SAXSMoW 2.0: Online calculator of the molecular weight of proteins in dilute solution from experimental SAXS data measured on a relative scale

Piiadov, Vassili; Araújo, Evandro Ares de; Neto, Mario Oliveira; Craievich, A. F.; Polikarpov, Igor

doi:10.1002/pro.3528

Cited by 132 publications

(136 citation statements)

References 30 publications

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“…Scattering contributions due to buffer were subtracted from protein peak using ScÅtter. SAXS based molecular weight estimates were done using SAXS MoW 2.0 [16]. Analysis of crystal structure fitting to scattering profiles was done using CRYSOL [17,18].…”

Section: Methodsmentioning

confidence: 99%

FMN-dependent oligomerization of putative lactate oxidase fromPediococcus acidilactici

Ashok

Maksimainen

Kallio

et al. 2019

Preprint

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13Lactate oxidases belong to a group of FMN-dependent enzymes and they catalyze a conversion 14 of lactate to pyruvate with a release of hydrogen peroxide. Hydrogen peroxide is also utilized 15 as a read out in biosensors to quantitate lactate levels in biological samples. Aerococcus 16 viridans lactate oxidase is the best characterized lactate oxidase and our knowledge of lactate 17 oxidases relies largely to studies conducted with that particular enzyme. Pediococcus 18 acidilactici lactate oxidase is also commercially available for e.g. lactate measurements, but 19 this enzyme has not been characterized before in detail. Here we report structural 20 characterization of the recombinant enzyme and its co-factor dependent oligomerization. The 21 crystal structures revealed two distinct conformations in the loop closing the active site, 22 consistent with previous biochemical studies implicating the role of loop in catalysis. Despite 23 the structural conservation of active site residues when compared to Aerococcus viridans 24 lactate oxidase we were not able to detect either oxidase or monooxygenase activity when L-25 lactate or other potential alpha hydroxyl acids were used as a substrate. Pediococcus 26 acidilactici lactate oxidase is therefore an example of a misannotation of an FMN-dependent 27 enzyme, which catalyzes likely a so far unknown oxidation reaction. 28 3 29 4 53 Pediococcus acidilactici proteome does not use lactate as a substrate. We describe here 54 structural and biophysical studies of PaLCTO, which revealed FMN-dependent folding and 55 oligomerization of the enzyme. 56 57 Materials and methods 58 Cloning and site directed mutagenesis 59 Pediococcus acidilactici proteome wide search for keywords lactate, lactate oxidase, α-60 hydroxy acid in Uniprot database gave only one hit that matched with AvLCTO. Similarly, 61 BLASTp search with AvLCTO against P. acidilactici DSM20284 revealed the same hit 62 annotated as putative L-lactate oxidase (Uniprot E0NE46). Genomic DNA of Pediococcus 63 acidilactici (DSM 20284) and Aerococcus viridans (DSM 20340) was obtained from DSMZ, 64 Germany. Gene encoding PaLCTO was cloned into pNH-TrxT (Structural Genomics 65 Consortium) vector using SLIC cloning. The vector encodes for an N-terminal thioredoxin tag 66 with a cleavable TEV protease recognition site. A94G mutant was obtained using standard site-67 directed mutagenesis protocol. All clones were verified using Sanger's dideoxy sequencing. 68 Untagged PaLCTO and AvLCTO were recombinantly expressed from pNIC-CH vector with a 69 stop codon added immediately after the native sequence. Commercial PaLCTO was purchased 70 from Sigma (catalog number LO638, lots STBG2905V and STBF3223V).71 Protein expression and purification 72 Proteins were expressed using BL21 (DE3). Overnight culture was inoculated into terrific broth 73 auto-induction media supplemented with 8 g/L glycerol and 50 μg/ml kanamycin. Cells were 74 grown at 37°C until OD 600 reached 1. The temperature was reduced to 18°C for overnight for 5 75 protein e...

