FEBS Open Bio Volume 9, Issue S1 Supplement: 44th FEBS Congress, From Molecules to Living Systems, Krakow, Poland, July 6‐11, 2019
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.
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.
The human epidermal growth factor (EGFR/HER) and growth hormone (GHR) receptors serve as excellent models of type I receptors to illustrate how ligand-induced conformational rearrangements and specific dimerization of extracellular domains lead to the allosteric activation of the cytoplasmic domains via single-span transmembrane domain (TMD). We determined the alternative dimeric conformations of the EGFR and GHR TMDs in different membrane-mimicking environments using high-resolution NMR spectroscopy combined with MD-relaxation in explicit lipid bilayer. Based on the location of pathogenic transmembrane mutations, observed conformations correspond to the dormant and active states of both receptors, assuming an impact of intramembrane interactions to the cell signaling dysfunction in human organism. Fine adaptation of intermolecular polar and hydrophobic contacts that we found to accompany the different EGFR TMD dimerizations suggests that certain membrane properties can govern the TMD helix-helix packing and, thus, their alteration can trigger the receptor state. Whereas two distinct dimeric modes of GHR TMD revealed the functional role of juxtamembrane region rearrangements in alternation between protein-protein and protein-lipid interactions that can be initiated by ligand binding. Observed the TMD helix-helix packing diversity appears in favor of the lipid-mediated rotationcoupled activation mechanism, which implies that the sequence of structural rearrangements of EGFR and GHR domains is associated with perturbations of the lipid bilayer in the course of ligand-induced receptor activation, considering the receptor together with its lipid environment as a self-consistent signal transduction system.
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.
Acid-sensing ion channels (ASICs), members of the family of amiloride-sensitive degenerin/epithelial Na + -channels, are expressed in neurons of both the peripheral and central nervous system. Among six currently known isoforms of mammalian ASICs, two, namely ASIC1a and ASIC3 are widespread and have the largest physiological contribution such as synaptic plas- ticity, learning and memory as well as pain perception and inflammation development. We have previously shown that seva- nol, a new lignan isolated from Thymus armeniacus, inhibits ASIC1a and ASIC3 currents and exerts analgesic and anti- inflammatory effects when administered intravenously. Here we present a scheme for the synthesis of sevanol, which was devel- oped for the first time. In addition, sevanol analogues were syn- thesized, in which the basic core of the molecule of epiphyllic acid remained unchanged, while substituents for carboxyl groups in positions 9,10 were modified. These analogues demonstrate a clear correlation between the activity of the molecule and the number of free carboxyl groups in it. Using molecular modeling and analysis of the activity of sevanol in the presence of ASIC1a potentiator, an RF-amide peptide, we established a possible bind- ing site for sevanol on the channel. We also showed that with intranasal administration, sevanol can have the same effective analgesic effect as with intravenous administration. Such struc- tural and functional analysis demonstrates a correlation between the inhibitory effect value and the number of functional groups of the molecule, which may be important for the rational design of biologically active sevanol-based compounds.
Receptor tyrosine kinases (RTK) are vital players in cell signaling governing growth and proliferation. These integral membrane proteins work only in dimeric states, so the conformation of transmembrane dimer determines the signal transferred into cell. Here, we used modern molecular modeling techniques to study details of protein-protein and protein-lipid interactions in model systems containing monomers and dimers of several receptor tyrosine kinases with glycophorin-like dimerization motifs. Comparison of structural and dynamic aspects of ErbB family members and glycophorin A (GpA) revealed similarities in their properties, especially, for ErbB1, ErbB2 and ErbB4 receptors utilizing the same GpA-like motif for dimerization in their basal state. We demonstrated that they all have similar organization of TM domain’s molecular surface in terms of both relief, hydrophobic properties and lipid binding sites resembling GpA pattern studied before. All these RTKs strongly interact with lipid acyl chains, forming stable binding sites both in monomeric and dimeric states, and the most prominent binding areas are located in monomers on the future GpA-like dimerization interfaces. Then, lipids distribution changes upon dimer formation. This is not the case for alternative packing geometries observed for the second state of ErbB1 and, especially, ErbB3. We found higher numbers of immobilized lipids near C-terminus in ErbB1 and ErbB2 active dimers, thus assuming that the existing structure of ErbB3 is also active. However, there is non-functional GpA-like motif in ErbB3 with some bound lipids present near the N-terminus, suspecting another structure for inactive receptor. However, despite considerable similarities, these RTKs have different hydrophobicity distributions along helices, that can be important in terms of preferable lipid environment. The work was funded by the Russian Academic Excellence Project ‘5-100’ and Russian Foundation for Basic Research grant 18-54-15007.
The worldwide rapid emergence of resistant bacteria put at threat the efficacy of antibiotics, thus the development of novel antibacterial agents is urgently needed. The cell wall precursor lipid II consisting the chemically conservative pyrophosphate group represents a promising pharmaceutical target. Antimicrobial peptides, that target lipid II, i.e. lantibiotic nisin, could be excellent prototypes for new generation antibiotics due to their low liability to develop resistance. Understanding of molecular mechanism of initial stages of membrane-bound lipid II recognition by water-soluble nisin is indispensable, in order to improve the peptide structure and properties into pharmaceutically applicable form. Here, we present a molecular dynamics simulation study of initial stages of the aforementioned recognition. In membrane environment, lipid II adopts very few conformations characterized by unique spatial arrangement of hydrogen bond acceptors in the pyrophosphate group at the bilayer surface. These acceptors are efficiently captured by NH groups of nisin, thus explaining its high selectivity to lipid II. Similarly, rings A and B of nisin, which are known to recognize lipid II, adopt the only stable conformation in the presence of dimethylpyrophosphate, which mimics the binding determinant of lipid II. Finally, we propose molecular model of nisin (rings A and B) / lipid II complex in bacterial membrane, which may be employed for design of novel antibiotic prototypes.