Signaling by the human C-type lectin-like receptor, natural killer (NK) cell inhibitory receptor NKR-P1, has a critical role in many immune-related diseases and cancer. C-type lectin-like receptors have weak affinities to their ligands; therefore, setting up a comprehensive model of NKR-P1-LLT1 interactions that considers the natural state of the receptor on the cell surface is necessary to understand its functions. Here we report the crystal structures of the NKR-P1 and NKR-P1:LLT1 complexes, which provides evidence that NKR-P1 forms homodimers in an unexpected arrangement to enable LLT1 binding in two modes, bridging two LLT1 molecules. These interaction clusters are suggestive of an inhibitory immune synapse. By observing the formation of these clusters in solution using SEC-SAXS analysis, by dSTORM super-resolution microscopy on the cell surface, and by following their role in receptor signaling with freshly isolated NK cells, we show that only the ligation of both LLT1 binding interfaces leads to effective NKR-P1 inhibitory signaling. In summary, our findings collectively support a model of NKR-P1:LLT1 clustering, which allows the interacting proteins to overcome weak ligand-receptor affinity and to trigger signal transduction upon cellular contact in the immune synapse.
- MeSH
- antigeny povrchové MeSH
- buňky NK * MeSH
- difrakce rentgenového záření MeSH
- lektinové receptory NK-buněk - podrodina B MeSH
- lektiny typu C MeSH
- lidé MeSH
- ligandy MeSH
- maloúhlový rozptyl MeSH
- receptory buněčného povrchu * MeSH
- shluková analýza MeSH
- synapse MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Unlike any protein studied so far, the active site of bilirubin oxidase from Myrothecium verrucaria contains a unique type of covalent link between tryptophan and histidine side chains. The role of this post-translational modification in substrate binding and oxidation is not sufficiently understood. Our structural and mutational studies provide evidence that this Trp396-His398 adduct modifies T1 copper coordination and is an important part of the substrate binding and oxidation site. The presence of the adduct is crucial for oxidation of substituted phenols and it substantially influences the rate of oxidation of bilirubin. Additionally, we bring the first structure of bilirubin oxidase in complex with one of its products, ferricyanide ion, interacting with the modified tryptophan side chain, Arg356 and the active site-forming loop 393-398. The results imply that structurally and chemically distinct types of substrates, including bilirubin, utilize the Trp-His adduct mainly for binding and to a smaller extent for electron transfer.
- MeSH
- bilirubin metabolismus MeSH
- Hypocreales metabolismus MeSH
- konformace proteinů MeSH
- molekulární modely * MeSH
- oxidace-redukce MeSH
- oxidoreduktasy působící na CH-CH vazby metabolismus MeSH
- transport elektronů fyziologie MeSH
- vazba proteinů fyziologie MeSH
- vazebná místa MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
The HelD is a helicase-like protein binding to Bacillus subtilis RNA polymerase (RNAP), stimulating transcription in an ATP-dependent manner. Here, our small angle X-ray scattering data bring the first insights into the HelD structure: HelD is compact in shape and undergoes a conformational change upon substrate analog binding. Furthermore, the HelD domain structure is delineated, and a partial model of HelD is presented. In addition, the unique N-terminal domain of HelD is characterized as essential for its transcription-related function but not for ATPase activity, DNA binding, or binding to RNAP. The study provides a topological basis for further studies of the role of HelD in transcription.
- MeSH
- Bacillus subtilis enzymologie MeSH
- bakteriální proteiny chemie metabolismus MeSH
- difrakce rentgenového záření MeSH
- DNA řízené RNA-polymerasy metabolismus MeSH
- maloúhlový rozptyl MeSH
- molekulární modely MeSH
- proteinové domény MeSH
- vazba proteinů MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
α-l-Fucosidase isoenzyme 1 from bacterium Paenibacillus thiaminolyticus is a member of the glycoside hydrolase family GH29 capable of cleaving l-fucose from nonreducing termini of oligosaccharides and glycoconjugates. Here we present the first crystal structure of this protein revealing a novel quaternary state within this family. The protein is in a unique hexameric assembly revealing the first observed case of active site complementation by a residue from an adjacent monomer in this family. Mutation of the complementing tryptophan residue caused changes in the catalytic properties including a shift of the pH optimum, a change of affinity to an artificial chromogenic substrate and a decreased reaction rate for a natural substrate. The wild-type enzyme was active on most of the tested naturally occurring oligosaccharides and capable of transglycosylation on a variety of acceptor molecules, including saccharides, alcohols or chromogenic substrates. Mutation of the complementing residue changed neither substrate specificity nor the preference for the type of transglycosylation acceptor molecule; however, the yields of the reactions were lower in both cases. Maltose molecules bound to the enzyme in the crystal structure identified surface carbohydrate-binding sites, possibly participating in binding of larger oligosaccharides.
