Toll-like receptor 3 transmembrane domain is able to perform various homotypic interactions: An NMR structural study

AbstractToll-like receptors (TLRs) take part in both the innate and adaptive immune systems. The role of the transmembrane domain in TLR signaling is still elusive, while its importance for the TLR activation was clearly demonstrated. In the present study the ability of the TLR3 transmembrane domain to form dimers and trimers in detergent micelles was shown by solution NMR spectroscopy. Spatial structures and free energy magnitudes were determined for the TLR3 transmembrane domain in dimeric and trimeric states, and two possible surfaces that may be used for the helix–helix interaction by the full-length TLR3 were revealed

NMR Dynamics of Transmembrane and Intracellular Domains of p75NTR in Lipid-Protein Nanodiscs

AbstractP75NTR is a type I integral membrane protein that plays a key role in neurotrophin signaling. However, structural data for the receptor in various functional states are sparse and controversial. In this work, we studied the spatial structure and mobility of the transmembrane and intracellular parts of p75NTR, incorporated into lipid-protein nanodiscs of various sizes and compositions, by solution NMR spectroscopy. Our data reveal a high level of flexibility and disorder in the juxtamembrane chopper domain of p75NTR, which results in the motions of the receptor death domain being uncoupled from the motions of the transmembrane helix. Moreover, none of the intracellular domains of p75NTR demonstrated a propensity to interact with the membrane or to self-associate under the experimental conditions. The obtained data are discussed in the context of the receptor activation mechanism

Structural Basis of p75 Transmembrane Domain Dimerization

Dimerization of single span transmembrane receptors underlies their mechanism of activation. p75 neurotrophin receptor plays an important role in the nervous system, but the understanding of p75 activation mechanism is still incomplete. The transmembrane (TM) domain of p75 stabilizes the receptor dimers through a disulfide bond, essential for the NGF signaling. Here we solved by NMR the three-dimensional structure of the p75-TM-WT and the functionally inactive p75-TM-C257A dimers. Upon reconstitution in lipid micelles, p75-TM-WT forms the disulfide-linked dimers spontaneously. Under reducing conditions, p75-TM-WT is in a monomer-dimer equilibrium with the Cys(257) residue located on the dimer interface. In contrast, p75-TM-C257A forms dimers through the AXXXG motif on the opposite face of the α-helix. Biochemical and cross-linking experiments indicate that AXXXG motif is not on the dimer interface of p75-TM-WT, suggesting that the conformation of p75-TM-C257A may be not functionally relevant. However, rather than mediating p75 homodimerization, mutagenesis of the AXXXG motif reveals its functional role in the regulated intramembrane proteolysis of p75 catalyzed by the γ-secretase complex. Our structural data provide an insight into the key role of the Cys(257) in stabilization of the weak transmembrane dimer in a conformation required for the NGF signaling.This work was supported in part by Russian Science Foundation Project 14-50-00131 (to A. S. A.) (NMR structural studies) and Spanish Ministry of Economy and Competitiveness (MINECO) Project BFU2013-42746-P (to M. V.). The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.S

Segregated Network Polymer Composites with High Electrical Conductivity and Well Mechanical Properties based on PVC, P(VDFTFE), UHMWPE, and rGO

The formation of a segregated network structure (wittingly uneven distribution of a filler) is one of the most promising strategies for the fabrication of electrically conductive polymer composites at present. However, the simultaneous achievement of high values of electrical conductivity with the retention of well mechanical properties within this approach remains a great challenge. Here, by means of X-ray photoelectron spectra (XPS), near-edge X-ray absorption fine structure (NEXAFS) spectra, scanning electron microscopy (SEM), dielectric spectroscopy, and compression engineering stress-strain curve analysis, we have studied the effect of a segregated network structure on the electrical conductivity and mechanical properties of a set of polymer composites. The composites were prepared by applying graphene oxide (GO) with ultralarge basal plane size (up to 150 μm) onto the surface of polymer powder particles, namely, poly(vinyl chloride) (PVC), poly(vinylidene fluoride-co-tetrafluoroethylene) (P(VDF-TFE)), and ultrahigh-molecular-weight poly(ethylene) (UHMWPE) with the subsequent GO reduction and composite hot pressing. A strong dependence of the segregated network polymer composites' physical properties on the polymer matrix was demonstrated. Particularly, 12 orders of magnitude rise of the polymers' electrical conductivity up to 0.7 S/m was found upon the incorporation of the reduced GO (rGO). A 17% increase in the P(VDF-TFE) elastic modulus filled by 1 wt % of rGO was observed. Fracture strength of PVC/rGO at 0.5 wt % content of the filler was demonstrated to decrease by fourfold. At the same time, the change in strength was not significant for P(VDF-TFE) and UHMWPE composites in comparison with pure polymers. Our results show a promise to accelerate the development of new composites for energy applications, such as metal-free supercapacitor plates and current collectors of lithium-ion batteries, bipolar plates of proton-exchange membrane fuel cells, antistatic elements of various electronic devices, etc. © 2020 American Chemical Society

