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生物計算資料

分子對接案例

《項(xiàng)目方案評估》

1. 蛋白序列

CRT:蛋白質(zhì)晶體結(jié)構(gòu)RCSB PDBID:3POS

A VYFKEQFLDG DGWTSRWIES KHKSDFGKFV LSSGKFYGDE EKDKGLQTSQ DARFYALSAS FEPFSNKGQT LVVQFTVKHE QNIDCGGGYV KLFPNSLDQT DMHGDSEYNI MFGPDICGPG TKKVHVIFNY KGKNVLINKD IRCKDDEFTH LYLIVRPDN TYEVKIDNSQ VES

IGG:蛋白晶體結(jié)構(gòu)2DLF、1WZQ、1GLC,此處挑選2DLF


 

Structure Obtain

The amino acid sequence of CRT and IGG were retrieved from the sequence database of NCBI (www.ncbi.nlm.nih. gov). The three-dimensional structures of CRT and IGG were both available in Protein Data Bank (PDB ID: 3POS, 2DLF).

 

2. 對接位點(diǎn)分析

根據(jù)文獻(xiàn)報道并結(jié)合模板蛋白晶體結(jié)構(gòu),進(jìn)行蛋白質(zhì)相互作用預(yù)估分析,整理其可能相互作用的結(jié)合模式。

在以文獻(xiàn)中對該蛋白所屬6類亞型蛋白進(jìn)行了序列比對,發(fā)現(xiàn)該蛋白序列特別是binding domain存在著極高的序列相似性,高達(dá)95%以上,說明該蛋白結(jié)合槽位置高度保守,這一推論表明我們所研究的蛋白質(zhì)在“結(jié)構(gòu)組成”和“結(jié)合模式”上都是較為統(tǒng)一。

因此,該文獻(xiàn)具有相對較為可靠的參考價值,參照本文獻(xiàn)可以進(jìn)行相似方法的蛋白分析。

分子對接案例1

 

3. 蛋白蛋白對接

結(jié)合已有相關(guān)資料與蛋白對接軟件Rossetta,對蛋白質(zhì)多聚體進(jìn)行構(gòu)建。

? 蛋白蛋白對接一直是分子模擬中非常重要同時非常難解決的話題,相較于小分子蛋白之間的聯(lián)系,蛋白蛋白對接如今更加不成熟,在蛋白蛋白對接之前,最好能夠搜集更多的文獻(xiàn)進(jìn)行支持,讓模擬的結(jié)果不空洞,才能保證對接的準(zhǔn)確性。
 

蛋白蛋白對接如今比較著名的軟件有Hex Protein Docking,ZDock,rDock,以及Rosetta等等。其中Hex Protein Docking雖算法比較復(fù)雜,但沒有評分功能(PS:可能是我沒有找到···),ZDock一般作為前期的初對接,rDock為ZDock的升級算法,一般將ZDock對接后的前幾個得分構(gòu)象使用rDock進(jìn)行進(jìn)一步對接。但是Hex Protein Docking,ZDock和rDock對接方法,目前均處于低級對接階段,對接結(jié)果基本不可信,特別是本文中的大體系跨膜蛋白分子對接項(xiàng)目。
 

Rosetta對接軟件,是由加州大學(xué)舊金山分校著名晶體結(jié)構(gòu)物理學(xué)家David Baker及其項(xiàng)目組所開發(fā)研究,進(jìn)展使接近原子精度的蛋白質(zhì)預(yù)測和設(shè)計成為可能。這一軟件,基于高精度高計算效率的全原子能量函數(shù)和用于搜索極端崎嶇勢能面的有效采樣策略的開發(fā),這兩者都由結(jié)構(gòu)預(yù)測和設(shè)計的測試結(jié)果的反饋所推動。Rosetta程序中統(tǒng)一的能量和運(yùn)動框架可廣泛用于大分子建模的問題中,從微纖結(jié)構(gòu)預(yù)測到RNA折疊再到設(shè)計新的蛋白質(zhì)界面,都易于進(jìn)行研究和突出區(qū)域的改進(jìn)設(shè)計。其對接結(jié)果相較于Hex Protein Docking,ZDock和rDock等幾個軟件,有著極大的提升與可靠性。

Rosetta中進(jìn)行對接包含兩個步驟。第一步,進(jìn)行積極(aggresive)采樣,方法使用的質(zhì)心模型。第二步采用全原子模型進(jìn)行小范圍的優(yōu)化。
 

Rosetta軟件最早是在華盛頓大學(xué)David Baker教授實(shí)驗(yàn)室開發(fā)的,目前軟件內(nèi)有多個應(yīng)用可供用戶使用,常用的應(yīng)用程序有同源建模(comparative modelling)、短片段模擬與重建(Loop modelling and rebuilding)、蛋白質(zhì)設(shè)計(protein design)、蛋白質(zhì)與蛋白質(zhì)對接(Protein-protein docking)、蛋白質(zhì)配體對接(Protein-ligand docking)等。Rosetta軟件對于學(xué)術(shù)界用戶是免費(fèi)的,只需要申請獲得一個許可證,就可以從Rosetta的官網(wǎng)中下載軟件了。

 

Protein-Protein Docking

A geometry-based molecular docking software called ROSETTA was used for docking the predicted three dimensional models of CRT and crystal structure of IG. The software predicts the docked transformations that produce good molecular shape complementarity. The algorithm divides the Connolly dot surface representation of the molecules into concave, convex, and flat patches. The patches were matched according to their complementarities in order to generate different transformations. A default value of 4 A? was used for clustering and redundant solutions were discarded by RMSD clustering. The Patch Dock output generates the geometric score, desolvation energy, interface area size, and the actual rigid transformation of the solutions. Twenty solutions, out of 60 predicted complexes, were sorted according to their geometric shape were again refined through the fire dock server. The complementarities scores were analyzed for identifying the residues involved in the protein-protein interface.


