Dynamic and conformational study of protein calgranulin C (S100A12) in interaction with ions and RAGE receptor

Author(s)

Renata Almeida Garcia Reis

• Document type: Master's Dissertation
• Press: Ribeirão Preto.
• Institution: Universidade de São Paulo (USP). Faculdade de Ciências Farmacêuticas de Ribeirão Preto (PCARP/BC)
• Defense date: 2012-05-03

Examining board members

Antonio Caliri; Pedro Geraldo Pascutti

• Advisor: Antonio Caliri

Abstract

Calgranulin C (S100A12) is a member of the S100 family of EF-hand calcium-binding proteins. Human S100A12 is predominantly expressed by granulocytes and is markedly overexpressed in inflammatory compartments. Elevated serum levels of S100A12 are found in patients suffering from various inflammatory, neurodegenerative, metabolic, and neoplasic disorders. Intracellular S100A12 exists as an anti-parallel homodimer. Each monomer is composed of a C-terminal, classic EF-hand (HI - LI - HII), an N-terminal, pseudo EF-hand (HIII - LIII - HIV). The motifs are linked by the hinge-region. Calgranulin C also binds zinc and copper ions in a site formed by both subunits of dimer. Changes in cytosolic ions concentrations regulate a wide variety of cellular process, and ions-binding proteins are the key molecules in signal transduction, differentiation, and cell cycle control. The mechanism by which calgranulin C modulates the course of inflammatory process is related to its interaction with the receptor for advanced glycated products (RAGE). In order to obtain details about the mechanism involved in cell signaling steps in which S100A12 participates, our goal was to qualify and quantify the activity conformational of S100A12 domains, induced by variations of intensive thermodynamic parameters, as changes in the concentration of ions. Furthermore we investigated the details of the interaction between S100A12 and RAGE in order to elucidate the region of the receptor which interacts with S100A12 and what are the residues involved in this interaction. In order to access the influence of the presence of ions over the conformational dynamics of S100A12, molecular dynamics simulations were performed using the GROMACS suite with the OPLS-AA force field and NVT ensemble. The initial structures used were experimentally determined by X-ray crystallography (PDB ID: 2WCE and 1E8A). They were separately submitted to different concentrations of sodium, calcium and zinc chloride and solvated with the SPC water model. Our results suggest that at low concentrations of Ca²?, LI remains occupied by Na?. During calcium-waves, it can reach the protein exclusively through LIII (in EF-2). As the Zn²? concentration rises, it contributes to the Na? unbinding from LI, an event that involves the residue ASP-25, which allows LI to open and the Na? to unbind. Furthermore, because of its high structural deformability, HIII is strongly influenced by both Na? and Ca²? ions which, in certain concentrations, leads to partial loss of this helix and of HIIa (Hinge-Region) and increases in the flexibility of this region, although only Ca²? is able to bind, through HIII, to the region near LIII. Regarding the RAGE studies, molecular docking essays and SMD (Steered Molecular Dynamics) simulations were performed. Our data analysis suggests that the interaction between S100A12 and RAGE takes place through both V and C1 RAGE domains and depends upon the interdomain region. Additionally, we observed that higher oligomeric states, e.g. S100A12 hexamers (PDB ID: 1GQM), have more interaction possibilites with RAGE and that, according to our results, in this case the interacting region of S100A12 comprises the N- and C-terminal portions of HI and Cterminal of HIV. (AU)

• FAPESP's process: 10/01538-6 - Dynamic and conformational study of protein calgranulin C (S100A12) in interaction with bivalent ions and RAGE receptor
• Grantee: Renata Almeida Garcia Reis
• Support Opportunities: Scholarships in Brazil - Master