Crystal structure of native and Cd/Cd-substituted Dioclea guianensis seed lectin. A novel manganese-binding site and structural basis of dimer-tetramer association

2001 ◽  
Vol 310 (4) ◽  
pp. 885-894 ◽  
Author(s):  
David A Wah ◽  
Antonio Romero ◽  
Francisca Gallego del Sol ◽  
Benildo S Cavada ◽  
Marcio V Ramos ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Binding Site ◽  
Structural Basis ◽  
Manganese Binding Site

Crystal structure and collagen-binding site of immune inhibitory receptor LAIR-1: unexpected implications for collagen binding by platelet receptor GPVI

Blood ◽  
2010 ◽  
Vol 115 (7) ◽  
pp. 1364-1373 ◽  
Author(s):  
T. Harma C. Brondijk ◽  
Talitha de Ruiter ◽  
Joost Ballering ◽  
Hans Wienk ◽  
Robert Jan Lebbink ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Binding Site ◽  
Cell Activation ◽  
Immune Cell ◽  
Structural Basis ◽  
Collagen Binding ◽  
Glycoprotein Vi ◽  
Platelet Receptor ◽  
Fundamental Insight ◽  
Key Residues

Abstract Leukocyte-associated immunoglobulin-like receptor-1 (LAIR-1), one of the most widely spread immune receptors, attenuates immune cell activation when bound to specific sites in collagen. The collagen-binding domain of LAIR-1 is homologous to that of glycoprotein VI (GPVI), a collagen receptor crucial for platelet activation. Because LAIR-1 and GPVI also display overlapping collagen-binding specificities, a common structural basis for collagen recognition would appear likely. Therefore, it is crucial to gain insight into the molecular interaction of both receptors with their ligand to prevent unwanted cross-reactions during therapeutic intervention. We determined the crystal structure of LAIR-1 and mapped its collagen-binding site by nuclear magnetic resonance (NMR) titrations and mutagenesis. Our data identify R59, E61, and W109 as key residues for collagen interaction. These residues are strictly conserved in LAIR-1 and GPVI alike; however, they are located outside the previously proposed GPVI collagen-binding site. Our data provide evidence for an unanticipated mechanism of collagen recognition common to LAIR-1 and GPVI. This fundamental insight will contribute to the exploration of specific means of intervention in collagen-induced signaling in immunity and hemostasis.

scholarly journals Phytophthora sojae effector Avr1d functions as E2 competitor and inhibits ubiquitination activity of GmPUB13 to facilitate infection

2020 ◽  
Author(s):  
Yachun Lin ◽  
Qinli Hu ◽  
Jia Zhou ◽  
Weixiao Yin ◽  
Deqiang Yao ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Binding Site ◽  
Ubiquitin Ligase ◽  
E3 Ligase ◽  
Phytophthora Sojae ◽  
Structural Basis ◽  
Susceptibility Factor ◽  
Ubiquitin Conjugating Enzyme ◽  
Ubiquitination System

AbstractOomycete pathogens such as Phytophthora secrete a repertoire of effectors to host cells to manipulate host immunity and benefit infection. In this study, we found that an RxLR effector, Avr1d, promoted Phytophthora sojae infection in soybean hairy-roots. Using a yeast two-hybrid screen, we identified the soybean E3 ubiquitin ligase GmPUB13 as a host target for Avr1d. By co-immunoprecipitation, gel infiltration and ITC assays, we confirmed that Avr1d interacts with GmPUB13 both in vivo and in vitro. Furthermore, we found that Avr1d inhibits the E3 ligase activity of GmPUB13. The crystal structure of Avr1d in complex with GmPUB13 was solved and revealed that Avr1d occupies the binding site for E2 ubiquitin conjugating enzyme on GmPUB13. In line with this, Avr1d competed with E2 ubiquitin conjugating enzymes for GmPUB13 binding in vitro, thereby decreasing the E3 ligase activity of GmPUB13. Meanwhile, we found that inactivation of the ubiquitin ligase activity of GmPUB13 stabilized GmPUB13 by blocking GmPUB13 degradation. Silencing of GmPUB13 in soybean hairy-roots decreased P. sojae infection, suggesting that GmPUB13 acts as a susceptibility factor, negatively regulating soybean resistance against P. sojae. Altogether, this study highlights a novel virulence mechanism of Phytophthora effectors, by which Avr1d competes with E2 for GmPUB13 binding to repress the GmPUB13 E3 ligase activity and thereby stabilizing the susceptibility factor GmPUB13 to facilitate Phytophthora infection. This is the first study to unravel the structural basis for modulation of host targets by Phytophthora effectors and will be instrumental for boosting plant resistance breeding.Significance StatementUbiquitination acts as a crucial regulator in plant immunity. Accordingly, microbial pathogens secrete effectors to hijak host ubiquitination system. However, the molecular mechanisms by which microbial effectors modulate host ubiquitination system are not yet clear. Here, we found that the Phytophthora sojae effector Avr1d physically binds to the U-box type E3 ligase GmPUB13, a susceptibility factor in soybean. The crystal structure of Avr1d in complex with GmPUB13 revealed that Avr1d occupies the binding site in GmPUB13 for the E2 ubiquitin conjugating enzyme and competes with E2 for physical binding to GmPUB13. Avr1d stabilized GmPUB13 by suppressing the self-ubiquitination activity of GmPUB13 and thereby promoting Phytophthora infection. This study provides structural basis for modulation of host targets by Phytophthora effectors.

