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Explication of interactions between HMGCR isoform 2 and various statins through In silico modeling and docking
Computers in Biology and Medicine 42 (2012) 156–163
Contents lists available at SciVerse ScienceDirect
Computers in Biology and Medicine journal homepage: www.elsevier.com/locate/cbm
Explication of interactions between HMGCR isoform 2 and various statins through In silico modeling and docking M.V.K. Karthik a, M.V.K.N. Satya Deepak b, Pratyoosh Shukla a,n a b
Enzyme Technology Laboratory, Department of Biotechnology, Birla Institute of Technology, Mesra, Ranchi 835215, Jharkhand, India Department of Pharmacy, SASTRA University, Thanjavur, Tamilnadu
a r t i c l e i n f o
a b s t r a c t
Article history: Received 29 April 2011 Accepted 7 November 2011
The enzyme 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGCR) catalyzes the conversion of HMG-CoA to mevalonate, a four-electron oxidoreduction that is the rate-limiting step in the synthesis of cholesterol and other isoprenoids. This study was designed to understand the mode of interactions of HMGCR isoform 2 with other statins. Hence, ligands such as Atorvastatin (DB01076), Lovastatin (DB00227), Fluvastatin (DB01095), Simvastatin (DB00641), Pravastatin (DB00175), Rosuvastatin (DB01098) and Cerivastatin (DB00439) were docked with enzymes HMGCR isoform 1 (pdb: 1DQ8) and modeled HMGCR isoform 2 (gi9196049380). Our homology modeling results were further processed to model the structure of human HMGCR isoform 2 and its accuracy was confirmed through RMS Z-scores (1.249). These interactions revealed that binding residues such as Arg515, Asp516, Tyr517 and Asn518 are found to be conserved in HMGCR isoform 2 with various statins. Our studies further concluded that Atorvastatin is most efficient inhibitor against both the isoforms of HMGCR whereas HMGCR isoform 2 shows less effectiveness with statins when compared with HMGCR isoform 1. & 2011 Elsevier Ltd. All rights reserved.
Keywords: Statins Docking Homology modeling HMGCR isoform 1 HMGCR isoform 2
1. Introduction The coronary artery diseases are one of the major health concerns in the contemporary world. 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGCR) inhibitors are revealed as plasma cholesterol lowering molecules. The innovation of statins had substantial impact on reducing the worldwide burden of cardiovascular disease. Earlier to statin treatment, lowering of elevated LDL cholesterol level was not easy even though clinical practice guidelines were generally recommended [1,2]. HMGCR is attached in the membrane of the endoplasmic reticulum and is long considered to have seven transmembrane domains. The active site of HMGCR is located in a long carboxyl terminal domain in cytosol. Moreover, some recent studies show it to comprise eight transmembrane domains [3]. HMGCR catalyzes the conversion of hydroxymethyl-glutarylCoA to mevalonate (EC 1.1.1.34) or vice versa (EC 1.1.1.82; in mevalonate-feeding bacteria). In eukaryotes, HMGCR catalyzes the rate-limiting reaction of isoprenoid biosynthesis and is the main target of the favorite cholesterol-lowering drugs, the statins [3,4].
n
Corresponding author. Tel.: þ91 9431171157; fax: þ91 651 2275401. E-mail addresses: [email protected], [email protected] (P. Shukla). 0010-4825/$ - see front matter & 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.compbiomed.2011.11.003
The reaction catalyzed by HMGCR is represented here [5] (S)-HMG-CoAþ2NADPHþ2H þ -(R)-mevalonate þ 2NADP þ þ CoA-SH. The above reaction occurs in subsequent three steps: Step 1: HMG-CoA þNADPHþH þ -[Mevaldyl-CoA]þNADP þ Step 2: [Mevaldyl-CoA]-[Mevaldehyde]þCoA-SH Step 3: [Mevaldehyde]þNADPH þH þ -Mevalonate þNADP þ The homologous isoforms of these enzymes are reported to have accompanying functions in specified sub-regions of the endoplasmic reticulum [6]. Keller et al. (1985) shows evidences indicating that mammals have a second organelle-specific HMGCR, which is restricted to peroxisomes [7]. HMGCR activity was detected for the first time in liver peroxisomes of rat identified by immunoelectron microscopy and enzyme assay. In this report the activity in peroxisome was reported as less than 5% of the total HMGCR in the cells in control animal. However it was observed that after treatment with cholestyramine it reached up to 30% [8]. Few other findings about the presence of peroxisomal HMGCR are noteworthy [9,10] and it is also well reported in other epithelial tissues of hamster ovary (CHO cells) [11]. By the detailed analysis and observations of the peroxisomal HMGCR activity it was noted that it may be coded by a second
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Fig. 1. Figure showing the involvement of peroxisomal HMGCR in mevalonate pathway (Reproduced from Krisans et al., 2002 [10] with his kind permission).
