scholarly journals Crystal structure of the LRR ectodomain of the plant immune receptor kinase SOBIR1

2019 ◽  
Author(s):  
Ulrich Hohmann ◽  
Michael Hothorn
Keyword(s):  
Crystal Structure ◽  
Membrane Receptor ◽  
Size Exclusion ◽  
Receptor Kinase ◽  
Linear Motif ◽  
Immune Receptor ◽  
Protein Protein Interaction ◽  
Exclusion Chromatography ◽  
Missense Allele ◽  
Positively Charged

AbstractPlant unique membrane receptor kinases with leucine-rich repeat (LRR) extracellular domains are key regulators of development and immune responses. Here we present the 1.55 Å resolution crystal structure of the immune receptor kinase SOBIR1 from Arabidopsis. The ectodomain structure reveals the presence of 5 LRRs sandwiched between non-canonical capping domains. The disulphide bond-stabilized N-terminal cap harbors an unusual β-hairpin structure. The C-terminal cap features a highly positively charged linear motif which we find largely disordered in our structure. Size-exclusion chromatography and right-angle light scattering experiments suggest that SOBIR1 is a monomer in solution. The protruding β-hairpin, a set of highly conserved basic residues at the inner surface of the SOBIR LRR domain and the presence of a genetic missense allele in LRR2, together suggest that the SOBIR1 ectodomain may mediate protein – protein interaction in plant immune signalling.SynopsisThe ectodomain structure of a novel plant membrane receptor kinase with unusual capping domains is reported.

scholarly journals Crystal structure of the leucine-rich repeat ectodomain of the plant immune receptor kinase SOBIR1

2019 ◽  
Vol 75 (5) ◽  
pp. 488-497 ◽  
Author(s):  
Ulrich Hohmann ◽  
Michael Hothorn
Keyword(s):  
Crystal Structure ◽  
Size Exclusion ◽  
Receptor Kinase ◽  
Leucine Rich Repeat ◽  
Linear Motif ◽  
Immune Receptor ◽  
Protein Protein Interaction ◽  
Exclusion Chromatography ◽  
Missense Allele ◽  
Positively Charged

Plant-unique membrane receptor kinases with leucine-rich repeat (LRR) extracellular domains are key regulators of development and immune responses. Here, the 1.55 Å resolution crystal structure of the immune receptor kinase SOBIR1 from Arabidopsis is presented. The ectodomain structure reveals the presence of five LRRs sandwiched between noncanonical capping domains. The disulfide-bond-stabilized N-terminal cap harbours an unusual β-hairpin structure. The C-terminal cap features a highly positively charged linear motif which was found to be largely disordered in this structure. Size-exclusion chromatography and right-angle light-scattering experiments suggest that SOBIR1 is a monomer in solution. The protruding β-hairpin, a set of highly conserved basic residues at the inner surface of the SOBIR LRR domain and the presence of a genetic missense allele in LRR2 together suggest that the SOBIR1 ectodomain may mediate protein–protein interaction in plant immune signalling.

scholarly journals Inhaled Anesthetics Promote Albumin Dimerization through Reciprocal Exchange of Subdomains

2010 ◽  
Vol 2010 ◽  
pp. 1-7 ◽  
Author(s):  
Benjamin J. Pieters ◽  
Eugene E. Fibuch ◽  
Joshua D. Eklund ◽  
Norbert W. Seidler
Keyword(s):  
Fluorescence Emission ◽  
Size Exclusion ◽  
Inhaled Anesthetics ◽  
Protein Protein Interaction ◽  
Reciprocal Exchange ◽  
Proposed Model ◽  
Exclusion Chromatography ◽  
Spectral Shifting ◽  
And Control ◽  
The Impact

Inhaled anesthetics affect protein-protein interaction, but the mechanisms underlying these effects are still poorly understood. We examined the impact of sevoflurane and isoflurane on the dimerization of human serum albumin (HSA), a protein with anesthetic binding sites that are well characterized. Intrinsic fluorescence emission was analyzed for spectral shifting and self-quenching, and control first derivatives (spectral responses to changes in HSA concentration) were compared against those obtained from samples treated with sevoflurane or isoflurane. Sevoflurane increased dimer-dependent self-quenching and both decreased oligomer-dependent spectral shifting, suggesting that inhaled anesthetics promoted HSA dimerization. Size exclusion chromatography and polarization data were consistent with these observations. The data support the proposed model of a reciprocal exchange of subdomains to form an HSA dimer. The open-ended exchange of subdomains, which we propose occuring in HSA oligomers, was inhibited by sevoflurane and isoflurane.

