Eudicot plant-specific sphingolipids determine host selectivity of microbial NLP cytolysins

Science ◽  
2017 ◽  
Vol 358 (6369) ◽  
pp. 1431-1434 ◽  
Author(s):  
Tea Lenarčič ◽  
Isabell Albert ◽  
Hannah Böhm ◽  
Vesna Hodnik ◽  
Katja Pirc ◽  
...  
Keyword(s):  
Binding Site ◽  
Conformational Changes ◽  
Pathogenic Bacteria ◽  
Head Group ◽  
Microbial Infection ◽  
Binding Studies ◽  
X Ray ◽  
Plant Pathogenic Bacteria ◽  
X Ray Crystallography ◽  
Host Selectivity

Necrosis and ethylene-inducing peptide 1–like (NLP) proteins constitute a superfamily of proteins produced by plant pathogenic bacteria, fungi, and oomycetes. Many NLPs are cytotoxins that facilitate microbial infection of eudicot, but not of monocot plants. Here, we report glycosylinositol phosphorylceramide (GIPC) sphingolipids as NLP toxin receptors. Plant mutants with altered GIPC composition were more resistant to NLP toxins. Binding studies and x-ray crystallography showed that NLPs form complexes with terminal monomeric hexose moieties of GIPCs that result in conformational changes within the toxin. Insensitivity to NLP cytolysins of monocot plants may be explained by the length of the GIPC head group and the architecture of the NLP sugar-binding site. We unveil early steps in NLP cytolysin action that determine plant clade-specific toxin selectivity.

scholarly journals Unusual Conformational Changes in 6-Hydroxymethyl-7,8-dihydropterin Pyrophosphokinase as Revealed by X-ray Crystallography and NMR

2001 ◽  
Vol 276 (43) ◽  
pp. 40274-40281 ◽  
Author(s):  
Bing Xiao ◽  
Genbin Shi ◽  
Jinhai Gao ◽  
Jaroslaw Blaszczyk ◽  
Qin Liu ◽  
...  
Keyword(s):  
Conformational Changes ◽  
X Ray ◽  
X Ray Crystallography

scholarly journals Engineered occluded apo-intermediate of LacY

2018 ◽  
Vol 115 (50) ◽  
pp. 12716-12721 ◽  
Author(s):  
Irina Smirnova ◽  
Vladimir Kasho ◽  
H. Ronald Kaback
Keyword(s):  
Binding Site ◽  
Lactose Permease ◽  
Binding Studies ◽  
X Ray Crystallography ◽  
Sugar Binding ◽  
Transport Cycle ◽  
Open Structures ◽  
Unrestricted Access ◽  
Tight Association ◽  
Alternating Access

The lactose permease of Escherichia coli (LacY) utilizes an alternating access symport mechanism with multiple conformational intermediates, but only inward (cytoplasmic)- or outward (periplasmic)-open structures have been characterized by X-ray crystallography. It is demonstrated here with sugar-binding studies that cross-linking paired-Cys replacements across the closed cytoplasmic cavity stabilize an occluded conformer with an inaccessible sugar-binding site. In addition, a nanobody (Nb) that stabilizes a periplasmic-open conformer with an easily accessible sugar-binding site in WT LacY fails to cause the cytoplasmic cross-linked mutants to become accessible to galactoside, showing that the periplasmic cavity is closed. These results are consistent with tight association of the periplasmic ends in two pairs of helices containing clusters of small residues in the packing interface between N- and C-terminal six-helix bundles of the symporter. However, after reduction of the disulfide bond, the Nb markedly increases the rate of galactoside binding, indicating unrestricted access to the Nb epitope and the galactoside-binding site from the periplasm. The findings indicate that the cross-linked cytoplasmic double-Cys mutants resemble an occluded apo-intermediate in the transport cycle.

scholarly journals Probing the Role of Divalent Metal Ions in a Bacterial Psychrophilic Metalloprotease: Binding Studies of an Enzyme in the Crystalline State by X-Ray Crystallography

2003 ◽  
Vol 185 (14) ◽  
pp. 4195-4203 ◽  
Author(s):  
Stephanie Ravaud ◽  
Patrice Gouet ◽  
Richard Haser ◽  
Nushin Aghajari
Keyword(s):  
Metal Ions ◽  
Calcium Ions ◽  
Calcium Ion ◽  
Crystalline State ◽  
Zinc Ion ◽  
Binding Studies ◽  
X Ray ◽  
X Ray Crystallography ◽  
Catalytic Zinc

