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 Tolllike receptor 4 (TLR4), TLR2, CD14, and CD40. This leads to activation of nuclear factor-kappa B (NF-κB), release of proinflammatory cytokines, enhancement of the phagocytic activity of innate immune cells, and stimulation of antigenspecific 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.

Extracellular HSP70 Activates ERK1/2, NF-kB and Pro-Inflammatory Gene Transcription Through Binding with RAGE in A549 Human Lung Cancer Cells

“…In this study, we used a human lung cancer-derived A549 cell line, which constitutively expresses RAGE, and validated the interaction between this receptor and eHSP70 by a cellular study-based approach. Since the interaction between eHSP70 and RAGE has already been reported [23, 24], our immunoprecipitation and protein proximity image assay results confirmed this specific binding around the cell, leading us to use this cell model to investigate functional signaling responses evoked by eHSP70-RAGE engagement. It is important to note that the evaluation of functional responses mediated by RAGE is a major step to establish eHSP70 as a RAGE agonist, since HSP70 is able to interact with a myriad of proteins due to its chaperone activity.…”

“…TLR, CD14) are strongly influenced by RAGE, such as ERK1/2-mediated activation of NF-kB. Furthermore, an ELISA screen assay proposed a potential interaction between RAGE and extracellular HSP70 [23], while a recent work by our group proposed an interaction model by molecular docking and evidenced a physical interaction between RAGE-eHSP70[24]. However, since the activity of HSP70 as molecular chaperone allows nonspecific interactions with diverse proteins, the aim of the current study was to investigate if the eHSP70-RAGE interaction is able to induce a functional activation of RAGE in a cellular model, in order to establish eHSP70 as a functional RAGE agonist.…”

Unveiling the Interplay between the TLR4/MD2 Complex and HSP70 in the Human Cardiovascular System: A Computational Approach

“…The utmost goal in molecular docking is to use algorithms to predict the likelihood that a given protein will bind to a target. Here, we identified the same pattern observed by Grunwald and collaborators [32] where the ATP-bound state of HSP70 formed a more stable interaction with the TLR4/MD2 complex (Table 2) with no significant deformability in the interface (Figure 4). This might be because the two domains of HSP70 in the ADP-bound state could rotate in opposite directions, making the protein a not very likely ligand, at least in this conformation, which was not observed in the ATP-bound state [32].…”

“…In a previous study, aiming at elucidating the complexation model between HSP70 and the receptor for advanced glycation end product [RAGE (IDs 3CJJ and 4LP5)], the authors showed that the ADP bound state (ID 2KHO) also formed a complex with the receptor although it was less energetically favorable [32]. In our molecular docking simulations, we aimed at characterizing HSP70 affinity towards the human TLR4/MD2 (ID 3FXI) complex using both ADP (ID 2KHO) and ATP (ID 4B9Q) states.…”

“…Besides changes in its 3D structure, the rearrangement cycle undergone by HSP70 affects the overall distribution of the electrostatic potential in the surface of the molecule. As shown in Figure 1, HSP70 has a negative surface potential, which might favor the interaction with positively charged receptors [32] such as the co-adaptor MD2 [33], a protein known for physically associating with TLR4 [34].…”