Atlas of interactions between TLR 5 and flagellin in the protein complex

Our team decided, that protein interactions in blood of vertebrates, especially associated with the immune system - is a broad topic with lots of interesting details. To choose an object (the complex of two proteins) we used “advanced search” in Protein Data Bank [1] with special parameters. The parameters were: X-Ray diffraction, good resolution, new annotated structure, and section “proteins of the immune system”. Thus, the crystal structure of a complex between Bacillus subtilis flagellin and zebrafish Toll-like receptor 5 (PDB ID: 5GY2) was selected [2]. See ill.1.


ill.1
There are the big variety of different proteins (protective, like antibodies, immunogenic, like toxins, etc.) in the immune system. Interactions between them vary too, so it isn’t easy to choose the most interesting one. One of the most important and promising classes of proteins – is ‘toll-like receptor’ class [3]. The Nobel Prize 2011 can only prove our suggestion, because that time scientific group headed by Bruce A. Beutler and Jules A. Hoffmann discovered, that TLR play a significant role in the activation of innate immunity and Ralph M. Steinman with his colleagues discovered the dendritic cell and its role in adaptive immunity [4]. To put it in a nutshell, different types of toll-like receptors of innate immunity recognize different pathogen-associated molecular patterns — PAMP, special “characteristics” of pathogens, such as components of bacterial murein and so on. Toll-like receptors – are transmembrane proteins of dendritic cell membrane. As well as PAMP contact it’s TLR, dendritic cell activates adaptive immunity. Adaptive immunity allows organism to kill pathogen more effective and form “immune memory” to this pathogen, or toxin.
Funny video :)
About our complex: In this work we chose the complex between Bacillus subtilis flagellin and zebrafish Toll-like receptor 5. It was released 2/15/2017 with X-ray Diffraction, has resolution 2.1 A.
Macromolecules [4]: Molecule 1 is a protein called Tlr5b protein, variable lymphocyte receptor B. It contains 2 chains (A and B). Uniprot ID of the 1ST protein: B3DIN1 . Organism - Eptatretus burger Danio Rerio. See iil.2 [5]
Molecule 2 is a protein called Flagellin. It also contains 2 chains (C and D), but they are shorter than A and B ones. Uniprot ID of the 2nd protein: Q4G1L2 . Organism - Bacillus subtilis. Flagellin [6] (a bacterial protein) forms the flagellar filament and provides bacterial motility. See ill.3 [7] When flagellated bacteria invade the host, flagellin is recognized by Toll-like receptor 5 (TLR5) as a pathogen invasion signal and eventually evokes the immune response.
Unique Ligands: 1). NAG (N-acetyl-d-glucosamine) [6], see ill.2 It is a monosaccharide (glucose derivative). NAG in a complex with N-acetylmuramic acid (MurNAc), cross-linked with oligopeptides at the lactic acid forms peptidoglycan called murein, which built bacteria cell wall. Also NAG is the monomer of polymer chitin (substance, which forms cell walls of fungi, external skeleton of most arthropoda, etc.
2). PG4 (Tetraethylene glycol) [7], see ill.3 It is also called tetraglyme and seems to be a polar aprotic solvent with excellent chemical and thermal stability. Its high boiling point and stability makes it an ideal candidate for separation processes and high temperature reactions.
Atlas of connections:
There are 4 buttons for start script under the applet: Protein-Ligand, Hydrophilic, Hydrogene, Salt Bridges. Each script has several visualisations, so press "Resume" to move between them. Button "Rotate" and " Rotate off" start and stop the rotation of the Macromolecule.

