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.

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.

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.

MpaA is a murein-tripeptide-specific zinc carboxypeptidase that functions as part of a catabolic pathway for peptidoglycan-derived peptides in γ-proteobacteria

2012 ◽  
Vol 448 (3) ◽  
pp. 329-341 ◽  
Author(s):  
Abbas Maqbool ◽  
Mireille Hervé ◽  
Dominique Mengin-Lecreulx ◽  
Anthony J. Wilkinson ◽  
Gavin H. Thomas
Keyword(s):  
Structural Similarity ◽  
Zinc Ion ◽  
Diaminopimelic Acid ◽  
Size Exclusion ◽  
Spectrometric Analysis ◽  
Catabolic Pathway ◽  
E Coli ◽  
Transporter Protein ◽  
Exclusion Chromatography ◽  
Binding Groove

The murein peptide amidase MpaA is a cytoplasmic enzyme that processes peptides derived from the turnover of murein. We have purified the enzyme from Escherichia coli and demonstrated that it efficiently hydrolyses the γ-D-glutamyl-diaminopimelic acid bond in the murein tripeptide (L-Ala-γ-D-Glu-meso-Dap), with Km and kcat values of 0.41±0.05 mM and 38.3±10 s−1. However, it is unable to act on the murein tetrapeptide (L-Ala-γ-D-Glu-meso-Dap-D-Ala). E. coli MpaA is a homodimer containing one bound zinc ion per chain, as judged by mass spectrometric analysis and size-exclusion chromatography. To investigate the structure of MpaA we solved the crystal structure of the orthologous protein from Vibrio harveyi to 2.17 Å (1Å=0.1 nm). Vh_MpaA, which has identical enzymatic and biophysical properties to the E. coli enzyme, has high structural similarity to eukaryotic zinc carboxypeptidases. The structure confirms that MpaA is a dimeric zinc metalloprotein. Comparison of the structure of MpaA with those of other carboxypeptidases reveals additional structure that partially occludes the substrate-binding groove, perhaps explaining the narrower substrate specificity of the enzyme compared with other zinc carboxypeptidases. In γ-proteobacteria mpaA is often located adjacent to mppA which encodes a periplasmic transporter protein previously shown to bind murein tripeptide. We demonstrate that MppA can also bind murein tetrapeptide with high affinity. The genetic coupling of these genes and their related biochemical functions suggest that MpaA amidase and MppA transporter form part of a catabolic pathway for utilization of murein-derived peptides that operates in γ-proteobacteria in addition to the established murein recycling pathways.

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 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 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.

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 Interrogation of 3D-swapped structure and functional attributes of quintessential Sortase A from Streptococcus pneumoniae

2020 ◽  
Vol 477 (24) ◽  
pp. 4711-4728
Author(s):  
Tora Biswas ◽  
Anurag Misra ◽  
Sreetama Das ◽  
Prity Yadav ◽  
Suryanarayanarao Ramakumar ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Streptococcus Pneumoniae ◽  
Biochemical Characterization ◽  
Surface Proteins ◽  
Size Exclusion ◽  
Sedimentation Equilibrium ◽  
Exclusion Chromatography ◽  
Distance Mapping ◽  
Regulatory Functions ◽  
Pilus Assembly

The anchoring of the surface proteins to the cell wall in gram-positive bacteria involves a peptide ligation reaction catalyzed by transpeptidase sortase. Most bacterial genomes encode multiple sortases with dedicated functions. Streptococcus pneumoniae (Sp) carries four sortases; a housekeeping sortase (SrtA), and three pilin specific sortases (SrtC1, C2, C3) dedicated to the biosynthesis of covalent pilus. Interestingly, SrtA, meant for performing housekeeping roles, is also implicated in pilus assembly of Sp. The allegiance of SpSrtA to the pathogenic pilus assembly makes it an ideal target for clinical inhibitor development. In this paper, we describe biochemical characterization, crystal structure and peptide substrate preference of SpSrtA. Transpeptidation reaction with a variety of substrates revealed that the enzyme preferred elongated LPXTG sequences and transferred them equally well to both Ala- and Gly-terminated peptides. Curiously, the crystal structure of both wild type and an active site (Cys to Ala) mutant of SpSrtA displayed inter-twined 3D-swapped dimers in which each protomer generated a classic eight-stranded beta-barrel ‘sortase fold'. Size-exclusion chromatography and sedimentation equilibrium measurements revealed the predominant presence of a dimer in equilibrium with its monomer. The crystal structure-based Cys–Cys distance mapping with defined chemical cross-linkers established the existence of 3D-swapped structure in solution. The swapping in SpSrtA, unprecedented for sortase family, may be physiologically relevant and meant to perform regulatory functions.

scholarly journals Oligomeric States and Hydrodynamic Properties of Lysyl Oxidase-Like 2

Biomolecules ◽  
2021 ◽  
Vol 11 (12) ◽  
pp. 1846
Author(s):  
Alex A. Meier ◽  
Hee-Jung Moon ◽  
Ronald Toth ◽  
Ewa Folta-Stogniew ◽  
Krzysztof Kuczera ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Amine Oxidase ◽  
Lysyl Oxidase ◽  
Scavenger Receptor ◽  
Size Exclusion ◽  
Ecm Remodeling ◽  
Physiological Importance ◽  
Exclusion Chromatography ◽  
The Difference

Lysyl oxidase-like 2 (LOXL2) has emerged as a promising therapeutic target against metastatic/invasive tumors and organ and tissue fibrosis. LOXL2 catalyzes the oxidative deamination of lysine and hydroxylysine residues in extracellular matrix (ECM) proteins to promote crosslinking of these proteins, and thereby plays a major role in ECM remodeling. LOXL2 secretes as 100-kDa full-length protein (fl-LOXL2) and then undergoes proteolytic cleavage of the first two scavenger receptor cysteine-rich (SRCR) domains to yield 60-kDa protein (Δ1-2SRCR-LOXL2). This processing does not affect the amine oxidase activity of LOXL2 in vitro. However, the physiological importance of this cleavage still remains elusive. In this study, we focused on characterization of biophysical properties of fl- and Δ1-2SRCR-LOXL2s (e.g., oligomeric states, molecular weights, and hydrodynamic radii in solution) to gain insight into the structural role of the first two SRCR domains. Our study reveals that fl-LOXL2 exists predominantly as monomer but also dimer to the lesser extent when its concentration is <~1 mM. The hydrodynamic radius (Rh) determined by multi-angle light scattering coupled with size exclusion chromatography (SEC-MALS) indicates that fl-LOXL2 is a moderately asymmetric protein. In contrast, Δ1-2SRCR-LOXL2 exists solely as monomer and its Rh is in good agreement with the predicted value. The Rh values calculated from a 3D modeled structure of fl-LOXL2 and the crystal structure of the precursor Δ1-2SRCR-LOXL2 are within a reasonable margin of error of the values determined by SEC-MALS for fl- and Δ1-2SRCR-LOXL2s in mature forms in this study. Based on superimposition of the 3D model and the crystal structure of Δ1-2SRCR-LOXL2 (PDB:5ZE3), we propose a configuration of fl-LOXL2 that explains the difference observed in Rh between fl- and Δ1-2SRCR-LOXL2s in solution.

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