Structural Basis for High Specificity of Amadori Compound and Mannopine Opine Binding in Bacterial Pathogens

ABSTRACT ADP-ribose (ADPR) is one of the main substrates of Nudix proteins. Among the eight Nudix proteins of Thermus thermophilus HB8, we previously determined the crystal structure of Ndx4, an ADPR pyrophosphatase (ADPRase). In this study we show that Ndx2 of T. thermophilus also preferentially hydrolyzes ADPR and flavin adenine dinucleotide and have determined its crystal structure. We have determined the structures of Ndx2 alone and in complex with Mg2+, with Mg2+ and AMP, and with Mg2+ and a nonhydrolyzable ADPR analogue. Although Ndx2 recognizes the AMP moiety in a manner similar to those for other ADPRases, it recognizes the terminal ribose in a distinct manner. The residues responsible for the recognition of the substrate in Ndx2 are not conserved among ADPRases. This may reflect the diversity in substrate specificity among ADPRases. Based on these results, we propose the classification of ADPRases into two types: ADPRase-I enzymes, which exhibit high specificity for ADPR; and ADPRase-II enzymes, which exhibit low specificity for ADPR. In the active site of the ternary complexes, three Mg2+ ions are coordinated to the side chains of conserved glutamate residues and water molecules. Substitution of Glu90 and Glu94 with glutamine suggests that these residues are essential for catalysis. These results suggest that ADPRase-I and ADPRase-II enzymes have nearly identical catalytic mechanisms but different mechanisms of substrate recognition.

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Crystal structure of Staphylococcus aureus exfoliative toxin D-like protein: Structural basis for the high specificity of exfoliative toxins

Biochemical and Biophysical Research Communications ◽

10.1016/j.bbrc.2015.08.083 ◽

2015 ◽

Vol 467 (1) ◽

pp. 171-177 ◽

Cited By ~ 3

Author(s):

Ricardo B. Mariutti ◽

Tatiana A.C.B. Souza ◽

Anwar Ullah ◽

Icaro P. Caruso ◽

Fábio R. de Moraes ◽

...

Keyword(s):

Crystal Structure ◽

Staphylococcus Aureus ◽

High Specificity ◽

Structural Basis ◽

Exfoliative Toxin

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Structural basis for high specificity of octopine binding in the plant pathogen Agrobacterium tumefaciens

Scientific Reports ◽

10.1038/s41598-017-18243-8 ◽

2017 ◽

Vol 7 (1) ◽

Cited By ~ 9

Author(s):

Armelle Vigouroux ◽

Abbas El Sahili ◽

Julien Lang ◽

Magali Aumont-Nicaise ◽

Yves Dessaux ◽

...

Keyword(s):

Agrobacterium Tumefaciens ◽

Plant Pathogen ◽

High Specificity ◽

Structural Basis

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Two distinct mechanisms of transcriptional regulation by the redox sensor YodB

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1604427113 ◽

2016 ◽

Vol 113 (35) ◽

pp. E5202-E5211 ◽

Cited By ~ 8

Author(s):

Sang Jae Lee ◽

In-Gyun Lee ◽

Ki-Young Lee ◽

Dong-Gyun Kim ◽

Hyun-Jong Eun ◽

...

Keyword(s):