scholarly journals Crystal structure of bacteriophage T4 Spackle as determined by native SAD phasing

2020 ◽  
Vol 76 (9) ◽  
pp. 899-904
Author(s):  
Ke Shi ◽  
Fredy Kurniawan ◽  
Surajit Banerjee ◽  
Nicholas H. Moeller ◽  
Hideki Aihara
Keyword(s):  
Crystal Structure ◽  
Bacteriophage T4 ◽  
Early Gene ◽  
Size Exclusion ◽  
Amino Terminal ◽  
Intramolecular Disulfide Bond ◽  
Exclusion Chromatography ◽  
T4 Bacteriophage ◽  
Sad Phasing ◽  
T4 Phage

The crystal structure of a bacteriophage T4 early gene product, Spackle, was determined by native sulfur single-wavelength anomalous diffraction (SAD) phasing using synchrotron radiation and was refined to 1.52 Å resolution. The structure shows that Spackle consists of a bundle of five α-helices, forming a relatively flat disc-like overall shape. Although Spackle forms a dimer in the crystal, size-exclusion chromatography with multi-angle light scattering shows that it is monomeric in solution. Mass spectrometry confirms that purified mature Spackle lacks the amino-terminal signal peptide and contains an intramolecular disulfide bond, consistent with its proposed role in the periplasm of T4 phage-infected Escherichia coli cells. The surface electrostatic potential of Spackle shows a strikingly bipolar charge distribution, suggesting a possible mode of membrane association and inhibition of the tail lysozyme activity in T4 bacteriophage superinfection exclusion.

scholarly journals Assembly of the Complex between Archaeal RNase P Proteins RPP30 and Pop5

Archaea ◽  
2011 ◽  
Vol 2011 ◽  
pp. 1-12 ◽  
Author(s):  
Brandon L. Crowe ◽  
Christopher J. Bohlen ◽  
Ross C. Wilson ◽  
Venkat Gopalan ◽  
Mark P. Foster
Keyword(s):  
Protein Interactions ◽  
Pyrococcus Furiosus ◽  
Resonance Assignments ◽  
Rnase P ◽  
Size Exclusion ◽  
Titration Calorimetry ◽  
Protein Protein Interaction ◽  
Model Complex ◽  
Exclusion Chromatography ◽  
P Proteins

RNase P is a highly conserved ribonucleoprotein enzyme that represents a model complex for understanding macromolecular RNA-protein interactions. Archaeal RNase P consists of one RNA and up to five proteins (Pop5, RPP30, RPP21, RPP29, and RPP38/L7Ae). Four of these proteins function in pairs (Pop5-RPP30 and RPP21–RPP29). We have used nuclear magnetic resonance (NMR) spectroscopy and isothermal titration calorimetry (ITC) to characterize the interaction between Pop5 and RPP30 from the hyperthermophilic archaeonPyrococcus furiosus(Pfu). NMR backbone resonance assignments of free RPP30 (25 kDa) indicate that the protein is well structured in solution, with a secondary structure matching that observed in a closely related crystal structure. Chemical shift perturbations upon the addition of Pop5 (14 kDa) reveal its binding surface on RPP30. ITC experiments confirm a net 1 : 1 stoichiometry for this tight protein-protein interaction and exhibit complex isotherms, indicative of higher-order binding. Indeed, light scattering and size exclusion chromatography data reveal the complex to exist as a 78 kDa heterotetramer with two copies each of Pop5 and RPP30. These results will inform future efforts to elucidate the functional role of the Pop5-RPP30 complex in RNase P assembly and catalysis.

scholarly journals Crystal structure of an anti-idiotype variable lymphocyte receptor

2017 ◽  
Vol 73 (12) ◽  
pp. 682-687
Author(s):  
Bernard C. Collins ◽  
Hiro Nakahara ◽  
Sharmistha Acharya ◽  
Max D. Cooper ◽  
Brantley R. Herrin ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Structural Similarity ◽  
Cell Receptor ◽  
Lymphocytic Leukemia ◽  
Size Exclusion ◽  
Antigen Receptors ◽  
Diagnostic Assays ◽  
Great Utility ◽  
Exclusion Chromatography ◽  
Leukemic Clone