ABSTRACT The psychrophilic alkaline metalloprotease (PAP) produced by a Pseudomonas bacterium isolated in Antarctica belongs to the clan of metzincins, for which a zinc ion is essential for catalytic activity. Binding studies in the crystalline state have been performed by X-ray crystallography in order to improve the understanding of the role of the zinc and calcium ions bound to this protease. Cocrystallization and soaking experiments with EDTA in a concentration range from 1 to 85 mM have resulted in five three-dimensional structures with a distinct number of metal ions occupying the ion-binding sites. Evolution of the structural changes observed in the vicinity of each cation-binding site has been studied as a function of the concentration of EDTA, as well as of time, in the presence of the chelator. Among others, we have found that the catalytic zinc ion was the first ion to be chelated, ahead of a weakly bound calcium ion (Ca 700) exclusive to the psychrophilic enzyme. Upon removal of the catalytic zinc ion, the side chains of the active-site residues His-173, His-179 and Tyr-209 shifted ∼4, 1.0, and 1.6 Å, respectively. Our studies confirm and also explain the sensitivity of PAP toward moderate EDTA concentrations and propose distinct roles for the calcium ions. A new crystal form of native PAP validates our previous predictions regarding the adaptation of this enzyme to cold environments as well as the proteolytic domain calcium ion being exclusive for PAP independent of crystallization conditions.

Guanine Binding Site of theNicotiana glutinosaRibonuclease NW Revealed by X-Ray Crystallography

Biochemistry ◽  
2002 ◽  
Vol 41 (51) ◽  
pp. 15195-15202 ◽  
Author(s):  
Shin Kawano ◽  
Yoshimitsu Kakuta ◽  
Makoto Kimura
Keyword(s):  
Binding Site ◽  
X Ray ◽  
X Ray Crystallography

scholarly journals Allosteric interactions of glycogen phosphorylase b. A crystallographic study of glucose 6-phosphate and inorganic phosphate binding to di-imidate-cross-linked phosphorylase b

1984 ◽  
Vol 218 (1) ◽  
pp. 45-60 ◽  
Author(s):  
A Lorek ◽  
K S Wilson ◽  
M S P Sansom ◽  
D I Stuart ◽  
E A Stura ◽  
...  
Keyword(s):  
Binding Site ◽  
Conformational Changes ◽  
Inorganic Phosphate ◽  
Glycogen Phosphorylase ◽  
Phosphate Binding ◽  
Binding Studies ◽  
Structural Explanation ◽  
Alpha Helices ◽  
Allosteric Effector ◽  
Glycogen Phosphorylase B

The binding to glycogen phosphorylase b of glucose 6-phosphate and inorganic phosphate (respectively allosteric inhibitor and substrate/activator of the enzyme) were studied in the crystal at 0.3 nm (3A) resolution. Glucose 6-phosphate binds in the alpha-configuration at a site that is close to the AMP allosteric effector site at the subunit-subunit interface and promotes several conformational changes. The phosphate-binding site of the enzyme for glucose 6-phosphate involves contacts to two cationic residues, Arg-309 and Lys-247. This site is also occupied in the inorganic-phosphate-binding studies and is therefore identified as a high-affinity phosphate-binding site. It is distinct from the weaker phosphate-binding site of the enzyme for AMP, which is 0.27 nm (2.7A) away. The glucose moiety of glucose 6-phosphate and the adenosine moiety of AMP do not overlap. The results provide a structural explanation for the kinetic observations that glucose 6-phosphate inhibition of AMP activation of phosphorylase b is partially competitive and highly co-operative. The results suggest that the transmission of allosteric conformational changes involves an increase in affinity at phosphate-binding sites and relative movements of alpha-helices. In order to study glucose 6-phosphate and phosphate binding it was necessary to cross-link the crystals. The use of dimethyl malondi-imidate as a new cross-linking reagent in protein crystallography is discussed.

scholarly journals Exploring ribozyme conformational changes with X-ray crystallography

Methods ◽  
2009 ◽  
Vol 49 (2) ◽  
pp. 87-100 ◽  
Author(s):  
Robert C. Spitale ◽  
Joseph E. Wedekind
Keyword(s):  
Conformational Changes ◽  
X Ray ◽  
X Ray Crystallography

