L.F. Castillo et al. (eds.), Advances in Computational Biology, Advances in Intelligent Systems and Computing 232,

29

DOI: 10.1007/978-3-319-01568-2_5, © Springer International Publishing Switzerland 2014

Analysis of Binding Residues between PDGF-BB and Epidermal Growth Factor Receptor:

A Computational Docking Study

Ricardo Cabezas, Daniel Torrente, Marco Fidel Avila, Jannet González*, and George Emilio Barreto

School of Sciences, Department of Nutrition and Biochemistry, Pontificia Universidad Javeriana

janneth.gonzalez@javeriana.edu.co, ricardocabe@gmail.com

Abstract. A directed docking was performed using Cluspro between human PDGF-BB and EGFR using specific templates obtained from PDB. Various con-served residues were found to be involved in the docking interaction of the com-plex by means of hydrophobic interactions and hydrogen bonds. An electrostatic potential evaluation of the PDGF-BB-EGFR complex was also performed to vali-date if the complex is electrostatically complementary in the binding area. Results suggested a possible binding mechanism which could explain the in vivo evidence of formation of heterodimeric receptors EGFR-PDGFR.

Keywords: PDGF-BB, EGFR, docking, Transactivation, interactions.

1 Introduction

The human EGF receptor (EGFR) is a transmembral glycoprotein of 1186 amino acids [1,2], that belongs to the ErbB family 2 which consist of four members: The epidermal growth factor receptor (EGFR, also referred to as ErbB1 or Her1),3 ErbB2 (p185Neu or Her2),4 ErbB3 (Her3)5 and ErbB4 (Her4),6 all of which have been iden-tified to be either over-expressed, amplified or altered in a wide range of human epi-thelial tumours [3]. EGFR has a 622 amino acid extracellular region, with 4 domains (I–IV), also known as the L1, S1, L2, and S2 domains respectively [4]. EGF binding by EGFR occurs at Domains I and III, which share 37% amino acid identity, while domains II and IV are homologous Cys-rich domains, denoted CR1 and CR2, respectively [5].

The transmembrane domain IV is involved in both receptor dimerization, as EGF binding [6]. This domain IV, includes residues 480-614 and contains a transmem-brane domain of 23 amino acids starting at Ile-622. EGFR binding to the receptor, activates receptor dimerisation, internalisation and autophosphorylation and down-stream signal transduction pathways including the RAS/MAP kinase, the prosurvival

* Corresponding author.

30 R. Cabezas et al.

phosphatidylinositol 3-kinase (PI-3K)/Akt, the PLCγ and the JAK-STAT pathways [7]. Aditionally, EGFR can be activated by other receptor tyrosine kinases including insulin-like growth factor-1 receptor, adhesion molecules (e.g. E- cadherin and inte-grins) and G-protein-coupled receptors (GPCR). Furthermore, it has been noticed that the EGFR is able to interact with other receptors and perform transactivavion signal-ing by virtue of the fact that it does not require binding of its ligand for tyrosine kinase activity and dimerization [8,9].

Previous research have shown that in certain types of tumors such as glioblasto-ma, Mul tiforme have shown overexpression of both EGFR and PDGFR (platelet derived growth factor) that can contribute to the malignant phenothype of glioblas-toma [10,11]. Importantly, both EGFR and PDGF receptors have been reported to be associated and exert its signaling via caveolin-containing membranes (caveolae) in the presence of ligand [12]. Interestingly these authors also founded that pretreatment of cells with EGF prevented PDGF-dependent phosphorylation of PDGF receptors and ERK1/2 kinase activation and that cells pretreated with PDGF prevent EGF de-pendent phosphorylation of EGF receptors and ERK1/2 kinase activation, suggesting that both receptors are physically located in the same membrane domains and an hete-rologous desensitization of the other receptor by sequestration of cholesterol caveolin-containing membranes [12]. Moreover, a recent study suggested that PDGFRA, PDGFRB, and EGFR were expressed and activated, in high levels of EGFR expression inducing an autocrine loop activation of these receptors along with their coactivation [13].

