The research in this
laboratory is geared towards understanding structural and mechanistic features
of molecules that transfer signals across biological membranes*. Projects in this group lie in the broad area
of inter-cell communication, transcriptional regulation and molecular
mechanisms of drug resistance in gram-positive bacteria. The techniques we employ include molecular
biology, biophysical and biochemical analysis and crystallography.
On-going Research
Projects:
1. Regulation of
transcription in Mycobacterium
tuberculosis
In the case of M. tuberculosis, we
aim to understand how environmental signals dictate transcriptional
regulation. The ability of M.
tuberculosis to survive in the hostile environmental conditions of the
host, the so-called latent phase, is brought about by adaptability to rapidly
changing environmental conditions. We
aim to understand how a class of proteins, the Extra-Cytoplasmic Function (ECF) s factors, synchronize changes in the
transcription profile with environmental stimuli. In general, ECF s factors are regulated by their interactions with
membrane associated proteins called anti s factors.
Studies from our group have shown that M. tuberculosis ECF s's
adopt several variations to this mechanism.
For example, sC does not appear to have a
regulatory anti-s
factor. The crystal structure and
biochemical analysis of this protein revealed that interaction between the
promoter-recognition domains in sC
regulates the activity of this protein even in the absence of an anti s factor. More
recently, we determined the crystal structure of sL in
complex with a Zinc-associated anti s factor, RslA. The structure and biophysical
characterization of this complex provides a rationale for the role of sL in the
oxidative stress response of M. tuberculosis.
http://www.scitopics.com/Sigma_Factors.html
Thakur, K.G., Joshi, A. M. & Gopal, B. (2007).J. Biol.
Chem 282, 4711-4718. Structural and biophysical studies on two promoter
recognition domains of the extra-cytoplasmic function s factor sC from Mycobacterium
tuberculosis
Thakur, K. G., Praveena,
T. & Gopal, B. (2010). J. Mol. Biol. 397:
1199-1208. Structural and biochemical basis for the redox sensitivity of Mycobacterium
tuberculosis RslA.
Jaiswal, R. K., Manjeera,
G. & Gopal, B. (2010). BBRC doi:10.1016/j.bbrc.2010.06.027.
Role of a PAS sensor
domain in the Mycobacterium tuberculosis
transcription regulator Rv1364c.
2. Role of cell wall
components and transport machinery in facilitating multi-drug resistance in Staphylococcus aureus
Our emphasis in this area is on
proteins involved in cell wall synthesis (the Lysine biosynthesis pathway),
membrane-associated penicillin binding proteins (PBPs)
and specific multi-drug pumps and receptor proteins involved in the efflux of
antibiotics. The structural studies on
proteins involved in lysine biosynthesis have revealed novel regulatory
mechanism(s) for these proteins- a finding that could potentially be employed
for the design of specific anti-microbial agents.
Girish, T. S., Sharma, E. & Gopal, B. (2008). FEBS Letts. 582:2923-2930. Structural basis for the regulation of
Dihydrodipicolinate Synthase
(DHDPS) activity from Staphylococcus aureus.
Navratna, V., Nadig, S., Sood, V.,
Prasad, K., Arakere, G. & Gopal, B. (2010). J. Bacteriology 192: 134-144. Molecular Basis for the role of Staphylococcus aureus
Penicillin Binding Protein 4 in antimicrobial resistance
The other projects in the
group include:
1. Synthesis of peptide
antibiotics
Bacilysin is a non-ribosomally
synthesized dipeptide antibiotic that is active
against a wide range of bacteria and some fungi. Synthesis of bacilysin
(L-alanine-[2,3-epoxycyclohexano-4]-L-alanine) is achieved by proteins in the bac operon,
also referred to as the bacABCDE (ywfBCDEF) gene cluster in Bacillus subtilis. Extensive genetic analysis from several
strains of B. subtilis
suggests that the bacABC
gene cluster encodes all the proteins that synthesize the epoxyhexanone
ring of L-anticapsin.
Recently, we could demonstrate that BacA is a decarboxylase that acts on prephenate. Further, based on the biochemical
characterization and the crystal structure of BacB,
we note that BacB is an oxidase
that catalyzes the synthesis of 2-oxo-3-(4-oxocyclohexa-2,5-dienyl)propanoic acid, a precursor to L-anticapsin. Studies are currently in progress to
understand the machinery that regulates the export of this antibiotic and the
regulation of bacilysin production by the quorum
sensing machinery.
Rajavel, M,
Mitra, A. & Gopal, B. (2009) J. Biol. Chem. 284:31882-31892. Structural basis for the role of Bacillus subtilis
BacB in the synthesis of the antibiotic bacilysin.
2. Studies on Receptor
Protein Tyrosine Phosphatases involved in axon
guidance in the fruit fly Drosophila melanogaster
Our interest in this system is to
determine the molecular basis for substrate specificity in this class of phosphatases. It is
envisaged that this information could lead to the design of specific inhibitors
that can then be employed for therapeutic intervention. We have recently made some progress in the
cloning and characterization of the five RPTPs from D. melanogaster
involved in the axon guidance mechanism.
Studies to understand the molecular basis for the activity, regulation
and interactions between these proteins are presently underway.
Madan,
L. L. & Gopal, B (2008). Prot. Exp. Purif. 57:234-243. Studies on the
construct-dependence in the expression of eukaryotic proteins in Escherichia coli: Case of the catalytic
domains of the Receptor Protein Tyrosine Phosphatases
from Drosophila melanogaster
3. Computational methods
for macromolecular crystallography
The
advent of structural genomics has led to a dramatic increase in the number of
structures deposited in the Protein Data Bank.
The number of new folds, however, still remains a very small fraction of
the total number of deposited structures.
Recent data on the progress of the structural genomics initiative
reveals that over 85 % of target proteins that progress to the stage of data
collection and structure determination have a known fold. Enzymes, which tend to exploit reaction space
while adopting a common stable scaffold, contribute significantly to this
observation. We were able to demonstrate
that a fold detection strategy based on secondary structure signatures followed
by Molecular Replacement using a minimalist model can be effectively employed
to solve the phase problem in X-ray crystallography without further recourse to
heavy atom derivatives or Multiple Anomalous Dispersion techniques. Three common folds- the triosephosphate isomerase
(TIM), adenine nucleotide alpha hydrolase-like (HUP)
and RNA recognition motif (RRM)
were examined using this approach. Our
current interest in this area lies in correlating diffraction intensity
statistics with the shape and, if feasible, the fold of the protein.
Rajavel, M., Warrier,
T., & Gopal, B. (2006). Proteins 64, 223-230. Old
fold in a new X-Ray diffraction dataset? Low-resolution molecular replacement
using representative structural templates can provide phase information.
A complete list of
publications may be found here-
http://www.ncbi.nlm.nih.gov/sites/entrez?term=gopal%20b%20&cmd=search&db=pubmed
*Research in this laboratory is funded by the Department of
Biotechnology, the Department of Science and Technology and the Council for
Scientific and Industrial Research, Government of India and the Wellcome Trust,