Fig. 2. Phylogenetic dendrogram for various HMGCR, red block highlighted represents HMGCR isoforms from Homo sapiens.
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independent gene. After the detection of HMGCR in peroxisomes it was showed that there is huge difference of specific activity of peroxisomal HMGCR. In this study they reported that cholestyramine treatment produced a 6–7 fold increase in peroxisomal HMGCR activity, whereas the activity increased only by 2-fold in microsomal HMGCR [8]. It was also demonstrated that the increase in activity is paralleled by an increase in immunolabeling of peroxisomes by HMGCR antibodies. A comprehensive overview by displaying the activity and immunoreactivity of HMGCR in microsome and peroxisomes has different diurnal rhythms is also well reported in literature [12]. Moreover, Aboushadi et al. (2000) provided additional incidental confirmations for a second peroxisome-specific gene for HMGCR [13]. They proved that peroxisomal reductase activity cannot be altered by inhibitors of cellular phosphatase. It is also noted that peroxisomal enzyme degradation is not enhanced by mevalonate and its degradation is not blocked by N-acetylleu-leu-norleucinal. This is more resistant to inhibition by statins [13]. Peroxisomal activity is decreased by mevalonate [14]. Involvement of peroxisomal HMGCR in mevalonate pathway is represented in Fig. 1, which clearly shows that peroxisomal activity of several enzymes takes place immediately downstream of HMGCR in the isoprenoid biosynthesis pathway [15]. In the present study we modeled HMGCR isoform 2, which plays an important role in mevalonate pathway along with HMGCR isoform 1. Efficacy of various statins was performed with both the HMGCR isoforms and important binding residues of HMGCR isoform were elucidated.
Fig. 3. (a) Systematic ribbon view representation of 3D structure of HMGCR isoform 2 of H. sapiens. (b) Structure composition of modeled HMGCR isoform 2 showing different specifications in a form of pie chart. (c) Detailed structure composition where different color represents different type of structure.
Table 1 Results obtained from SWISS-MODEL server for modeled for Hydroxymethylglutaryl-CoA reductase isoform2 structure. Structure and sequence specifications
Results
NCBI Gene identity Number of HMGCR Isoform 2 protein sequences Sequence identity PDB ID of the template retrieved for modeling Modeled residue range Final total energy in kJ/mol (modeling log)
gi9196049380 87.264% 2q6bA 441–811 12429.743
2. Material and methods Protein sequence of HMGCR isoforms (gi94557643 and gi9196049380) of Homo sapiens were obtained from NCBI (http://www.ncbi.nlm.nih.gov/) and pdb structure of HMGCR isoform 1 (pdb: 1DQ8) was retrieved from RSCB protein data bank (http://www.pdb.org/pdb/home/home.do). Statins viz. Atorvastatin (DB01076), Lovastatin (DB00227), Fluvastatin (DB01095), Simvastatin (DB00641), Pravastatin (DB00175), Rosuvastatin (DB01098) and Cerivastatin (DB00439) were downloaded from Drugbank (www.drugbank.ca/). 2.1. Phylogenetic analysis Sequences of similarity was obtained from BlastP by taking HMGCR isoforms as a template and their multiple sequence alignment is done by using ClustalX 2.0 [16]. Using TreeView 1.6.6 [17] phylogenetic tree was analyzed. Conserved regions from HMGCR isoforms of Homo sapiens were identified by ClustalX alignment. 2.2. Homology modeling Homology modeling was performed with the SWISS-MODEL online server for automated protein homology modeling (http:// swissmodel.expasy.org) [18–22]. The resulting structure that coordinates with the HMGCR isoform 2 model was obtained as an online article from SWISS-MODEL online server. Structure obtained from homology modeling was verified by PROCHECK [23] and PROVE [24]. PROCHECK checks the stereo chemical quality of a protein structure and analyzes residue by residue geometry from overall structure geometry. PROVE calculates the volumes of atoms in HMGCR isoform 2 using an algorithm, which treats the atoms like hard spheres and calculates a statistical Z-score deviation. For the accuracy of the structure Z-score should be 0, most of the time if it is negative it means worse than average and positive better than average. An RMS Z-score should be close to 1.0.
Fig. 4. Ramachandran’s Map of HMGCR isoform 2 model from Homo sapiens. The Plot calculation was done with PROCHECK program.