Furthermore, other studies have shown that the epidermal growth factor creceptor (EGFR) may become transactivated by specific ligands of other membrane creceptors, such as angiotensin II, carbachol, thrombin, endothelin and others [9,14]. Additionally various researchers have shown that the EGFR becames transactivated in response to PDGF [9,15-17]. This last study, performed in vascular smooth muscle cells from rat also shows the formation of PDGFBR-EGFR heterodimers, induced by PDGF-BB, which are important for cell migration processes [9].

Taken together, these results suggest a fundamental cross talk between these tyrosine kinase coupled receptors, in signal transduction [9]. However, at the present moment there is no information regarding the possible interaction between the PDGF-BB and the EGFR at the molecular level. The structural analysis of complexes between EGFR in complex with PDGFB is unknown, due to the lack of suitable crys-tal structure and difficulties of protein multimerization in solution [1,9,18]. In the present study, molecular modeling, protein–protein docking simulations and docking validation with bioinformatics tools were assessed to predict the structure of PDGF-B and its interaction with EGFR. Furthermore, the interacting residues of the EGFR-PDGFB complex were analyzed, suggesting the existence of a possible binding between the PDGF-B and the EGFR, which could be of importance in a biological contex.

Analysis of Binding Residues between PDGF-BB and Epidermal Growth Factor Receptor 31

2 Materials and Methods

2.1 Data Set

The crystallized structures of PDGF-BB and EGFR, 1PDG and 3NJP respectively [1,6,18] were obtained from the PDB at the Broohaven National Laboratory [19], the details of which are given in Table 1. The EGFR structure was solved with < 3.3A˚ resolutions and the PDGF-BB was solved with < 3.0A˚. These are the best templates for modeling the PDGF-BB and EGFR as on date.

2.2 Protein–Protein Docking

We used ClusPro’s [20] algorithm for obtaining the bound structures of EGFR receptor with PDGF-BB. ClusPro has the option of selecting either DOT or ZDOCK to perform rigid body docking and both are based on the fast Fourier transform (FFT) correlation techniques [20]. We selected DOT for our work, since this selection al-lows for the use of an static potential in the scoring function and on the surface com-plementarity between the two structures. DOT runs on a 128A˚ × 128A˚ × 128A grid, using a grid spacing ˚ of 1 Å. It performs 13 000 rotations, initially obtaining over 2.7×10 10 structures and finally retaining only 20 000 structures with the best surface complementarity scores. All the 20 000 docked structures are then filtered using dis-tance-dependent electrostatics and an empirical potential energy. The 2000 conforma-tions retained after filtering is clustered based on the pairwise RMSD (root mean square deviation) and selects the best conformational structure. Finally, the represent-ative conformation selected is refined using CHARMM minimization [21,22]. An initial set of 30 conformations were obtained, based on the coefficient weights following the application of the formula of Kozakov et. al. 2010:

E=0.40Erep−0.40Eatt+600Eelec+1.00EDARS

Biochemical and literature data was used to filter the docking results, using the resi-dues on the surface of the interacting proteins as candidates, as well the experimental information on PDGFRβ-PDGFB and EGFR-EGF complexes previously reported [1,6,18].

2.3 Docking Validation

The best structural model for the complexes EGFRα-PDGFB obtained from the dock-ing analysis were validated using the following methods: Ramachandran plots from Mol Probity [23], and PROCHECK [24] were employed to examine the stereochem-ical quality of the built model. Results indicates that more than 76.7% of the residues have phi and psi angles in the most favored regions, 21.1% in additionally allowed regions, 1.3% in generally allowed regions and 0.8% in disallowed regions. An

32 R. Cabezas et al.

overall G-factor of –0.09 indicates a good quality of the built model. Additionally, the residues in the disallowed regions were located outside of the binding pocket, and so, no further action was taken to further improve the backbone folding of these residues.

