The human cis-prenyltransferase (hcis-PT) is an enzymatic complex essential for protein N-glycosylation. Synthesizing the precursor of the glycosyl carrier dolichol-phosphate, mutations in hcis-PT cause severe human diseases. Here, we reveal that hcis-PT exhibits a heterotetrameric assembly in solution, consisting of two catalytic dehydrodolichyl diphosphate synthase (DHDDS) and inactive Nogo-B receptor (NgBR) heterodimers. Importantly, the 2.3 Å crystal structure reveals that the tetramer assembles via the DHDDS C-termini as a dimer-of-heterodimers. Moreover, the distal C-terminus of NgBR transverses across the interface with DHDDS, directly participating in active-site formation and the functional coupling between the subunits. Finally, we explored the functional consequences of disease mutations clustered around the active-site, and in combination with molecular dynamics simulations, we propose a mechanism for hcis-PT dysfunction in retinitis pigmentosa. Together, our structure of the hcis-PT complex unveils the dolichol synthesis mechanism and its perturbation in disease.

• MeSH
• alkyltransferasy a aryltransferasy chemie   genetika   metabolismus   MeSH
• aminokyselinové motivy MeSH
• dimerizace MeSH
• katalytická doména MeSH
• lidé MeSH
• mutace MeSH
• receptory buněčného povrchu chemie   genetika   metabolismus   MeSH
• retinopathia pigmentosa enzymologie   genetika   MeSH
• transferasy chemie   genetika   metabolismus   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Three-dimensional structure models refined using low-resolution data from crystallographic or electron cryo-microscopy experiments can benefit from high-quality restraints derived from quantum-chemical methods. However, nonperiodic atom-centered quantum-chemistry codes do not inherently account for nearest-neighbor interactions of crystallographic symmetry-related copies in a satisfactory way. Here, these nearest-neighbor effects have been included in the model by expanding to a super-cell and then truncating the super-cell to only include residues from neighboring cells that are interacting with the asymmetric unit. In this way, the fragmentation approach can adequately and efficiently include nearest-neighbor effects. It has previously been shown that a moderately sized X-ray structure can be treated using quantum methods if a fragmentation approach is applied. In this study, a target protein (PDB entry 4gif) was partitioned into a number of large fragments. The use of large fragments (typically hundreds of atoms) is tractable when a GPU-based package such as TeraChem is employed or cheaper (semi-empirical) methods are used. The QM calculations were run at the HF-D3/6-31G level. The models refined using a recently developed semi-empirical method (GFN2-xTB) were compared and contrasted. To validate the refinement procedure for a non-P1 structure, a standard set of crystallographic metrics were used. The robustness of the implementation is shown by refining 13 additional protein models across multiple space groups and a summary of the refinement metrics is presented.

• MeSH
• konformace proteinů MeSH
• krystalografie rentgenová metody   MeSH
• molekulární modely * MeSH
• receptory buněčného povrchu chemie   MeSH
• software * MeSH
• vápníkové kanály chemie   MeSH
• Publikační typ
• časopisecké články MeSH

Multistep phosphorelay (MSP) cascades mediate responses to a wide spectrum of stimuli, including plant hormonal signaling, but several aspects of MSP await elucidation. Here, we provide first insight into the key step of MSP-mediated phosphotransfer in a eukaryotic system, the phosphorylation of the receiver domain of the histidine kinase CYTOKININ-INDEPENDENT 1 (CKI1RD) from Arabidopsis thaliana We observed that the crystal structures of free, Mg2+-bound, and beryllofluoridated CKI1RD (a stable analogue of the labile phosphorylated form) were identical and similar to the active state of receiver domains of bacterial response regulators. However, the three CKI1RD variants exhibited different conformational dynamics in solution. NMR studies revealed that Mg2+ binding and beryllofluoridation alter the conformational equilibrium of the β3-α3 loop close to the phosphorylation site. Mutations that perturbed the conformational behavior of the β3-α3 loop while keeping the active-site aspartate intact resulted in suppression of CKI1 function. Mechanistically, homology modeling indicated that the β3-α3 loop directly interacts with the ATP-binding site of the CKI1 histidine kinase domain. The functional relevance of the conformational dynamics observed in the β3-α3 loop of CKI1RD was supported by a comparison with another A. thaliana histidine kinase, ETR1. In contrast to the highly dynamic β3-α3 loop of CKI1RD, the corresponding loop of the ETR1 receiver domain (ETR1RD) exhibited little conformational exchange and adopted a different orientation in crystals. Biochemical data indicated that ETR1RD is involved in phosphorylation-independent signaling, implying a direct link between conformational behavior and the ability of eukaryotic receiver domains to participate in MSP.

