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The times of the hords will come, the Carcharodon is here!
We'll dance out everything Gapon didn't dare to dream about
Compsognathi will arise taking to arms against their foes
They are led into the battle by a Tzar of prehistoric times!
Mare And The Corpse-Eyed Toads, "Molokh"

Interaction mapping of 4DKL

1. Introduction

**Picture 1.** 4DKL. Source: Protein DataBase.

μ-opioid receptor is a receprot for endogenous opioids such as beta-endorphin and morphin. Agonist binding to receptor triggers bonding to an inactive GDP-bound heterotrimeric G-protein complex and subsequent exchange of GDP for GTP in the G-protein alpha subunit leading to dissociation of the G-protein complex with the free GTP-bound G-protein alpha and the G-protein beta-gamma dimer activating downstream cellular effectors^[1,2]. It mediates an array of downstream cellular responses, including inhibition of adenylate cyclase activity and calcium channels, activation of mitogen-activated protein kinase (MAPK)^[3-5], phospholipase C^[3-5], regulation of NF-κB^[3-5] and many more^[6]. Phosphorylation by Ser/Thr protein kinases and association with β-arrestins is involved in short-term receptor desensitization. β-arrestins associate with the phosphorylated receptor and uncouple it from the G-protein thus terminating signal transduction^[6,7]. Endogenous ligands induce rapid desensitization, endocytosis and recycling whereas morphine induces only low desensitization and endocytosis^[6-8].It is also stated that the protein is involved in neurogenesis^[9].
Crystalized protein is a dimeric structure consisting of two similar subunits. Comparison of the μOR-Gi complex to previously determined structures of other GPCRs (G-protein coupled receptors) bound to the stimulatory G protein Gs reveals differences in the interactions between the G protein α subunit and the receptor core: the ligand binds in a deep and open pocket^[2].

2. Ligands

You can see the ligands interacting with the protein in the application by choosing a ligand of interest in the menu.

**Table 1.** 4DKL ligands. Click on a molecular structure picture to see a ball-rod model. Source - RCSB PDB.
Ligand	Structure	Chemical formula	Name by IUPAC	Molecular weight, g/mol	PubChem ID
SO4		SO₄	SULFATE ION	96.07	1117
Cl		Cl	CHLORIDE ION	35.45	312
MPG		C₂₁H₄₀O₄	[(Z)-octadec-9-enyl] (2R)-2,3-bis(oxidanyl)propanoate	356.5	17754086
BF0		C₂₅H₃₂N₂O₆	methyl 4-{[(5beta,6alpha)-17-(cyclopropylmethyl)- 3,14-dihydroxy-4,5-epoxymorphinan-6-yl]amino}-4-oxobutanoate	456.5	137348997
1PE		C₁₀H₂₂O₆	PENTAETHYLENE GLYCOL	238.28	62551
CLR		C₂₇H₄₆O	CHOLESTEROL	386.7	5997

Choose the script you want to see by clicking it on the menu |

3. Inner protein interactions

3.1. Covalent bonding

To find covalent sulfur bonds we isolated all the cysteine aminoacids in the protein and manually searched for cysteines forming a bond.
We found one cystine bridge formed by Cys 140 and Cys 217. It can be seen in applet if you choose the «Cystine bridge» script. The bond stabilizes the extracellular domain of the protein while leaving the active site open.

3.2. Hydrogen bonds

The hydrogen bonds were calculated after restricting all the atoms not involved in the secondary structures like helices and sheets.
In total, the protein has a lot of alpha-helices supported by hydrogen bonds. Beside those hydrogen bonds, there are ones between aminoacid residues not involved in helices and sheets. For example: Thr 1054 and Val 1057, Asn 1020 and Tyr 1024. There are a total of 14 hydrogen bonds not involved in secondary structures. Also there is a bond between Thr 249 of two subunits. These bonds can be seen in the applet when the «Hydrogen bonds» script is chosen. The characteristics of those bonds (Table 2) are supported by those already estimate in reliable sources.^{[12, 13]}
The functional role of these bonds is in stabilizing the dimer structure of the receptor.