2.4 Docking Interface Analysis

Protein-protein interactions were analyzed with LigPlot+ v1.4 [25], using the DIMPLOT program for protein-protein interactions. This program shows hydrophob-ic and hydrogens bonds interactions between proteins. Electrostatic potential surface and interactions of the complexes were calculated and represented by using Pymol v1.5 [26]. Electrostatic potential surface and interactions of the complexes were calculated and represented by using Pymol v1.5 [26].

3 Results

3.1 Sequence Analysis of Human PDGF-BB and EGFR

Initially a BLAST [27] was performed in the RefSeq database to the NCBI human PDGFB. Found 3 access numbers for human PDGFB, reported for June 2011: CAG46606, CAG46606.1 and NP_002599.1. These correspond to the PDGF protein to establish a sequence of 241 amino acids which is accurate to that reported by the Uniprot database with the accession number P01127.

Meanwhile NP_002599.1 sequence corresponds to the preprotein form of PDGF-B. This was the most recent sequence (July, 2012) reported for the PDGF-B and for this reason, further analysis was conducted with this sequence. During maturation, the PDGF is a secreted protein, suffers removing its signal peptide of 20 amino acids and 2 additional regions located at the N terminus (61 amino acids) and the C terminus (51 amino acids). The mature form of this growth factor, corresponds to the central region of the preprotein, and has a size of 109 amino acids. EGFR sequence was ob-tained after performing a search against the whole PDB to select the templates which could be used to generate receptor model. Information regarding the templates are summarized in table 1.

Table 1. Detailed information of the human EGFR and PDGFBB used in this work as a templates

No of aminoacids

Molecular weight

Source organism

Swiss-Pro accession number

PDB ID

EGFR 1210 120,692 Homo sapiens Q504U8 3NJP

PDGF-BB 109 30 Homo sapiens P01127 1PDG

Analysis of Binding Residues

3.2 Sequence Edition o

The crystallized structures were initially edited in Pymchains, glycosylated residuthe QMEAN6 from Swisscomposite scoring functionmodel edited in Pymol and in a model. The results shofor the modified EGFR (fifrom 0 to 1.

Fig. 1. Modified structure of troman numeration, along with

Fig. 2. Modified structure of mers are in grey and darker gre

3.3 PDGF-BB Docked

Based on the output of ClPDGF complex showed a gwas chosen based on factorand stability of the model.PDGF-BB-EGFR complex cally complementary in th

between PDGF-BB and Epidermal Growth Factor Receptor

of Human PDGF-BB and EGFR Templates

of PDGF-BB and EGFR, 1PDG and 3NJP respectivmol [26] for the elimination of redundant and unnecesses and hydrogens. To validate the edited models, we u-model structure assessment tool [28,29]. QMEAN6 i

n used for the estimation of the global quality of the enfor the local per-residue analysis of different regions w

owed a value of 0.546 for the PDGF-BB (fig. 1) and 0.6g. 2). Those are relatively good scores in a scale rang

the EGFR (From Lu et al 2010). The four domains are showthe predicted site for EGF binding.

the EGFR (From Oefner et al 1992). Different PDGF-B moey. The cysteine knot dimerization domain is shown.

with EGFR

lusPro’s [20] algorithm, we report here that the EGFgood docking behavior (fig. 3). The selected docking mors such as the area of surface contact, extent of interacti. Additionally, an electrostatic potential evaluation of was performed to validate if the complex is electrost

he binding area. Our results show that the EGFR, in

33

ely, sary used is a

ntire with-

696 ging

wn in

ono-

FR-odel ions the

tati-the

34 R. Cabezas et al.

interacting regions of domakT/e and -36.616 kT/e; ) (+60.813 kT/e and -60.813 with the EGFR (fig. 4).