Distemper, extinction, and vaccination of the Amur tiger

Canine distemper virus (CDV) has recently emerged as an extinction threat for the endangered Amur tiger (Panthera tigris altaica). CDV is vaccine-preventable, and control strategies could require vaccination of domestic dogs and/or wildlife populations. However, vaccination of endangered wildlife remains controversial, which has led to a focus on interventions in domestic dogs, often assumed to be the source of infection. Effective decision making requires an understanding of the true reservoir dynamics, which poses substantial challenges in remote areas with diverse host communities. We carried out serological, demographic, and phylogenetic studies of dog and wildlife populations in the Russian Far East to show that a number of wildlife species are more important than dogs, both in maintaining CDV and as sources of infection for tigers. Critically, therefore, because CDV circulates among multiple wildlife sources, dog vaccination alone would not be effective at protecting tigers. We show, however, that low-coverage vaccination of tigers themselves is feasible and would produce substantive reductions in extinction risks. Vaccination of endangered wildlife provides a valuable component of conservation strategies for endangered species

Spatial Structure of NanoFAST in the Apo State and in Complex with its Fluorogen HBR-DOM2

NanoFAST is a fluorogen-activating protein and can be considered one of the smallest encodable fluorescent tags. Being a shortened variant of another fluorescent tag, FAST, nanoFAST works nicely only with one out of all known FAST ligands. This substantially limits the applicability of this protein. To find the reason for such a behavior, we investigated the spatial structure and dynamics of nanoFAST, both in the apo state and in the complex with its fluorogen molecule, using the solution NMR spectroscopy. We showed that the truncation of FAST did not affect the structure of the remaining part of the protein. Our data suggest that the deleted N-terminus of FAST destabilizes the C-terminal domain in the apo state. While it does not contact the fluorogen directly, it serves as a free energy reservoir that enhances the ligand binding propensity of the protein. The structure of nanoFAST/HBR-DOM2 complex reveals the atomistic details of nanoFAST interactions with the rhodanine-based ligands and explains the ligand specificity. NanoFAST selects ligands with the lowest dissociation constants, 2,5-disubstituted 4-hydroxybenzyldienerhodainines, which allow the non-canonical intermolecular CH–N hydrogen bonding and provide the optimal packing of the ligand within the hydrophobic cavity of the protein

Revising the mechanism of p75NTR activation: intrinsically monomeric state of death domains invokes the "helper" hypothesis

Abstract The neurotrophin receptor p75NTR plays crucial roles in neuron development and regulates important neuronal processes like degeneration, apoptosis and cell survival. At the same time the detailed mechanism of signal transduction is unclear. One of the main hypotheses known as the snail-tong mechanism assumes that in the inactive state, the death domains interact with each other and in response to ligand binding there is a conformational change leading to their exposure. Here, we show that neither rat nor human p75NTR death domains homodimerize in solution. Moreover, there is no interaction between the death domains in a more native context: the dimerization of transmembrane domains in liposomes and the presence of activating mutation in extracellular juxtamembrane region do not lead to intracellular domain interaction. These findings suggest that the activation mechanism of p75NTR should be revised. Thus, we propose a novel model of p75NTR functioning based on interaction with “helper” protein