• MeSH
• Arabidopsis enzymologie   genetika   MeSH
• krystalografie rentgenová MeSH
• nukleární magnetická rezonance biomolekulární MeSH
• proteinkinasy chemie   genetika   MeSH
• proteinové domény MeSH
• proteiny huseníčku chemie   genetika   MeSH
• receptory buněčného povrchu chemie   genetika   MeSH
• sekundární struktura proteinů MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

While the molecular basis for cytokinin action is quite well understood in flowering plants, little is known about the cytokinin signal transduction in early diverging land plants. The genome of the bryophyte Physcomitrella patens (Hedw.) B.S. encodes three classical cytokinin receptors, the CHASE domain-containing histidine kinases, CHK1, CHK2, and CHK3. In a complementation assay with protoplasts of receptor-deficient Arabidopsis thaliana as well as in cytokinin binding assays, we found evidence that CHK1 and CHK2 receptors can function in cytokinin perception. Using gene targeting, we generated a collection of CHK knockout mutants comprising single (Δchk1, Δchk2, Δchk3), double (Δchk1,2, Δchk1,3, Δchk2,3), and triple (Δchk1,2,3) mutants. Mutants were characterized for their cytokinin response and differentiation capacities. While the wild type did not grow on high doses of cytokinin (1 µM benzyladenine), the Δchk1,2,3 mutant exhibited normal protonema growth. Bud induction assays showed that all three cytokinin receptors contribute to the triggering of budding, albeit to different extents. Furthermore, while the triple mutant showed no response in this bioassay, the remaining mutants displayed budding responses in a diverse manner to different types and concentrations of cytokinins. Determination of cytokinin levels in mutants showed no drastic changes for any of the cytokinins; thus, in contrast to Arabidopsis, revealing only small impacts of cytokinin signaling on homeostasis. In summary, our study provides a first insight into the molecular action of cytokinin in an early diverging land plant and demonstrates that CHK receptors play an essential role in bud induction and gametophore development.

• MeSH
• biotest MeSH
• butadieny farmakologie   MeSH
• cytokininy farmakologie   MeSH
• fenotyp MeSH
• fyziologická adaptace MeSH
• genový knockout MeSH
• hemiterpeny farmakologie   MeSH
• mechy účinky léků   genetika   metabolismus   MeSH
• mutace genetika   MeSH
• pentany farmakologie   MeSH
• receptory buněčného povrchu chemie   metabolismus   MeSH
• regulace genové exprese u rostlin MeSH
• rostlinné proteiny chemie   metabolismus   MeSH
• terciární struktura proteinů MeSH
• vztahy mezi strukturou a aktivitou MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

The discovery of effective ligands for DC-SIGN receptor is one of the most challenging concepts of antiviral drug design due to the importance of this C-type lectin in infection processes. DC-SIGN recognizes mannosylated and fucosylated oligosaccharides but glycosidic linkages are accessible to both chemical and enzymatic degradations. To avoid this problem, the synthesis of stable glycoside mimetics has attracted increasing attention. In this work we establish for the first time mono- and divalent C-glycosides based on d-manno and l-fuco configurations as prospective DC-SIGN ligands. In particular, the l-fucose glycomimetics were more active than the respective d-mannose ones. The highest affinity was assessed for simple 1,4-bis(α-l-fucopyranosyl)butane (SPR: IC50 0.43 mM) that displayed about twice higher activity than natural ligand Le(x). Our results make C-glycosides attractive candidates for multivalent presentations.

