Light-driven proton pumps are present in many organisms. Here, we present a high-resolution structure of a proteorhodopsin from a permafrost bacterium, Exiguobacterium sibiricum rhodopsin (ESR). ...Contrary to the proton pumps of known structure, ESR possesses three unique features. First, ESR's proton donor is a lysine side chain that is situated very close to the bulk solvent. Second, the α-helical structure in the middle of the helix F is replaced by 3 ₁₀- and π-helix–like elements that are stabilized by the Trp-154 and Asn-224 side chains. This feature is characteristic for the proteorhodopsin family of proteins. Third, the proton release region is connected to the bulk solvent by a chain of water molecules already in the ground state. Despite these peculiarities, the positions of water molecule and amino acid side chains in the immediate Schiff base vicinity are very well conserved. These features make ESR a very unusual proton pump. The presented structure sheds light on the large family of proteorhodopsins, for which structural information was not available previously.
Fibroblast growth factor receptor 3 (FGFR3) transduces biochemical signals via lateral dimerization in the plasma membrane, and plays an important role in human development and disease. Eight ...different pathogenic mutations, implicated in cancers and growth disorders, have been identified in the FGFR3 transmembrane segment. Here, we describe the dimerization of the FGFR3 transmembrane domain in membrane-mimicking DPC/SDS (9/1) micelles. In the solved NMR structure, the two transmembrane helices pack into a symmetric left-handed dimer, with intermolecular stacking interactions occurring in the dimer central region. Some pathogenic mutations fall within the helix-helix interface, whereas others are located within a putative alternative interface. This implies that although the observed dimer structure is important for FGFR3 signaling, the mechanism of FGFR3-mediated transduction across the membrane is complex. We propose an FGFR3 signaling mechanism that is based on the solved structure, available structures of isolated soluble FGFR domains, and published biochemical and biophysical data.
Display omitted
•FGFR3 TM domains associate in a left-handed parallel helical dimer via a heptad motif•The solved NMR structure likely corresponds to the FGFR3 basal phosphorylation state•Some, but not all, pathogenic mutations fall within the FGFR3 TM dimer interface•An FGFR3 signaling mechanism based on two alternative TM dimerization modes is proposed
The transmembrane domain (TMD) of FGFR3 is notorious for housing different mutations linked to diverse human pathologies. Bocharov et al. describe the dimerization of TMD and solve an NMR structure to provide testable hypotheses about the mechanism of signaling and the effects of pathogenic mutations on function.
Toll-like receptors (TLRs) are key players in the innate immune system. Despite the great efforts in TLR structural biology, today we know the spatial structures of only four human TLR intracellular ...TIR domains. All of them belong to one of five subfamilies of receptors. One of the main bottlenecks is the high-level production of correctly folded proteins in soluble form. Here we used a rational approach to find the optimal parameters to produce TIR domains of all ten human TLR family members in soluble form in E. coli cells. We showed that dozens of milligrams of soluble His-tagged TLR2/3/6/7.sub.TIR and MBP-tagged TLR3/5/7/8.sub.TIR can be produced. We also developed the purification protocols and demonstrated by CD and NMR spectroscopy that purified TLR2/3/7.sub.TIR demonstrate a structural organization inherent to TIR domains. This illustrates the correct folding of produced proteins and their suitability for further structural and functional investigations.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
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.
Thionins are the family of small (∼5 kDa) cationic cysteine-rich peptides involved in the immune response in plants. Viscotoxin A3 (VtA3) is the thionin from mistletoe (Viscum album) demonstrating ...antimicrobial and cytotoxic activity against cancer cells in vitro. VtA3 (charge +6) interacts with the membranes containing anionic lipids and forms cation-selective ion channels. Here we studied the VtA3 structure in membrane-mimicking media by NMR spectroscopy. Spatial structure of VtA3, consisting of a helical hairpin and a short β-sheet, was stable and did not undergo significant changes during micelle binding. VtA3 molecule bound with high affinity to the surface of zwitterionic dodecylphosphocholine (DPC) micelle by hydrophobic patch in the helical hairpin. Oligomerization of VtA3 was observed in the anionic micelles of sodium dodecylsulphate (SDS). No direct contacts between the peptide molecules were observed and the possible interfaces of detergent-assisted oligomerization were revealed. The data obtained suggest that the VtA3 membrane activity, depending on the concentration, obeys the ‘toroidal’ pore model or the ‘carpet’ mechanism. The model of the membrane disrupting complex, which explains the ion channel formation in the partially anionic membranes, was proposed.
Display omitted
•Spatial structure of VtA3 was studied by NMR in membrane-mimicking micelles.•VtA3 binds to the surface of zwitterionic DPC micelle by helical hairpin.•Oligomerization of VtA3 was observed in the presence of anionic SDS micelles.•No direct contacts between the peptide molecules were observed in SDS.•Configuration of the membrane-embedded toroidal pore was suggested.
