Three Dimensional Structure Modeling and Analysis of Ribulose 1,5-Bisphosphate Carboxylase of Hypericum perforatum

A. Ashish Gupta1, B. Anshika Malaviya1, C. Sharada M. Potukuchi2, and D. Jitendra Narayan1 1 Amity Institute of Biotechnology, Amity University, Noida,U.P., India 2 School of Biotechnology, Shri Mata Vaishno Devi University, Katra, J&K, India

Abstract - Ribulose 1,5 bisphosphate carboxylase (RuBP Carboxylase) is an enzyme that is used in the Calvin cycle to catalyze the first major step of carbon fixation, it catalyzes either the carboxylation or the oxygenation of ribulose-1,5 bisphosphate with carbon dioxide or oxygen. RuBP Carboxylase is very important in terms of biological impact because it catalyzes the most commonly used chemical reaction by which inorganic carbon enters the biosphere. Using homology modelling the three dimensional structure of RuBP Carboxylase has been designed with the help of known three dimensional (3D) structure of a crystal form enzyme from Nicotiana tabacum. The reliability of the model was confirmed by obtaining scores from variety of inslico scoring tools, including Verify 3D, Errat. The 3-D model of RuBP Carboxylase predicted from this study has been successfully submitted into Protein Model Database (PMDB ID PM0075653), which can provide a platform to further study this important plant enzyme/protein. Keywords: St. Johns wort, homology modeling, PMDB, Nicotiana tabacum, 3D

One of the earliest discoveries of RuBP Carboxylase made by Wildman & Bonner [7], of a major protein in green leaves called Fraction I protein due to its electrophoretic homogeneity. Several reviews on the properties of the RuBP Carboxylase have since appeared [8, 9, 10, 11] and much is now known concerning the biosynthesis of the enzyme and its genetic regulation by both nuclear and chloroplast genomes [12]. As a genetic marker it has been useful for probing many nucleocytoplasmic relationships in plants. It has also proven to be an excellent tool to follow the evolution of autotrophy [13]. Homology modeling (HM), also known as comparative modeling, is a class of methods for constructing an atomic resolution model of a protein from its amino acid sequence i.e. "query sequence" or "target [14]. HM can produce high quality structural models when the target and template are closely related. HM is becoming a useful technique in the field of bioinformatics because knowledge of the 3-D structure of a protein would be an invaluable aid to understand the details of a particular protein [15, 16]. Since the structure of this important plant protein has not been reported, the main aim of this study was to predict the 3-D structure of RuBP Carboxylase and establish some important analysis.

Introduction

In recent years Hypericum perforatum, also known as St Johns Wort, has received special attention due to its pharmacological properties, including antiseptic, spasmolytic, tonic, diuretic, and anesthetic remedies [1, 2]. Extracts from this plant are utilized in the treatment of mild to moderately severe depression [3, 4]. P27SJ which is isolated from this plant have an important role in regulation of HIV-1 genome [5]. RuBP Carboxylase is very important in terms of biological impact because it catalyzes the most commonly used chemical reaction by which inorganic carbon enters the biosphere. It is also the most abundant protein in leaves, and it may be the most abundant protein on earth. Given its important role in the biosphere, there are efforts currently to genetically engineer crop plants so as to contain more efficient RuBP Carboxylase. It is a key enzyme that converts atmospheric carbon to food and supports life on this planet. Its low catalytic activity and specificity for oxygen leads to photorespiration, severely limiting photosynthesis and crop productivity [6].

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2.1

Material and Method

Retrieval of Target sequence

The amino acid sequence of the RuBP Carboxylase, was obtained from the sequence database of NCBI (accession no.: AAL35668.1, entry name RuBP Carboxylase, Hypericum perforatum and GeneID: 17135961). It was ascertained that the 3-D structure of the protein was not available in PDB (Protein Data Bank) http://www.rcsb.org/pdb/home/home.do hence the present exercise of developing the 3-D model of RuBP Carboxylase of Hypericum perforatum was undertaken. The protein consists of 467 amino acids.

2.2

Template Searching

An attempt was made to find a suitable template protein for the modeling of the target protein. The template protein

was searched through Geno3D server [17], http://geno3dpbil.ibcp.fr/cgi-bin/d3_geno3d2.pl (an online tool for searching similar sequences, based on sequence and structure-wise similarity). From the homology searching DING protein of Nicotiana tabacum 4RUB Chain A [18] was selected as template protein.