**Table 2.** Hydrogen bonds characteristics
Interacting aminoacids	Atoms participating in a bond	Length, Å	Angle, deg
Thr 249:1 - Thr 249:2	O - O	2.6	134.5
Thr 1054 - Val 1057	O - N	3.2	145
Asn 1020 - Tyr 1024	N - O	2.8	142.8
Asn 1020 - Tyr 1024	O - N	2.4	111.7

3.3. Salt bridges

**Picture 2.** Hypothetical ionic bridge between Glu (on the right) and arginine, which side chain is missing in a *.pdb file.

Salt bridges were found by restricting all aminoacids that can exist in a charged form and calculating the distances between them to see if a salt bridge can be formed.
Thorough protein structure research resulted in us finding 4 salt bridges: between Glu 341 and Lys 344, Glu 229 and Lys 233, Glu 270 and Arg 263, Glu 1128 and Arg 1125. The distances between atoms that tend to form a salt bridge (i.e. nitrogen atom from lysine or arginine amino group and oxygen atom from glutamate carboxyl group) are shown in Table 3. However we want to point out that .pdb file lacks Arg 263 side group (its salt bridge is between the subunits of the dimer), but it most likely forms a salt bridge with Glu 270, because the distance between ꞵ-carbon atom of Arg and the oxygen atom of Glu is 3.29 Å (Pic. 3). Acquired lengths support the data previously known^{[14, 15]}. Every salt bridge can be seen in the application by choosing the «Salt bridges» script. Every bridge from 1 to 4 (according to Table 3) has its own function: the first stabilizes the helix it’s in after the turn; the second stabilizes its helix; third one orients its helix into the right way; the fourth bridge stabilizes the bond between subunits.

**Table 3.** Distances in salt bridges. A bridge can be formed if residues are less than 4 Å apart.
	Residues that form bridge	Distance, Å
1	Glu 341 - Lys 344	3.5 и 2.9
2	Glu 229 - Lys 233	3.2
3	Glu 1128 - Arg 1125	3.5 и 2,8
4	Glu 270/1 - Arg 263/2	<3.3

3.4. Hydrophobic agglutination

Since the receptor is a transmembrane and dimeric it seems reasonable to propose that its bulk is represented by hydrophobic aminoacids. This becomes clear when restricting the protein’s hydrophobic aminoacids. They form a massive agglutination cluster. There are also a lot of aminoacids in the area where two subunits interact that can be found inside or outside the cell. This hydrophobic interaction seems to have a key role in the quaternary structure of the protein. The agglutination can be seen in the application by choosing the «Hydrophobic agglutination».

Choose the script you want to see by clicking it on the menu |

4. Hydrophobic core

Phe 1153 was chosen to demonstrate the hydrophobic core.
When the script from the application has worked you can see that the distance of 7 Å is the least distance to fully cover the aminoacids with other atoms. On average, the distance between the nuclei of neighboring non-covalently bound atoms in the protein is not more than 6.1 Å, meaning that a water molecule cannot fit into it (distance between the surfaces of two atoms is not more than 2.2 Å, and the diameter of the oxygen atom is 2.8 Å^[16]. However there is a hole that is not occupied by anything and that can fit a water molecule in proximity of the sulfur atom of Met 1102.

область без атомов — **Picture 3.** “Atomless” space.

To activate the application click here, or “Hydrophobic core” button under the application | Script

5. Transmembraneity

The protein is a GPCR. As stated by the TMHMM prediction (Pic. 4) the protein has 7 transmembrane domains in froms of alpha-helices pointing with the N-end outside the cell. The aminoacids distribution near the membrane is shown in the Table 4. Obviously the protein has inner, outer and transmembrane domains. Inner domains are needed to connect to the G-protein. The part of the protein between the 206 and 384 residue is the largest of all the inner domains and is most likely to be the binding site of the G-protein. Free energy of transmembrane helix association is −65 kcal/mol, which is energetically beneficial — that’s why the protein is neatly packed in the membrane. Visualizations of the protein in the membrane can be seen in the pictures after the TMHMM prediction.

**Table 4.** Protein distribution near the membrane. There are 152.5 residues in the membrane.
Extracellular aminoacids	1- 19				77- 90				156- 182				405- 418
Transmembrane aminoacids		20- 42		54- 76		91- 112		135- 155		183- 205		385- 404		419- 441
Intracellular aminoacids			43- 53				113- 132				206- 384				442- 464

Visualiztion of protein in membrane from diferent databases.

