Crystal structure of a MarR family protein from the psychrophilic bacterium Paenisporosarcina sp. TG-14 in complex with a lipid-like molecule

MarR family proteins regulate the transcription of multiple antibiotic-resistance genes and are widely found in bacteria and archaea. Recently, a new MarR family gene was identified by genome analysis of the psychrophilic bacterium Paenisporosarcina sp. TG-14, which was isolated from sediment-laden basal ice in Antarctica. In this study, the crystal structure of the MarR protein from Paenisporosarcina sp. TG-14 (PaMarR) was determined at 1.6 Å resolution. In the crystal structure, a novel lipid-type compound (palmitic acid) was found in a deep cavity, which was assumed to be an effector-binding site. Comparative structural analysis of homologous MarR family proteins from a mesophile and a hyperthermophile showed that the DNA-binding domain of PaMarR exhibited relatively high mobility, with a disordered region between the β1 and β2 strands. In addition, structural comparison with other homologous complex structures suggests that this structure constitutes a conformer transformed by palmitic acid. Biochemical analysis also demonstrated that PaMarR binds to cognate DNA, where PaMarR is known to recognize two putative binding sites depending on its molar concentration, indicating that PaMarR binds to its cognate DNA in a stoichiometric manner. The present study provides structural information on the cold-adaptive MarR protein with an aliphatic compound as its putative effector, extending the scope of MarR family protein research.

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Crystal structure of a novel putative sugar isomerase from the psychrophilic bacterium Paenibacillus sp. R4

Biochemical and Biophysical Research Communications ◽

10.1016/j.bbrc.2021.11.026 ◽

2021 ◽

Author(s):

Sunghark Kwon ◽

Hyun Ji Ha ◽

Yong Jun Kang ◽

Ji Hye Sung ◽

Jisub Hwang ◽

...

Keyword(s):

Crystal Structure ◽

Psychrophilic Bacterium ◽

Paenibacillus Sp

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Crystal Structure of African Swine Fever Virus pS273R Protease and Implications for Inhibitor Design

Journal of Virology ◽

10.1128/jvi.02125-19 ◽

2020 ◽

Vol 94 (10) ◽

Author(s):

Guobang Li ◽

Xiaoxia Liu ◽

Mengyuan Yang ◽

Guangshun Zhang ◽

Zhengyang Wang ◽

...

Keyword(s):

Crystal Structure ◽

Structural Information ◽

African Swine Fever Virus ◽

African Swine Fever ◽

Hydrolytic Activity ◽

Viral Disease ◽

Fever Virus ◽

Dna Virus ◽

Core Domain ◽

The Core

ABSTRACT African swine fever (ASF) is a highly contagious hemorrhagic viral disease of domestic and wild pigs that is responsible for serious economic and production losses. It is caused by the African swine fever virus (ASFV), a large and complex icosahedral DNA virus of the Asfarviridae family. Currently, there is no effective treatment or approved vaccine against the ASFV. pS273R, a specific SUMO-1 cysteine protease, catalyzes the maturation of the pp220 and pp62 polyprotein precursors into core-shell proteins. Here, we present the crystal structure of the ASFV pS273R protease at a resolution of 2.3 Å. The overall structure of the pS273R protease is represented by two domains named the “core domain” and the N-terminal “arm domain.” The “arm domain” contains the residues from M1 to N83, and the “core domain” contains the residues from N84 to A273. A structure analysis reveals that the “core domain” shares a high degree of structural similarity with chlamydial deubiquitinating enzyme, sentrin-specific protease, and adenovirus protease, while the “arm domain” is unique to ASFV. Further, experiments indicated that the “arm domain” plays an important role in maintaining the enzyme activity of ASFV pS273R. Moreover, based on the structural information of pS273R, we designed and synthesized several peptidomimetic aldehyde compounds at a submolar 50% inhibitory concentration, which paves the way for the design of inhibitors to target this severe pathogen. IMPORTANCE African swine fever virus, a large and complex icosahedral DNA virus, causes a deadly infection in domestic pigs. In addition to Africa and Europe, countries in Asia, including China, Vietnam, and Mongolia, were negatively affected by the hazards posed by ASFV outbreaks in 2018 and 2019, at which time more than 30 million pigs were culled. Until now, there has been no vaccine for protection against ASFV infection or effective treatments to cure ASF. Here, we solved the high-resolution crystal structure of the ASFV pS273R protease. The pS273R protease has a two-domain structure that distinguishes it from other members of the SUMO protease family, while the unique “arm domain” has been proven to be essential for its hydrolytic activity. Moreover, the peptidomimetic aldehyde compounds designed to target the substrate binding pocket exert prominent inhibitory effects and can thus be used in a potential lead for anti-ASFV drug development.