3.4 Interacting Residue

The Protein-protein interacractions but present some hcomplex PDGFB-EGFR ar

Fig. 3. Structure of the dockindomain. Predicted binding sitregion for PDGF-BB are show

Fig. 4. Electrostatic potential 58.551 kT/e ] .The darked colracts with EGFR.

ains I and III (fig. 3) has strong negative charges (+36. 6and might be electrostatic complementary to PDGF- kT/e) due to the positive charges in the interacting reg

es

ctions of the solved complex are mostly hydrophobic inhydrogen bond interactions. The residues implicated in re: ASN 128, SER 127, LYS 86, ASP 155, ILE 13, L

ng between PDGF-BB and the EGFR. At the EGFR extracellte of EGF at domain I (orange color) and the putative bind

wn.

generated for PDGF-BB. Electrostatic scale [+58.551 kT/e anlored structure represents the positive charge, where PDGFs i

616 BB

gion

nte-the

LEU

lular ding

nd -inte-

Analysis of Binding Residues

Fig. 5. PDGF-B binding to th(PDGF-B chain B). Terminal 151 by polar contact ( dotted li

Fig. 6. PDGF-B binding to (PDGF-B chain A). Terminal by polar contact (yellow dotted

14, THR 15, TRP 40, TYRPHE 84. ARG 73, LYS 10sults show that SER 127, A105 are present in the hydrimportance of both hydropPDGF-BB to EGFR. The spin fig. 5 and the specific int

4 Discussion

The main purpose of this sthomodimer with the EGFscope of these results is to the best protein conformatio

PDGF is a disulfide-linklypeptide chains A and B,

between PDGF-BB and Epidermal Growth Factor Receptor

he EGFR. Interaction between ASN 151 (EGFR) and LYSNitrogen of the LYS 81 interacts with an oxygen of the Aines) of 2.48 Å.

the EGFR. Interaction between TYR 42 (EGFR) and ILEoxigen of the TYR 42 interacts with an hydrogen of the ILE

d lines) of 2.3 Å.

R 45, ASN 151, LYS 81, ARG 125, LEU 69, TYR 15, GLN 71, SER92, GLU90, ASN91, PRO82, ILE77.

ASP 155, LYS 86, LYS 81, ASN 151, GLN 71 and Logen bond formation within this complex, suggesting

phobic interactions and hydrogen bond for the bindingpecific interaction between ASN 151 and LYS 81 is shoteraction between TYR 42 and ILE 13 is shown in fig. 6

tudy was to determine the complex structure of PDGF-FR, based on previously reported structures. The ove

consider computationally a suitable protocol for predictonal structure using resolved templates from the PDB. ked dimeric protein, which is composed of two related expressed from different genes and assembled as homo

35

S 81 ASN

E 13 E 13

101, Re-

LYS the

g of own 6.

-BB erall ting

po-o or

36 R. Cabezas et al.

heterodimers [30]. Active PDGF exhibits multiple forms and ranges in size from 28-35 kDa [31]. The most studied isoforms of PDGF; AA, BB and AB, have been shown to bind with different affinities to homo- and heterodimers of their receptor gene products, denoted a and B [18,32]. Depending on ligand configuration and the pattern of receptor expression, different receptor dimers may form, including homodimers and heterodimers of PDGFA-R and PDGFB-R [33].

Previous research has shown that all PDGFs have a highly conserved conserved Cystine knot-fold growth factor domain of approximately 100 amino acids (aa), called the PDGF/VEGF homology domain [34]. This domain, denoted the PDGF/ VEGF homology domain, is involved in forming inter- and intramolecular disulphide bridges to form the PDGF dimers that activate the receptor [35].

PDGFs act via two RTKs (PDGFR-α and PDGFR-β) which share common domain structures, including five extracellular immunoglobulin (Ig) loops and an intracellular tyrosine kinase (TK) domain. This structure is shared with other receptors like VEGFR, c-Fms, c-Kit, and Flt3 [34]. Ligand binding promotes receptor dimeriza-tion, which initiates signaling through various pathways became activated including: Mitogen activated protein kinase (MAPK), PI3K phosphatidylinositol 3- kinase, PLC-y, Src TK, and activation of transcription factors such as STATs, ELK-1, c-jun/c-fos and NFkB [34,36].

