Mutation D816V Alters the Internal Structure and Dynamics of c-KIT Receptor Cytoplasmic Region: Implications for Dimerization and Activation Mechanisms

The type III receptor tyrosine kinase (RTK) KIT plays a crucial role in the transmission of cellular signals through phosphorylation events that are associated with a switching of the protein conformation between inactive and active states. D816V KIT mutation is associated with various pathologies including mastocytosis and cancers. D816V-mutated KIT is constitutively active, and resistant to treatment with the anti-cancer drug Imatinib. To elucidate the activating molecular mechanism of this mutation, we applied a multi-approach procedure combining molecular dynamics (MD) simulations, normal modes analysis (NMA) and binding site prediction. Multiple 50-ns MD simulations of wild-type KIT and its mutant D816V were recorded using the inactive auto-inhibited structure of the protein, characteristic of type III RTKs. Computed free energy differences enabled us to quantify the impact of D816V on protein stability in the inactive state. We evidenced a local structural alteration of the activation loop (A-loop) upon mutation, and a long-range structural re-organization of the juxta-membrane region (JMR) followed by a weakening of the interaction network with the kinase domain. A thorough normal mode analysis of several MD conformations led to a plausible molecular rationale to propose that JMR is able to depart its auto-inhibitory position more easily in the mutant than in wild-type KIT and is thus able to promote kinase mutant dimerization without the need for extra-cellular ligand binding. Pocket detection at the surface of NMA-displaced conformations finally revealed that detachment of JMR from the kinase domain in the mutant was sufficient to open an access to the catalytic and substrate binding sites

Resistance variation of conductive ink applied by the screen printing technique on different substrates

This research study focuses on the application of conductive ink by the screen printing technique to evaluate the potential of creating printed electrodes and to investigate the effect of washing upon electrical resistance and flexibility. Two conductive inks were applied by a conventional screen printing method on four different textile substrates, 100% cotton, 50%/50% cotton/polyester, 100% polyester and 100% polyamide. The inks were also applied on a multifibre fabric. Atmospheric plasma treatment was applied to improve the adhesion to the samples, and the resistance values were compared with those of non-treated samples. The values were measured before and after cleaning and washing tests, which were performed to simulate domestic treatment for garments to predict the behaviour of the inks after normal usage of the fabrics. Comfort properties like stiffness of the fabrics were also evaluated after five and 10 washing cycles. It was observed that PE 825 ink forms a thicker film on the fabric surface, contributing to the loss of flexibility of the textile. However, PE 825 ink also produced the best results in terms of durability and lower values of resistance. Polyamide fabrics lost their conductive property after five washing cycles due to weak bonding between the ink and the fibres, whereas cotton fibres provided the best results.This work is financed by Project“Deus ex Machina”, NORTE-01-0145-FEDER-000026, funded by CCDRN, through Sistema de Apoio à Investigação Cientifica e Tecnológica (Projetos Estruturados I&D&I) of Programa Operacional Regional do Norte, from Portugal 2020 and by Project UID/CTM/00264/2019 of 2C2T –Centro de Ciência e Tecnologia Têxtil, funded by National Founds through FCT/MCTES.Derya Tama thanks FCT for fellowship 2C2T-BPD-08-2017

Evaluating the effect of mutations and ligand binding on transthyretin homotetramer dynamics

<div><p>Native transthyretin (TTR) homotetramer dissociation is the first step of the fibrils formation process in amyloid disease. A large number of specific point mutations that destabilize TTR quaternary structure have shown pro-amyloidogenic effects. Besides, several compounds have been proposed as drugs in the therapy of TTR amyloidosis due to their TTR tetramer binding affinities, and therefore, contribution to its integrity. In the present paper we have explored key positions sustaining TTR tetramer dynamical stability. We have identified positions whose mutations alter the most the TTR tetramer equilibrium dynamics based on normal mode analysis and their response to local perturbations. We have found that these positions are mostly localized at β-strands E and F and EF-loop. The monomer-monomer interface is pointed out as one of the most vulnerable regions to mutations that lead to significant changes in the TTR-tetramer equilibrium dynamics and, therefore, induces TTR amyloidosis. Besides, we have found that mutations on residues localized at the dimer-dimer interface and/or at the T4 hormone binding site destabilize the tetramer more than the average. Finally, we were able to compare several compounds according to their effect on vibrations associated to the ligand binding. Our ligand comparison is discussed and analyzed in terms of parameters and measurements associated to TTR-ligand binding affinities and the stabilization of its native state.</p></div