αIIbβ3 Modeling Simulation and Design of Cyclic RGD Peptide

CHINESE JOURNAL OF BIOTECHNOLOGY Volume 24, Issue 2, February 2008 Online English edition of the Chinese language journal RESEARCH PAPER

Cite this article as: Chin J Biotech, 2008, 24(2), 297í301.

DIIbE3 Modeling Simulation and Design of Cyclic RGD Peptide Mingyan Luo1, Meizong Chen1, and Imshik Lee1,2,3 1

College of Physics, Nankai University, Tianjin 300071, China

2

TEDA Applied Physics School, Nankai University, Tianjin 300457, China

3

Key Laboratory of Bioactive Materials of Ministry of Education, Nankai University, Tianjin 300071, China

Abstract:

Integrin DIIbE3 of the platelet surfaces regulates the thrombosis formation. DIIbE3 binds to the RGD sequence

(Arg-Gly-Asp) of fibrinogen, promotes the platelet aggregation and finally leads to the thrombus. We obtained the three-dimensional molecular structure of DIIbE3 using homology-modeling (modeller8v2 software), with integrin DvE3 (pdb code 1JV2) as the template. Accordingly, a cyclic RGD(RGD-c) peptide was designed to bind DIIbE3 as an antagonist and to block the formation of thrombus. We added two amino acids X, Y to both sides of RGD-c. X and Y could bind to each other by disulfide bond that finally made RGD-c a cyclic peptide. The optimum structure of RGD-c was obtained from the energetic optimization processes. All amino acids were placed at the X and Y to conduct molecular docking to the integrin DIIbE3. We got the optimum structure of RGD-c by energetic optimization and the antagonistic combination analysis. The results might provide an insight into designing and screening integrin DIIbE3 antagonists. Keywords: DIIbE3; homology modeling; dock; RGD-c

Introduction  DIIbE3 is an important receptor protein on the platelet[1], and it is composed of two subunits. DIIbE3 can bind to the specific RGD sequence (Arg-Gly-Asp) of fibrinogen, promotes the platelet aggregation and finally leads to the thrombus. In the past two decades, the researchers have had a lot of recognition about the 3-dimensional (3D) structure of DIIbE3, but hadn’t got the exact structure of it. The structure of the homology protein DvE3 had been gotten by X-ray diffraction[2]. One can make some structural information about DIIbE3 from DvE3 by homology simulation[3]. To design the steady small molecules[4,5] which are to mimic the active part in fibrinogen. Those moleculars are used as the antagonistic ligand of active protein in the

thrombosis signal pathway. Consequently, it can be prevented the thrombosis, with reducing the side effects in vivo. For example, PKC(protein kinase C) can activate DIIbE3 and make it to bind to the active sites of fibrinogen. Though the blocker of PKC can decrease the aggregation of platelet and reduce thrombus formation, this blocker can also affect other function of the PKC family in the cell. So it can be chosen another method to make the blocker of DIIbE3, preventing it to bind to the fibrinogen. At present, there are many antagonistic drug of the DIIbE3. These kinds of drug are still needed to improve the effective time of blocking the thrombus and preventing the formation of thrombus. DIIbE3 plays an important role in the processing of cruor; the antagonist drug can block its activation. However, currently, the blocker is mostly non-peptide inverse

Received: June 13, 2007; Accepted: August 6, 2007. Supported by: the doctoral fund of the Ministry of Education (No. 20050055037). Corresponding author: Imshik Lee. Tel: +86-22-23494411; E-mail: [email protected] Copyright © 2008, Institute of Microbiology, Chinese Academy of Sciences and Chinese Society for Microbiology. Published by Elsevier BV. All rights reserved.

Mingyan Luo et al. / Chinese Journal of Biotechnology, 2008, 24(2): 297–301

molecular. Though they can block the activation, they also produce other effects. In contrast, peptide molecular can block specified receptor and have strong affinity; the strongest advantage is that they can’t affect the whole signal pathway. They are composed of a few peptides which aren’t complex. They can effectively evade the adventure. In the processing of platelet aggregation, there are two kind of sequences which can bind to DIIbE3, KQAGDV and RGD[6]. Some researchers have made RGD-mSAK to prevent the aggregation of platelet[7]. In this work, we tried to design systemically the model of cyclic x-RGD-y, to maximize the binding stabilization and specialization to DIIbE3 by molecular docking. Subsequently, the active sites and binding mechanism were analyzed. Finally, the most effective binding model was suggested. The results could be used for screening the empirical effectiveness in preventing thrombus.