• MeSH
• biomimetika MeSH
• fukosa chemie   MeSH
• glykosidy chemická syntéza   chemie   MeSH
• lektiny typu C chemie   metabolismus   MeSH
• lidé MeSH
• mannosa chemie   MeSH
• molekulární struktura MeSH
• molekuly buněčné adheze chemie   metabolismus   MeSH
• receptory buněčného povrchu chemie   metabolismus   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH

DCL-1 (CD302) is a single-pass type one transmembrane protein which is predominantly expressed on myeloid cell lines. It possess the ability of endocytosis and is assumed to play a role in cell adhesion and migration. It has been also connected to several illnesses but more on the level of mRNA than on the protein expression level. More interestingly it is alternatively expressed in the form of a fusion protein with another single-pass type one transmembrane protein DEC205 (CD205) which is normally involved in antigen-uptake and endocytosis. The fusion protein has been assigned to have altered function compared to the wild type proteins. We have performed NMR structural analysis of the 16.2 kDa extracellular domain of DCL-1 to get a better insight onto this molecule. We have been able to assign nearly 97 % of resonance frequencies for the (15)N and (13)C labeled recombinant protein. The assignments have been deposited into Biological Magnetic Resonance Data Bank under the accession number 25802.

• MeSH
• extracelulární prostor * MeSH
• lektiny typu C chemie   MeSH
• lidé MeSH
• nukleární magnetická rezonance biomolekulární * MeSH
• proteinové domény MeSH
• receptory buněčného povrchu chemie   MeSH
• sekundární struktura proteinů MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Human LLT1 is a C-type lectin-like ligand of NKR-P1 (CD161, gene KLRB1), a C-type lectin-like receptor of natural killer cells. Using X-ray diffraction, the first experimental structures of human LLT1 were determined. Four structures of LLT1 under various conditions were determined: monomeric, dimeric deglycosylated after the first N-acetylglucosamine unit in two forms and hexameric with homogeneous GlcNAc2Man5 glycosylation. The dimeric form follows the classical dimerization mode of human CD69. The monomeric form keeps the same fold with the exception of the position of an outer part of the long loop region. The hexamer of glycosylated LLT1 consists of three classical dimers. The hexameric packing may indicate a possible mode of interaction of C-type lectin-like proteins in the glycosylated form.

• MeSH
• glykosylace MeSH
• kvarterní struktura proteinů MeSH
• lektinové receptory NK-buněk - podrodina B chemie   genetika   metabolismus   MeSH
• lektiny typu C chemie   genetika   metabolismus   MeSH
• lidé MeSH
• multimerizace proteinu * MeSH
• receptory buněčného povrchu chemie   genetika   metabolismus   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Dolichol is an obligate carrier of glycans for N-linked protein glycosylation, O-mannosylation, and GPI anchor biosynthesis. cis-prenyltransferase (cis-PTase) is the first enzyme committed to the synthesis of dolichol. However, the proteins responsible for mammalian cis-PTase activity have not been delineated. Here we show that Nogo-B receptor (NgBR) is a subunit required for dolichol synthesis in yeast, mice, and man. Moreover, we describe a family with a congenital disorder of glycosylation caused by a loss of function mutation in the conserved C terminus of NgBR-R290H and show that fibroblasts isolated from patients exhibit reduced dolichol profiles and enhanced accumulation of free cholesterol identically to fibroblasts from mice lacking NgBR. Mutation of NgBR-R290H in man and orthologs in yeast proves the importance of this evolutionarily conserved residue for mammalian cis-PTase activity and function. Thus, these data provide a genetic basis for the essential role of NgBR in dolichol synthesis and protein glycosylation.