2.3

Sequence Alignment

A, pdb|4RUB|A, which is a crystal form of RuBP Carboxylase from Nicotiana tabacum in the activated state, was selected as template (Figure 1). For 4RUB, E value 0.0, identity 94% and alignment score 904 bits was obtained. MODELLER 9v5 was used for building the model and global energy minimization. The sequence was obtained from sequence database and was submitted to blastp search. After the BLAST analysis [26], modeller software was used to build the models.

Amino acid sequence alignment of target and template proteins was derived using the Swiss-PdbViewer package [19]. Default parameters were applied and the aligned sequences were inspected and adjusted manually to minimize the number of gaps and insertions.

2.4

Rough Model

Rough 3-D models were constructed from the sequence alignment between RuBP Carboxylase and the template proteins using MODELLER 9v5 [20] with parameters of energy minimization value.

2.5

Refinement

The rough model constructed was solvated and subjected to constraint energy minimization with a harmonic constraint of 100 kJ/mol/2, applied for all protein atoms, using the steepest descent and conjugate gradient technique to eliminate bad contacts between protein atoms and structural water molecules. Computations were carried out in vacuo with the GROMOS96 43B1 [21] parameters set, implementation of Swiss-PdbViewer.

2.6

Evaluation of Refined Model

Figure 1. Alignment between template and target sequence shows that most residues are conserved and target sequence is aligned upto its full length. With the help of MODELLER software 20 models were generated, by using PROCHECK 3.0 and for each model (pdb file) 10 plots were obtained and after analysis the best model was selected. Refined model thus obtained was analyzed by different protein analysis programs including PROCHECK for the evaluation of the Ramachandran plot quality (Table 1, Figure 2) it was found that maximum residues of this protein lies in the most favored region. WHATIFserverhttp://swift.cmbi.ru.nl/servers/html/index.html for the proline puckering analysis of the refined model showed that all proline puckers were in normal range [27]. Table 1. Ramachandran Plot Statistics for Best Model. Number of Percentage of Residues Amino amino acids acids involved involved

In the last step of HM the refined structure of the model was subjected to series of tests for testing its internal consistency and reliability. Backbone conformation was evaluated by the inspection of the Psi/Phi Ramachandran plot obtained from PROCHECK analysis. The Swiss-PdbViewer energy minimization test was applied to check for energy criteria in comparison with the potential of mean force derived from a large set of known protein structures. Packing quality of the refined structure was investigated by the calculation of PROCHECK Quality Control value [22].

Conclusion

From the prot param tool it was found that molecular weight of RuBP Carboxylase was 51851.9 and theoretical pI ( iso electric point) was 6.14 [23]. In general, 30% sequence homology is required for generating useful models. Here, the sequence alignment score was 2852 as calculated by ClustalW2 [24]. Based on the results obtained from mGenTHREADER [25] program and geno3D server, the X-ray structure of the Chain

Residues in most favoured region [A,B,L] Residues in additional allowed region [a,b,l,p] Residues in generously allowed regions [~a,~b,~l,~p] Residues in disallowed regions Number of non-glycine and non proline residues Number of end-residues (excl. Gly and Pro) Number of glycine residues (shown as triangles) Number of proline residues Total number of residues

373 19 1

94.2% 4.8% .2%

3 396 1 49

.8% 100.0%

21 467

Figure 3. - Ribbon form representation of 3D structure of modelled protein. By using Yasara (Yet Another Scientific Artificial Reality Application) http://www.yasara.org/ ribbon representation. The overall model quality was calculated by using ProSA server(ProteinStructureAnalysis)https://prosa.services.came.s bg.ac.at/prosa.php. From this analysis Z score for the refined model was -10.05 obtained [29], for calculation of packing quality. This structure (Figure 3; PMDB ID - PM0075653 http://mi.caspur.it/PMDB/ for the corresponding coordinates in pdb format) was found to be satisfactory based on the above results [30]. Insilico study of proteins and nucleic acids are helpful in almost all research fields. It not only saves money but also valuable time. Bioinformatics is commonly used in drug design and molecular modelling. The model generated for RuBP Carboxylase in the present study has been successfully submitted in PMDB (Protein Model Database) with PMDB ID PM0075653. The 3D structure of RuBP Carboxylase may be further used in characterizing the protein in wet laboratory experimentations.

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Figure 2. Ramachandran Plot generated by PROCHECK (3.0) for best model shows that maximum residues lies in the most favored region. From the SAVES server analysis (Structural Analysis and Verification Server) http://nihserver.mbi.ucla.edu/SAVES; Verify3D result for the refined model showed that 92.95% of the residues had an averaged 3D-1D score greater than 0.2 and the overall quality factor for the refined model was 75.381 [28].

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