Download COMPARATIVE MODELING AND MOLECULAR

COMPARATIVE MODELING AND MOLECULAR DYNAMICS SIMULATION STUDY
OF MAMMALIAN ASPARTYLASPARTYL-tRNA SYNTHETASES
Waqasuddin Khan, Rabia Sattar and Zaheer-ul-Haq
Dr. Panjwani Center for Molecular Medicine and Drug Research, International Center for Chemical & Biological Sciences, University of Karachi, Pakistan.
Email: [email protected]
RESULTS & DISCUSSION
ABSTRACT
COMPUTATIONAL METHODS
The Aspartyl-tRNA synthetase (AspRS) belonging to the ligase family of enzymes has an
important role not only in the protein fidelity by specifically recognizing its cognate amino
acid but also in the aminoacylation of tRNAAsp. Several crystal structures of AspRS have
been determined. None of these structures is mammalian and yet there in no structural
information available about mammalian AspRS. The recognition of homology between
protein sequences provides valuable information about the biological behavior and
biochemical function of uncharacterized sequences. The homology modeling was done
by using yeast AspRS-tRNAAsp-ATP complex structure as our template. The resultant
models have excellent stereochemistry and a C-alpha trace similar to the crystal
structure. Molecular dynamics (MD) simulations were also performed to study the
conformational changes in the active site when an ATP molecule resides in the AspRS to
accomplish the first aminoacylation step. MD simulations reproduced some of the key
hydrogen bonds observed crystal structure. The rmsf graphs show most movements in
the catalytic site and in the flipping loop region while the main secondary structure
maintained the fairly stable conformations.
The homology modeling was performed by SYBYL® (version 7.3). Validation of
protein structures was determined by PROCHECK, VERIFY3D and ERRATA.
Molecular dynamics simulations (MD) was performed by AMBER and trajectory
analayis was studied by Ptraj command.
INTRODUCTION
Different catalytic strategies adapted by enzymes make proteins all-rounder. One such
enzyme is “Aminoacyl-tRNA Synthetases”, whose commitment to genetic translation is
interesting to explore. Aminoacyl-tRNA synthetases (aaRSs) constitute a family of
cytosolic enzymes of class ligases that play a vital role in protein biosynthesis. The
organization of aaRSs in mammalian cells as a supramolecular assemblage may reveal
the evolutionary pressure on the organization of protein biosynthetic machinery. This
ubiquitous assemblage consists of 11 polypeptide subunits. This complex comprises 9
aminoacyl-tRNA synthetases particular for their corresponding amino acid of class I and
class II tRNA synthetases, that are monomers (IleRS, LeuRS, MetRS, GlnRS, ArgRS)
and dimmers (LysRS, AspRS), and a bifunctional polypeptide (GluProRS), which
recently was found out to exist as fusion protein (and three auxiliary proteins of 18, 38
and 43kDa.The aspartate system offers a comprehensive explanation of the diverse
states that exists for an aminoacylation system of class II synthetases. The Yeast
Aspartyl-tRNA synthetase is a homodimeric protein enzyme (one monomer = 557 amino
acids) with a molecular weight range of 125 kDa.
Elucidation of structural determinants of ATP binding specificity is very crucial to gain the
structure-function relationship. As currently no structure data are available for
mammalian aspartyl tRNA synthetase (AspRS), this work reveals the homology
modeling and molecular dynamics (MD) simulation study of Homo sapiens and Mus
musculus AspRSs. In the order to understand the mechanism of ATP-induced
conformational changes, we used the molecular dynamics (MD) simulation method.
Analysis at different time frames revealed the persistence of motions in flipping loop
region (203-209) in both species during ATP-binding.
SYBYL uses Biopolymer module operated by FUGUE method to
find similarity between a target sequence and the sequence of
proteins of known structures
Biopolymer works with ORCHESTRAR suite of applications which
is specifically designed for homology protein modeling
Fig.2: From left to right; Cartoon representation of yeast AspRS, Homo sapiens AspRS and Mus musculus AspRS showing the domain architecture. Fig.6: RMSF for each C-alpha atoms of Homo sapiens AspRS (Black line) and Mus musculus AspRS (Red line).
The highly mobile flipping loop, the hinge region and some other important regions are highlighted. We are
interested in the flipping loop region and to some extent to hinge region which accounts for the twisting of or
The geometrical and structural consistencies of both the modeled
proteins were evaluated by the Procheck program
noticeably flexibility of both domain.