The Gram-negative bacterium Legionella pneumophila is one of the known opportunistic human pathogens with a gene coding for a zinc-dependent S1-P1 type nuclease. Bacterial zinc-dependent 3'-nucleases/nucleotidases are little characterized and not fully understood, including L. pneumophila nuclease 1 (Lpn1), in contrast to many eukaryotic representatives with in-depth studies available. To help explain the principle properties and role of these enzymes in intracellular prokaryotic pathogens we have designed and optimized a heterologous expression protocol utilizing E. coli together with an efficient purification procedure, and performed detailed characterization of the enzyme. Replacement of Ni2+ ions by Zn2+ ions in affinity purification proved to be a crucial step in the production of pure and stable protein. The production protocol provides protein with high yield, purity, stability, and solubility for structure-function studies. We show that highly thermostable Lpn1 is active mainly towards RNA and ssDNA, with pH optima 7.0 and 6.0, respectively, with low activity towards dsDNA; the enzyme features pronounced substrate inhibition. Bioinformatic and experimental analysis, together with computer modeling and electrostatics calculations point to an unusually high positive charge on the enzyme surface under optimal conditions for catalysis. The results help explain the catalytic properties of Lpn1 and its substrate inhibition.
- MeSH
- adenosinmonofosfát metabolismus MeSH
- bakteriální proteiny chemická syntéza chemie metabolismus MeSH
- jednovláknová DNA metabolismus MeSH
- koncentrace vodíkových iontů MeSH
- konformace proteinů MeSH
- Legionella pneumophila enzymologie MeSH
- molekulární modely MeSH
- nukleotidasy chemická syntéza chemie metabolismus MeSH
- proteiny - lokalizační signály fyziologie MeSH
- rekombinantní proteiny chemie izolace a purifikace metabolismus MeSH
- RNA metabolismus MeSH
- sekvence aminokyselin MeSH
- sekvenční homologie aminokyselin MeSH
- sekvenční seřazení MeSH
- statická elektřina MeSH
- substrátová specifita MeSH
- teplota MeSH
- zinek chemie MeSH
- Publikační typ
- časopisecké články MeSH
3'-nucleases/nucleotidases of the S1-P1 family (EC 3.1.30.1) are single-strand-specific or non-specific zinc-dependent phosphoesterases present in plants, fungi, protozoan parasites, and in some bacteria. They participate in a wide variety of biological processes and their current biotechnological applications rely on their single-strand preference, nucleotide non-specificity, a broad range of catalytic conditions and high stability. We summarize the present and potential utilization of these enzymes in biotechnology and medicine in the context of their biochemical and structure-function properties. Explanation of unanswered questions for bacterial and trypanosomatid representatives could facilitate development of emerging applications in medicine.