• MeSH
• bodová mutace MeSH
• dolichol metabolismus   MeSH
• genový knockout MeSH
• glykosylace MeSH
• kultivované buňky MeSH
• lidé MeSH
• metabolické nemoci genetika   metabolismus   MeSH
• molekulární evoluce MeSH
• molekulární sekvence - údaje MeSH
• myši MeSH
• receptory buněčného povrchu chemie   genetika   metabolismus   MeSH
• Saccharomyces cerevisiae - proteiny chemie   genetika   metabolismus   MeSH
• Saccharomyces cerevisiae chemie   genetika   metabolismus   MeSH
• sekvence aminokyselin MeSH
• transferasy chemie   genetika   metabolismus   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• mužské pohlaví MeSH
• myši MeSH
• ženské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Research Support, N.I.H., Extramural MeSH

In this study, in silico mutagenesis and docking in Ralstonia solanacearum lectin (RSL) were carried out, and the ability of several docking software programs to calculate binding affinity was evaluated. In silico mutation of six amino acid residues (Agr17, Glu28, Gly39, Ala40, Trp76, and Trp81) was done, and a total of 114 in silico mutants of RSL were docked with Me-α-L-fucoside. Our results show that polar residues Arg17 and Glu28, as well as nonpolar amino acids Trp76 and Trp81, are crucial for binding. Gly39 may also influence ligand binding because any mutations at this position lead to a change in the binding pocket shape. The Ala40 residue was found to be the most interesting residue for mutagenesis and can affect the selectivity and/or affinity. In general, the docking software used performs better for high affinity binders and fails to place the binding affinities in the correct order.

• MeSH
• krystalografie rentgenová MeSH
• lektiny chemie   genetika   metabolismus   MeSH
• molekulární modely MeSH
• molekulární sekvence - údaje MeSH
• mutageneze MeSH
• počítačová simulace MeSH
• Ralstonia solanacearum chemie   genetika   metabolismus   MeSH
• receptory buněčného povrchu chemie   MeSH
• sacharidové sekvence MeSH
• software MeSH
• vazebná místa MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

N-methyl-D-aspartate (NMDA) receptors are glutamate ion channels that are critically involved in excitatory synaptic transmission and plasticity. The functional NMDA receptor is a heterotetramer composed mainly of GluN1 and GluN2 subunits. It is generally thought that only correctly assembled NMDA receptors can pass the quality control checkpoint in the endoplasmic reticulum (ER) and are transported to the cell surface membranes. The molecular mechanisms underlying these processes remain poorly understood. Using chimeric and mutated GluN1 subunits expressed in heterologous cells, we identified a single amino acid residue within the fourth membrane domain (M4) of GluN1 subunit, L830, that regulates the surface number of NMDA receptors. Our experiments show that this residue is not critical for the interaction between GluN1 and GluN2 subunits or for the formation of functional receptors, but rather that it regulates the forward trafficking of the NMDA receptors. The surface expression of both GluN2A- and GluN2B-containing receptors is regulated by the L830 residue in a similar manner. We also found that the L830 residue is not involved in the trafficking of individually expressed GluN1 subunits. Our data reveal a critical role of the single amino acid residue within the GluN1 M4 domain in the surface delivery of functional NMDA receptors.

• MeSH
• aminokyseliny genetika   MeSH
• Cercopithecus aethiops MeSH
• COS buňky MeSH
• endoplazmatické retikulum metabolismus   MeSH
• HEK293 buňky MeSH
• kvarterní struktura proteinů fyziologie   MeSH
• lidé MeSH
• mutageneze fyziologie   MeSH
• receptory buněčného povrchu chemie   genetika   metabolismus   MeSH
• receptory N-methyl-D-aspartátu chemie   genetika   metabolismus   MeSH
• terciární struktura proteinů fyziologie   MeSH
• transport proteinů fyziologie   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH