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Objectives:
The
underlying principle of the function of a macromolecule is its
structure or in other words structure dictates
function. Our laboratory aims at understanding the
function of biomolecules by combining experimentally obtained
structural information using X-ray crystallography with other
biophysical and biochemical data.
Projects:
1)
Translational accuracy
Aminoacyl-tRNA
synthetases (aaRSs) establish the rules of the genetic code by
attaching the correct amino acid to the cognate tRNA. A major
goal of our laboratory is in trying to understand how very
similar amino acid substrates are recognized with a very high
accuracy during translation of the genetic code. Structural
data over the past few years is throwing light on
editing/proof-reading mechanisms of aaRSs, which are
responsible for the high fidelity. We are focusing our efforts
on threonyl-tRNA synthetases (ThrRSs) from some pathogenic
organisms, which showed interesting features with respect to
amino acid recognition and the editing mechanism. Most of the
archaeal ThrRSs possess a unique editing domain when compared
to its counterparts in eubacteria and eukaryotes. We are
focusing our efforts in understanding the structural basis of
the editing mechanism in archaea.
2)
Structural basis of function of Polyketide Synthases and Fatty
Acyl Synthases from Mycobacterium tuberculosis (in
collaboration with the group of Dr. Rajesh Gokhale, NII, New
Delhi)
Mycobacterium
tuberculosis
(M.tb) possesses a complex lipid composition and
constitutes 50% of the dry cell weight. The genome sequence of
M.tb revealed a battery of PKS and Fatty acid synthase
genes and many of them have been shown to be or implicated in
the virulence of the mycobacteria. In the lab, we are looking
at the structure-function relationship of several of these
proteins. We recently determined the structure of PKS18 in
order to understand its unusual substrate specificity. The
structure led us to identify a novel tunnel in the molecule
and provided a clear structural basis for PKS18 function. At
present, several of the PKS and FAS from M. tb are
expressed and we are in the process of crystallizing and
determining their structures to understand their function.
3)
Structural basis of enhanced thermostability of Bacillus
subtilis lipase (in collaboration with the group of Dr. N.
Madhusudana Rao, CCMB)
A
triple mutant of Bacillus subtilis lipase
(LipA) was obtained by directed evolution approaches with
approximately 300-fold increased thermostability compared to
the wild-type enzyme. Structural information was used to
combine mutants for obtaining a minimalist solution for
enhancing thermostability. To gain insights into the
structural basis of the observed enhancement in
thermostability and to improve it further we crystallized and
solved the structure of a double and triple mutant of LipA.
High-resolution crystal structures of the mutants show subtle
changes, which include stacking of tyrosines, peptide plane
flipping and a better anchoring of the terminus, that
challenge rational design and explain the structural basis for
enhanced thermostability. The approach offers an efficient and
minimalist solution for the enhancement of a desired property
of a protein.
4)
Structure-function analysis of virulence factors of
Xanthomonas oryzae pv.
oryzae, the bacterial leaf blight pathogen of rice
(in collaboration with the group of Dr. Ramesh V.
Sonti, CCMB)
Xanthomonas
oryzae pv.
oryzae causes bacterial leaf blight, a serious
disease of rice. We are analyzing some of its virulence
factors via structural biology. One of the virulence factors
being studied is XadA (Xanthomonas
adhesin-like protein), a 1265 amino acid-long
outer-membrane located protein. XadA shows significant
sequence homology to the non-fimbrial adhesins of animal
pathogenic bacteria. XadA may have a critical role in the
early stages of infection. We hypothesize that XadA promotes
attachment to a host ligand at the hydathodal openings. Our
initial efforts are focused in understanding the structural
basis of XadA function. Sequence analysis of XadA indicates
that it contains four HIM motifs (variations of TDAVNVAQL)
that are present in multiple copies in all the related
non-fimbrial adhesins. This motif was shown to be in the
‘neck’ region essential for homotrimerisation of the
Yersinia enterocolitica adhesin YadA, whose structure
has been solved. Alignment of XadA with YadA revealed that
XadA sequence could be divided into four modules, which are
highly homologous to each other and the YadA sequence. We are
in the process of cloning, expressing and purifying each of
these modules individually and in combinations as soluble
His-tagged proteins in E. coli for structural
studies.
5)
Structural characterization of RNA degradation machinery in
Pseudomonas syringae (in collaboration with
the group of Dr. Malay K Ray, CCMB)
The
post-transcriptional RNA degradation plays a significant role
in gene regulation and protein expression. In prokaryotes the
RNA degrading machinery is made up of a multi component
protein complex where the endoribonuclease RNaseE plays a
significant role in providing structural scaffold for binding
of other proteins of the complex. In the mesophilic E.coli the degradosome
is made up of endoribnonuclease RNase E, exoribnuclease
PNPase, and helicaseRhlE. A novel RNA degradation complex has
been identified in cold adapted bacterium Pseudomonas syringae.
This complex is made up of RNase E, RNaseR and SrmB. The main
objective of the work is to characterize P.syringae degradosomal
proteins by crystallizing the proteins involved in the complex
to provide insight into the functioning of this novel
complex.
6) Calcium
binding proteins (in collaboration with the group of Dr.
Yogendra Sharma, CCMB)
This
project deals with understanding structural, metal binding and
functional aspects of calcium binding proteins like neuronal
calcium sensors and βγ-crystallins.
The neuronal calcium sensors are a group of conventional
EF-hand calcium binding proteins and play sensory roles in
various Ca2+ dependent cellular functions. The
βγ-crystallin
super family comprises of proteins involved in rendering the
eye lens transparent and helps microbes counter stressful
conditions. We found the domain to have calcium binding
properties and are involved in trying to establish it as a
universal calcium binding superfamily.
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