Modulation of mice pulmonary allergic responses by the sthaphylococcal enterotoxin...
| Grant number: | 13/26313-5 |
| Support type: | Regular Research Grants |
| Duration: | March 01, 2015 - February 28, 2017 |
| Field of knowledge: | Biological Sciences - Biophysics |
| Principal Investigator: | Eduardo Horjales Reboredo |
| Grantee: | Eduardo Horjales Reboredo |
| Home Institution: | Instituto de Física de São Carlos (IFSC). Universidade de São Paulo (USP). São Carlos , SP, Brazil |
| Assoc. researchers: | Ilana Lopes Baratella da Cunha Camargo |
Abstract
Enterococcus faecalis is an important nosocomial pathogen associated to hospital infections in the Americas and Europe. ElrA is a virulence factor related to cellular invasion, given that the elrA gene suppression reduces the virulence in a mouse peritonitis model. ElrA is positively regulated by ElrR. ElrR is considered a member of the RGG family of transcriptional regulators. As all the proteins in this family, ElrR has a helix-turn-helix domain (which binds specifically to DNA) in its N-terminal end. The C-terminal domain is an alpha-helical domain, probably responsible for the conformational changes associated with the biological activity.In 2012 we began a research collaboration with Dr. Ilana Camargo (IFSC/USP) and Dr. Pascale Serror (Unité des Bactéries Lactiques et Pathogènes Opportunistes UR13888, INRA, Jouy-en-Josas, France) that was supported by USP/COFECUB (Comité Français d'Evaluation de la Coopéracion Universiaire et Scientifique avec le Brasil). In this context, our laboratory assumed the task of determining the crystallographic structure of these two proteins that participate in the process of infection of Enterococcus fecalis: ElrA, which is homologous to internalin proteins from de Listeria monocytogenes and ElrR its transcriptional activator.We have got crystals from ElrR (some with spacial group P 41 21 2 or P 43 21 2 at 3.3 Å resolution and other with spacial group P 1 at 2.7 Å resolution).To solve the crystallographic structure we will use SAD (single anomalous diffraction) on the protein with Met residues substituted by selenio-methionines (SeMetSAD). We will also try SIRAS or MIRAS.The crystallographic studies seeking to determine the structure of these proteins have two objectives: 1) It will help to understand the biological function of these proteins and their role in the infection process. 2) In a longer term, it will be essential to build a strategy of infection inhibition, through the design of specific inhibitors.We will also develop molecular dynamics simulations and normal modes calculations of transcriptional regulators, to study the relation between molecular flexibility and allosteric activation in this protein superfamily and especially in ElrR.Allosteric systems present more than one equilibrium state (or native state). Thus, an allosteric protein (a homodimeric transcriptional regulator, t. ex.) undergoes modifications in its biological activity when it is in complex with an allosteric ligand which establishes a displacement of the populational equilibrium. NMR experiment determining relaxation times, have shown that only a fraction of the enzymes accessible states (or conformations) are catalytic. Molecular simulations as molecular dynamics or normal modes calculations can generate a more detailed description because these present the process as function of time and not only the equilibrium states.The computational simulations will calculate the larger possible conformational ensemble, taking into account the computer limitations and the low frequency of the protein movements associated to biological activity.We will analyze the structural flexibilities during dynamics or normal mode oscillations, searching for low frequency movements with large amplitudes which are the biologically interesting ones when studying the allosteric transformation. We will identify the protein domains, which behave as quasi-rigid bodies during the simulations. The analysis and detailed characterization of those low frequency motions should help to obtain the dynamical properties needed for a protein to present a certain conformational change (and biological function). The control and modification of these properties is a tool to modify the regulation of the enzymatic activity. (AU)