scholarly journals Cryo-EM structure of the B cell co-receptor CD19 bound to the tetraspanin CD81

Science ◽  
2021 ◽  
Vol 371 (6526) ◽  
pp. 300-305
Author(s):  
Katherine J. Susa ◽  
Shaun Rawson ◽  
Andrew C. Kruse ◽  
Stephen C. Blacklow
Keyword(s):  
B Cell ◽  
Rational Design ◽  
Cell Receptor ◽  
Binding Pocket ◽  
Structural Basis ◽  
Transmembrane Helices ◽  
Critical Determinant ◽  
Cell Dysfunction ◽  
Cryo Electron Microscopy ◽  
Binding Surface

Signaling through the CD19-CD81 co-receptor complex, in combination with the B cell receptor, is a critical determinant of B cell development and activation. It is unknown how CD81 engages CD19 to enable co-receptor function. Here, we report a 3.8-angstrom structure of the CD19-CD81 complex bound to a therapeutic antigen-binding fragment, determined by cryo–electron microscopy (cryo-EM). The structure includes both the extracellular domains and the transmembrane helices of the complex, revealing a contact interface between the ectodomains that drives complex formation. Upon binding to CD19, CD81 opens its ectodomain to expose a hydrophobic CD19-binding surface and reorganizes its transmembrane helices to occlude a cholesterol binding pocket present in the apoprotein. Our data reveal the structural basis for CD19-CD81 complex assembly, providing a foundation for rational design of therapies for B cell dysfunction.

scholarly journals Structural Basis for Galectin-1-dependent Pre-B Cell Receptor (Pre-BCR) Activation

2012 ◽  
Vol 287 (53) ◽  
pp. 44703-44713 ◽  
Author(s):  
Latifa Elantak ◽  
Marion Espeli ◽  
Annie Boned ◽  
Olivier Bornet ◽  
Jeremy Bonzi ◽  
...  
Keyword(s):  
B Cell ◽  
Cell Receptor ◽  
B Cell Receptor ◽  
Structural Basis

scholarly journals Structural basis for VIPP1 oligomerization and maintenance of thylakoid membrane integrity

2020 ◽  
Author(s):  
Tilak Kumar Gupta ◽  
Sven Klumpe ◽  
Karin Gries ◽  
Steffen Heinz ◽  
Wojciech Wietrzynski ◽  
...  
Keyword(s):  
Membrane Integrity ◽  
Point Mutations ◽  
Binding Pocket ◽  
Thylakoid Membranes ◽  
Lipid Binding ◽  
Structural Basis ◽  
Amphipathic Helices ◽  
Cryo Electron Microscopy

AbstractVesicle-inducing protein in plastids (VIPP1) is essential for the biogenesis and maintenance of thylakoid membranes, which transform light into life. However, it is unknown how VIPP1 performs its vital membrane-shaping function. Here, we use cryo-electron microscopy to determine structures of cyanobacterial VIPP1 rings, revealing how VIPP1 monomers flex and interweave to form basket-like assemblies of different symmetries. Three VIPP1 monomers together coordinate a non-canonical nucleotide binding pocket that is required for VIPP1 oligomerization. Inside the ring’s lumen, amphipathic helices from each monomer align to form large hydrophobic columns, enabling VIPP1 to bind and curve membranes. In vivo point mutations in these hydrophobic surfaces cause extreme thylakoid swelling under high light, indicating an essential role of VIPP1 lipid binding in resisting stress-induced damage. Our study provides a structural basis for understanding how the oligomerization of VIPP1 drives the biogenesis of thylakoid membranes and protects these life-giving membranes from environmental stress.

scholarly journals Crystal structure of Arabidopsis thaliana HPPK/DHPS, a bifunctional enzyme and target of the herbicide asulam

2021 ◽  
Author(s):  
Grishma Vadlamani ◽  
Kirill V Sukhoverkov ◽  
Joel Haywood ◽  
Karen J Breese ◽  
Mark F Fisher ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Arabidopsis Thaliana ◽  
Mode Of Action ◽  
Rational Design ◽  
Binding Pocket ◽  
Structural Basis ◽  
Metabolic Resistance ◽  
Folate Biosynthesis ◽  
Limited Opportunity ◽  
Regulatory Scrutiny