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The detailed view of HMGCR isoform 2 model structure was obtained using STRIDE software [25]. The baseline homology model resulting structures were used as an input for docking calculations. 2.3. Molecular docking Hex 5.1 docking program [26] was used for protein ligand docking calculations. In Hex’s calculations each molecule resembled 3D parametric functions describing surface shape, electrostatic charge and potential distributions. So, electrostatic and van-der-Waals interactions are taken into account in our calculations. By mutual overlapping score for parametric functions, the expression for docking score is derived as function of the six degrees of freedom in rigid body docking search. With the appropriate scaling factors, this docking score could be interpreted as interaction energy to minimize the simulation. In this study we used various statins viz. Atorvastatin, Cerivastatin, Fluvastatin, Lovastatin, Pravastatin, Rosuvastatin and Simvastatin were taken as ligands and docked with modeled HMGCR isoform 1 and HMGCR isoform 2. Amino acids involved in interactions were identified through docking.
3. Results and discussions: The objective of this work was characterization of HMGCR isoform 2 involved in the conversion of HMG-CoA to mevalonate in peroxisomes. From our results of docking we found HMGCR isoform 2 showed mild interaction with statins when compared with HMGCR isoform 1. For validation of evolutionary status of the HMGCR isoform 2, we constructed a phylogenetic tree using gi9196049380. A rooted phylogenetic tree with a unique node corresponding to the most recent common ancestor was found using the evolutionary analysis study. Danio rerio isoform-A shows close relation of 77.9% with HMGCR isoform 1 and 77.3% with HMGCR isoform 2 (Fig. 2). Model construction by the ProModII program [27] included complete backbone and side chain building, loop building, verification of model quality, including packing and subsequent energy minimization, using the Gromos96 force field [28]. The stereo-chemical and energetic parameters of the initial 3D protein models were evaluated by the WHATCHECK [29], PROSAII [30], ANOLEA [31] and Verify3D [32] analysis reports provided by the SWISS-MODEL server (Fig. 3). The ribbon presentation of structural models were built using SWISSMODEL server, based on crystal structure of 2q6bA (Protein Data Bank code). Different colored ribbons represent different peptide helices. Structure composition of modeled HMGCR isoform 2 shows different specifications in a form of pie chart. Data generated from SWISS-MODEL for HMGCR isoform 2 is represented in Table 1. The structure obtained contains 165 alpha helix and 67 turns and is verified by Ramachandran Plot (PROCHECK) where 87.9% residues are in favored region and where RMS Z-score is nearly 1 i.e. 1.249 using PROVE (Figs. 4 and 5) (Table 1). Interaction energies for the docked-complexes were calculated by Hex 5.1 and summarized in Table 2. A higher value of negative interaction energy is an indicator of more efficient interaction between the HMGCR isoforms and various statins. This shows that HMGCR isoform 2 has less interaction with statins when compared with HMGCR isoform 1. From the binding site interaction between HMGCR isoform 2 and various statins we found common residues Arg515, Asp516, Tyr517 and Asn518 involved in binding and is summarized in Table 3. Binding site interaction between HMGCR isoform 1 and various statins were represented
Fig. 5. Validation of HMGCR isoform 2 modeled structure using PROVE where it calculates the volume of atoms in HMGCR isoform 2 using an algorithm, which treats the atoms like hard spheres and calculates a statistical Z-score deviation.
Table 2 Interaction energies of HMGCR isoform 1 and HMGCR isoform 2 with various statins. Statins
Isoform 1 E-shape
Atorvastatin Cerivastatin Lovastatin Pravastatin Rosuvastatin Simvastatin Fluvastatin
Isoform 2 E-force
E-total (kJ/mol)
161.14 545.95 707.09 202.60 206.04 408.64 102.68 432.91 535.59 105.19 370.33 475.52 175.05 269.55 444.60 119.53 348.30 467.82 138.11 310.21 448.32
E-shape
E-force
E-total (kJ/mol)
185.80 75.69 129.48 127.31 139.08 40.73 138.47
110.14 169.71 105.22 111.47 92.51 197.90 86.41
295.94 245.40 234.70 238.78 231.58 238.63 224.88
in Table 4. Binding interaction of HMGCR isoform 1 and HMGCR isoform 2 with statins was represented in Figs. 6 and 7.
