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Citation: Subramanian R, Muthurajan R, Ayyanar M (2008) Comparative Modeling and Analysis of 3-D Structure of EMV2, a
Late Embryogenesis Abundant Protein of Vigna Radiata (Wilczek). J Proteomics Bioinform 1: 401-407.
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Copyright: © 2008 Subramanian R, etal. This is an open-access article distributed under the terms of the Creative Commons Attribution
License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and
source are credited.
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Abstract
LEA proteins are ubiquitous among photosynthetic organisms and have been reported in mono- and dicot
plants as well as in nematodes, yeast, bacteria and cyanobacteria. EMV2 is a Group 1 LEA protein isolated fromVigna radiata, which is speculated to impart desiccation tolerance in plants. The homology model of this protein
was generated by using the LOOPP software based on available structural homologues in protein databases. The
final model obtained by molecular mechanics and dynamics method was assessed by PROCHECK that showed
that the final refined model is reliable. The model could prove useful in further functional characterization of this
protein.
Keywords
LEA proteins; Vigna radiate; desiccation; Homology modeling; validation
Abbreviations
SCOP-Structural Classification Of Proteins, PDB-Protein Data Bank, LOOPP -Learning, Observing and
Outputting Protein Patterns
Introduction
Late embryogenesis abundant (LEA) protein genes are
highly expressed during late stages of seed development at
normal growth condition, but many of the LEA class genes
are also frequently expressed in vegetative tissues when
plants are exposed to environmental stress (Bray et al., 2000).
Several groups of LEA protein genes have been demonstrated
to confer water-deficit and salt-stress tolerance.
On the basis of sequence similarities, LEA proteins have
been classified in six groups (Dure, 1993; Bray, 1993). Group
2 LEA proteins or dehydrins are by far the most frequently
described LEA protein family and have been classified in
distinct groups (Close, 1997) that differ in the arrangement
and number of conserved motifs: the lysine-rich repeat (KIKEKLPG) or K segment, the stretch of serine or S segment
and the V/T DEYGNP motif or Y segment. Some of
these structural motifs are predicted to form amphipathic
alpha helices, which may be important for their function in
protecting plant cells against dehydration. Evidence of functional
links between LEA protein accumulation and improved
stress tolerance of transgenic yeast and plants support this
hypothesis (Imai, 1996; Xu et al., 1996; Sivamani et al.,
2000). It was therefore proposed that most LEA and
dehydrin proteins exist as largely unfolded structures in their
native state, although a few members exist as dimers or
tetramers (Ceccardi et al., 1994; Kazuoka and Oeda, 1994).
Hydrophilicity is a common characteristic of LEA-type and
other osmotic stress-responsive proteins. Our earlier work on the occurrence of LEA proteins in the embryonic axes
of Vigna radiata (L.) Wilczek referred as EMV proteins,
the first ever report in the Fabaceae family (Manickam and
Carlier, 1980). cDNA encoding these proteins were isolated,
characterized (Manickam et al., 1996). In silico analysis of
the 20-mer motif of this EMV2 categorize this protein to
Group1 LEA and hypothesize to function as DNA/RNA
binding proteins in stabilizing membranes/macromolecules
at the time of dehydration process (Rajesh and Manickam,
2006, Gilles et al., 2007).
In the present study, effort was made to generate the
three-dimensional (3D) structure of the EMV2 protein based
on the available template structural homologues from Protein
Data Bank and SCOP databases and the model validated
with standard parameters. This study could prove
useful in further functional characterization of this important
group of proteins.
Material and Methods
Datasets
The peptide sequences of Vigna radiata, EMV2 (NCBI
GenBank accession number U31211; UniProt acc. Nos.
Q41685 ) and other sequences examined in this study were
retrieved from the public databases, http://www.ncbi.nlm.nih.gov and http://www.ebi.ac.uk. Structurally homologous subsets of the experimentally determined
3D structures of the EMV proteins were retrieved from
PDB and SCOP databases. The template used for comparative
modeling of EMV2 is a DNA Binding Protein from
Homo sapiens (1IG6_A.pdb) and a bifunctional inhibitor/
lipid-transfer protein/seed storage 2S albumin (1RZL.pdb)
with the sequence similarity of 56.44 and 56.67 % at the
loop/coiled coil regions. Similarity at these regions was considered
because LEA proteins are generally loosely structured
with predominantly random coiled regions. However
structure was further refined with sequence of 1RZL.pdb
as it showed overall 40 % sequence identity in the sequence
length of 89% to a bifunctional inhibitor/lipid transfer protein/
seed storage 2S albumin of rice.
Comparative Modeling of EMV2 Protein
Tertiary structure of the Vigna radiata LEA protein, EMV2
was modeled by submitting the deduced amino acid sequences
to the Computational Biology Service Unit, Cornell
Theory Center, Cornell University. The Atomic coordinates
for the protein models were generated by aligning to the
structural homologues in the fold recognition program of
LOOPP v3.0 server (Teodorescu et al., 2004).