Herbicides are vital for modern agriculture, but their utility is threatened by genetic or metabolic resistance in weeds as well as heightened regulatory scrutiny. Of the known herbicide modes of action, 6-hydroxymethyl-7,8-dihydropterin synthase (DHPS) which is involved in folate biosynthesis, is targeted by just one commercial herbicide, asulam. A mimic of the substrate para-aminobenzoic acid, asulam is chemically similar to sulfonamide antibiotics - and while still in widespread use, asulam has faced regulatory scrutiny. With an entire mode of action represented by just one commercial agrochemical, we sought to improve the understanding of its plant target. Here we solve a 2.6 Å resolution crystal structure for Arabidopsis thaliana DHPS that is conjoined to 6-hydroxymethyl-7,8-dihydropterin pyrophosphokinase (HPPK) and reveal a strong structural conservation with bacterial counterparts at the sulfonamide-binding pocket of DHPS. We demonstrate asulam and the antibiotics sulfacetamide and sulfamethoxazole have herbicidal as well as antibacterial activity and explore the structural basis of their potency by modelling these compounds in mitochondrial HPPK/DHPS. Our findings suggest limited opportunity for the rational design of plant selectivity from asulam and that pharmacokinetic or delivery differences between plants and microbes might be the best approaches to safeguard this mode of action.

scholarly journals Identification of MALT1 feedback mechanisms enables rational design of potent antilymphoma regimens for ABC-DLBCL

Blood ◽  
2021 ◽  
Vol 137 (6) ◽  
pp. 788-800 ◽  
Author(s):  
Lorena Fontan ◽  
Rebecca Goldstein ◽  
Gabriella Casalena ◽  
Matthew Durant ◽  
Matthew R. Teater ◽  
...  
Keyword(s):  
B Cell ◽  
Rational Design ◽  
Tumor Regression ◽  
B Cell Lymphoma ◽  
Cell Receptor ◽  
Pi3k Pathway ◽  
Irreversible Inhibitor ◽  
Feedback Mechanisms

Abstract MALT1 inhibitors are promising therapeutic agents for B-cell lymphomas that are dependent on constitutive or aberrant signaling pathways. However, a potential limitation for signal transduction–targeted therapies is the occurrence of feedback mechanisms that enable escape from the full impact of such drugs. Here, we used a functional genomics screen in activated B-cell–like (ABC) diffuse large B-cell lymphoma (DLBCL) cells treated with a small molecule irreversible inhibitor of MALT1 to identify genes that might confer resistance or enhance the activity of MALT1 inhibition (MALT1i). We find that loss of B-cell receptor (BCR)- and phosphatidylinositol 3-kinase (PI3K)-activating proteins enhanced sensitivity, whereas loss of negative regulators of these pathways (eg, TRAF2, TNFAIP3) promoted resistance. These findings were validated by knockdown of individual genes and a combinatorial drug screen focused on BCR and PI3K pathway–targeting drugs. Among these, the most potent combinatorial effect was observed with PI3Kδ inhibitors against ABC-DLBCLs in vitro and in vivo, but that led to an adaptive increase in phosphorylated S6 and eventual disease progression. Along these lines, MALT1i promoted increased MTORC1 activity and phosphorylation of S6K1-T389 and S6-S235/6, an effect that was only partially blocked by PI3Kδ inhibition in vitro and in vivo. In contrast, simultaneous inhibition of MALT1 and MTORC1 prevented S6 phosphorylation, yielded potent activity against DLBCL cell lines and primary patient specimens, and resulted in more profound tumor regression and significantly improved survival of ABC-DLBCLs in vivo compared with PI3K inhibitors. These findings provide a basis for maximal therapeutic impact of MALT1 inhibitors in the clinic, by disrupting feedback mechanisms that might otherwise limit their efficacy.

scholarly journals Structural basis for membrane insertion by the human ER membrane protein complex

Science ◽  
2020 ◽  
pp. eabb5008 ◽  
Author(s):  
Tino Pleiner ◽  
Giovani Pinton Tomaleri ◽  
Kurt Januszyk ◽  
Alison J. Inglis ◽  
Masami Hazu ◽  
...  
Keyword(s):  
Membrane Protein ◽  
Protein Complex ◽  
Structural Basis ◽  
Membrane Insertion ◽  
Transmembrane Helices ◽  
Membrane Protein Complex ◽  
Cryo Electron Microscopy ◽  
Membrane Thinning ◽  
Positively Charged ◽  
Er Membrane

A defining step in the biogenesis of a membrane protein is the insertion of its hydrophobic transmembrane helices into the lipid bilayer. The nine-subunit ER membrane protein complex (EMC) is a conserved co- and post-translational insertase at the endoplasmic reticulum. We determined the structure of the human EMC in a lipid nanodisc to an overall resolution of 3.4 Å by cryo-electron microscopy, permitting building of a nearly complete atomic model. We used structure-guided mutagenesis to demonstrate that substrate insertion requires a methionine-rich cytosolic loop and occurs via an enclosed hydrophilic vestibule within the membrane formed by the subunits EMC3 and EMC6. We propose that the EMC uses local membrane thinning and a positively charged patch to decrease the energetic barrier for insertion into the bilayer.