4. Conclusion In the present study, we have successfully carried out the enzyme modeling of hydroxymethyl-glutaryl-CoA reductase isoform2 and further its structure was verified through various bioinformatics software viz. PROCHECK and PROVE. There are few recent studies on Streptococcus pneumoniae HMGCR and interaction studies between Streptococcus pneumoniae HMGCR and four
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Table 3 Interaction table of HMGCR isoform 2 with various statins. Atorvastatin
Cerivastatin
Fluvastatin
Lovastatin
Pravastatin
Rosuvastatin
Simvastatin
Tyr514 Arg515 Asp516 Tyr517 Asn518
Tyr514 Arg515 Asp516 Tyr517 Asn518
Tyr514 Arg515 Asp516 Tyr517 Asn518
Tyr514 Arg515 Asp516 Tyr517 Asn518
Arg515 Asp516 Tyr517 Asn518
Tyr514 Arg515 Asp516 Tyr517 Asn518 Tyr519
Tyr514 Arg515 Asp516 Tyr517 Asn518
Ala706 Ile707 Ile709 Ala710
Ala706
Ala706
Ala710
Ile709 Ala710
Ile709 Ala710
Ile709 Ala710 Leu759 Pro760 Ala763
Cys764 Met767
Met767
Cys764 Met767
Cys764 Met767
Leu759 Pro760 Ala763 Cys764
Table 4 Interaction table of HMGCR isoform 1 with various statins. Atorvastatin
Cerivastatin
Fluvastatin
Lovastatin
IIe536 Ala556 Thr557 Thr558 Glu559
Ala556 Thr557 Thr558 Glu559 Gly560 Leu562
Leu562 Leu584 Ala585 Trp698 IIe699 Ser705 Val707
Pravastatin
Rosuvastatin IIe536 Ala556 Thr557 Thr558 Glu559
Simvastatin
Ala556 Thr557 Thr558 Glu559 Gly560
Leu562
Leu584 Trp698
Val707 Cys708 Lys735 Ser740 Ala743 Ser745 IIe746 Gly747 Gly748 Tyr749 Asn750 Ala751 His752 Ala753 Ala754 Asn755
Asn753 Ala754
Ala754 Asn755 IIe756 Thr758 Ala759
Thr758 Ala759 IIe762 Asn771
Ala751 His752
His752
Ala754 Asn755
Ala754 Asn755
Thr758
Thr758 Ala759
Asn771 Asn771 Ser775
Val772 Ser775 Asn776 Cys777 IIe778 Thr779 Leu780 Glu782 Tyr792 IIe793 Ser794 Cys795 Thr796 Pro798
IIe778 Leu780 Tyr792 Ser794 Thr796 Pro798 Leu853
Leu853
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Fig. 6. Systematic representation of Binding site of HMGCR isoform 1 with various statins generated by Discovery studio 2.5.
annonaceous acetogenins revealing new inhibitor candidates of the Streptococcus pneumonia II HMGCR verified through kinetic studies and some noteworthy structure-activity studies on hepatoselective HMGCR inhibitors [33,34]. Few more docking studies are also conducted for HMGCR inhibitors [35]. Adding to this in
our studies the Ramachandran plot obtained through PROCHECK shows that 87.9% residues are in favored region and RMS Z-score was to be 1.249 (nearly 1), which shows the accuracy and efficacy of our model. Our molecular docking studies also reported HMGCR isoform 2 is less interactive to various statins as
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Fig. 7. Systematic representation of Binding site of HMGCR isoform 2 with various statins generated by Discovery studio 2.5.
compared to HMGCR isoform 1. Hence the present study concludes that binding site residues viz. Arg515, Asp516, Tyr517 and Asn518 were found to be conserved in the interaction of HMGCR isoform 2 with various statins. Nevertheless more kinetic and structure-activity studies are required in HMGCR isoform 2 towards achieving success in statin therapy.
Acknowledgments We hereby acknowledge BTIS Sub-DIC, DBT, Government of India and Department of Agriculture, Government of Jharkhand, India for financial support to our department towards infrastructure facilities.
Mr. MVKN Satya Deepak acknowledges Dr. P. Shukla for the help and support towards completion of his virtual project. References [1] M.D. Bethesda, Third Report of the National Cholesterol Education Program (NCEP) Expert Panel on Detection, Evaluation, and Treatment of High Blood Cholesterol in Adults (Adult Treatment Panel III): Executive Summary: National Institutes of Health. National Heart, Lung, and Blood Institute, National Cholesterol Education Program, NIH Publication no. 01-3670, 2001, pp. 40. [2] National Collaborating Centre for Primary Care, NICE Clinical Guideline 67: Lipid Modification, National Institute for Health and Clinical Excellence, London, 2010, pp. 38. [3] M. Farnier, J. Davignon, Current and future treatment of hyperlipidemia: the role of statins, Am. J. Cardiol. 82 (1998) 3J–10J.
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MVK Karthik is pursuing M.Sc. (Bioinformatics) from Birla Institute of Technology, Mesra, Ranchi, India. His current research interests are molecular docking, protein interactions, simulation studies and pathway modeling.