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Crystal Structure of a Plant Multidrug and Toxic Compound Extrusion Family Protein

Structure ◽

10.1016/j.str.2017.07.009 ◽

2017 ◽

Vol 25 (9) ◽

pp. 1455-1460.e2 ◽

Cited By ~ 19

Author(s):

Yoshiki Tanaka ◽

Shigehiro Iwaki ◽

Tomoya Tsukazaki

Keyword(s):

Crystal Structure ◽

Toxic Compound ◽

Family Protein

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Abstract 304: Crystal Structure Of Fak/mef2 Complex Reveals The Role Of Fak In The Regulation Of Mef2 Transcription Factor.

Circulation Research ◽

10.1161/res.115.suppl_1.304 ◽

2014 ◽

Vol 115 (suppl_1) ◽

Author(s):

Alisson C Cardoso ◽

Ana H Pereira ◽

Andre L Ambrosio ◽

Silvio R Consonni ◽

Sandra M Dias ◽

...

Keyword(s):

Crystal Structure ◽

Mechanical Stress ◽

Focal Adhesion ◽

Molecular Mechanisms ◽

Structural Information ◽

Mechanistic Model ◽

Focal Adhesions ◽

Saxs Data ◽

X Ray ◽

Gene Assay

Members of MEF2 (Myocyte Enhancer Factor 2) family of transcription factors are major regulators of cardiac development and homeostasis. Their functions are regulated at several levels, including the association with a variety of protein partners. We have previously shown that FAK (Focal Adhesion Kinase) regulates the stretch-induced activation of MEF2 in cardiomyocytes. But, the molecular mechanisms, involved in this process, are unclear. Here, we integrated biochemical, imaging and structural analyses to characterize a novel interaction between MEF2 and FAK. An association between MEF2 and FAK was detected by co-immunoprecipitation in the extracts of stretched cardiomyocytes (10%, 60Hz, 2 hours). MEF2 and FAK staining were co-localized in the nuclei of stretched cells. Pull down assays indicated that the Focal Adhesion Targeting (FAT) domain is sufficient to confer FAK interaction with MEF2. Gene reporter assays indicated that the interaction with FAK enhances the MEF2C transcriptional activity in cultured cardiomyocytes. Also, we present a 2.9-Å X-ray crystal structure for the FAK_FAT domain bound to MEF2C (1-95), comprised by the MADS box/MEF2 domain. The structural information, when used in combination with biochemical studies, small-angle X-ray scattering (SAXS) data and reporter gene assay, lead to a mechanistic model describing how FAK binds to MEF2C and stimulates its transcription function in cardiomyocytes. We further validated this model by showing that the binding of FAK to MEF2C is essential for the hypertrophy of cardiomyocyte in response to mechanical stress. Our results present FAK as a new positive regulator of MEF2, implicated in the fine control of the signal transduction between focal adhesions and the nucleus of cardiac myocytes during mechanical stress.

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Crystal structure of a YeeE/YedE family protein engaged in thiosulfate uptake

Science Advances ◽

10.1126/sciadv.aba7637 ◽

2020 ◽

Vol 6 (35) ◽

pp. eaba7637

Author(s):

Yoshiki Tanaka ◽

Kunihito Yoshikaie ◽

Azusa Takeuchi ◽

Muneyoshi Ichikawa ◽

Tomoyuki Mori ◽

...

Keyword(s):

Crystal Structure ◽

Sulfur Cycle ◽

Sulfur Source ◽

Bacterial Membrane ◽

Density Maps ◽

Family Protein ◽

Cysteine Synthesis ◽

Plausible Mechanism ◽

Dynamics Simulations ◽

Positively Charged

We have demonstrated that a bacterial membrane protein, YeeE, mediates thiosulfate uptake. Thiosulfate is used for cysteine synthesis in bacteria as an inorganic sulfur source in the global biological sulfur cycle. The crystal structure of YeeE at 2.5-Å resolution reveals an unprecedented hourglass-like architecture with thiosulfate in the positively charged outer concave side. YeeE is composed of loops and 13 helices including 9 transmembrane α helices, most of which show an intramolecular pseudo 222 symmetry. Four characteristic loops are buried toward the center of YeeE and form its central region surrounded by the nine helices. Additional electron density maps and successive molecular dynamics simulations imply that thiosulfate can remain temporally at several positions in the proposed pathway. We propose a plausible mechanism of thiosulfate uptake via three important conserved cysteine residues of the loops along the pathway.

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Structural Insights into 6-Hydroxypseudooxynicotine Amine Oxidase from Pseudomonas geniculata N1, the Key Enzyme Involved in Nicotine Degradation

Applied and Environmental Microbiology ◽

10.1128/aem.01559-20 ◽

2020 ◽

Vol 86 (19) ◽

Cited By ~ 1

Author(s):

Gongquan Liu ◽

Weiwei Wang ◽

Fangyuan He ◽

Peng Zhang ◽

Ping Xu ◽

...