► We determined the left-handed dimeric structure of transmembrane domain of amyloid precursor protein. ► The dimerization mechanism of APP is important for understanding the molecular mechanism of ...production of amyloid-β peptide. ► Familial mutations of amyloid precursor protein can affect its lateral dimerization.
Some pathogenic mutations associated with Alzheimer’s disease are thought to affect structural-dynamic properties and the lateral dimerization of amyloid precursor protein (APP) in neuron membrane. Dimeric structure of APP transmembrane fragment Gln686-Lys726 was determined in membrane-mimicking dodecylphosphocholine micelles using high-resolution NMR spectroscopy. The APP membrane-spanning α-helix Lys699-Lys724 self-associates in a left-handed parallel dimer through extended heptad repeat motif I702X3M706X2G709X3A713X2I716X3I720X2I723, whereas the juxtamembrane region Gln686-Val695 constitutes the nascent helix, also sensing the dimerization. The dimerization mechanism of APP transmembrane domain has been described at atomic resolution for the first time and is important for understanding molecular events of APP sequential proteolytical cleavage resulting in amyloid-β peptide.
APPjmtm and APPjmtmbind by comigration in gel electrophoresis (View interaction)
APPjmtm and APPjmtmbind by nuclear magnetic resonance (View interaction).
The Chemical Basis of Fungal Bioluminescence Purtov, Konstantin V.; Petushkov, Valentin N.; Baranov, Mikhail S. ...
Angewandte Chemie (International ed.),
July 6, 2015, Letnik:
54, Številka:
28
Journal Article
Recenzirano
Many species of fungi naturally produce light, a phenomenon known as bioluminescence, however, the fungal substrates used in the chemical reactions that produce light have not been reported. We ...identified the fungal compound luciferin 3‐hydroxyhispidin, which is biosynthesized by oxidation of the precursor hispidin, a known fungal and plant secondary metabolite. The fungal luciferin does not share structural similarity with the other eight known luciferins. Furthermore, it was shown that 3‐hydroxyhispidin leads to bioluminescence in extracts from four diverse genera of luminous fungi, thus suggesting a common biochemical mechanism for fungal bioluminescence.
Getting the green light: Hispidin is shown to be a luciferin precursor in at least four evolutionary distant genera of luminous fungi. However, its biosynthesis alone does not result in fungal bioluminescence. Two enzymes are required: first a hydroxylase converts the hispidin into 3‐hydroxyhispidin, which acts as the luciferin, which undergoes a subsequent oxidation in the presence of a luciferase.
Alzheimer’s disease is the most common type of neurodegenerative disease in the world. Genetic evidence strongly suggests that aberrant generation, aggregation, and/or clearance of neurotoxic ...amyloid-β peptides (Aβ) triggers the disease. Aβ accumulates at the points of contact of neurons in ordered cords and fibrils, forming the so-called senile plaques. Aβ isoforms of different lengths are found in healthy human brains regardless of age and appear to play a role in signaling pathways in the brain and to have neuroprotective properties at low concentrations. In recent years, different substances have been developed targeting Aβ production, aggregation, interaction with other molecules, and clearance, including peptide-based drugs. Aβ is a product of sequential cleavage of the membrane glycoprotein APP (amyloid precursor protein) by β- and γ-secretases. A number of familial mutations causing an early onset of the disease have been identified in the APP, especially in its transmembrane domain. The mutations are reported to influence the production, oligomerization, and conformational behavior of Aβ peptides. This review highlights the results of structural studies of the main proteins involved in Alzheimer’s disease pathogenesis and the molecular mechanisms by which perspective therapeutic substances can affect Aβ production and nucleation.
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.
One of the essential characteristics of any tag used in bioscience and medical applications is its size. The larger the label, the more it may affect the studied object, and the more it may distort ...its behavior. In this paper, using NMR spectroscopy and X-ray crystallography, we have studied the structure of fluorogen-activating protein FAST both in the apo form and in complex with the fluorogen. We showed that significant change in the protein occurs upon interaction with the ligand. While the protein is completely ordered in the complex, its apo form is characterized by higher mobility and disordering of its N-terminus. We used structural information to design the shortened FAST (which we named nanoFAST) by truncating 26 N-terminal residues. Thus, we created the shortest genetically encoded tag among all known fluorescent and fluorogen-activating proteins, which is composed of only 98 amino acids.
We solved the structure of a fluorogen-activating protein FAST and synthesized the library of potential fluorogens. Using these data, we designed the shortest genetically encoded fluorescent tag among all known.
PDF
