Identifying Relationships between Structure and Function of the Bacterial Metabolic Pathway TR-TRX-TPX

VAZQUEZ DS ; ISERTE J; GONZÁLEZ LEBRERO MC; AGUDELO WA; FERRER-SUETA G; MANTA B; MARINO BULJE CB ; SANTOS J

Conferencia; Argentinian Conference on Bioinformatics and Computational Biology; 2014

Throughout all the kingdoms, the cellular antioxidant and redox homeostasis are regulated by the thioredoxin and glutathione systems [1,2] which comprise several TRX-like fold proteins such as glutaredoxins, thiol-dependent peroxidases (PRXs), thioredoxin, among others. Our interest in this system, from a biophysical viewpoint, is mainly based on (i) the plasticity in the thioredoxin (TRX) substrate recognition process. TRX has different targets and is only reduced by the FAD- dependent thioredoxin reductase in vivo; (ii) the existence of large conformational changes in PRX family (helix-coil transitions) that take place coupled to catalysis and may impact over the catalytic rate; (iii) electron canalization in TR. These aspects among others prompted us to hypothesize the existence of an evolutionary preserved interaction network involved in protein-protein contact and substrate recognition as well as in internal dynamics and thermodynamic stability. For this, we performed an exhaustive bioinformatic and structural analysis of three well-characterized proteins: thioredoxin reductase (TR), thioredoxin 1 (TRX) and the thiol-dependent peroxidase (TPX, an atypical 2-Cys- peroxiredoxin). Methodology Mutual co-evolutionary relationships between positions in a multiple sequence alignment containing the TR, TRX and TPX protein sequences from the bacterial kingdom were performed using the MISTIC on line server (http://mistic.leloir.org.ar [3]). The most promised inter- and intra- protein pairs-of-residues obtained by mutual information (MI) were subjected to in silico mutation, molecular dynamic simulations and principal components analysis (PCA) in order to explore the role in the dynamic/thermodynamic in each protein. MDs were performed in the AMBER14 ? GPU package [4] using the ff14SB force field. PCA were performed and post-processed with the ccptraj module of AmberTools13. bioinformatics was complemented by biophysical experimental results. Results Preliminary results from mutual information analysis suggest the existence of qualitatively different pair of residues: (i) located near of the active site suggesting a role in catalysis, (ii) residues with high accessible surface area suggesting a role in protein-protein interaction (see Figure 1). In addition, to characterize the quaternary structure of TPX, the protein was purified and analyzed by light-scattering techniques and circular dichroism.