Phase Transitions in Small Isotropic Bicelles

Isotropic phospholipid bicelles are one of the most prospective membrane mimetics for the structural studies of membrane proteins in solution. Recent works provided an almost full set of data regarding the properties of isotropic bicelles; however, one major aspect of their behavior is still under consideration: the possible mixing between the lipid and detergent in the bilayer area. This problem may be resolved by studying the lipid phase transitions in bicelle particles. In the present work, we investigate two effects: phase transitions of bilayer lipids and temperature-induced growth of isotropic bicelles using the NMR spectroscopy. We propose an approach to study the phase transitions in isotropic bicelles based on the properties of ³¹P NMR spectra of bilayer-forming lipids. We show that phase transitions in small bicelles are “fractional”, particles with the liquid-crystalline and gel bilayers coexist in solution at certain temperatures. We study the effects of lipid fatty chain type and demonstrate that the behavior of various lipids in bilayers is reproduced in the isotropic bicelles. We show that the temperature-induced growth of isotropic bicelles is not related directly to the phase transition but is the result of the reversible fusion of bicelle particles. In accordance with our data, rim detergents also have an impact on phase transitions: detergents that resist the temperature-induced growth provide the narrowest and most expressed transitions at higher temperatures. We demonstrate clearly that phase transitions take place even in the smallest bicelles that are applicable for structural studies of membrane proteins by solution NMR spectroscopy. This last finding, together with other data draws a thick line under the long-lasting argument about the relevance of small isotropic bicelles. We show with certainty that the small bicelles can reproduce the most fundamental property of lipid membranes: the ability to undergo phase transition

Structural basis of the transmembrane domain dimerization and rotation in the activation mechanism of the TRKA receptor by nerve growth factor

Artículo 10 páginas, 6 figuras. Contiene en material suplementario: Tabla S1 y Figs. S1–S7. This research was originally published in the Journal of Biological Chemistry. María L. Franco, Kirill D. Nadezhdin, Sergey A. Goncharuk, Konstantin S. Mineev, Alexander S. Arseniev, and Marçal Vilar. Structural basis of the transmembrane domain dimerization and rotation in the activation mechanism of the TRKA receptor by nerve growth factor. J. Biol. Chem. 2020; 295(1):275-286. © the American Society for Biochemistry and Molecular Biology.Tropomyosin-receptor kinases (TRKs) are essential for the development of the nervous system. The molecular mechanism of TRKA activation by its ligand nerve growth factor (NGF) is still unsolved. Recent results indicate that at endogenous levels most of TRKA is in a monomer-dimer equilibrium and that the binding of NGF induces an increase of the dimeric and oligomeric forms of this receptor. An unsolved issue is the role of the TRKA transmembrane domain (TMD) in the dimerization of TRKA and the structural details of the TMD in the active dimer receptor. Here, we found that the TRKA-TMD can form dimers, identified the structural determinants of the dimer interface in the active receptor, and validated this interface through site-directed mutagenesis together with functional and cell differentiation studies. Using in vivo cross-linking, we found that the extracellular juxtamembrane region is reordered after ligand binding. Replacement of some residues in the juxtamembrane region with cysteine resulted in ligand-independent active dimers and revealed the preferred dimer interface. Moreover, insertion of leucine residues into the TMD helix induced a ligand-independent TRKA activation, suggesting that a rotation of the TMD dimers underlies NGF-induced TRKA activation. Altogether, our findings indicate that the transmembrane and juxtamembrane regions of TRKA play key roles in its dimerization and activation by NGF.This work was supported by the Ministerio de Economía, Industria y Competitividad Projects BFU2013-42746-P and SAF2017-84096-R and by Generalitat Valenciana Prometeo Grant 2018/055 (to M. V.). The studies of TRKA-TM homodimerization were supported by Russian Science Foundation Grant 19-74-30014 (to A. S. A). The authors declare that they have no conflicts of interest with the contents of this articlePeer reviewe

A Combination of Library Screening and Rational Mutagenesis Expands the Available Color Palette of the Smallest Fluorogen-Activating Protein Tag nanoFAST

NanoFAST is the smallest fluorogen-activating protein, consisting of only 98 amino acids, used as a genetically encoded fluorescent tag. Previously, only a single fluorogen with an orange color was revealed for this protein. In the present paper, using rational mutagenesis and in vitro screening of fluorogens libraries, we expanded the color palette of this tag. We discovered that E46Q is one of the key substitutions enabling the range of possible fluorogens to be expanded. The introduction of this and several other substitutions has made it possible to use not only orange but also red and green fluorogens with the modified protein