Further minimizations and MD simulations were performed to study
the interacting active site residues using the AMBER suite of
programs
Fig. 3: C‐alpha based superimposition of the template and target models. The red, yellow and blue ribbon representations show the yeast, Homo sapiens STEREOCHEMICAL PROPERTIES
OF MODELED PROTEINS
and Mus musculus AspRSs, respectively. From left to right; motif 1, motif 2 and motif 3.
Fig.7
Fig.7: Variations of the gyration radii for the different systems: Homo sapiens AspRS (Black line) and Mus musculus (Red line).
CONCLUSION
Fig.4: Flipping loop movement deviation between the 1EOV (open state‐red ribbon) and the two predicted 3D models of AspRSs (closed state‐ yellow; Homo sapiens, blue; Mus musculus). The amino acid residues having the CPK representation show conserved substitution.
MULTIPLE SEQUENCE ALIGNMENT
Fig.5: Active site of the mammalian AspRSs showing the class II specific key amino acid residues with its bound substrate ATP. The ATP is shown in ball and stick model. The amino acid residues (represented as their color IDs) are involved in the substrate
positioning. In silico-developed homology protein structure modeling builds a threedimensional model of a given protein sequence based on its similarity to one
or more known structures belonging to the same member of a protein family.
The purpose of this study is strongly related to a drug discovery strategy
against yeast AspRS to cease the uncontrolled growth of yeast causing
infections. Candida, a genus of yeast, of which Candida albicans is the most
occurring species, is responsible for Candidiases. Candidiases includes a
wide range of yeast infections, including mycosis. Recently, the probable
sequence of candida albicans AspRS is revealed. This sequence has 69%
with another yeast species, Saccharomyces cerevisiae AspRS, the only
protein molecule with its x-ray determined 3D crystal structure. This 3D
AspRS was used as a template to guide the exploration of our homolog
models. Since the sequence identity in both yeast AspRSs is high, it would
be a better opportunity for us to take 1ASZ as our template. In order to
develop a drug targeting yeast AspRS against yeast infections, likeness
between eukaryotic AspRSs creates a problem to drug discovery. Drug
should be well enough conformationally intelligent that it would be able to
identify its true target rather than inhibiting the host system. So, the predicted
drug could be well enough to discriminate between these two closely related
protein molecules.
Fig.1 : From top to bottom; Compatibility score of verify3D of Homo sapiens AspRS and Mus musculus : From top to bottom; Compatibility score of verify3D of Homo sapiens AspRS and Mus musculus AspRS, and errata error value plot of Homo sapiens AspRS and Mus musculus AspRS.
ATP
contacts
Regions
AspRS Homo
sapiens
AspRS Mus
musculus
1ASZ
(Template)
% of residues in most favored regions
83.5
85.2
91.0
% residues in most additional allowed regions
13.9
12.8
8.3
% of residues in generously allowed regions
1.2
0.5
0.7
% of residues in disallowed regions
1.4
1.4
0.0
Table 1. Results of PROCHECK of evaluation by Modeled Protein Structures
ATP
ATP
ATP
ATP
ATP
ATP
Protein
contacts
OP2 α
N1
N6
O3’
O3’
O2’
Arg273
Leu283
Leu283
Ile425
Gly472
Arg475
Nη 1
NH
CO
CO
NH
Nη 1
Donor-acceptor
distances (Predicted this
work)
Homo sapiens Mus
musculus
AspRS
AspRS
2.64
2.21
3.39
3.38
2.72
2.52
2.74
1.82
3.68
3.65
2.55
2.30
Predicted
(Ruff et
al., 1994)
2.64
3.34
2.83
2.73
3.80
2.77
Accuracy (%)*
Homo sapiens Mus
musculus
AspRS
AspRS
100
84
98.5
100
96
89
100
66.6
97
96.0
92
83.0
REFERENCES
•Cavarelli, J., Rees, B., Eriani, G., Ruff, M., Boeglin, M., Gangloff, J., Thierry,
J. C. and Moras, D. (1994) EMBO. J., 13, 327-337.
•Fiser, A. and Sali, A. (2002) Methods Enzymol., Ref Type: Journal (Full).
ACKNOWLEDGEMENT
We are greatly thankful to AMBER supporting team for providing AMBER
suite of applications.
Table 2. ATP‐Homo sapiens AspRS Interatomic Distances (Hydrogen bonding‐Å)
11th International Symposium on Natural Product Chemistry on 29th October-01st November 2008