scholarly journals The structural basis of T-cell receptor (TCR) activation: An enduring enigma

2019 ◽  
Vol 295 (4) ◽  
pp. 914-925 ◽  
Author(s):  
Roy A. Mariuzza ◽  
Pragati Agnihotri ◽  
John Orban
Keyword(s):  
T Cell ◽  
T Cell Receptor ◽  
T Cell Activation ◽  
Intracellular Signaling ◽  
Rational Design ◽  
Cell Receptor ◽  
Structural Basis ◽  
Specific Information ◽  
Antigenic Peptides ◽  
Tcr Activation

T cells are critical for protective immune responses to pathogens and tumors. The T-cell receptor (TCR)–CD3 complex is composed of a diverse αβ TCR heterodimer noncovalently associated with the invariant CD3 dimers CD3ϵγ, CD3ϵδ, and CD3ζζ. The TCR mediates recognition of antigenic peptides bound to MHC molecules (pMHC), whereas the CD3 molecules transduce activation signals to the T cell. Whereas much is known about downstream T-cell signaling pathways, the mechanism whereby TCR engagement by pMHC is first communicated to the CD3 signaling apparatus, a process termed early T-cell activation, remains largely a mystery. In this review, we examine the molecular basis for TCR activation in light of the recently determined cryoEM structure of a complete TCR–CD3 complex. This structure provides an unprecedented opportunity to assess various signaling models that have been proposed for the TCR. We review evidence from single-molecule and structural studies for force-induced conformational changes in the TCR–CD3 complex, for dynamically-driven TCR allostery, and for pMHC-induced structural changes in the transmembrane and cytoplasmic regions of CD3 subunits. We identify major knowledge gaps that must be filled in order to arrive at a comprehensive model of TCR activation that explains, at the molecular level, how pMHC-specific information is transmitted across the T-cell membrane to initiate intracellular signaling. An in-depth understanding of this process will accelerate the rational design of immunotherapeutic agents targeting the TCR–CD3 complex.

scholarly journals Interaction of Pleuromutilin Derivatives with the Ribosomal Peptidyl Transferase Center

2006 ◽  
Vol 50 (4) ◽  
pp. 1458-1462 ◽  
Author(s):  
Katherine S. Long ◽  
Lykke H. Hansen ◽  
Lene Jakobsen ◽  
Birte Vester
Keyword(s):  
Drug Targets ◽  
Rational Design ◽  
Ribosomal Subunit ◽  
Binding Pocket ◽  
Chain Extension ◽  
Side Chain ◽  
Peptidyl Transferase ◽  
Peptidyl Transferase Center ◽  
Binding Surface ◽  
The Common

ABSTRACT Tiamulin is a pleuromutilin antibiotic that is used in veterinary medicine. The recently published crystal structure of a tiamulin-50S ribosomal subunit complex provides detailed information about how this drug targets the peptidyl transferase center of the ribosome. To promote rational design of pleuromutilin-based drugs, the binding of the antibiotic pleuromutilin and three semisynthetic derivatives with different side chain extensions has been investigated using chemical footprinting. The nucleotides A2058, A2059, G2505, and U2506 are affected in all of the footprints, suggesting that the drugs are similarly anchored in the binding pocket by the common tricyclic mutilin core. However, varying effects are observed at U2584 and U2585, indicating that the side chain extensions adopt distinct conformations within the cavity and thereby affect the rRNA conformation differently. An Escherichia coli L3 mutant strain is resistant to tiamulin and pleuromutilin, but not valnemulin, implying that valnemulin is better able to withstand an altered rRNA binding surface around the mutilin core. This is likely due to additional interactions made between the valnemulin side chain extension and the rRNA binding site. The data suggest that pleuromutilin drugs with enhanced antimicrobial activity may be obtained by maximizing the number of interactions between the side chain moiety and the peptidyl transferase cavity.

scholarly journals Generation of an Analog-sensitive Syk Tyrosine Kinase for the Study of Signaling Dynamics from the B Cell Antigen Receptor

2007 ◽  
Vol 282 (46) ◽  
pp. 33760-33768 ◽  
Author(s):  
Hyunju Oh ◽  
Elif Ozkirimli ◽  
Kavita Shah ◽  
Marietta L. Harrison ◽  
Robert L. Geahlen
Keyword(s):  
Catalytic Activity ◽  
Tyrosine Kinase ◽  
B Cell ◽  
Protein Tyrosine Kinase ◽  
Cell Receptor ◽  
Binding Pocket ◽  
Transient Nature ◽  
Activation Of Transcription ◽  
Short Period ◽  
Syk Protein Tyrosine Kinase