← OPM MemProt GLMol PDBTM →

Autors contribution

Dmitry Bosov: ligands application scripts, formulated the priceless contribution of Evgeny Egorov, report translation to the emoji-language

Evgeniy Egorov: most part of the work is done by our Dota 2 esports coach. It is hard to point out any key moments in his actions because any of his advice steers us. For a long time we could not understand how to move on the map. His qualitative analysis was viewing all our solo matches (with random players in the usual game mode) and also our team games. He gave us many tips regarding this aspect, these include:
– arrangement of lines: he placed the roles. He explained to us that carry should go to not to the forest, but to the easy lane.
– explained (especially to the 4th position — Artemiy or Shaker) the features of moving on the map during first moments of the game to supports.
– showed the importance of walking with the team after the lining stage.
Not only he debunked our massive mistakes, but the smaller ones too. He confidently underlined all the mistakes. We were sure that such kind of mistakes were familiar to the Tier 1 players, but when he heard this claim he showed us EvilArthas, the best Dota 2 player.
This is the best player who feels the gaming situation very well. He is able to win a game in solo. Besides a quality game situation analysis he never undrestimates his contribution to the game. He was the man who proved the existence of the hidden pool on his example.
When watching through professional plays (like The International) Arthas (EvilArthas) pointed out that there are 3 important aspects in the game: the first and the most important is heroes pick, the second being the team interactions, and the third being a player’s personal skills (e.g. skill to press BKB in time — as EvilArthas said: Bought BKB, didn’t buy BKB press).
The hero picking stage was mentioned intentionnaly — it was this exact stage that Evgeniy Egorov worked on most of the time. We have many combinations of heroes, thanks to his advice on choosing characters we confidently defend the line.
Most important advice of Coach are:
– Don’t pick Коротышка
– Ban Технари and Коротышка
– icking Рубик as support in 2045 makes you go down (EvilArthas is also sure about that)
– Командир Легиона is the worst hero (Best appearance, worst performance)
– Тень has been losing mid lane for 10 years already
Aside from defining weak heroes Coach also picks out Король Гнева, Луна, Ветрорейнджер.
It is hard to imagine where we would be if Evgeniy Egorov wouldn’t agree to train us. My arguments with the support Vladimir N. sometimes would almost come to fights, but thanks to Evgeniy Egorov the arguments stopped at all. When he first came to us we were on the verge of breakup: constant hidden pool, misunderstandings in the team, bad performance from Призрачный Улан. But he alone managed to set us up for the game, he managed to unlock the potential if every one of us. He wasn’t just a coach — he was a psychologist.
We couldn’t get onto the first qualification matches for Dota 2 Major because of the control work on chemistry, but obviously we were top 1 in the CIS. Besides from chemistry we understood that if we go to Chengdu (China) we would miss the colloquium on the calculus. It’s good we took it into account and chose to stay giving a chance to Na`Vi.
I remember him telling me how to defend the hard lane playing Ветрорейнджер in the right way. I understood that I was making a lot of mistakes and had no idea what I was doing. He showed Artemiy P. how to play Землетряс. I won’t be surprised if after Evgeniy’s guidance Artemiy gives a million dollar Echo Slam (that is a reference to The International when the north american team EG made a combination of Echo Slam + Ice Blast killing the whole enemy’s team when they were in the decisive battle on Roshan.
A reasonable question: how does this all connect to our report of Interaction mapping of the protein?
You know, all that was said above isn’t connected to the report, Evgeniy Egorov is just a good coach. In fact, if it was not for his experience, we would hardly have organized and so harmoniously done this work.

Vladimir Nozdrin: inner protein interactions search, inner protein interactions and hydrophobic core applications scripts, JMol screenshots to accompany the report, creation of the report webpage.

Artemiy Pigidanov: work with TMHMM, visualization of the protein in the membrane by using the OPM, MemProt, GLMol, PDBTM databases, hypotheses considering the functional role of the inner protein interactions found.

Daniil Khlebnikov: writing the report (Ru + En), description of the acquired results, source search, hydrogen bonds search.

Sources

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