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.

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

2021 ◽  
Vol 118 (10) ◽  
pp. e2018312118
Author(s):  
Yachun Lin ◽  
Qinli Hu ◽  
Jia Zhou ◽  
Weixiao Yin ◽  
Deqiang Yao ◽  
...  
Keyword(s):  
Hairy Roots ◽  
Ubiquitin Ligase ◽  
E3 Ligase ◽  
Phytophthora Sojae ◽  
Host Cells ◽  
Structural Basis ◽  
Titration Calorimetry ◽  
Susceptibility Factor

Oomycete pathogens such as Phytophthora secrete a repertoire of effectors into 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 coimmunoprecipitation (Co-IP), gel infiltration, and isothermal titration calorimetry (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 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. Altogether, this study highlights a 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 study unravels the structural basis for modulation of host targets by Phytophthora effectors and will be instrumental for boosting plant resistance breeding.

scholarly journals Structural analysis of the LDL receptor–interacting FERM domain in the E3 ubiquitin ligase IDOL reveals an obscured substrate-binding site

2020 ◽  
Vol 295 (39) ◽  
pp. 13570-13583
Author(s):  
Luca Martinelli ◽  
Athanassios Adamopoulos ◽  
Patrik Johansson ◽  
Paul T. Wan ◽  
Jenny Gunnarsson ◽  
...  
Keyword(s):  
Binding Site ◽  
Ubiquitin Ligase ◽  
Substrate Recognition ◽  
E3 Ubiquitin Ligase ◽  
Full Length ◽  
Scattering Data ◽  
Lipid Lowering ◽  
Structural Basis ◽  
Ferm Domain

Hepatic abundance of the low-density lipoprotein receptor (LDLR) is a critical determinant of circulating plasma LDL cholesterol levels and hence development of coronary artery disease. The sterol-responsive E3 ubiquitin ligase inducible degrader of the LDLR (IDOL) specifically promotes ubiquitination and subsequent lysosomal degradation of the LDLR and thus controls cellular LDL uptake. IDOL contains an extended N-terminal FERM (4.1 protein, ezrin, radixin, and moesin) domain, responsible for substrate recognition and plasma membrane association, and a second C-terminal RING domain, responsible for the E3 ligase activity and homodimerization. As IDOL is a putative lipid-lowering drug target, we investigated the molecular details of its substrate recognition. We produced and isolated full-length IDOL protein, which displayed high autoubiquitination activity. However, in vitro ubiquitination of its substrate, the intracellular tail of the LDLR, was low. To investigate the structural basis for this, we determined crystal structures of the extended FERM domain of IDOL and multiple conformations of its F3ab subdomain. These reveal the archetypal F1-F2-F3 trilobed FERM domain structure but show that the F3c subdomain orientation obscures the target-binding site. To substantiate this finding, we analyzed the full-length FERM domain and a series of truncated FERM constructs by small-angle X-ray scattering (SAXS). The scattering data support a compact and globular core FERM domain with a more flexible and extended C-terminal region. This flexibility may explain the low activity in vitro and suggests that IDOL may require activation for recognition of the LDLR.

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.

scholarly journals Inhibition of Ubiquitination and Stabilization of Human Ubiquitin E3 Ligase PIRH2 by Measles Virus Phosphoprotein

2005 ◽  
Vol 79 (18) ◽  
pp. 11824-11836 ◽  
Author(s):  
Mingzhou Chen ◽  
Jean-Claude Cortay ◽  
Ian R. Logan ◽  
Vasileia Sapountzi ◽  
Craig N. Robson ◽  
...  
Keyword(s):  
Binding Site ◽  
Measles Virus ◽  
Fluorescent Protein ◽  
E3 Ligase ◽  
Yeast Two Hybrid ◽  
Ubiquitin E3 Ligase ◽  
P Protein ◽  
Two Hybrid