Conformational Changes ◽

Disulfide Bonds ◽

Structural Changes ◽

High Specificity ◽

Redox Balance ◽

Structural Basis ◽

X Ray Crystallography ◽

Redox Sensor ◽

Regulatory Systems ◽

Sense And Respond

For bacteria, cysteine thiol groups in proteins are commonly used as thiol-based switches for redox sensing to activate specific detoxification pathways and restore the redox balance. Among the known thiol-based regulatory systems, the MarR/DUF24 family regulators have been reported to sense and respond to reactive electrophilic species, including diamide, quinones, and aldehydes, with high specificity. Here, we report that the prototypical regulator YodB of the MarR/DUF24 family from Bacillus subtilis uses two distinct pathways to regulate transcription in response to two reactive electrophilic species (diamide or methyl-p-benzoquinone), as revealed by X-ray crystallography, NMR spectroscopy, and biochemical experiments. Diamide induces structural changes in the YodB dimer by promoting the formation of disulfide bonds, whereas methyl-p-benzoquinone allows the YodB dimer to be dissociated from DNA, with little effect on the YodB dimer. The results indicate that B. subtilis may discriminate toxic quinones, such as methyl-p-benzoquinone, from diamide to efficiently manage multiple oxidative signals. These results also provide evidence that different thiol-reactive compounds induce dissimilar conformational changes in the regulator to trigger the separate regulation of target DNA. This specific control of YodB is dependent upon the type of thiol-reactive compound present, is linked to its direct transcriptional activity, and is important for the survival of B. subtilis. This study of B. subtilis YodB also provides a structural basis for the relationship that exists between the ligand-induced conformational changes adopted by the protein and its functional switch.

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Affimer proteins inhibit immune complex binding to FcγRIIIa with high specificity through competitive and allosteric modes of action

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1707856115 ◽

2017 ◽

Vol 115 (1) ◽

pp. E72-E81 ◽

Cited By ~ 20

Author(s):

James I. Robinson ◽

Euan W. Baxter ◽

Robin L. Owen ◽

Maren Thomsen ◽

Darren C. Tomlinson ◽

...

Keyword(s):

Protein Interactions ◽

High Specificity ◽

Structural Basis ◽

Protein Protein Interactions ◽

Cellular Functions ◽

High Sequence Identity ◽

Downstream Effector ◽

Igg Binding ◽

Genes Encoding ◽

Dynamics Simulations

Protein–protein interactions are essential for the control of cellular functions and are critical for regulation of the immune system. One example is the binding of Fc regions of IgG to the Fc gamma receptors (FcγRs). High sequence identity (98%) between the genes encoding FcγRIIIa (expressed on macrophages and natural killer cells) and FcγRIIIb (expressed on neutrophils) has prevented the development of monospecific agents against these therapeutic targets. We now report the identification of FcγRIIIa-specific artificial binding proteins called “Affimer” that block IgG binding and abrogate FcγRIIIa-mediated downstream effector functions in macrophages, namely TNF release and phagocytosis. Cocrystal structures and molecular dynamics simulations have revealed the structural basis of this specificity for two Affimer proteins: One binds directly to the Fc binding site, whereas the other acts allosterically.

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Staphylopine, pseudopaline, and yersinopine dehydrogenases: A structural and kinetic analysis of a new functional class of opine dehydrogenase

Journal of Biological Chemistry ◽

10.1074/jbc.ra118.002007 ◽

2018 ◽

Vol 293 (21) ◽

pp. 8009-8019 ◽

Cited By ~ 17

Author(s):

Jeffrey S. McFarlane ◽

Cara L. Davis ◽

Audrey L. Lamb

Keyword(s):

Kinetic Analysis ◽

Active Sites ◽

Bacterial Pathogens ◽

Functional Class ◽

Structural Basis ◽

Keto Acid ◽

Burn Wound ◽

New Class ◽

Bound Variation ◽

Steady State Kinetic

Opine dehydrogenases (ODHs) from the bacterial pathogens Staphylococcus aureus, Pseudomonas aeruginosa, and Yersinia pestis perform the final enzymatic step in the biosynthesis of a new class of opine metallophores, which includes staphylopine, pseudopaline, and yersinopine, respectively. Growing evidence indicates an important role for this pathway in metal acquisition and virulence, including in lung and burn-wound infections (P. aeruginosa) and in blood and heart infections (S. aureus). Here, we present kinetic and structural characterizations of these three opine dehydrogenases. A steady-state kinetic analysis revealed that the three enzymes differ in α-keto acid and NAD(P)H substrate specificity and nicotianamine-like substrate stereoselectivity. The structural basis for these differences was determined from five ODH X-ray crystal structures, ranging in resolution from 1.9 to 2.5 Å, with or without NADP+ bound. Variation in hydrogen bonding with NADPH suggested an explanation for the differential recognition of this substrate by these three enzymes. Our analysis further revealed candidate residues in the active sites required for binding of the α-keto acid and nicotianamine-like substrates and for catalysis. This work reports the first structural kinetic analyses of enzymes involved in opine metallophore biosynthesis in three important bacterial pathogens of humans.