Our results show that PDGF-B homodimer shares a large group of residues in the interaction with EGFR (ASN 128, SER 127, LYS 86, ASP 155, ILE 13, LEU 14, THR 15, TRP 40, ASN 151, LYS 81, ARG 125, LEU 69, TYR 101, PHE 84. ARG 73, LYS 105, GLN 71, SER92, GLU90, ASN91, PRO82, ILE77), Previous mutation-al studies have shown that mutations in ILE 30 and ARG 27, reduce the binding interaction between PDGF-BB and it´s receptor PDGF-bR. Furthermore, the study of Ostman et al., [37], indicates that residues from segment 73-77, specially Arg73 and Ile77, are involved in receptor binding. Oefner et al. [18] had also reported that posi-tively charged amino acid residues have an important role in the binding of PDGF-BB to its receptors, including residues from the loop III (Val78-Arg79-Lys8O-Lys81) which are conserved in the human PDGF-BB homodimers. Finally, the crystallo-graphic structure of Oefner et al [18], recognizes an hydrophobic patch, adjacent to loop I, that includes Arg27, Leu38, Pro82 and Phe84 which is also conserved in the PDGF-A sequence.

Interestingly, in the present study, we have reported ILE13, ARG73, ILE77, LYS 81, PRO82 and PHE84, as part of the binding interactions between PDGF-BB and EGFR. These results suggests a possible conserved motif in the interaction between PDGFB and EGFR which could be important in the binding of PDGF-BB with other receptors such as PDGFRα, that can associate with PDGFRβ and form the heterodimeric PDGFRαβ.

It has been previously reported that both hydrophobic interactions and hydrogen bonds play an important role in the recognition of the PDGFR to its ligand [35], Our results show that both of these interactions are important for the binding interaction of PDGF-BB to EGFR. Importantly, both of these interactions are also important in the interaction between EGF and its receptor [1]. It´s important to notice that many tyro-sine kinase receptors including PDGFR or EGFR are often able to form heterodimeric

Analysis of Binding Residues between PDGF-BB and Epidermal Growth Factor Receptor 37

or hybrid receptors with differential affinity for their ligands [38,39]. Regarding EGFR, in addition to its cognate ligands, this receptor has been reported to become activated by stimuli that do not directly interact with the EGFR ectodomain including other RTK agonists, chemokines, cytokines GPCR ligands, and cell adhesion ele-ments that involves important transactivation mechanisms [40]. The existence of PDGFBR-EGFR heterodimers have been previously reported in a vascular model from rat [9]. In it, EGFR transactivation does not require PDGFBR kinase activity, and the disruption of this heterodimeric receptor significantly inhibited PDGF-mediated ERK activation, which could have an important effect in biological processes such as cell migration cell during wound healing [9,14].

Results presented in this study, shown the possibility of an interaction between EGFR and PDGF-BB in the interacting region of the EGFR (domains I and III), by means of conserved aminoacids from the PDGF homology domain (cysteine Knot). Additionally, these results may be used as an output for a better understanding of the signaling mechanism exerted by the PDGF-B in the context of human pathologies that shown aberrant expression of the PDGF and EGF ligands and receptor such as brain diseases or cancer. Further biological information will be needed in order to corroborate this model and it´s regulation over cell signaling.

5 Conclusions

A directed docking was performed using Cluspro between human PDGF-BB and EGFR using the templates 1PDG and 3NJP from PDB. Various conserved residues were found to be involved in the docking intereaction of the complex. Additionally, an electrostatic potential evaluation of the PDGF-BB-EGFR complex was performed to validate if the complex is electrostatically complementary in the binding area. Results suggested a possible binding mechanism which could explain the in vivo evidence of formation of heterodimeric receptors EGFR-PDGFRB.

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