MVKN Satya Deepak is M. Pharm. (Pharmacology) working for an online project with Dr. P. Shukla who is associated with Birla Institute of Technology, Mesra, Ranchi, India. His working area is on clinical trials for lowering of CRP levels and protein interactions.
Pratyoosh Shukla is Associate Professor at Department of Biotechnology, Birla Institute of Technology, Mesra, Ranchi, India. His field of research is enzyme technology and protein bioinformatics.
Contents lists available at SciVerse ScienceDirect
Computers in Biology and Medicine journal homepage: www.elsevier.com/locate/cbm
Explication of interactions between HMGCR isoform 2 and various statins through In silico modeling and docking M.V.K. Karthik a, M.V.K.N. Satya Deepak b, Pratyoosh Shukla a,n a b
Enzyme Technology Laboratory, Department of Biotechnology, Birla Institute of Technology, Mesra, Ranchi 835215, Jharkhand, India Department of Pharmacy, SASTRA University, Thanjavur, Tamilnadu
a r t i c l e i n f o
a b s t r a c t
Article history: Received 29 April 2011 Accepted 7 November 2011
The enzyme 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGCR) catalyzes the conversion of HMG-CoA to mevalonate, a four-electron oxidoreduction that is the rate-limiting step in the synthesis of cholesterol and other isoprenoids. This study was designed to understand the mode of interactions of HMGCR isoform 2 with other statins. Hence, ligands such as Atorvastatin (DB01076), Lovastatin (DB00227), Fluvastatin (DB01095), Simvastatin (DB00641), Pravastatin (DB00175), Rosuvastatin (DB01098) and Cerivastatin (DB00439) were docked with enzymes HMGCR isoform 1 (pdb: 1DQ8) and modeled HMGCR isoform 2 (gi9196049380). Our homology modeling results were further processed to model the structure of human HMGCR isoform 2 and its accuracy was confirmed through RMS Z-scores (1.249). These interactions revealed that binding residues such as Arg515, Asp516, Tyr517 and Asn518 are found to be conserved in HMGCR isoform 2 with various statins. Our studies further concluded that Atorvastatin is most efficient inhibitor against both the isoforms of HMGCR whereas HMGCR isoform 2 shows less effectiveness with statins when compared with HMGCR isoform 1. & 2011 Elsevier Ltd. All rights reserved.
Keywords: Statins Docking Homology modeling HMGCR isoform 1 HMGCR isoform 2
1. Introduction The coronary artery diseases are one of the major health concerns in the contemporary world. 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGCR) inhibitors are revealed as plasma cholesterol lowering molecules. The innovation of statins had substantial impact on reducing the worldwide burden of cardiovascular disease. Earlier to statin treatment, lowering of elevated LDL cholesterol level was not easy even though clinical practice guidelines were generally recommended [1,2]. HMGCR is attached in the membrane of the endoplasmic reticulum and is long considered to have seven transmembrane domains. The active site of HMGCR is located in a long carboxyl terminal domain in cytosol. Moreover, some recent studies show it to comprise eight transmembrane domains [3]. HMGCR catalyzes the conversion of hydroxymethyl-glutarylCoA to mevalonate (EC 1.1.1.34) or vice versa (EC 1.1.1.82; in mevalonate-feeding bacteria). In eukaryotes, HMGCR catalyzes the rate-limiting reaction of isoprenoid biosynthesis and is the main target of the favorite cholesterol-lowering drugs, the statins [3,4].
n
Corresponding author. Tel.: þ91 9431171157; fax: þ91 651 2275401. E-mail addresses: [email protected], [email protected] (P. Shukla). 0010-4825/$ - see front matter & 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.compbiomed.2011.11.003
The reaction catalyzed by HMGCR is represented here [5] (S)-HMG-CoAþ2NADPHþ2H þ -(R)-mevalonate þ 2NADP þ þ CoA-SH. The above reaction occurs in subsequent three steps: Step 1: HMG-CoA þNADPHþH þ -[Mevaldyl-CoA]þNADP þ Step 2: [Mevaldyl-CoA]-[Mevaldehyde]þCoA-SH Step 3: [Mevaldehyde]þNADPH þH þ -Mevalonate þNADP þ The homologous isoforms of these enzymes are reported to have accompanying functions in specified sub-regions of the endoplasmic reticulum [6]. Keller et al. (1985) shows evidences indicating that mammals have a second organelle-specific HMGCR, which is restricted to peroxisomes [7]. HMGCR activity was detected for the first time in liver peroxisomes of rat identified by immunoelectron microscopy and enzyme assay. In this report the activity in peroxisome was reported as less than 5% of the total HMGCR in the cells in control animal. However it was observed that after treatment with cholestyramine it reached up to 30% [8]. Few other findings about the presence of peroxisomal HMGCR are noteworthy [9,10] and it is also well reported in other epithelial tissues of hamster ovary (CHO cells) [11]. By the detailed analysis and observations of the peroxisomal HMGCR activity it was noted that it may be coded by a second
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Fig. 1. Figure showing the involvement of peroxisomal HMGCR in mevalonate pathway (Reproduced from Krisans et al., 2002 [10] with his kind permission).
