title: Putative model for heat shock protein 70 complexation with receptor of advanced glycation end products through fluorescence proximity assays and normal mode analyses.
abstract: Extracellular heat shock protein 70 (HSP70) is recognized by receptors on the plasma membrane, such as Toll-like receptor 4 (TLR4), TLR2, CD14, and CD40. This leads to activation of nuclear factor-kappa B (NF-κB), release of pro-inflammatory cytokines, enhancement of the phagocytic activity of innate immune cells, and stimulation of antigen-specific responses. However, the specific characteristics of HSP70 binding are still unknown, and all HSP70 receptors have not yet been described. Putative models for HSP70 complexation to the receptor for advanced glycation endproducts (RAGEs), considering both ADP- and ATP-bound states of HSP70, were obtained through molecular docking and interaction energy calculations. This interaction was detected and visualized by a proximity fluorescence-based assay in A549 cells and further analyzed by normal mode analyses of the docking complexes. The interacting energy of the complexes showed that the most favored docking situation occurs between HSP70 ATP-bound and RAGE in its monomeric state. The fluorescence proximity assay presented a higher number of detected spots in the HSP70 ATP treatment, corroborating with the computational result. Normal-mode analyses showed no conformational deformability in the interacting interface of the complexes. Results were compared with previous findings in which oxidized HSP70 was shown to be responsible for the differential modulation of macrophage activation, which could result from a signaling pathway triggered by RAGE binding. Our data provide important insights into the characteristics of HSP70 binding and receptor interactions, as well as putative models with conserved residues on the interface area, which could be useful for future site-directed mutagenesis studies.
authors.affiliation: Department of Biochemistry, Institute of Basic Health Sciences, Federal University of Rio Grande do Sul, Porto Alegre, Rio Grande do Sul, Brazil. marcelo.sartori@yahoo.com.br.
authors.forename: Marcelo Sartori
authors.initials: MS
authors.lastname: Grunwald
authors.affiliation: Center of Biotechnology, Federal University of Rio Grande do Sul, Porto Alegre, Rio Grande do Sul, Brazil.
authors.forename: Rodrigo
authors.initials: R
authors.lastname: Ligabue-Braun
authors.affiliation: Department of Biochemistry, Institute of Basic Health Sciences, Federal University of Rio Grande do Sul, Porto Alegre, Rio Grande do Sul, Brazil.
authors.forename: Cristiane Santos
authors.initials: CS
authors.lastname: Souza
authors.affiliation: Department of Biochemistry, Institute of Basic Health Sciences, Federal University of Rio Grande do Sul, Porto Alegre, Rio Grande do Sul, Brazil.
authors.forename: Luana
authors.initials: L
authors.lastname: Heimfarth
authors.affiliation: Center of Biotechnology, Federal University of Rio Grande do Sul, Porto Alegre, Rio Grande do Sul, Brazil.
authors.forename: Hugo
authors.initials: H
authors.lastname: Verli
authors.affiliation: Department of Biochemistry, Institute of Basic Health Sciences, Federal University of Rio Grande do Sul, Porto Alegre, Rio Grande do Sul, Brazil.
authors.forename: Daniel Pens
authors.initials: DP
authors.lastname: Gelain
authors.affiliation: Department of Biochemistry, Institute of Basic Health Sciences, Federal University of Rio Grande do Sul, Porto Alegre, Rio Grande do Sul, Brazil.
authors.forename: José Cláudio Fonseca
authors.initials: JC
authors.lastname: Moreira
mesh_terms: D000072283:A549 Cells; D001665:Binding Sites; D005456:Fluorescent Dyes / Q000737:chemistry; D018840:HSP70 Heat-Shock Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D008856:Microscopy, Fluorescence; D062105:Molecular Docking Simulation; D016297:Mutagenesis, Site-Directed; D011485:Protein Binding; D055550:Protein Stability; D020836:Protein Structure, Quaternary; D017434:Protein Structure, Tertiary; D000067759:Receptor for Advanced Glycation End Products / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D013816:Thermodynamics
chemical_list: D005456:Fluorescent Dyes; D018840:HSP70 Heat-Shock Proteins; D000067759:Receptor for Advanced Glycation End Products
keywords: Complexation; Heat shock protein 70; Molecular docking; Receptor of advanced glycation end products; Signal transduction pathways