The role of conformational entropy in molecular evolution of bacterial transcriptional repressors

DAVID GIEDROC; CAPDEVILA, DAIANA A

Holderness

Congreso; Proteins Gordon Research Conference; 2019

Gordon Research Conferences

Allosteric communication between two ligand-binding sites in a protein is a central aspect of biological regulation that remains mechanistically unclear. Over the years, we have developed a new regulatory model for stressor sensing in bacterial allosteric transcriptional repressors that relies nearly exclusively on a redistribution of atomic motions to regulate gene transcription.In our previous work, we identified a subset of fast internal motions that increase flexibility upon DNA binding (entropy reservoir) in the ArsR (arsenic repressor) family Zn efflux repressor CzrA (chromosomal zinc-regulated repressor)1. Hence, Zn binding inhibits DNA binding by restricting access to such an entropy reservoir, via entropy redistribution1,2. We propose that driving forces arising from dynamics can be harnessed by nature to evolve new allosteric ligand specificities. To test this hypothesis, we are currently by investigating the contribution of entropy reservoirs to a wide range of sensors from the ArsR family that share a common ancestor but respond to a binding event in a distinct recognition site. In this work, I will present a structural and mechanistic study on a sulfide-responsive transcriptional repressor, SqrR, that functions as a master regulator of sulfide-dependent gene expression in the purple photosynthetic bacterium Rhodobacter capsulatus3. We conducted an extensive crystallographic study of SqrR and have solved the crystal structures of the reduced -DNA binding competent- and several oxidized forms -DNA binding incompetent- SqrRs. This includes, to our knowledge, the first crystal structure of a tetrasulfide crosslink within proteins. These studies strongly suggest that this allostery may be inherently dynamic (all structures are globally nearly identical); moreover, the NMR spectra of SqrR (≈24.6kDa) are of remarkably high quality allowing the determination of fast internal side-chain dynamics in the sidechains. Finally, I will present a kinetics study of the formation of tetrasulfide bridge to understand the determinants of inducer recognition in this sensor, using a combination of ratiometric pulsed alkylation-mass spectrometry and site-directed mutagenesis. (1) Capdevila, D. A.; Braymer, J. J.; Edmonds, K. A.; Wu, H.; Giedroc, D. P. Entropy Redistribution Controls Allostery in a Metalloregulatory Protein. Proc. Natl. Acad. Sci. U. S. A. 2017, 114 (17), 4424?4429.(2) Capdevila, D. A.; Edmonds, K. A.; Campanello, G. C.; Wu, H.; Gonzalez-Gutierrez, G.; Giedroc, D. P. Functional Role of Solvent Entropy and Conformational Entropy of Metal Binding in a Dynamically Driven Allosteric System. J. Am. Chem. Soc. 2018, 140 (29), 9108?9119.(3) Shimizu, T.; Shen, J.; Fang, M.; Zhang, Y.; Hori, K.; Trinidad, J. C.; Bauer, C. E.; Giedroc, D. P.; Masuda, S.; Bauer, C. E.; et al. Sulfide-Responsive Transcriptional Repressor SqrR Functions as a Master Regulator of Sulfide-Dependent Photosynthesis. Proc. Natl. Acad. Sci. 2017, 114 (9), 2355?2360.