Variable lymphocyte receptors (VLRs), the leucine-rich repeat (LRR)-based antigen receptors of jawless fish, have great utility in a wide variety of biochemical and biological applications, similar to classical Ig-based antibodies. VLR-based reagents may be particularly useful when traditional antibodies are not available. An anti-idiotype lamprey VLR, VLR39, has previously been identified that recognizes the heavy-chain CDR3 of the B-cell receptor (BCR) of a leukemic clone from a patient with chronic lymphocytic leukemia (CLL). VLR39 was used successfully to track the re-emergence of this clone in the patient following chemotherapy. Here, the crystal structure of VLR39 is presented at 1.5 Å resolution and compared with those of other protein-specific VLRs. VLR39 adopts a curved solenoid fold and exhibits substantial structural similarity to other protein-binding VLRs. VLR39 has a short LRRCT loop that protrudes outwards away from the concave face and is similar to those of its protein-specific VLR counterparts. Analysis of the VLR39–BCR interaction by size-exclusion chromatography and biolayer interferometry using the scFv version of the BCR confirms that VLR39 recognizes the BCR Fv region. Such VLR-based reagents may be useful for identifying and monitoring leukemia in CLL patients and in other clinical diagnostic assays.

Crystal and solution structures of fragments of the human leucocyte common antigen-related protein

2020 ◽  
Vol 76 (5) ◽  
pp. 406-417
Author(s):  
Joachim Vilstrup ◽  
Amanda Simonsen ◽  
Thea Birkefeldt ◽  
Dorthe Strandbygård ◽  
Jeppe Lyngsø ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Size Exclusion ◽  
Receptor Protein ◽  
Type I ◽  
Common Antigen ◽  
Heparin Binding ◽  
Related Protein ◽  
Saxs Data ◽  
Exclusion Chromatography ◽  
The Individual

Leucocyte common antigen-related protein (LAR) is a post-synaptic type I transmembrane receptor protein that is important for neuronal functionality and is genetically coupled to neuronal disorders such as attention deficit hyperactivity disorder (ADHD). To understand the molecular function of LAR, structural and biochemical studies of protein fragments derived from the ectodomain of human LAR have been performed. The crystal structure of a fragment encompassing the first four FNIII domains (LARFN1–4) showed a characteristic L shape. SAXS data suggested limited flexibility within LARFN1–4, while rigid-body refinement of the SAXS data using the X-ray-derived atomic model showed a smaller angle between the domains defining the L shape compared with the crystal structure. The capabilities of the individual LAR fragments to interact with heparin was examined using microscale thermophoresis and heparin-affinity chromatography. The results showed that the three N-terminal immunoglobulin domains (LARIg1–3) and the four C-terminal FNIII domains (LARFN5–8) both bound heparin, while LARFN1–4 did not. The low-molecular-weight heparin drug Innohep induced a shift in hydrodynamic volume as assessed by size-exclusion chromatography of LARIg1–3 and LARFN5–8, while the chemically defined pentameric heparin drug Arixtra did not. Together, the presented results suggest the presence of an additional heparin-binding site in human LAR.

scholarly journals Structure of the Prx6-subfamily 1-Cys peroxiredoxin from Sulfolobus islandicus

2019 ◽  
Vol 75 (6) ◽  
pp. 428-434 ◽  
Author(s):  
Sander Stroobants ◽  
Inge Van Molle ◽  
Queen Saidi ◽  
Karl Jonckheere ◽  
Dominique Maes ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Quaternary Structure ◽  
Size Exclusion ◽  
Molecular Replacement ◽  
Oligomeric State ◽  
Data Set ◽  
Cell Parameters ◽  
Aeropyrum Pernix ◽  
Sulfolobus Islandicus ◽  
Exclusion Chromatography

