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DR. RAVISHANKAR RAMACHANDRAN |
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Group leader ( Scientist E- II )
Xray lab
Molecular & Structural Biology Division
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We elucidate the crystal structures
of proteins from pathogens like M. tuberculosis and
P. falciparum. Our work encompasses cloning, expression,
purification and biochemical/functional studies on proteins,
crystallization and 3D structure elucidation by x-ray crystallography
approaches. Subsequently the structures are analyzed and novel
inhibitors identified using state-of-the-art techniques like
virtual and high-throughput screening. The lab is well equipped
and has state-of-the-art instruments for protein purification
and characterization in addition to a modern x-ray data collection
facility. We also have several graphics workstations and servers
for molecular modeling and analysis of protein structures.
The projects in the lab are funded by CSIR and Department
of Biotechnology, India.
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Nucleic acid ligases
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Nucleic acid ligases are
important proteins which primarily join breaks in DNA or RNA.
We have initiated a long-term project to elucidate the crystal
structures of nucleic acid ligases and have cloned and expressed
several ligases, primarily from M. tuberculosis. |
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We recently elucidated the
3D structure of the ~35 kD adenylation domain of the NAD+
-dependent DNA ligase (MtbLigA) from the pathogen with bound
AMP co-factor. LigA is an ~70 kD multi-domain enzyme which
seals nicks in dsDNA. The enzyme is recognized as a novel
target as it is found exclusively in bacteria and some viral
sources. We have now generated several mutants of the enzyme
and are probing various aspects of the enzyme mechanism. We
use the structure in virtual screening approaches and have
identified novel inhibitors with diverse scaffolds. These
are being optimized in collaboration with medicinal chemistry
groups. |
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| ACT/RAM domain containing
regulatory proteins |
Many global and local regulatory
proteins are involved in controlling the changes to the lifestyle
of M. tuberculosis. The RAM domain (Regulator of
Amino acid Metabolism) and ACT domains share structural homology
while ligand binding sites occur in different regions. They
consist of about 80 amino acids and exhibit a ßaßßaß
topology. Proteins with this domain appear to be involved
in amino acid regulation and interact with amino acids. Many
aspects of their mechanism are unclear. How ACT/RAM domains
can control the function of a variety of proteins? The way
by which they translate a ligand-binding event to the protein-DNA
binding site/active site of enzymes is largely unknown. Some
proteins are inhibited by the ligand-binding event while others
are activated. |
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We have identified about
16 proteins with this domain from the analysis of the M.
tuberculosis genome sequence. Some of them had been found
earlier to be essential for in vitro/ in vivo survival
of the pathogen. We have cloned several proteins containing
this domain and have solved the crystal structure of one of
them. |
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| Proteins involved
in tuberculosis persistence |
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M. tuberculosis
is a successful pathogen primarily because of its ability
to persist in the human host for years avoiding the host-immune
system. We have chosen to work on many proteins which are
up-regulated in tuberculosis models designed to mimic the
persistent state. |
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Recently we elucidated the
crystal structure of Lysine e-aminotransferase (MtbLAT), an
enzyme which is up-regulated over 40-fold in a nutrient starvation
model of tuberculosis. We elucidated structurally how the
enzyme can distinguish between structurally similar substrates
through an elegant mechanism involving a glutamate ‘switch’.
We are dissecting aspects of the enzyme action through a series
of structures as also a mutational analysis. Other proteins
are in various stages of the structure elucidation pipeline. |
P. falciparum
proteins |
The apicoplast is a relic
plastid of plant origin which is an essential organelle of
P.falciparum. We are in the process of elucidating
structures of proteins which are coded for by the apicoplast
or which help nuclear encoded proteins to interact with the
organelle. |
Virtual Screening |
The 3D structure of a protein
is a powerful tool which helps in the design of novel inhibitors;
much the same as a wax-imprint of the keyhole of a lock helps
the design of a duplicate key. |
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| SELECTED RECENT PUBLICATIONS |
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Malik SS,
Luthra A & Ravishankar Ramachandran
(2009) Interactions of the M. tuberculosis UsfX with the
cognate sigma factor SigF and the anti-anti sigma factor
RsfA Biochem. Biophys. Acta, 1794, 541-553,
2009 |
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Misra G, Aggarwal
A, Dube D, Zaman MS, Singh Y & Ravishankar
Ramachandran (2009) Crystal structure of the
Bacillus anthracis nucleoside diphosphate kinase and its
characterization reveals an enzyme adapted to perform
under stress conditions PROTEINS, doi:
10.1002/PROT.22364, 2009 |
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Misra G &
Ravishankar Ramachandran (2009) Hsp70-1
from Plasmodium falciparum: Protein stability, domain
analysis and chaperone activity Biophys. Chem.