Fig. 2. Phylogenetic dendrogram for various HMGCR, red block highlighted represents HMGCR isoforms from Homo sapiens.
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independent gene. After the detection of HMGCR in peroxisomes it was showed that there is huge difference of specific activity of peroxisomal HMGCR. In this study they reported that cholestyramine treatment produced a 6–7 fold increase in peroxisomal HMGCR activity, whereas the activity increased only by 2-fold in microsomal HMGCR [8]. It was also demonstrated that the increase in activity is paralleled by an increase in immunolabeling of peroxisomes by HMGCR antibodies. A comprehensive overview by displaying the activity and immunoreactivity of HMGCR in microsome and peroxisomes has different diurnal rhythms is also well reported in literature [12]. Moreover, Aboushadi et al. (2000) provided additional incidental confirmations for a second peroxisome-specific gene for HMGCR [13]. They proved that peroxisomal reductase activity cannot be altered by inhibitors of cellular phosphatase. It is also noted that peroxisomal enzyme degradation is not enhanced by mevalonate and its degradation is not blocked by N-acetylleu-leu-norleucinal. This is more resistant to inhibition by statins [13]. Peroxisomal activity is decreased by mevalonate [14]. Involvement of peroxisomal HMGCR in mevalonate pathway is represented in Fig. 1, which clearly shows that peroxisomal activity of several enzymes takes place immediately downstream of HMGCR in the isoprenoid biosynthesis pathway [15]. In the present study we modeled HMGCR isoform 2, which plays an important role in mevalonate pathway along with HMGCR isoform 1. Efficacy of various statins was performed with both the HMGCR isoforms and important binding residues of HMGCR isoform were elucidated.
Fig. 3. (a) Systematic ribbon view representation of 3D structure of HMGCR isoform 2 of H. sapiens. (b) Structure composition of modeled HMGCR isoform 2 showing different specifications in a form of pie chart. (c) Detailed structure composition where different color represents different type of structure.
Table 1 Results obtained from SWISS-MODEL server for modeled for Hydroxymethylglutaryl-CoA reductase isoform2 structure. Structure and sequence specifications
Results
NCBI Gene identity Number of HMGCR Isoform 2 protein sequences Sequence identity PDB ID of the template retrieved for modeling Modeled residue range Final total energy in kJ/mol (modeling log)
gi9196049380 87.264% 2q6bA 441–811 12429.743
2. Material and methods Protein sequence of HMGCR isoforms (gi94557643 and gi9196049380) of Homo sapiens were obtained from NCBI (http://www.ncbi.nlm.nih.gov/) and pdb structure of HMGCR isoform 1 (pdb: 1DQ8) was retrieved from RSCB protein data bank (http://www.pdb.org/pdb/home/home.do). Statins viz. Atorvastatin (DB01076), Lovastatin (DB00227), Fluvastatin (DB01095), Simvastatin (DB00641), Pravastatin (DB00175), Rosuvastatin (DB01098) and Cerivastatin (DB00439) were downloaded from Drugbank (www.drugbank.ca/). 2.1. Phylogenetic analysis Sequences of similarity was obtained from BlastP by taking HMGCR isoforms as a template and their multiple sequence alignment is done by using ClustalX 2.0 [16]. Using TreeView 1.6.6 [17] phylogenetic tree was analyzed. Conserved regions from HMGCR isoforms of Homo sapiens were identified by ClustalX alignment. 2.2. Homology modeling Homology modeling was performed with the SWISS-MODEL online server for automated protein homology modeling (http:// swissmodel.expasy.org) [18–22]. The resulting structure that coordinates with the HMGCR isoform 2 model was obtained as an online article from SWISS-MODEL online server. Structure obtained from homology modeling was verified by PROCHECK [23] and PROVE [24]. PROCHECK checks the stereo chemical quality of a protein structure and analyzes residue by residue geometry from overall structure geometry. PROVE calculates the volumes of atoms in HMGCR isoform 2 using an algorithm, which treats the atoms like hard spheres and calculates a statistical Z-score deviation. For the accuracy of the structure Z-score should be 0, most of the time if it is negative it means worse than average and positive better than average. An RMS Z-score should be close to 1.0.