PLP undergoes conformational changes during the course of an enzymatic reaction

2014 ◽  
Vol 70 (2) ◽  
pp. 596-606 ◽  
Author(s):  
Ho-Phuong-Thuy Ngo ◽  
Nuno M. F. S. A. Cerqueira ◽  
Jin-Kwang Kim ◽  
Myoung-Ki Hong ◽  
Pedro Alexandrino Fernandes ◽  
...  
Keyword(s):  
Conformational Change ◽  
Conformational Changes ◽  
Catalytic Mechanism ◽  
Enzymatic Reaction ◽  
Nucleophilic Attack ◽  
The Novel ◽  
X Ray ◽  
X Ray Crystallography ◽  
Starting Point ◽  
High Level

Numerous enzymes, such as the pyridoxal 5′-phosphate (PLP)-dependent enzymes, require cofactors for their activities. Using X-ray crystallography, structural snapshots of the L-serine dehydratase catalytic reaction of a bacterial PLP-dependent enzyme were determined. In the structures, the dihedral angle between the pyridine ring and the Schiff-base linkage of PLP varied from 18° to 52°. It is proposed that the organic cofactor PLP directly catalyzes reactions by active conformational changes, and the novel catalytic mechanism involving the PLP cofactor was confirmed by high-level quantum-mechanical calculations. The conformational change was essential for nucleophilic attack of the substrate on PLP, for concerted proton transfer from the substrate to the protein and for directing carbanion formation of the substrate. Over the whole catalytic cycle, the organic cofactor catalyzes a series of reactions, like the enzyme. The conformational change of the PLP cofactor in catalysis serves as a starting point for identifying the previously unknown catalytic roles of organic cofactors.

scholarly journals Identification of the Calcium Binding Site and a Novel Ytterbium Site in Blood Coagulation Factor XIII by X-ray Crystallography

1999 ◽  
Vol 274 (8) ◽  
pp. 4917-4923 ◽  
Author(s):  
Brian A. Fox ◽  
Vivien C. Yee ◽  
Lars C. Pedersen ◽  
Isolde Le Trong ◽  
Paul D. Bishop ◽  
...  
Keyword(s):  
Binding Site ◽  
Blood Coagulation ◽  
Calcium Binding ◽  
Factor Xiii ◽  
Coagulation Factor ◽  
Calcium Binding Site ◽  
X Ray ◽  
X Ray Crystallography ◽  
Coagulation Factor Xiii

scholarly journals The C-terminal tail of the bacterial translocation ATPase SecA modulates its activity

2018 ◽  
Author(s):  
Mohammed Jamshad ◽  
Timothy J. Knowles ◽  
Scott A. White ◽  
Douglas G. Ward ◽  
Fiyaz Mohammed ◽  
...  
Keyword(s):  
Conformational Changes ◽  
Metal Binding ◽  
Cytoplasmic Membrane ◽  
Protein Recognition ◽  
X Ray ◽  
Ribosome Binding ◽  
X Ray Crystallography ◽  
Substrate Protein ◽  
Metal Binding Domain

AbstractIn bacteria, the translocation of a subset of proteins across the cytoplasmic membrane by the Sec machinery requires SecA. Although SecA can recognise nascent polypeptides, the mechanism of cotranslational substrate protein recognition is not known. Here, we investigated the role of the C-terminal tail (CTT) of SecA, which consists of a flexible linker (FLD) and a small metal-binding domain (MBD), in its interaction with nascent polypeptides. Phylogenetic analysis and ribosome binding experiments indicated that the MBD interacts with 70S ribosomes. Disruption of the entire CTT or the MBD alone had opposing effects on ribosome binding, substrate-protein binding, ATPase activity and in vivo function. Autophotocrosslinking, mass spectrometry, x-ray crystallography and small-angle x-ray scattering experiments provided insight into the CTT-mediated conformational changes in SecA. Finally, photocrosslinking experiments indicated that binding of SecA to substrate protein affected its interaction with the ribosome. Taken together, our results suggest a mechanism for substrate protein recognition.Impact StatementSecA is an evolutionarily conserved ATPase that is required for the translocation of a subset of proteins across the cytoplasmic membrane in bacteria. We investigated how SecA recognises its substrate proteins at the ribosome as they are still being synthesised (i.e. cotranslationally).

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