Validation of EMV2 Protein Model
PROCHECK, a versatile protein structure analysis program (Laskowski et al., 1993) available at the Joint Centre
for Structural Genomics, Bioinformatics core, University of
California, San Diego; was used in validation of protein structure
and models by verifying the parameters like
Ramachandran plot quality, peptide bond planarity, Bad nonbonded
interactions, main chain hydrogen bond energy, Calpha
chirality and over-all G factor and the side chain parameters
like standard deviations of chi1 gauche minus, trans
and plus, pooled standard deviations of chi1 with respect to
refined structures( Morris et al., 1992).
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Figure 1: Predicted 3-Dimensional Structure of the EMV2 protein. Ribbon view of EMV2 structure for residues 1-99.
Numbers represent the order of helices. N and C termini of the protein are labeled.
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Results
Comparative Modeling of EMV2 Protein
Tertiary structure of a protein is build by packing of its
secondary structure elements to form discrete domains or autonomous folding units. Comparative modeling to build
3D structure of the EMV2 protein was made based on the
experimentally solved structural homologues. The amino acid
sequences of EMV2 were submitted to LOOPP server,
Cornell Bioinformatics Structural Unit (CBSU) and atomic
coordinates for the proteins were generated based on Hidden
Markov Model. The hypothetical protein models created were stored as PDB output file. The hypothetical proteins
were visualized and computed by Swiss PDB Viewer
and Rastop. The 3D structure of the proteins were represented
by cartoon display and colored based on the secondary
structure (Fig. 1).
Validation of Protein Structures of EMV2
The hypothetical protein models generated were analyzed
online by submitting to Joint Center for Structural Genomics
(JCSG), Bioinformatics core, University of California, San
Diego. Accuracy of the protein model generated was judged
by validity report generated by PROCHECK. Parameter
comparisons of these proteins were made with well-refined
structures that have similar resolution.
The main chain parameters plotted are Ramachandran
plot quality, peptide bond planarity, Bad non-bonded interactions,
main chain hydrogen bond energy, C-alpha chirality
and over-all G factor. In the Ramamchandran plot analysis,
the residues were classified according to its regions in the
quadrangle. The Ramachandran map for EMV2 (Fig. 2)
and the plot statistics (Table 1) is represented. Non-bonded
interactions check revealed the value of 30 bad contacts per 100 residues for EMV2 and the chirality measure of 0.1
for the zeta torsion angle is observed. The over-all G factor
computed for the EMV2 is 0.1, which is higher than the
ideal value (-0.4).
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Figure 2: Ramachandran map of EMV2 protein. The Plot calculation was done with PROCHECK program.
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Table 1: Ramachandran plot statistics of EMV2 computed by PROCHECK.
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The protein models were analyzed for the side chain parameters
like standard deviations of chi1 gauche minus, trans
and plus, pooled standard deviations of chi1 with respect to
refined structure. The standard deviations of Chi1 gauche
minus, trans and plus are in better range and within limits
for EMV2 hypothetical protein model.
Computations Performed in the EMV2 Model
Computation of Electrostatic Potential
The electrostatic potential of the residues in the hypothetical
protein was computed based on Coulomb’s method.
The cloud of charged residues is represented in blue and
red colors and the proteins visualized as density map of
EMV2 (Fig. 3).
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Figure 3: Electron density map of EMV2.
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Computation of Force Field Energy
Force field energy was computed for EMV2 protein model.
Positive values were observed (64x108 forEMV2). Model
refinement was done by energy minimization. Energy minimization
was carried out to reduce clashing amino acids,
using GROMOS96 force field algorithm. Decrease in the
force field energy was observed for both the protein models
after successive energy minimization (-1718 for EMV2).
The energy minimized models will however needs further
refinement in order to reduce the non-bonded interactions
for the model to be judged as a good homology model.
Discussion
3D Modeling of EMV2, LEA Protein from Vigna Radiata
Prediction of tertiary structure of a protein molecule signifies
an important step towards understanding the structure–function relationships in the concerned protein family.
Recently, the first solution structure of a LEA protein, LEA14
from Arabidopsis thaliana has been reported (Singh et al.,
2005). In the present study, model of EMV2 LEA protein of
Vigna radiata was generated from the LOOPP server, based
on the structural homologues derived from the SCOP and
protein data banks.
There exists biological sequence-structure deficit with
more than 3 lakhs protein sequences and millions of partial
nucleotide sequences, available in the public non-redundant
databases (Boguski et al., 1994); and by contrast, the number
of unique 3D structures in the protein data bank is still
less than 1500 (Attwood and Parry-Smith, 2005). The difference
of scale in sequence and structural information is
an important factor to be considered when assigning functions
to hypothetical proteins. Structure based functional
implications of such proteins have always been speculative.
Generally, under stress situations the plants may induce
formation of coiled coil / folding of the natively unfolded
proteins into more rigid structures upon binding to the partner
molecules. Since all natively unfolded proteins have
defined partner molecules that can be as small as nucleotide
or cations or a macromolecule, LEA proteins being
natively unfolded is believed to have such binding partners
to attain a rigid structures.