142, 55-64, 2009 |
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Luthra A,
Mahmood, A, Arora A & Ravishankar Ramachandran
(2008) Characterization of Rv3868: an essential hypothetical
protein of the ESX-1 secretion system in M. tuberculosis
J. Biol. Chem., 283, 36532-36541, 2008 |
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Malik SS, Luthra A, Srivastava
S & Ravishankar Ramachandran (2008)
The M. tuberculosis UsfX (Rv3287c) exhibits novel nucleotide
binding and hydrolysis properties Biochem. Biophys.
Res. Comm. 375, 465-470, 2008 |
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Luthra A, Malik SS &
Ravishankar Ramachandran (2008) Comparative
structural analysis of two hypothetical proteins from
Mycobacterium tuberculosis found in the human granuloma
during persistence and highly up-regulated under carbon-starvation
conditions Prot. Exp. Purif. 62, 64-74,
2008 |
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Tripathi SM & Ravishankar
Ramachandran (2008) Crystal structures of the
Mycobacterium tuberculosis secretory antigen Alanine dehydrogenase
(Rv2780) in apo and ternary complex forms captures ‘open’
and ‘closed’ enzyme conformations PROTEINS
72, 1089-1095, 2008 |
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Tripti, S & Ravishankar
Ramachandran (2007) Mechanistic insights from
the crystal structures of a feast/famine regulatory protein
from Mycobacterium tuberculosis H37Rv Nucleic
Acids Res. 35, 7324-7335, 2007 |
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Namrata Dwivedi, Divya
Dube, Jyoti Pandey, Biswajit Singh, Vandna Kukshal, Ravishankar
Ramachandran* & Rama Pati Tripathi* (2008)
NAD+ -dependent DNA ligase: A novel target waiting for
the right inhibitor Medicinal Research Reviews
28, 545-568, 2008
*(Joint corresponding authors) |
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Srivastava SK, Dube D,
Vandna, K, Jha AK, Hajela K & Ravishankar
Ramachandran (2007) NAD+-dependent DNA ligase
(Rv3014c) from Mycobacterium tuberculosis: Novel structure-function
relationship and identification of a specific inhibitor
PROTEINS: 69, 97-111, 2007 |
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Divya Dube, Sarvind Mani
Tripathi & Ravishankar Ramachandran (2007)
Identification of inhibitors of M. tuberculosis Lysine
e-aminotransferase by virtual screening Med. Chem.
Res. 15, 181, 2007 |
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Tripathi, SM & Ravishankar
Ramachandran (2007) Direct evidence for a glutamate
switch necessary for substrate recognition: Crystal structures
of Lysine e-aminotransferase (Rv3290c) from Mycobacterium
tuberculosis H37Rv J. Mol. Biol. 362,
877-886, 2006
(Suggests a common mechanism for a sub-class of aminotransferases) |
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Srivastava SK, Dube D,
Tewari N, Dwivedi, N, Tripathi RP & Ravishankar
Ramachandran (2005)
Mycobacterium tuberculosis NAD+ -dependent DNA ligase
is selectively inhibited by glycosylamines compared with
human DNA ligase I. Nucleic Acids Res.
33, 7090-7101, 2005 |
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Srivastava SK, Tripathi
RP, Ravishankar Ramachandran (2007) NAD+
-dependent DNA ligase (rv3014c) from M. tuberculosis:
Crystal structure of the adenylation domain and identification
of novel inhibitors. J. Biol. Chem. 280,
30273-30281, 2005 |
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