GARCIA-PINO's TEAM @CM2
The major focus of the CM2 lab is to study bacterial survival mechanisms from molecules to the cells. We are particularly interested in toxin-antitoxin systems, antibiotic-tolerance and bacterial metabolic regulation. Our long-term ambition is to understand metabolic processes involved in bacterial survival from a molecular mechanistic perspective. My group together with Van Melderen’s team is considered one of the leading laboratories working in the field of toxin-antitoxin systems and has a strong track record in the field of structural and molecular biology.
At the methodological level, in addition to the classical biochemical and structural biology methods such as X-ray crystallography, SAXS, calorimetry and other molecular recognition techniques, we are embracing single particle analysis methods by FRET and cryo-electron microscopy as part of the new wave in structural biology. The use of these techniques allow us studying complex regulatory mechanism at the molecular level with atomic resolution.
Toxin-antitoxin (TA) modules are ubiquitous in the genomes of prokaryotes. They are organized in operons containing two genes that code for an antitoxin and a toxin. If the action of the toxin is not damped, the cell will receive a ‘toxin overdose’ and by consequence the action of the toxin will lead to cell death. My group has a long standing track record in field of TA modules. We use structural biology, biophysics and biochemical techniques to study TA toxin functions. We are specially interested in discovering novel functions involving TA toxins.
Discovery of novel toxin-antitoxin (TA) functions and regulatory mechanisms
Allostery and intrinsic disorder mediate transcription regulation of type II TA modules
As part of its regulatory cycle, antitoxins controls their own transcription by binding DNA at its own operon. In many cases, increasing the ratio of toxin/antitoxin initially inhibits transcription, and as the ratio exceeds a certain biological threshold, transcription is activated again, exhibiting ‘conditional cooperativity'. This regulatory mechanism results from the delicate balance of the ensemble of states present in solution.
Many antitoxins have a locally unfolded N- or C- terminus, which undergoes a disorder-to-order transition that exposes high-affinity (H) and low-affinity (L) binding sites. Increasing the ratio of toxin/antitoxin leads to partial saturation of the H and L sites. Finally, increasing the toxin/antitoxin ratio further leads to saturation of the H and L binding sites and dissociation from DNA. The work on the regulation of transcription by the intrinsically disordered regions of antitoxin constitutes the first experimental demonstration that an allosteric signal could be transmitted between two intrinsically disordered regions.
Regulation of (p)ppGpp synthesis and hydrolysis
Our long time ambition is to understand key steps in the molecular mechanisms of ppGpp synthesis and hydrolysis across bacterial species, which ultimately lead to the formation of antibiotic-tolerant persister cells. We will study RSHs enzymes and their regulatory interplay that controls ppGpp levels, a crucial aspect of the stringent response. We want to further characterize the molecular mechanism of Rel/RelA/SpoT regulation. Our research is mainly focused on the structural and biophysical characterization of GTP-pyrophosphokinases/hydrolases from different bacteria E. coli, T. thermophylus, M. tuberculosis, C. tepidum, S. aureus and S. typhimurium. It includes the determination of the 3D structure of these enzymes together with classical biochemistry and biophysical measurements to characterize its interaction with the ribosome, tRNA and nucleotides. We are also very interested in discovering new compounds that can modulate the activity of RSHs enzymes.