3.1 Rosetta對接結(jié)果挑選

利用Rosetta軟件共對接出10000中蛋白相互作用位相。利用Rosetta自帶聚類分析模塊進(jìn)行聚類分析并根據(jù)分子對接能量打分進(jìn)行排名。其中位置比較靠譜,能量打分較好的前10位蛋白質(zhì)對接能量如下:
分子對接案例2

我們?nèi)∑渲心芰孔詈玫慕Y(jié)構(gòu)Conformation 1 (-265.1Kcal/mol)進(jìn)行后續(xù)分析。

 

4. 蛋白分子動力學(xué)優(yōu)化

Molecular Dynamics Simulation

Molecular dynamics (MD) simulations were performed using the GROMACS 4.5 package [67]. The GROMACS program package (http://www. gromacs.org) adopting the OPLSAA force field parameters were used for EM and MD simulations. The protein-protein complex were analyzed in three separate system to obtain the stable conformation of the protein and protein-protein complex for analyzing three dynamic behavior of these structures. The bad contacts from the 3D structure of the proteins were refined and solvated with the solvent [68]. The system was further relaxed by energy minimization and for the MD simulation studies, the structures were solvated using the TIP3P water model and the solvated structures were energy minimized using the steepest descent method, terminating when maximum force was found smaller than 100 KJ/mol-1/nm-1. All the simulations were performed in the NPT ensemble at constant temperature (300 K) and pressure (1 bar) with a time step of 2 fs. NVT were performed for 1ns (nanoseconds) and the minimized structure were equilibrated with timescale of 10 ns (nanoseconds). Trajectory conversion and RMSD scripts were used for analysis of MD simulation.

經(jīng)過結(jié)構(gòu)疊合與補(bǔ)齊,并對構(gòu)象蛋白質(zhì)進(jìn)行6ns分子動力學(xué)優(yōu)化,直至結(jié)構(gòu)平衡。


6.1 方法

選取最優(yōu)構(gòu)象,用AMBER14軟件做分子動力學(xué)模擬。整個蛋白體系都采用gaff和ff14SB力場,以蛋白為中心,加10A的立方水盒子,加Na+使體系呈電中性,保存拓?fù)浜妥鴺?biāo)結(jié)構(gòu),然后進(jìn)行模擬,模擬流程如下:

(1)兩步能量最小化,先限制蛋白,最小化水分子的能量,然后放開蛋白,整個體系的能量最小化。第一次能量最小化一共5000次循環(huán),先采用最速下降法做1500次循環(huán),第二次能量最小化一共5000次循環(huán),先采用最速下降法做了2000次循環(huán)。

(2)體系平衡,首先是體系的升溫平衡過程,采用郎之萬控溫方法平衡了100ps。 然后進(jìn)行升壓平衡過程,一共平衡了100ps,采用了各向同性的 Berendsen 控壓方法。

(3)動力學(xué)模擬,無限制自由模擬階段??販乜貕悍椒ㄅc前一階段相同,范德華能和短程靜電能的截斷距離為10?,采用PME方法計算長程靜電能。
 

6.2 結(jié)果分析

6.2.1  RMSD值分析

對該蛋白進(jìn)行4次分子動力學(xué)優(yōu)化,從四個體系骨架原子的均方根波動值(RMSD值)可以看出,在2ns后,四個體系RMSD值的波動都小于0.5A,均已經(jīng)達(dá)到平衡狀態(tài),可以用來結(jié)構(gòu)分析。說明該蛋白質(zhì),無論始發(fā)點(diǎn)出于什么狀態(tài),初始參數(shù)均能使蛋白質(zhì)得到相應(yīng)的結(jié)構(gòu)優(yōu)化,并達(dá)到較好的平均結(jié)構(gòu)狀態(tài)。
分子對接案例3

4次體系隨時間變化的RMSD值

取出平均結(jié)構(gòu)進(jìn)行后續(xù)分析,得到的平均結(jié)構(gòu)如下:

分子對接案例4
分子對接案例5

Protein interactions (CRT-IGG)

H-bonds

Pi-Pi

Salt-bridges

disulphide bonds

ASN53-ND2OH-TYR49

None


None

LYS143-NZ---OD2-ASP60

LYS143-NZ---OD2-ASP60

LYS153-NZOH-TYR102


LEU156-NOG-SER56


ILE157-ONH1-ARG54


ASN158-ND2O-ARG54


ASN158-ND2OH-TYR49


LYS159-NZOD1-ASN53


ASP160-OD1NH2-ARG54

ASP160-OD1NH2-ARG54

GLU197-OOH-TYR99


GLU197-OD2ND1-HIS98

GLU197-OD2ND1-HIS98

ASP198-OOH-TYR49


ASP198-OD1NZ-LYS50

ASP198-OD1NZ-LYS50

ASP198-OD2NZ-LYS50

ASP198-OD2NZ-LYS50

TRP200-OOH-TYR99


PHE202-NOH-TYR99


PRO204-ON-THR28


ASP302-OD1NE2-HIS98

ASP302-OD1NE2-HIS98