1. Protein-protein
Salt bridges
First, we selected chains of one protein and with the help of command “within” saw atoms not farther than 2.5 A. There were found only two atoms (one at the distance of 2.42A, the other at the distance of 2,47A). This led us to the conclusion that there is no covalent bond between proteins. We also noticed that there is an interaction between ARG and ASP - salt bond. In chemistry, a salt bridge is a combination of two noncovalent interactions: hydrogen bonding and electrostatic interactions. Usually they contribute stability of tertiary structure of proteins. The salt bridge most often arises from the anionic carboxylate (RCOO-) of either aspartic acid or glutamic acid and the cationic ammonium (RNH3+) from lysine or the guanidinium (RNHC(NH2)2+) of arginine. The distance between the residues participating in the salt bridge is also cited as being important. The distance required is less than 4 A (400 pm) We suppose that in our case salt bonds between flagellin and TLR5 are needed to amplify the contact that will not collapse under external influence (such as thermal, for example).
Hydrofobic interactions
In order to fold into energetically favorable conformation hydrophobic radicals of aminoacids(such as valine, leucine, isoleucine, proline, methionine and phenylalanine) Strive to unite into a globular structure in the center of the protein. Thus, hydrophobic interaction, and van der Waals interactions between closely adjacent to each other atoms appear. As a result, they form the protein globule inside the protein called hydrophobic core. Hydrophobical interactions must play a significant role in our complex, because TLR - is trasmembrane protein. The process of recognision and visualisation of hydrophobic cores can briefle be outlined as: 1). With the usage of CluD we found all cores in our complex. Next, we selected 4 biggest cores and analysed them: 1). Core 1 - atoms, chain A of TLR 2). Core 2 - atoms, chains B and D 3). Core 3 - atoms, chains A and C 4). Core 25 - atoms, chain B Than we wrote a script to visualize them We can see, that huge hydrophobic cores form the structure of our complex. Mostly they are parts of TLR (cores 1 and 25 totally, and cores 2 and 3 bond chains of TLR and flagellin). So, our supposions were proved. And hydrophobic interactions not only form the structure of TLR, but also bond two proteins in the complex. Hydrogen bonds
2. Protein – ligand
Analysis of the structure of our complex (the description in PDB file) revealed the presence of two types of ligands: NAG and PG4. The first step of ligand-protein bonds’ search was to determine the location of the hydrophobic core and the possibility of hydrophobic bonds formation. We found out, that it is impossible because of large distance between ligands and nucleus. The next step was to look through all atoms within a radius of 3.5 angstroms, which could potentially form hydrogen bonds. We managed to find such bond, moreover we found out, that PG4 molecules are located within a radius of 2.9 angstroms, near the positively charged amino acids (His, and Arg). Thus, we could suggest, that the formation of a negative charge in the ligand takes place, so as the formation of a salt bridge. The exploration of NAG ligands’ surroundings showed, that there are distinct covalent bonds between NAG and A and B chains of TLR 5 (mostly with Asn). To see the visualization, press the button “Protein-ligand”. The most interesting detail about protein-ligand interactions is its biological sense. All ligands are binded with A and B chains of TLR5, so we suppose, that ligands (as PAMPs) could be coactivators of the receptor.
Conclusions:
During the research, we discovered and described different types of protein- protein interactions and protein- ligands interactions. In prot-prot there are hydrophobic bonds, salt bridges and hydrogen bonds. Covalent, hydrogen and salt bonds – are main types of bonds, which take place in protein-ligand interactions. Moreover, we found out that both NAG and PG4 are bonded only with A and B chains of Toll-like receptor 5. We found information about our complex, its macromolecules and ligands (functions, structure, nomenclature). Also we understood how innate inmunity and adaptive immunity are connected and comprehensed the scheme of this mechanism.

The contribution of team members:
1. Maria Selifanova: discovered and described hydrophobic cores and hydrophobic interactions, discribed hydrogene bonds, author of the script “Hydrophobic interactions”. Author of the text, author of html code and web-page design.
2. Eugenia Elisarova: found the protein complex to explore in PDB, discovered and described all protein-ligand interactions, specialist at bonds’ searching, the author of the script “Protein-Ligand interactions” and "Hydrogene". Script editor.
3. Maria Gurileva – found information about structure, function and nomenclature of macromolecules in our complex, discovered and described salt bridges, the author of the script “Salt bridges”, translated the text into Russian.
Acknowledgements:
Authors thank FBB student Zaira Seferbekova for editing the English version of the article.
References:
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