The Syk protein-tyrosine kinase is an essential component of the signaling machinery that couples the B cell receptor for antigen to multiple downstream signal transduction pathways. Syk is phosphorylated and activated rapidly and transiently following receptor engagement, but many signaling events, such as the activation of transcription factors occur over the course of several minutes or hours. To investigate a role for the continued activation of Syk in these processes, we generated an analog-sensitive mutant with an engineered ATP-binding pocket to render the kinase uniquely sensitive to an orthogonal inhibitor. Mutation of the gatekeeper residue in Syk yielded an enzyme with very low activity. Second-site mutations, selected based on structural comparisons between Syk and Src, were introduced that restored catalytic activity to the mutant Syk. Syk-deficient DT40 B cells were prepared expressing the analog-sensitive Syk (Syk-AQL). Inhibition of the activity of Syk prior to, concomitant with or shortly following receptor engagement led to the rapid inhibition of receptor-mediated tyrosine phosphorylation and blocked the activation of extracellular signal-regulated kinase, NF-κB, and NFAT. The receptor-mediated activation of NF-κB required active Syk for a relatively short period of time, whereas the activation of NFAT required active kinase for a prolonged (>1 h) period. Receptor cross-linking led to the recruitment of Syk to the clustered receptor. Retention of these receptor-kinase complexes on the cell surface was dependent on the continued activity of Syk. Thus, despite the apparent transient nature of the activation of Syk, the catalytic activity of the Syk was required for sustained signaling from ligated receptors.

scholarly journals Murine pre–B-cell ALL induces T-cell dysfunction not fully reversed by introduction of a chimeric antigen receptor

Blood ◽  
2018 ◽  
Vol 132 (18) ◽  
pp. 1899-1910 ◽  
Author(s):  
Haiying Qin ◽  
Kazusa Ishii ◽  
Sang Nguyen ◽  
Paul P. Su ◽  
Chad R. Burk ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
B Cell ◽  
T Cell Receptor ◽  
Chimeric Antigen Receptor ◽  
Cell Receptor ◽  
Cell Dysfunction ◽  
Key Points ◽  
T Cell Dysfunction

Key Points Pre–B-cell ALL induces T-cell dysfunction in vivo, mediated in part by a non–T-cell receptor–linked mechanism. Prior exposure of T cells to pre–B-cell ALL in vivo impairs subsequent functionality of CAR-expressing T cells.

scholarly journals Structural analysis of the catalytic domain of Artemis endonuclease/SNM1C reveals distinct structural features

2020 ◽  
Vol 295 (35) ◽  
pp. 12368-12377 ◽  
Author(s):  
Md Fazlul Karim ◽  
Shanshan Liu ◽  
Adrian R. Laciak ◽  
Leah Volk ◽  
Mary Koszelak-Rosenblum ◽  
...  
Keyword(s):  
B Cell ◽  
Catalytic Domain ◽  
Substrate Binding ◽  
Binding Pocket ◽  
Structural Features ◽  
Zinc Ion ◽  
Dna Double Strand Breaks ◽  
Repair Gene ◽  
Dna Hairpins ◽  
Binding Surface

The endonuclease Artemis is responsible for opening DNA hairpins during V(D)J recombination and for processing a subset of pathological DNA double-strand breaks. Artemis is an attractive target for the development of therapeutics to manage various B cell and T cell tumors, because failure to open DNA hairpins and accumulation of chromosomal breaks may reduce the proliferation and viability of pre-T and pre-B cell derivatives. However, structure-based drug discovery of specific Artemis inhibitors has been hampered by a lack of crystal structures. Here, we report the structure of the catalytic domain of recombinant human Artemis. The catalytic domain displayed a polypeptide fold similar overall to those of other members in the DNA cross-link repair gene SNM1 family and in mRNA 3′-end-processing endonuclease CPSF-73, containing metallo-β-lactamase and β-CASP domains and a cluster of conserved histidine and aspartate residues capable of binding two metal atoms in the catalytic site. As in SNM1A, only one zinc ion was located in the Artemis active site. However, Artemis displayed several unique features. Unlike in other members of this enzyme class, a second zinc ion was present in the β-CASP domain that leads to structural reorientation of the putative DNA-binding surface and extends the substrate-binding pocket to a new pocket, pocket III. Moreover, the substrate-binding surface exhibited a dominant and extensive positive charge distribution compared with that in the structures of SNM1A and SNM1B, presumably because of the structurally distinct DNA substrate of Artemis. The structural features identified here may provide opportunities for designing selective Artemis inhibitors.

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