Aerobic thermoacidophilic archaea belonging to the genus Sulfolobus harbor peroxiredoxins, thiol-dependent peroxidases that assist in protecting the cells from oxidative damage. Here, the crystal structure of the 1-Cys peroxiredoxin from Sulfolobus islandicus, named 1-Cys SiPrx, is presented. A 2.75 Å resolution data set was collected from a crystal belonging to space group P212121, with unit-cell parameters a = 86.8, b = 159.1, c = 189.3 Å, α = β = γ = 90°. The structure was solved by molecular replacement using the homologous Aeropyrum pernix peroxiredoxin (ApPrx) structure as a search model. In the crystal structure, 1-Cys SiPrx assembles into a ring-shaped decamer composed of five homodimers. This quaternary structure corresponds to the oligomeric state of the protein in solution, as observed by size-exclusion chromatography. 1-Cys SiPrx harbors only a single cysteine, which is the peroxidatic cysteine, and lacks both of the cysteines that are highly conserved in the C-terminal arm domain in other archaeal Prx6-subfamily proteins such as ApPrx and that are involved in the association of dimers into higher-molecular-weight decamers and dodecamers. It is thus concluded that the Sulfolobus Prx6-subfamily protein undergoes decamerization independently of arm-domain cysteines.

scholarly journals Crystal structure of Pseudomonas aeruginosa FabB C161A, a template for structure-based design for new antibiotics

F1000Research ◽  
2021 ◽  
Vol 10 ◽  
pp. 1102
Author(s):  
Vladyslav Yadrykhins'ky ◽  
Charis Georgiou ◽  
Ruth Brenk
Keyword(s):  
Crystal Structure ◽  
Pseudomonas Aeruginosa ◽  
Acyl Carrier Protein ◽  
Recombinant Expression ◽  
Acid Synthesis ◽  
Size Exclusion ◽  
Carrier Protein ◽  
Efficient System ◽  
Tev Protease ◽  
Exclusion Chromatography

Background: FabB (3-oxoacyl-[acyl-carrier-protein] synthase 1) is part of the fatty acid synthesis II pathway found in bacteria and a potential target for antibiotics. The enzyme catalyses the Claisen condensation of malonyl-ACP (acyl carrier protein) with acyl-ACP via an acyl intermediate. Here, we report the crystal structure of the intermediate-mimicking Pseudomonas aeruginosa FabB (PaFabB) C161A variant. Methods: His-tagged PaFabB C161A was expressed in E.coli Rosetta DE3 pLysS cells, cleaved by TEV protease and purified using affinity and size exclusion chromatography. Commercial screens were used to identify suitable crystallization conditions which were subsequently improved to obtain well diffracting crystals. Results: We developed a robust and efficient system for recombinant expression of PaFabB C161A. Conditions to obtain well diffracting crystals were established. The crystal structure of PaFabB C161A was solved by molecular replacement at 1.3 Å resolution. Conclusions: The PaFabB C161A crystal structure can be used as a template to facilitate the design of FabB inhibitors.

scholarly journals Structure of Human Cyclophilin A in Complex with the Novel Immunosuppressant Sanglifehrin A at 1.6 Å Resolution

2005 ◽  
Vol 280 (23) ◽  
pp. 21965-21971 ◽  
Author(s):  
Joerg Kallen ◽  
Richard Sedrani ◽  
Gerhard Zenke ◽  
Juergen Wagner
Keyword(s):  
Crystal Structure ◽  
Three Dimensional ◽  
Cyclophilin A ◽  
The Other ◽  
Size Exclusion ◽  
Dimensional Structure ◽  
The Novel ◽  
Immunosuppressive Activity ◽  
Exclusion Chromatography ◽  
Sanglifehrin A

Sanglifehrin A (SFA) is a novel immunosuppressant isolated from Streptomyces sp. that binds strongly to the human immunophilin cyclophilin A (CypA). SFA exerts its immunosuppressive activity through a mode of action different from that of all other known immunophilin-binding substances, namely cyclosporine A (CsA), FK506, and rapamycin. We have determined the crystal structure of human CypA in complex with SFA at 1.6 Å resolution. The high resolution of the structure revealed the absolute configuration at all 17 chiral centers of SFA as well as the details of the CypA/SFA interactions. In particular, it was shown that the 22-membered macrocycle of SFA is deeply embedded in the same binding site as CsA and forms six direct hydrogen bonds with CypA. The effector domain of SFA, on the other hand, has a chemical and three-dimensional structure very different from CsA, already strongly suggesting different immunosuppressive mechanisms. Furthermore, two CypA·SFA complexes form a dimer in the crystal as well as in solution as shown by light scattering and size exclusion chromatography experiments. This observation raises the possibility that the dimer of CypA·SFA complexes is the molecular species mediating the immunosuppressive effect.

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