1

Methods

1.1 DIibE3 homology simulation 1.1.1 The active part in the D subunit DIIbE3 and DvE3 are consisted of two subunits. Esubunit of them are the same. D subunit of DIIbE3 has 1039 amino acids, the other is 927(except for intracellular part). Dv is composed by 4 domains[8,9]: thigh ˈ calf-1 ˈ calf-2 and E-propeller. E-propeller is the most important domain in the D subunit. The E-propeller is formed from the NH2-terminal seven-fold 60 residue sequence and consists of seven radials arranged “blades,” each formed from a four-stranded antiparallel sheet. The two subunits have 37% similarity by modeller sequence analysis program. The simulation structure of DIIbE3 is built by modeller8v2 software(http://salilab.org/ modeller/). D subunit contains 4 domains, The main domain is Epropeller. The others are similar with Dv. The two subunits include a transmembrane part. Due to this part hasn’t clear structure in the template, the simulation structure of DIIbE3 still contains several undefined structures, the final simulation structure is got only by selfoptimization. For the following docking work, we used the only functional domain in the D subunit after optimizing it. Fig. 1 shows E-propeller[10]. Using the Arguslab software, the structure makes the steepest-descend optimization in the UFF force-field, repeatedly 1000 steps. BFGS optimization is applied for 1000 steps further. Non-band interaction force is calculated at the range of 8–10 A. Finally, the minimum energy optimization is operated. After optimization, there were no significant changes in the E-sheet, but in the structure of the strand, appearing the obvious D helix. The simulation structure of E-propeller is basically similar with the template

Fig. 1 Look-up and look-down structure of E-propeller

structure. 1.1.2 the active part of E subunit The structure of Eis composed by five domains: EAˈ EGF-3ˈEGF-4ˈET and hybrid. EA is link to E-propeller of Dsubunit. Both of them are the most active parts in the formation of thrombosis. All active sites of DIIbE3 are allocated in these two parts, including the metal ion dependent-support sites (MIDAS––Asp119, Ser121, Ser123, Glu220, Asp251) and a bind-site of the metal ion. As for the structure of E subunit in DIIbE3 has 98% similarity with DvE3 in the amino acid sequence, even the functional part EA is absolutely same. So we direct used EA in the DvE3 as the docking basis. Fig. 2 shows the EAdomain of the template DvE3.

Fig. 2

Structure of subunitEand EA

Blue regions stead for E$(in the left picture) and MIDAS (right), respectively

In this work the effects of X, Y residues, which located in the both sides of the RGD, is tested in terms of the selectivity and stabilization of their binding. In the following docking simulation, the domain including the possible binding sites is considered. 1.2 Modeling of cyclic x-RGD-y 1.2.1 Simulation of the guest molecular structure All the guest molecular structure is built by Moderler8v2 software, the detail operating method is the same to the DIIbE3. For the short peptide of 5 amino acids, the simulation of Moderler8v2 is roughness. But the target of this operation is to connect the five amino acids; the final accurate structure can be gotten by decorating.

Mingyan Luo et al. / Chinese Journal of Biotechnology, 2008, 24(2): 297–301

1.2.2 Adding the disulfide bond Using the ‘add atom’ function in the Arguslab software, firstly the –OH is deleted in the C-terminal and the –H in the N-terminal, then two sulfide atoms are added in the appropriate position. At last the C in the C-terminal S-S, and N in the N-terminal are connected.

active part in the DIIb subunit. The ligands consisted by hydrophobic amino acids are embed in the inside of E-propeller. There only exists van-der-Waals force in the Eblade. So in the first phase, all hydrophobic amino acids residues are deleted. In the following simulation, the hydrophilic residues are placed at x and y of the cyclic x-RGD-y. A number of the hydrophilic amino acid is 12, permutation, and combinational models are 132 x-RGD-y. Docking them with the Dsubunit (E-propeller), return 1320 PDB files. Because of the active sites of this domain is not clear, so only retain the results, which bind to the outside of E-propeller, and perform the statistic analysis. Fig. 4.