The LEA proteins from Vigna radiata are being classified
as Group 1 LEA protein because of its extreme hydrophilicity
and adoption of helical conformation as revealed by ab
initio secondary structure predictions, in combination with the predominant random-coiled arrangement of the residues
of Vigna LEA protein, is hypothesized to function as water
replacement molecule. Such a property may facilitate hydrogen
bonding of this EMV proteins with essentially any
macromolecular or membrane surface. However, additional
experiments on physico-chemical analyses including examination
of hydration properties of these proteins need to be
done to determine if EMV proteins can adopt certain structures
upon interaction with other macromolecules.
Structure homologues identified for these EMV proteins
show closest structural homology to proteins with helical
bundles of small proteins and DNA/RNA binding proteins.
These observations are contradictory to the earlier findings
from our group that the low molecular weight protein isolated
from Vigna was believed to be located in the cytoplasm
The structural motifs of these proteins are predicted
to form amphipathic a-helices which may be important for
their function in protecting cells against dehydration. However,
not all LEA proteins are folded and structured. Group
1 LEA proteins are reported to be very hydrophilic, loosely
structured with predominantly random-coiled structures.
These proteins are reported to form regular a-helical structure
when subjected to altered physiological conditions.
Observations from ab initio predictions of these Em proteins
of Vigna indicate 32.32% of EMV2 proteins attains
helical conformation as represented by helical blocks in the
3D hypothetical models (Manickam and Carlier, 1980).
Temperature-induced extended helix/random coil transition
was reported for a Group 1 LEA protein from soybean.
These proteins are by native, largely unstructured but attained
6- 14% helical conformation under temperature stress
or at high salt concentrations (Soulages et al., 2002). Similar
reports from (Goyal et al. 2003) for AavLEA1, a Group
3 LEA protein from the nematode, Aphelenchus avenae indicate
oligomerization of these proteins in immunoblotting
and cross-linking experiments, however majority of these
proteins was found to be monomeric in analytical ultracentrifugation
and gel filtration studies. Also, formation of
a-helical structures on drying was reported in partially characterized
protein from Typha latifolia, probably a Group 3
LEA protein based on Fourier transform-Infra red (FT-IR)
spectroscopy studies (Wolkers et al., 2001).
The LEA proteins from Vigna radiata are being classified
as Group 1 LEA protein because of its extreme hydrophilicity
and adoption of helical conformation as revealed by ab
initio secondary structure predictions, in combination with
the predominant random-coiled arrangement of the residues
of Vigna LEA protein, is hypothesized to function as water replacement molecule. Such a property may facilitate hydrogen
bonding of this EMV proteins with essentially any
macromolecular or membrane surface. However, additional
experiments on physico-chemical analyses including examination
of hydration properties of these proteins need to be
done to determine if EMV proteins can adopt certain structures
upon interaction with other macromolecules.
Validation of the Model
The hypothetical protein model generated was subjected
to structure validation, for testing the accuracy of the model.
The quality of the final ensemble of conformers was assessed
using PROCHECK, a protein structure validation
program. The visual displays of the models were performed
with either the Swiss PDB viewer (Guex and Peitsch, 1997)
or RasTop (Sayle and Milner-white, 1995).
Stereochemical parameters of the proteins like main-and
side chains data of EMV2 was considered for determining
the quality of the model. The main chain parameters like
Ramachandran plot quality; peptide bond planarity, C-alpha
chirality and over-all G factor are found to be within the
limits for the model. However, the bad contacts per 100
residues are high. The side chain parameters are in better
range and within the limits for EMV2. These parameters
are compared to essentially satisfy the generated models
with well-refined structures at similar resolution as described
by (Morris et al. 1992).
The validation reports for the protein models are analyzed,
and energy minimization of the models was made after
checking the force field energy of the models. For a model
to be validated based on quality, a good quality protein model
should have 90% or more residues in the most favored regions
of quadrangle in the Ramachandran plot. In the generated
model of EMV2, the distribution of residues in the
most favored regions is 87.6 and 93 %, respectively. This
infers that EMV2 as good hypothetical protein model. Also,
a tertiary structure of a protein can be worth considering
only from its solution structures, obtained from the experimentations
using either NMR or crystallographic studies.
The homology model of mungbean LEA proteins, thus generated
in this study, could some extent stimulate investigations
at determining the mechanistic function of this stress
associated proteins.
Future Perspectives
Functional implications based on the structural homologues
hypothesize EMV2 to act as a water replacement molecule.
LEA proteins generally are reported to posses multiple functions
like salt, drought, heat and cold tolerance. Hence, the present study will be useful in further in vitro studies by
over expression in model systems like E.coli or yeast cells
and the recombinant protein can be subjected to Salinity,
Cold shock, thermal stability analysis and the stress induced
structural changes can be monitored to ascertain the possible
functions of this important class of proteins.
Acknowledgements
S.Rajesh is grateful to the Council of Scientific and Industrial
Research, New Delhi for grant of Research Fellowship.
We are grateful to the crews of NCBI, EBI, MRC
Lab-UK and SIB for making computational biology data/
tools publicly available.
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