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Section: Methodsmentioning

confidence: 99%

FMN-dependent oligomerization of putative lactate oxidase fromPediococcus acidilactici

Ashok

Maksimainen

Kallio

et al. 2019

Preprint

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“…The pair distribution function, P(r), and maximum particle dimension, D max , were determined in GNOM (55). Molecular weight was determined using the SAXSMoW2 webserver (http://www.saxs.ifsc.usp.br) (56). Theoretical scattering of the crystal structures was computed and fitted with the experimental data using CRYSOL (57).…”

Section: Crystallization Data Collection and Structurementioning

confidence: 99%

Structural basis of cell-surface signaling by a conserved sigma regulator in Gram-negative bacteria

Jensen

Jernberg

Sinha

et al. 2020

Journal of Biological Chemistry

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Cell-surface signaling (CSS) in Gram-negative bacteria involves highly conserved regulatory pathways that optimize gene expression by transducing extracellular environmental signals to the cytoplasm via inner-membrane sigma regulators. The molecular details of ferric siderophore-mediated activation of the iron import machinery through a sigma regulator are unclear. Here, we present the 1.56 Å resolution structure of the periplasmic complex of the C-terminal CSS domain (CCSSD) of PupR, the sigma regulator in the Pseudomonas capeferrum pseudobactin BN7/8 transport system, and the N-terminal signaling domain (NTSD) of PupB, an outer-membrane TonB-dependent transducer. The structure revealed that the CCSSD consists of two subdomains: a juxta-membrane subdomain, which has a novel all-β-fold, followed by a secretin/TonB, short N-terminal subdomain at the C terminus of the CCSSD, a previously unobserved topological arrangement of this domain. Using affinity pulldown assays, isothermal titration calorimetry, and thermal denaturation CD spectroscopy, we show that both subdomains are required for binding the NTSD with micromolar affinity and that NTSD binding improves CCSSD stability. Our findings prompt us to present a revised model of CSS wherein the CCSSD:NTSD complex forms prior to ferric-siderophore binding. Upon siderophore binding, conformational changes in the CCSSD enable regulated intramembrane proteolysis of the sigma regulator, ultimately resulting in transcriptional regulation.

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“…Data processing was performed using ScÅtter 39 and Primus 40 to obtain the radius of gyration (R g ), the maximum particle dimension (D max ), the excluded particle volume (Vp) and the pair distribution function (P(r)). In addition, Rg and MW were derived using the SAXS MoW server 41 . Theoretical scattering curves from PDB coordinates and their fits to the experimental scattering curves were calculated using the FOXS server 42 .…”

Section: Size Exclusion Chromatography (Sec)mentioning

confidence: 99%

Oligomerization and characteristics of phosphoenolpyruvate carboxylase in Synechococcus PCC 7002

Durall

Selmer

et al. 2020

Sci Rep

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phosphoenolpyruvate carboxylase (pepc) is an essential enzyme in plants. A photosynthetic form is present both as dimer and tetramer in C4 and CAM metabolism. Additionally, non-photosynthetic PEPcs are also present. The single, non-photosynthetic PEPc of the unicellular cyanobacterium Synechococcus PCC 7002 (Synechococcus), involved in the tcA cycle, was examined. Using size exclusion chromatography (SEC) and small angle X-ray scattering (SAXS), we observed that PEPc in Synechococcus exists as both a dimer and a tetramer. This is the first demonstration of two different oligomerization states of a non-photosynthetic PEPc. High concentration of Mg 2+ , the substrate pep and a combination of low concentration of Mg 2+ and HCO 3 − induced the tetramer form of the carboxylase. Using SEC-SAXS analysis, we showed that the oligomerization state of the carboxylase is concentration dependent and that, among the available crystal structures of pepc, the scattering profile of PEPc of Synechococcus agrees best with the structure of pepc from Escherichia coli. in addition, the kinetics of the tetramer purified in presence of Mg 2+ using Sec, and of the mixed population purified in presence of Mg 2+ using a Strep-tagged column were examined. Moreover, the enzyme showed interesting allosteric regulation, being activated by succinate and inhibited by glutamine, and not affected by either malate, 2-oxoglutarate, aspartic acid or citric acid.

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SAXSMoW 2.0: Online calculator of the molecular weight of proteins in dilute solution from experimental SAXS data measured on a relative scale

Cited by 132 publications

References 30 publications

FMN-dependent oligomerization of putative lactate oxidase fromPediococcus acidilactici

FMN-dependent oligomerization of putative lactate oxidase fromPediococcus acidilactici

Structural basis of cell-surface signaling by a conserved sigma regulator in Gram-negative bacteria

Oligomerization and characteristics of phosphoenolpyruvate carboxylase in Synechococcus PCC 7002

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