Fig. 4 Binding structure of RGD and E-propeller Fig. 3

Initial structure (a) adding sulfur atoms (b) the optimal by the steepest descent method (c) by BFGS (d)

1.2.3 Optimization of the structure Every x-RGD-y has the same optimize processing with the steepest-descend optimization under the UFF force-field, repeatedly 1000 steps. Subsequently, BFGS optimization is applied, repeatedly 1000 steps. At last the minimum energy optimization is performed Fig. 3 shows the cyclic structure of x-RGD-y. 1.3 Molecular docking All docking works is performed in the Gramm service (http://www.bioinformatics.ku.edu), it can return the PDB file of the docking result. This service adopts the precision docking simulation program, searching all the possible binding manners and returning the 10 highest scored docking results.

2

Results

2.1 Docking of cyclic x-RGD-y to DIib subunit DIIb binds to ligand cyclic x-RGD-y, the main binding sites are located in the outside of E-propeller and the E -turn in every blade. But the detail sites haven’t been found. In this work, we try to discover the possible binding sites. In the simulation, all the amino acid is separated two groups: hydrophilic and hydrophobic. The results demonstrate that hydrophilic amino acids can bind to the

a): the ligand binding with the inner of ȕ-propeller, as there are 7 groups of ȕ brand in inner place, no special binding site; (b): the binding sites located in the outer of ȕ brand

All the binding sites are statistic, 124Ala, 129Trp, 200Phe, 205Thr, 206Gln, 242Leu-244His, 266Tyr, 329Gly331Gly, 387Ser, 393Gly, 401Asn, 448Phe, 453Ala, 454Val, in these amino acids, the time of binding is most, 10–20% in the all binding sites. In addition, except for 242Leu-244His and 329Gly-331Gly, all the others are the amino acids located in the both sides of the E–turn. The possibility of becoming the binding sites is small. So, if there are the binding sites in this domain, it should be located in the vicinity of 242Leu-244His and 329Gly-331Gly. Because of the structure of this subunit is based on the simulation and supposing, so in this research, we can only guarantee that the results are the most precision in this basis. The more accurate structure needs more information to prove. 2.2 Docking of x-RGD-y to E3 subunit In this simulation, 20 amino acids, after permutation and combination, get 400 x-RGD-y. As for the binding sites of this subunit is clear, so in the 4000 results, binding sites which are located in E$ and the connectional part with the next domain are deleted. In the existed active sites, we get some results and analyze them, because the ligands have only 5 amino acids, so we can study the binding sites one by one. In this processing, we exclude the wrong binding manners. Fig. 5 shows the manners of binding.

Mingyan Luo et al. / Chinese Journal of Biotechnology, 2008, 24(2): 297–301

The non-bond binding energy and hydrogen bond are two important criterions in the checking up the effect of docking. So we calculate the non-bond binding energy[11] by SPDBV and GROMOS96 software. Fig. 6 shows the energy distribution.

Fig. 5 Binding structure of RGD and EA A: no binding sites in the connection and the next domain in the theory, so this kind of binging is deleted; B: the right sites but the wrong amino acid (the open place towards up, the binding amino acid is RGD)

aspartate with negative charge, phenylalanine consist the x-RGD-y, which have the average -120kJ binding energy.

3

Prospect

In a word, the structure of DIIbE3 is built by homology simulation. The cyclic RGD was designed and computed. It’s docking to DIIbE3 in this paper. From the results of docking x-RGD-y to Dsubunit, all the binding sites that x-RGD-y binds to Dsubunit are statistic. 10-20% of x-RGD-y located in 124Ala, 129Trp, 200Phe, 205Thr, 206Gln, 242Leu-244His, 266Tyr, 329Gly-331Gly, 387Ser, 393Gly, 401Asn, 448Phe, 453Ala, 454Val. all the others are located in the both sides of the E–turn. The possibility of becoming the binding sites is small. So, if there are the binding sites in this domain, it should be located in the vicinity of 242Leu-244His and 329Gly-331Gly. This result can direct us to find those possible binding sites, and then research the property of them, which help to design the special antagonist ligand. From the non-bind energy, the most steady x-RGD-y located the active sites in the Esubunit are discovered, as the upper text said. At last, though some information has been statisticed, some other useful information may be leaked. For the experiment, the work of testing the binding sites of the receptor can be lessened a lot. Directly use the simulation result as the basis of experiments, which can save a lot time.

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