- MeSH
- antitumorózní látky farmakologie MeSH
- biotechnologie metody MeSH
- cílená molekulární terapie metody MeSH
- endonukleasy specifické pro jednořetězcové nukleové kyseliny chemie genetika metabolismus farmakologie MeSH
- fungální proteiny chemie metabolismus MeSH
- interakce hostitele a patogenu MeSH
- lidé MeSH
- mutační analýza DNA metody MeSH
- nukleotidasy metabolismus MeSH
- substrátová specifita MeSH
- vztahy mezi strukturou a aktivitou MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- přehledy MeSH
The single-strand-specific S1 nuclease from Aspergillus oryzae is an archetypal enzyme of the S1-P1 family of nucleases with a widespread use for biochemical analyses of nucleic acids. We present the first X-ray structure of this nuclease along with a thorough analysis of the reaction and inhibition mechanisms and of its properties responsible for identification and binding of ligands. Seven structures of S1 nuclease, six of which are complexes with products and inhibitors, and characterization of catalytic properties of a wild type and mutants reveal unknown attributes of the S1-P1 family. The active site can bind phosphate, nucleosides, and nucleotides in several distinguished ways. The nucleoside binding site accepts bases in two binding modes-shallow and deep. It can also undergo remodeling and so adapt to different ligands. The amino acid residue Asp65 is critical for activity while Asn154 secures interaction with the sugar moiety, and Lys68 is involved in interactions with the phosphate and sugar moieties of ligands. An additional nucleobase binding site was identified on the surface, which explains the absence of the Tyr site known from P1 nuclease. For the first time ternary complexes with ligands enable modeling of ssDNA binding in the active site cleft. Interpretation of the results in the context of the whole S1-P1 nuclease family significantly broadens our knowledge regarding ligand interaction modes and the strategies of adjustment of the enzyme surface and binding sites to achieve particular specificity.
- MeSH
- aminokyseliny metabolismus MeSH
- Aspergillus oryzae enzymologie metabolismus MeSH
- endonukleasy specifické pro jednořetězcové nukleové kyseliny metabolismus MeSH
- fungální proteiny metabolismus MeSH
- katalytická doména fyziologie MeSH
- katalýza MeSH
- kinetika MeSH
- sekvence aminokyselin MeSH
- sekvenční seřazení MeSH
- substrátová specifita MeSH
- vazebná místa fyziologie MeSH
- Publikační typ
- časopisecké články MeSH
Bacterial RNA polymerase (RNAP) is an essential multisubunit protein complex required for gene expression. Here, we characterize YvgS (HelD) from Bacillus subtilis, a novel binding partner of RNAP. We show that HelD interacts with RNAP-core between the secondary channel of RNAP and the alpha subunits. Importantly, we demonstrate that HelD stimulates transcription in an ATP-dependent manner by enhancing transcriptional cycling and elongation. We demonstrate that the stimulatory effect of HelD can be amplified by a small subunit of RNAP, delta. In vivo, HelD is not essential but it is required for timely adaptations of the cell to changing environment. In summary, this study establishes HelD as a valid component of the bacterial transcription machinery.
- MeSH
- adenosintrifosfát metabolismus MeSH
- Bacillus subtilis enzymologie genetika MeSH
- bakteriální proteiny izolace a purifikace metabolismus MeSH
- DNA řízené RNA-polymerasy chemie izolace a purifikace metabolismus MeSH
- DNA metabolismus MeSH
- elongace genetické transkripce MeSH
- fenotyp MeSH
- genetická transkripce * MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Interactions between C-type lectin-like NK cell receptors and their protein ligands form one of the key recognition mechanisms of the innate immune system that is involved in the elimination of cells that have been malignantly transformed, virally infected, or stressed by chemotherapy or other factors. We determined an x-ray structure for the extracellular domain of mouse C-type lectin related (Clr) protein g, a ligand for the activation receptor NKR-P1F. Clr-g forms dimers in the crystal structure resembling those of human CD69. This newly reported structure, together with the previously determined structure of mouse receptor NKR-P1A, allowed the modeling and calculations of electrostatic profiles for other closely related receptors and ligands. Despite the high similarity among Clr-g, Clr-b, and human CD69, these molecules have fundamentally different electrostatics, with distinct polarization of Clr-g. The electrostatic profile of NKR-P1F is complementary to that of Clr-g, which suggests a plausible interaction mechanism based on contacts between surface sites of opposite potential.
- MeSH
- CD antigeny chemie imunologie MeSH
- diferenciační antigeny T-lymfocytů chemie imunologie MeSH
- krystalografie rentgenová MeSH
- lektiny typu C chemie imunologie MeSH
- lidé MeSH
- ligandy MeSH
- membránové proteiny chemie imunologie MeSH
- myši MeSH
- receptory imunologické chemie imunologie MeSH
- statická elektřina MeSH
- strukturní homologie proteinů MeSH
- terciární struktura proteinů MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- myši MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
