Modelagem estrutural do canal de sódio 1.4 e estudo teórico da ligação de alfa e beta toxinas de escorpião

• Ano de defesa: 2019
• Autor(a) principal: João Ronaldo Clemente Fernandes
• Tipo de documento: Tese
• Tipo de acesso: Acesso aberto
• Idioma: por
• Instituição de defesa: Universidade Federal de Minas Gerais; Brasil; ICB - INSTITUTO DE CIÊNCIAS BIOLOGICAS; Programa de Pós-Graduação em Bioinformatica; UFMG
• Palavras-chave em Português: Bioinformática; Biologia computacional; Canais de sódio disparados por voltagem; Simulação de acoplamento molecular
• Link de acesso: http://hdl.handle.net/1843/35074

Voltage-gated sodium channels (VGSD) are proteins responsible for the propagation of information between neurons, skeletal muscle fibers, cardiac muscle fibers and other types of excitable cells. They are rapidly activated when the cell membrane is depolarized, followed by inactivation, which leads to a transient sodium influx. Until recently there was no complete structural model of the human sodium channel. In this work, we performed the comparative modeling of the hNav1.4 channel, using as template the rabbit calcium channel (Cav1.1), cockroach sodium channel (NavPaS) and the electric eel sodium channel (EeNav1.4), with the Rosetta and SWISS-MODEL programs. These models were analyzed in two ways: the fidelity of their folding in relation to their templates using the TM-Align tool, and by angles of the peptide bonds between the amino acid residues, using the Ramachandran plot. All models were considered successful based on these criteria. More accurate analysis of the structures showed that the hNav1.4/Cav model has inconsistencies when compared to the functioning of a sodium channel, making the model inappropriate. The pore of the channel is closed and its voltage sensitive domains (VSD) are activated, which makes this model faulty in relation to the functional properties of the channel. On the other hand, the hNav1.4/PaS and hNav1.4/Ee models were able to represent closed and open conformational states, respectively, and were consistent with known biophysical and pharmacological data. As a criterion to identify the conformational state the channels, the analysis of the segments S4 was performed checking its position in relation to the hydrophobic constriction and the interactions of the charged residues with segments S1, S2 and S3. Considering this criterion, it was possible to count the elementary charges necessary to activate the hNav1.4 channel, reaching an approximate value of 6.5. Completing the analysis of the channel characteristics, the docking of alpha- and beta-scorpion toxins to the canal was performed. Beta-toxin Ts1 bound to channel site 4 with high affinity and binding free energy (ΔG) of -9.4 kcal/mol, thus confirming that the segment S4 of the DII domain is in the activated position, although the pore of the channel is closed state (as shown by the analysis by the MOLEonline program). The alpha toxin AaHII bound to site 3 of the closed-state model hNav1.4/PaS with a free energy of binding of -10.7 kcal/mol. A new docking was tested with the open-state model, hNav1.4/Ee, without success. This result is in agreement with the toxin's ability to bind to the VSDIV of the channel, but not when it is activated. Further analysis showed that the D1435 residue of the channel, considered to be of great importance for the toxin-channel interaction, is at an unfavorable position for toxin binding. This change results from the outward movement of the S4 segment, which causes a rotation of S3, placing the D1435 in unfavorable position. This observation can account for one characteristic of the alpha-toxins, which is their displacement from the channel when strong depolarizing pulses are applied to the membrane. The results presented by the docking of the toxins can explain the results obtained experimentally. Our results show that these theoretical models may provide structural basis to understand the physiological and pharmacological characteristics of these channels, thus enabling a new range of theoretical researches involving the hNav1.4 channel at different conformational states.