ABSTRACT Using a C-terminal domain (PCT) of the measles virus (MV) phosphoprotein (P protein) as bait in a yeast two-hybrid screen, a cDNA identical to the recently described human p53-induced-RING-H2 (hPIRH2) cDNA was isolated. A glutathione S-transferase-hPIRH2 fusion protein expressed in bacteria was able to pull down P protein when mixed with an extract from P-expressing HeLa cells in vitro, and myc-tagged hPIRH2 could be reciprocally coimmunoprecipitated with MV P protein from human cells. Additionally, immunoprecipitation experiments demonstrated that hPIRH2-myc, MV P, and nucleocapsid (N) proteins form a ternary complex. The hPIRH2 binding site was mapped to the C-terminal X domain region of the P protein by using a yeast two-hybrid assay. The PCT binding site was mapped on hPIRH2 by using a novel yeast two-hybrid tagged PCR approach and by coimmunoprecipitation of hPIRH2 cysteine mutants and mouse/human PIRH2 chimeras. The hPIRH2 C terminus could mediate the interaction with MV P which was favored by the RING-H2 motif. When coexpressed with an enhanced green fluorescent protein-tagged hPIRH2 protein, MV P alone or in a complex with MV N was able to redistribute hPIRH2 to outside the nucleus, within intracellular aggregates. Finally, MV P efficiently stabilized hPIRH2-myc expression and prevented its ubiquitination in vivo but had no effect on the stability or ubiquitination of an alternative ubiquitin E3 ligase, Mdm2. Thus, MV P protein is the first protein from a pathogen that is able to specifically interact with and stabilize the ubiquitin E3 ligase hPIRH2 by preventing its ubiquitination.

scholarly journals SLAP2 adaptor binding disrupts c-CBL autoinhibition to activate ubiquitin ligase function

2020 ◽  
Author(s):  
Leanne E. Wybenga-Groot ◽  
Andrea J. Tench ◽  
Craig D. Simpson ◽  
Jonathan St. Germain ◽  
Brian Raught ◽  
...  
Keyword(s):  
Tyrosine Kinase ◽  
Binding Site ◽  
Ubiquitin Ligase ◽  
Adaptor Proteins ◽  
Negative Regulator ◽  
Alternative Mechanism ◽  
Kinase Signaling ◽  
Tyrosine Kinase Signaling ◽  
Ligase Function

AbstractCBL is a RING type E3 ubiquitin ligase that functions as a negative regulator of tyrosine kinase signaling and loss of CBL E3 function is implicated in several forms of leukemia. The Src-like adaptor proteins (SLAP/SLAP2) bind to CBL and are required for CBL-dependent downregulation of antigen receptor, cytokine receptor, and receptor tyrosine kinase signaling. Despite the established role of SLAP/SLAP2 in regulating CBL activity, the nature of the interaction and the mechanisms involved are not known. To understand the molecular basis of the interaction between SLAP/SLAP2 and CBL, we solved the crystal structure of CBL tyrosine kinase binding domain (TKBD) in complex with SLAP2. The carboxy-terminal region of SLAP2 adopts an α-helical structure which binds in a cleft between the 4H, EF-hand, and SH2 domains of the TKBD. This SLAP2 binding site is remote from the canonical TKBD phospho-tyrosine peptide binding site but overlaps with a region important for stabilizing CBL in its autoinhibited conformation. In addition, binding of SLAP2 to CBL in vitro activates the ubiquitin ligase function of autoinhibited CBL. Disruption of the CBL/SLAP2 interface through mutagenesis demonstrated a role for this protein-protein interaction in regulation of CBL E3 ligase activity in cells. Our results reveal that SLAP2 binding to a regulatory cleft of the TKBD provides an alternative mechanism for activation of CBL ubiquitin ligase function.

scholarly journals Protein polyglutamylation catalyzed by the bacterial calmodulin-dependent pseudokinase SidJ

eLife ◽  
2019 ◽  
Vol 8 ◽  
Author(s):  
Alan Sulpizio ◽  
Marena E Minelli ◽  
Min Wan ◽  
Paul D Burrowes ◽  
Xiaochun Wu ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Mass Spectrometry ◽  
Ubiquitin Ligase ◽  
Legionella Pneumophila ◽  
Kinase Activity ◽  
Effector Protein ◽  
Dependent Manner ◽  
Biochemical Analyses ◽  
Human And Mouse