Critical residues for ligand binding in blade 2 of the propeller domain of the integrin αIIb subunit

2004 ◽  
Vol 91 (01) ◽  
pp. 111-118 ◽  
Author(s):  
Tatsushiro Tamura ◽  
Jun Yamanouchi ◽  
Shigeru Fujita ◽  
Takaaki Hato
Keyword(s):  
Crystal Structure ◽  
Ligand Binding ◽  
Binding Site ◽  
Thrombus Formation ◽  
Direct Interaction ◽  
Binding Pocket ◽  
Crystal Structure Analysis ◽  
Site Directed Mutagenesis ◽  
Structural Basis ◽  
Critical Residues

SummaryLigand binding to integrin αIIbβ3 is a key event of thrombus formation. The propeller domain of the αIIb subunit has been implicated in ligand binding. Recently, the ligand binding site of the αV propeller was determined by crystal structure analysis. However, the structural basis of ligand recognition by the αIIb propeller remains to be determined. In this study, we conducted site-directed mutagenesis of all residues located in the loops extending above blades 2 and 4 of the αIIb propeller, which are spatially close to, but distinct from, the loops that contain the binding site for an RGD ligand in the crystal structure of the αV propeller. Replacement by alanine of Q111, H112 or N114 in the loop within the blade 2 (the W2:2-3 loop in the propeller model) abolished binding of a ligand-mimetic antibody and fibrinogen to αIIbβ3 induced by different types of integrin activation including activation of αIIbβ3 by β3 cytoplasmic mutation. CHO cells stably expressing recombinant αIIbβ3 bearing Q111A, H112A or N114A mutation did not exhibit αIIbβ3mediated adhesion to fibrinogen. According to the crystal structure of αVβ3, the αV residue corresponding to αIIbN114 is exposed on the integrin surface and close to the RGD binding site. These results suggest that the Q111, H112 and N114 residues in the loop within blade 2 of the αIIb propeller are critical for ligand binding, possibly because of direct interaction with ligands or modulation of the RGD binding pocket.

scholarly journals How HIV-1 Nef hijacks the AP-2 clathrin adaptor to downregulate CD4

eLife ◽  
2014 ◽  
Vol 3 ◽  
Author(s):  
Xuefeng Ren ◽  
Sang Yoon Park ◽  
Juan S Bonifacino ◽  
James H Hurley
Keyword(s):  
Crystal Structure ◽  
Cell Surface ◽  
Binding Site ◽  
Indirect Interactions ◽  
Structural Basis ◽  
Clathrin Adaptor ◽  
Central Loop ◽  
Hiv 1

The Nef protein of HIV-1 downregulates the cell surface co-receptor CD4 by hijacking the clathrin adaptor complex AP-2. The structural basis for the hijacking of AP-2 by Nef is revealed by a 2.9 Å crystal structure of Nef bound to the α and σ2 subunits of AP-2. Nef binds to AP-2 via its central loop (residues 149–179) and its core. The determinants for Nef binding include residues that directly contact AP-2 and others that stabilize the binding-competent conformation of the central loop. Residues involved in both direct and indirect interactions are required for the binding of Nef to AP-2 and for downregulation of CD4. These results lead to a model for the docking of the full AP-2 tetramer to membranes as bound to Nef, such that the cytosolic tail of CD4 is situated to interact with its binding site on Nef.

scholarly journals Structural basis for the neutralization of SARS-CoV-2 by an antibody from a convalescent patient