Fig. 4. Ramachandran’s Map of HMGCR isoform 2 model from Homo sapiens. The Plot calculation was done with PROCHECK program.
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The detailed view of HMGCR isoform 2 model structure was obtained using STRIDE software [25]. The baseline homology model resulting structures were used as an input for docking calculations. 2.3. Molecular docking Hex 5.1 docking program [26] was used for protein ligand docking calculations. In Hex’s calculations each molecule resembled 3D parametric functions describing surface shape, electrostatic charge and potential distributions. So, electrostatic and van-der-Waals interactions are taken into account in our calculations. By mutual overlapping score for parametric functions, the expression for docking score is derived as function of the six degrees of freedom in rigid body docking search. With the appropriate scaling factors, this docking score could be interpreted as interaction energy to minimize the simulation. In this study we used various statins viz. Atorvastatin, Cerivastatin, Fluvastatin, Lovastatin, Pravastatin, Rosuvastatin and Simvastatin were taken as ligands and docked with modeled HMGCR isoform 1 and HMGCR isoform 2. Amino acids involved in interactions were identified through docking.
3. Results and discussions: The objective of this work was characterization of HMGCR isoform 2 involved in the conversion of HMG-CoA to mevalonate in peroxisomes. From our results of docking we found HMGCR isoform 2 showed mild interaction with statins when compared with HMGCR isoform 1. For validation of evolutionary status of the HMGCR isoform 2, we constructed a phylogenetic tree using gi9196049380. A rooted phylogenetic tree with a unique node corresponding to the most recent common ancestor was found using the evolutionary analysis study. Danio rerio isoform-A shows close relation of 77.9% with HMGCR isoform 1 and 77.3% with HMGCR isoform 2 (Fig. 2). Model construction by the ProModII program [27] included complete backbone and side chain building, loop building, verification of model quality, including packing and subsequent energy minimization, using the Gromos96 force field [28]. The stereo-chemical and energetic parameters of the initial 3D protein models were evaluated by the WHATCHECK [29], PROSAII [30], ANOLEA [31] and Verify3D [32] analysis reports provided by the SWISS-MODEL server (Fig. 3). The ribbon presentation of structural models were built using SWISSMODEL server, based on crystal structure of 2q6bA (Protein Data Bank code). Different colored ribbons represent different peptide helices. Structure composition of modeled HMGCR isoform 2 shows different specifications in a form of pie chart. Data generated from SWISS-MODEL for HMGCR isoform 2 is represented in Table 1. The structure obtained contains 165 alpha helix and 67 turns and is verified by Ramachandran Plot (PROCHECK) where 87.9% residues are in favored region and where RMS Z-score is nearly 1 i.e. 1.249 using PROVE (Figs. 4 and 5) (Table 1). Interaction energies for the docked-complexes were calculated by Hex 5.1 and summarized in Table 2. A higher value of negative interaction energy is an indicator of more efficient interaction between the HMGCR isoforms and various statins. This shows that HMGCR isoform 2 has less interaction with statins when compared with HMGCR isoform 1. From the binding site interaction between HMGCR isoform 2 and various statins we found common residues Arg515, Asp516, Tyr517 and Asn518 involved in binding and is summarized in Table 3. Binding site interaction between HMGCR isoform 1 and various statins were represented
Fig. 5. Validation of HMGCR isoform 2 modeled structure using PROVE where it calculates the volume of atoms in HMGCR isoform 2 using an algorithm, which treats the atoms like hard spheres and calculates a statistical Z-score deviation.
Table 2 Interaction energies of HMGCR isoform 1 and HMGCR isoform 2 with various statins. Statins
Isoform 1 E-shape
Atorvastatin Cerivastatin Lovastatin Pravastatin Rosuvastatin Simvastatin Fluvastatin
Isoform 2 E-force
E-total (kJ/mol)
161.14 545.95 707.09 202.60 206.04 408.64 102.68 432.91 535.59 105.19 370.33 475.52 175.05 269.55 444.60 119.53 348.30 467.82 138.11 310.21 448.32
E-shape
E-force
E-total (kJ/mol)
185.80 75.69 129.48 127.31 139.08 40.73 138.47
110.14 169.71 105.22 111.47 92.51 197.90 86.41
295.94 245.40 234.70 238.78 231.58 238.63 224.88
in Table 4. Binding interaction of HMGCR isoform 1 and HMGCR isoform 2 with statins was represented in Figs. 6 and 7.
