Preparation and Validation of Homology modeling protein

Protein and ligand preparation protocol

GRID generation for analysis of the binding pocket

Peptide docking

System Builder and Dynamic Simulation Set up

Result and Discussion

Preparation and Validation of Homology modeling protein

References

Additional Declarations

Status:

Version 1