Keyword(s):

Crystal Structure ◽

Structural Information ◽

Amine Oxidase ◽

Substrate Binding ◽

Docking Study ◽

Site Directed Mutagenesis ◽

Flavin Mononucleotide ◽

Structure Comparison ◽

Content Type ◽

Key Enzyme

ABSTRACT Bacteria degrade nicotine mainly using pyridine and pyrrolidine pathways. Previously, we discovered a hybrid of the pyridine and pyrrolidine pathways (the VPP pathway) in Pseudomonas geniculata N1 and characterized its key enzyme, 6-hydroxypseudooxynicotine amine oxidase (HisD). It catalyzes oxidative deamination of 6-hydroxypseudooxynicotine to 6-hydroxy-3-succinoylsemialdehyde-pyridine, which is the crucial step connecting upstream and downstream portions of the VPP pathway. We determined the crystal structure of wild-type HisD to 2.6 Å. HisD is a monomer that contains a flavin mononucleotide, an iron-sulfur cluster, and ADP. On the basis of sequence alignment and structure comparison, a difference has been found among HisD, closely related trimethylamine dehydrogenase (TMADH), and histamine dehydrogenase (HADH). The flavin mononucleotide (FMN) cofactor is not covalently bound to any residue, and the FMN isoalloxazine ring is planar in HisD compared to TMADH or HADH, which forms a 6-S-cysteinyl flavin mononucleotide cofactor and has an FMN isoalloxazine ring in a “butterfly bend” conformation. Based on the structure, docking study, and site-directed mutagenesis, the residues Glu60, Tyr170, Asp262, and Trp263 may be involved in substrate binding. The expanded understanding of the substrate binding mode from this study may guide rational engineering of such enzymes for biodegradation of potential pollutants or for bioconversion to generate desired products. IMPORTANCE Nicotine is a major tobacco alkaloid in tobacco waste. Pyridine and pyrrolidine pathways are the two best-elucidated nicotine metabolic pathways; Pseudomonas geniculata N1 catabolizes nicotine via a hybrid between the pyridine and pyrrolidine pathways. The crucial enzyme, 6-hydroxypseudooxynicotine amine oxidase (HisD), links the upstream and downstream portions of the VPP pathway; however, there is little structural information about this important enzyme. In this study, we determined the crystal structure of HisD from Pseudomonas geniculata N1. Its basic insights about the structure may help us to guide the engineering of such enzymes for bioremediation and bioconversion applications.

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Protein Dynamics in F-like Bacterial Conjugation

Biomedicines ◽

10.3390/biomedicines8090362 ◽

2020 ◽

Vol 8 (9) ◽

pp. 362

Author(s):

Nicholas Bragagnolo ◽

Christina Rodriguez ◽

Naveed Samari-Kermani ◽

Alice Fours ◽

Mahboubeh Korouzhdehi ◽

...

Keyword(s):

Antibiotic Resistance ◽

Protein Interactions ◽

Drug Targets ◽

Dynamic Models ◽

Structural Information ◽

Imaging Techniques ◽

Antibiotic Resistance Genes ◽

Protein Protein Interactions ◽

Secretion Systems ◽

Type Iv

Efficient in silico development of novel antibiotics requires high-resolution, dynamic models of drug targets. As conjugation is considered the prominent contributor to the spread of antibiotic resistance genes, targeted drug design to disrupt vital components of conjugative systems has been proposed to lessen the proliferation of bacterial antibiotic resistance. Advancements in structural imaging techniques of large macromolecular complexes has accelerated the discovery of novel protein-protein interactions in bacterial type IV secretion systems (T4SS). The known structural information regarding the F-like T4SS components and complexes has been summarized in the following review, revealing a complex network of protein-protein interactions involving domains with varying degrees of disorder. Structural predictions were performed to provide insight on the dynamicity of proteins within the F plasmid conjugative system that lack structural information.

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Crystal structure of the M5muscarinic acetylcholine receptor

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1914446116 ◽

2019 ◽

Vol 116 (51) ◽

pp. 26001-26007 ◽

Cited By ~ 5

Author(s):

Ziva Vuckovic ◽

Patrick R. Gentry ◽

Alice E. Berizzi ◽

Kunio Hirata ◽

Swapna Varghese ◽

...

Keyword(s):

Crystal Structure ◽

Acetylcholine Receptor ◽

Rational Design ◽

Structural Information ◽

Family Members ◽

Muscarinic Acetylcholine Receptor ◽

Receptor Subtype ◽

Ligand Interaction ◽

Related Family ◽

Allosteric Sites

The human M5muscarinic acetylcholine receptor (mAChR) has recently emerged as an exciting therapeutic target for treating a range of disorders, including drug addiction. However, a lack of structural information for this receptor subtype has limited further drug development and validation. Here we report a high-resolution crystal structure of the human M5mAChR bound to the clinically used inverse agonist, tiotropium. This structure allowed for a comparison across all 5 mAChR family members that revealed important differences in both orthosteric and allosteric sites that could inform the rational design of selective ligands. These structural studies, together with chimeric swaps between the extracellular regions of the M2and M5mAChRs, provided structural insight into kinetic selectivity, where ligands show differential residency times between related family members. Collectively, our study provides important insights into the nature of orthosteric and allosteric ligand interaction across the mAChR family that could be exploited for the design of selective drugs.

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