Single-point mutations in the transmembrane (TM) region of receptor tyrosine kinases (RTKs) can lead to abnormal ligand-independent activation. We use a combination of computational modeling, NMR spectroscopy and cell experiments to analyze in detail the mechanism of how TM domains contribute to the activation of wild-type (WT) PDGFRA and its oncogenic V536E mutant. Using a computational framework, we scan all positions in PDGFRA TM helix for identification of potential functional mutations for the WT and the mutant and reveal the relationship between the receptor activity and TM dimerization via different interfaces. This strategy also allows us design a novel activating mutation in the WT (I537D) and a compensatory mutation in the V536E background eliminating its constitutive activity (S541G). We show both computationally and experimentally that single-point mutations in the TM region reshape the TM dimer ensemble and delineate the structural and dynamic determinants of spontaneous activation of PDGFRA via its TM domain. Our atomistic picture of the coupling between TM dimerization and PDGFRA activation corroborates the data obtained for other RTKs and provides a foundation for developing novel modulators of the pathological activity of PDGFRA.
Получено разложение коэффициента диффузии иона в жидкости при низкой концентрации ионов на сумму “коллективной” и “структурной” диффузии (коллективная диффузия–диффузия иона вместе с сольватной оболочкой как единого целого, структурная диффузия – диффузия иона, связанная с обменами атомов в сольватной оболочке, которые приводят к изменению ее конфигурации). Показано, что область применимости такого разложения соответствует ионам, сильно взаимодействующим с сольватной оболочкой в силу их малого размера. Результаты верифицированы сравнением с молекулярно-динамическими расчетами диффузии ионов в воде и жидком ксеноне. Показано хорошее согласие с имеющимися экспериментальными данными.
Bitopic proteins having only one helical transmembrane (TM) domain are a class of biologically significant membrane proteins, including the type I receptors and amyloid precursor protein (APP), which are involved in regulating the homeostasis of human organism and recognized as substrate by c-secretase. Amyloid Ab-peptides forming plaques in brain during Alzheimer disease (AD) are the products of sequential intramembrane cleavage of APP. A lot of mutations associated with AD familial forms were found in the APP transmembrane (TM) domain and juxtamembrane (JM) regions. We designed highly productive systems of bacterial and cell-free expression and easy purification procedure for 13C/15N-isotope labeled APP JM-TM fragments of different length (corresponding to the sequential cleavage steps of APP) and with several familial AD mutations, as well as the TM fragments of c-secretase. The fragments were solubilized in membrane-mimicking complexes (detergent micelles and lipid bicelles), which allows to acquire proper high-resolution NMR spectra despite low sample stability and to characterize their structural-dynamic properties. Molecular Dynamics relaxation of obtained NMR structures of the fragments in hydrated explicit lipid bilayers provided a detailed atomistic picture of the intra- and intermolecular interactions within membrane. The mutant APP JM-TM fragments are shown to be promising objects for elaboration the molecular aspects of the abnormal recognition and sequential proteolysis by c-secretase, revealing a straightforward mechanism of the pathogenesis associated with some familial AD mutation as well as with aging.
Insulin receptor (IR) family is represented by three membrane proteins participating in organism development, growth, and vital activity. Modulation of the functioning of these receptors by external agents looks very perspective from a pharmaceutical point of view. Although IR is well studied, little is known about the role of its transmembrane (TM) domain in receptor activity. Nowadays, the major model of signal transfer by these receptors describes ligand-triggered conformational changes in the extracellular domain, bringing together TM domains that dimerize. This allows trans-autophosphorylation of intracellular domains followed by activation of secondary messengers. However, the conformation of the TM dimeric state is still unknown. Here, we studied in silico dimerization of TM segments of two closest members of the family: IR and IGF-1R. As a result, TM dimeric structures were predicted. This was done taking into account available structural data on extra- and intracellular parts of the receptors. Inspection of the extracellular segment mobility in the basal state revealed several modes of protein motion, although none of them allow TM domain dimerization. The calculated molecular dynamics of TM helices linked to intracellular domain led to a conclusion about autonomic behavior of the TM domain. Based on this data, the dimerization of TM domains was further simulated without extramembrane parts. The most probable models of TM dimeric structures were predicted and the free energy of helix-helix association in explicit lipid bilayers was evaluated. Two most energetically favorable models for IR and one for IGF-1R were delineated. Despite the lack of sequence homology, TM segments in both receptors pack in similar parallel dimers, thus suggesting a close activation mechanism.
В рамках теории функционала плотности исследовано изменение структуры твёрдого водорода при сжатии вдоль изотермы 100 К в области перехода в проводящее состояние. Рассчитаны давление, парная корреляционная функция протонов, плотность электронных состояний и электропроводность в диапазоне плотностей водорода от 1,14 до 2,11 г/см3. Обнаружен переход моноклинной структуры молекулярного твёрдого водорода в ромбоэдрическую с симметрией Cmca с 12 атомами водорода в элементарной ячейке. При этом наблюдается увеличение электропроводности, хотя водород остаётся молекулярным. При сжатии до плотности 1,563 г/см3 наблюдается распад молекул водорода. Элементарная ячейка, возникающий при этом, — квазитетраэдр, образован пятью протонами с расстоянием 0,92 Å от центрального протона до четырёх остальных.
Elastin is an essential component of numerous human tissues and plays a critical role in elasticity of skin, lungs and arteries. During vascular aging, the elastin network is degraded generating elastin-derived peptides (EDP). The ERC (Elastin Receptor Complex) is a membrane heterotrimeric receptor composed, amongst others, of a membrane-bound neuraminidase, NEU-1. Binding of EDP to the ERC induces the activation of signaling pathways associated with biological effects notably the development of diseases such as atherosclerosis, cancer and diabetes. Previous studies of our laboratory have shown that NEU-1 catalytic activity is linked to its ability to homodimerize. Thus, NEU-1 constitutes a key pharmacological target to fight against deleterious effects of EDP. The aim of this work is to develop by biological/biochemical experiments and molecular dynamic (MD) simulations a transmembrane interfering peptide (pI) able to inhibit specifically NEU-1 dimerization. Peptides are delivered into cells using two strategies, TAT peptides, which are cell-penetrating peptides, or lithium dodecyl sulfate micelles. No cellular toxicity was observed in both approaches. Confocal microscopy underlines a colocalization between pI and NEU-1 at the plasma membrane and coimmunoprecipitation experiments show an interaction between pI and NEU-1. Furthermore, sialidase activity assays point out the ability of pI to inhibit NEU-1 homodimerization (47%; 51%) and its associated sialidase activity (21%; 47%). Preliminary MD simulation studies emphasize that both pI and the transmembrane domain of NEU-1 are stable and helix integrity is conserved in lipid bilayer environments. Moreover, the formation of a spontaneous dimer between NEU-1 and pI was identified. Further MD analyses underline the bio- logical relevance of our membrane model. These results reveal the ability of pI to bind to NEU-1, inhibit its dimerization and sialidase activity.