2020 ◽  
Author(s):  
Daming Zhou ◽  
Helen ME Duyvesteyn ◽  
Cheng-Pin Chen ◽  
Chung-Guei Huang ◽  
Ting-Hua Chen ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Binding Site ◽  
Economic Impact ◽  
Receptor Binding ◽  
Late Stage ◽  
Structural Basis ◽  
Receptor Binding Domain ◽  
Spike Glycoprotein ◽  
Receptor Binding Site ◽  
Conserved Epitope

AbstractThe COVID-19 pandemic has had unprecedented health and economic impact, but currently there are no approved therapies. We have isolated an antibody, EY6A, from a late-stage COVID-19 patient and show it neutralises SARS-CoV-2 and cross-reacts with SARS-CoV-1. EY6A Fab binds tightly (KD of 2 nM) the receptor binding domain (RBD) of the viral Spike glycoprotein and a 2.6Å crystal structure of an RBD/EY6A Fab complex identifies the highly conserved epitope, away from the ACE2 receptor binding site. Residues of this epitope are key to stabilising the pre-fusion Spike. Cryo-EM analyses of the pre-fusion Spike incubated with EY6A Fab reveal a complex of the intact trimer with three Fabs bound and two further multimeric forms comprising destabilized Spike attached to Fab. EY6A binds what is probably a major neutralising epitope, making it a candidate therapeutic for COVID-19.

scholarly journals Crystal Structure of IlvC, a Ketol-Acid Reductoisomerase, from Streptococcus Pneumoniae

Crystals ◽  
2019 ◽  
Vol 9 (11) ◽  
pp. 551 ◽  
Author(s):  
Gyuhee Kim ◽  
Donghyuk Shin ◽  
Sumin Lee ◽  
Jaesook Yun ◽  
Sangho Lee
Keyword(s):  
Crystal Structure ◽  
Streptococcus Pneumoniae ◽  
Binding Site ◽  
Conformational Changes ◽  
Bacterial Pathogen ◽  
Crystallographic Analysis ◽  
Structural Basis ◽  
Isoleucine Leucine ◽  
X Ray Scattering ◽  
Branched Chain

Biosynthesis of branched-chain amino acids (BCAAs), including isoleucine, leucine and valine, is required for survival and virulence of a bacterial pathogen such as Streptococcus pneumoniae. IlvC, a ketol-acid reductoisomerase (E.C. 1.1.1.86) with NADP(H) and Mg2+ as cofactors from the pathogenic Streptococcus pneumoniae (SpIlvC), catalyzes the second step in the BCAA biosynthetic pathway. To elucidate the structural basis for the IlvC-mediated reaction, we determined the crystal structure of SpIlvC at 1.69 Å resolution. The crystal structure of SpIlvC contains an asymmetric dimer in which one subunit is in apo-form and the other in NADP(H) and Mg2+-bound form. Crystallographic analysis combined with an activity assay and small-angle X-ray scattering suggested that SpIlvC retains dimeric arrangement in solution and that D83 in the NADP(H) binding site and E195 in the Mg2+ binding site are the most critical in the catalytic activity of SpIlvC. Crystal structures of SpIlvC mutants (R49E, D83G, D191G and E195S) revealed local conformational changes only in the NADP(H) binding site. Taken together, our results establish the molecular mechanism for understanding functions of SpIlvC in pneumococcal growth and virulence.

scholarly journals Structural basis of Naa20 activity towards a canonical NatB substrate

2021 ◽  
Vol 4 (1) ◽  
Author(s):  
Dominik Layer ◽  
Jürgen Kopp ◽  
Miriam Fontanillo ◽  
Maja Köhn ◽  
Karine Lapouge ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Drug Development ◽  
Catalytic Mechanism ◽  
Peptide Binding ◽  
Competitive Inhibitor ◽  
Structural Basis ◽  
Substrate Affinity ◽  
Protein Homeostasis ◽  
Auxiliary Subunit

AbstractN-terminal acetylation is one of the most common protein modifications in eukaryotes and is carried out by N-terminal acetyltransferases (NATs). It plays important roles in protein homeostasis, localization, and interactions and is linked to various human diseases. NatB, one of the major co-translationally active NATs, is composed of the catalytic subunit Naa20 and the auxiliary subunit Naa25, and acetylates about 20% of the proteome. Here we show that NatB substrate specificity and catalytic mechanism are conserved among eukaryotes, and that Naa20 alone is able to acetylate NatB substrates in vitro. We show that Naa25 increases the Naa20 substrate affinity, and identify residues important for peptide binding and acetylation activity. We present the first Naa20 crystal structure in complex with the competitive inhibitor CoA-Ac-MDEL. Our findings demonstrate how Naa20 binds its substrates in the absence of Naa25 and support prospective endeavors to derive specific NAT inhibitors for drug development.

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