4. Conclusion In the present study, we have successfully carried out the enzyme modeling of hydroxymethyl-glutaryl-CoA reductase isoform2 and further its structure was verified through various bioinformatics software viz. PROCHECK and PROVE. There are few recent studies on Streptococcus pneumoniae HMGCR and interaction studies between Streptococcus pneumoniae HMGCR and four
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Table 3 Interaction table of HMGCR isoform 2 with various statins. Atorvastatin
Cerivastatin
Fluvastatin
Lovastatin
Pravastatin
Rosuvastatin
Simvastatin
Tyr514 Arg515 Asp516 Tyr517 Asn518
Tyr514 Arg515 Asp516 Tyr517 Asn518
Tyr514 Arg515 Asp516 Tyr517 Asn518
Tyr514 Arg515 Asp516 Tyr517 Asn518
Arg515 Asp516 Tyr517 Asn518
Tyr514 Arg515 Asp516 Tyr517 Asn518 Tyr519
Tyr514 Arg515 Asp516 Tyr517 Asn518
Ala706 Ile707 Ile709 Ala710
Ala706
Ala706
Ala710
Ile709 Ala710
Ile709 Ala710
Ile709 Ala710 Leu759 Pro760 Ala763
Cys764 Met767
Met767
Cys764 Met767
Cys764 Met767
Leu759 Pro760 Ala763 Cys764
Table 4 Interaction table of HMGCR isoform 1 with various statins. Atorvastatin
Cerivastatin
Fluvastatin
Lovastatin
IIe536 Ala556 Thr557 Thr558 Glu559
Ala556 Thr557 Thr558 Glu559 Gly560 Leu562
Leu562 Leu584 Ala585 Trp698 IIe699 Ser705 Val707
Pravastatin
Rosuvastatin IIe536 Ala556 Thr557 Thr558 Glu559
Simvastatin
Ala556 Thr557 Thr558 Glu559 Gly560
Leu562
Leu584 Trp698
Val707 Cys708 Lys735 Ser740 Ala743 Ser745 IIe746 Gly747 Gly748 Tyr749 Asn750 Ala751 His752 Ala753 Ala754 Asn755
Asn753 Ala754
Ala754 Asn755 IIe756 Thr758 Ala759
Thr758 Ala759 IIe762 Asn771
Ala751 His752
His752
Ala754 Asn755
Ala754 Asn755
Thr758
Thr758 Ala759
Asn771 Asn771 Ser775
Val772 Ser775 Asn776 Cys777 IIe778 Thr779 Leu780 Glu782 Tyr792 IIe793 Ser794 Cys795 Thr796 Pro798
IIe778 Leu780 Tyr792 Ser794 Thr796 Pro798 Leu853
Leu853
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Fig. 6. Systematic representation of Binding site of HMGCR isoform 1 with various statins generated by Discovery studio 2.5.
annonaceous acetogenins revealing new inhibitor candidates of the Streptococcus pneumonia II HMGCR verified through kinetic studies and some noteworthy structure-activity studies on hepatoselective HMGCR inhibitors [33,34]. Few more docking studies are also conducted for HMGCR inhibitors [35]. Adding to this in
our studies the Ramachandran plot obtained through PROCHECK shows that 87.9% residues are in favored region and RMS Z-score was to be 1.249 (nearly 1), which shows the accuracy and efficacy of our model. Our molecular docking studies also reported HMGCR isoform 2 is less interactive to various statins as
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Fig. 7. Systematic representation of Binding site of HMGCR isoform 2 with various statins generated by Discovery studio 2.5.
compared to HMGCR isoform 1. Hence the present study concludes that binding site residues viz. Arg515, Asp516, Tyr517 and Asn518 were found to be conserved in the interaction of HMGCR isoform 2 with various statins. Nevertheless more kinetic and structure-activity studies are required in HMGCR isoform 2 towards achieving success in statin therapy.
Acknowledgments We hereby acknowledge BTIS Sub-DIC, DBT, Government of India and Department of Agriculture, Government of Jharkhand, India for financial support to our department towards infrastructure facilities.
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MVK Karthik is pursuing M.Sc. (Bioinformatics) from Birla Institute of Technology, Mesra, Ranchi, India. His current research interests are molecular docking, protein interactions, simulation studies and pathway modeling.
MVKN Satya Deepak is M. Pharm. (Pharmacology) working for an online project with Dr. P. Shukla who is associated with Birla Institute of Technology, Mesra, Ranchi, India. His working area is on clinical trials for lowering of CRP levels and protein interactions.
Pratyoosh Shukla is Associate Professor at Department of Biotechnology, Birla Institute of Technology, Mesra, Ranchi, India. His field of research is enzyme technology and protein bioinformatics.