Pseudokinases are considered to be the inactive counterparts of conventional protein kinases and comprise approximately 10% of the human and mouse kinomes. Here, we report the crystal structure of the Legionella pneumophila effector protein, SidJ, in complex with the eukaryotic Ca2+-binding regulator, calmodulin (CaM). The structure reveals that SidJ contains a protein kinase-like fold domain, which retains a majority of the characteristic kinase catalytic motifs. However, SidJ fails to demonstrate kinase activity. Instead, mass spectrometry and in vitro biochemical analyses demonstrate that SidJ modifies another Legionella effector SdeA, an unconventional phosphoribosyl ubiquitin ligase, by adding glutamate molecules to a specific residue of SdeA in a CaM-dependent manner. Furthermore, we show that SidJ-mediated polyglutamylation suppresses the ADP-ribosylation activity. Our work further implies that some pseudokinases may possess ATP-dependent activities other than conventional phosphorylation.

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

scholarly journals Structural Basis of VSIG3: The Ligand for VISTA

2021 ◽  
Vol 12 ◽  
Author(s):  
Xiaoxue Xie ◽  
Caiping Chen ◽  
Wenting Chen ◽  
Jingwei Jiang ◽  
Lanlan Wang ◽  
...  
Keyword(s):  
Crystal Structure ◽  
T Cell ◽  
T Cell Activation ◽  
Cell Activation ◽  
Biological Activities ◽  
Protein Docking ◽  
Structural Basis ◽  
Cell Functions ◽  
B7 Family

B7 family members and their receptors play key roles in regulating T cell responses, and constitute very attractive targets for developing immunotherapeutic drugs. V-Set and Immunoglobulin domain containing 3 (VSIG3), a ligand for the novel B7 family immune checkpoint V-domain immunoglobulin suppressor of T cell activation (VISTA), can significantly inhibit T cell functions. Inhibitors targeting the VISTA/VSIG3 pathway are of great significance in tumor immunology. Here, we show the crystal structure of the extracellular domain (ECD) of the human VSIG3 protein at 2.64 angstrom resolution, and we produce recombinant human VSIG-3 ECD in both CHO cells and E. coli. Furthermore, we demonstrated the interaction of VISTA and VSIG3 by coimmunoprecipitation (Co-IP). Based on protein-protein docking for VISTA and VSIG3, we report a small molecule inhibitor of VSIG3 K284-3046 and evaluate its biological activities in vitro. This study was the first to reveal the crystal structure of VSIG3, and provides the structural basis for designing antibodies or compounds for the unique VSIG3/VISTA coinhibitory pathway in the treatment of cancers, autoimmune diseases and may be beneficial of designing vaccines.

scholarly journals Protein polyglutamylation catalyzed by the bacterial Calmodulin-dependent pseudokinase SidJ

2019 ◽  
Author(s):  
Alan Sulpizio ◽  
Marena E. Minelli ◽  
Min Wan ◽  
Paul D. Burrowes ◽  
Xiaochun Wu ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Mass Spectrometry ◽  
Ubiquitin Ligase ◽  
Legionella Pneumophila ◽  
Biochemical Analysis ◽  
Kinase Activity ◽  
Effector Protein ◽  
Dependent Manner ◽  
Human And Mouse

AbstractPseudokinases are considered to be the inactive counterparts of conventional protein kinases and comprise approximately 10% of the human and mouse kinomes. Here we report the crystal structure of theLegionella pneumophilaeffector protein, SidJ, in complex with the eukaryotic Ca2+-binding regulator, Calmodulin (CaM). The structure reveals that SidJ contains a protein kinase-like fold domain, which retains a majority of the characteristic kinase catalytic motifs. However, SidJ fails to demonstrate kinase activity. Instead, mass spectrometry and in vitro biochemical analysis demonstrate that SidJ modifies anotherLegionellaeffector SdeA, an unconventional phosphoribosyl ubiquitin ligase, by adding glutamate molecules to a specific residue of SdeA in a CaM-dependent manner. Furthermore, we show that SidJ-mediated polyglutamylation suppresses the ADP-ribosylation activity. Our work further implies that some pseudokinases may possess ATP-dependent activities other than conventional phosphorylation.

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