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Structural Analyses on the Deamidation of N-Terminal Asn in the Human N-Degron Pathway

Biomolecules ◽

10.3390/biom10010163 ◽

2020 ◽

Vol 10 (1) ◽

pp. 163 ◽

Cited By ~ 6

Author(s):

Joon Sung Park ◽

Jae-Young Lee ◽

Yen Thi Kim Nguyen ◽

Nae-Won Kang ◽

Eun Kyung Oh ◽

...

Keyword(s):

High Specificity ◽

Catalytic Triad ◽

Structural Basis ◽

Side Chain ◽

Peptide Substrates ◽

Hydrophobic Residues ◽

Structural Principles ◽

Peptide Complexes ◽

Biochemical Analyses

The N-degron pathway is a proteolytic system in which a single N-terminal amino acid acts as a determinant of protein degradation. Especially, degradation signaling of N-terminal asparagine (Nt-Asn) in eukaryotes is initiated from its deamidation by N-terminal asparagine amidohydrolase 1 (NTAN1) into aspartate. Here, we have elucidated structural principles of deamidation by human NTAN1. NTAN1 adopts the characteristic scaffold of CNF1/YfiH-like cysteine hydrolases that features an α-β-β sandwich structure and a catalytic triad comprising Cys, His, and Ser. In vitro deamidation assays using model peptide substrates with varying lengths and sequences showed that NTAN1 prefers hydrophobic residues at the second-position. The structures of NTAN1-peptide complexes further revealed that the recognition of Nt-Asn is sufficiently organized to produce high specificity, and the side chain of the second-position residue is accommodated in a hydrophobic pocket adjacent to the active site of NTAN1. Collectively, our structural and biochemical analyses of the substrate specificity of NTAN1 contribute to understanding the structural basis of all three amidases in the eukaryotic N-degron pathway.

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Structural basis of the myosin X PH1N-PH2-PH1C tandem as a specific and acute cellular PI(3,4,5)P3 sensor

Molecular Biology of the Cell ◽

10.1091/mbc.e11-04-0354 ◽

2011 ◽

Vol 22 (22) ◽

pp. 4268-4278 ◽

Cited By ~ 29

Author(s):

Qing Lu ◽

Jiang Yu ◽

Jing Yan ◽

Zhiyi Wei ◽

Mingjie Zhang

Keyword(s):

Molecular Mechanisms ◽

Lipid Membrane ◽

High Specificity ◽

Cell Types ◽

Lipid Binding ◽

Structural Basis ◽

Ph Domain ◽

Tail Region ◽

Binding Pockets ◽

Myosin X

Myosin X (MyoX) is an unconventional myosin that is known to induce the formation and elongation of filopodia in many cell types. MyoX-induced filopodial induction requires the three PH domains in its tail region, although with unknown underlying molecular mechanisms. MyoX's first PH domain is split into halves by its second PH domain. We show here that the PH1N-PH2-PH1C tandem allows MyoX to bind to phosphatidylinositol (3,4,5)-triphosphate [PI(3,4,5)P3] with high specificity and cooperativity. We further show that PH2 is responsible for the specificity of the PI(3,4,5)P3 interaction, whereas PH1 functions to enhance the lipid membrane–binding avidity of the tandem. The structure of the MyoX PH1N-PH2-PH1C tandem reveals that the split PH1, PH2, and the highly conserved interdomain linker sequences together form a rigid supramodule with two lipid-binding pockets positioned side by side for binding to phosphoinositide membrane bilayers with cooperativity. Finally, we demonstrate that disruption of PH2-mediated binding to PI(3,4,5)P3 abolishes MyoX's function in inducing filopodial formation and elongation.

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