ThreaDomEx: a unified platform for predicting continuous and discontinuous protein domains by multiple-threading and segment assembly

Wang, Yan; Wang, Jian; Li, Ruiming; Shi, Qiang; Xue, Zhidong; Zhang, Yang

doi:10.1093/nar/gkx410

Cited by 27 publications

(16 citation statements)

References 50 publications

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“…Modeling and Docking of Albumin and Individual Fibrinogen Domains into Fibrin. Predictions of the location of individual domains were performed with the web services provided by ThreaDomEx (26) and Robetta (27). Additional template searches were performed with HHalign-Kbest (28).…”

Section: Methodsmentioning

confidence: 99%

Missing regions within the molecular architecture of human fibrin clots structurally resolved by XL-MS and integrative structural modeling

Klykov

Zwaan

Heck

et al. 2020

Proc. Natl. Acad. Sci. U.S.A.

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Upon activation, fibrinogen forms large fibrin biopolymers that coalesce into clots which assist in wound healing. Limited insights into their molecular architecture, due to the sheer size and the insoluble character of fibrin clots, have restricted our ability to develop novel treatments for clotting diseases. The, so far resolved, disparate structural details have provided insights into linear elongation; however, molecular details like the C-terminal domain of the α-chain, the heparin-binding domain on the β-chain, and other functional domains remain elusive. To illuminate these dark areas, we applied cross-linking mass spectrometry (XL-MS) to obtain biochemical evidence in the form of over 300 distance constraints and combined this with structural modeling. These restraints additionally define the interaction network of the clots and provide molecular details for the interaction with human serum albumin (HSA). We were able to construct the structural models of the fibrinogen α-chain (excluding two highly flexible regions) and the N termini of the β-chain, confirm these models with known structural arrangements, and map how the structure laterally aggregates to form intricate lattices together with the γ-chain. We validate the final model by mapping mutations leading to impaired clot formation. From a list of 22 mutations, we uncovered structural features for all, including a crucial role for βArg’169 (UniProt: 196) in lateral aggregation. The resulting model can potentially serve for research on dysfibrinogenemia and amyloidosis as it provides insights into the molecular mechanisms of thrombosis and bleeding disorders related to fibrinogen variants. The structure is provided in the PDB-DEV repository (PDBDEV_00000030).

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Section: Methodsmentioning

confidence: 99%

Missing regions within the molecular architecture of human fibrin clots structurally resolved by XL-MS and integrative structural modeling

Klykov

Zwaan

Heck

et al. 2020

Proc. Natl. Acad. Sci. U.S.A.

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“…In addition, homolog detection and structural prediction by HHPRED [84], SWISS-MODEL [85] and, to some extent, PHYRE2 [86] were used to further investigate the function of the predicted structural proteins in S144. ThreaDomEx [87] was used for domain prediction in the putative tail fiber (ORF31). The sequence alignment and structural superposition of tail fibers of S144 and of Mu G+ (gpS) were conducted in CLC Main Workbench 20 and the structures were visualized in Pymol (version 2.3, DeLano Scientific, San Carlos, CA, USA).…”

Section: Genome Assembly and Bioinformatic Analysismentioning

confidence: 99%

Phage S144, a New Polyvalent Phage Infecting Salmonella spp. and Cronobacter sakazakii

Gambino

Sørensen

Ahern

et al. 2020

IJMS

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Phages are generally considered species- or even strain-specific, yet polyvalent phages are able to infect bacteria from different genera. Here, we characterize the novel polyvalent phage S144, a member of the Loughboroughvirus genus. By screening 211 Enterobacteriaceae strains, we found that phage S144 forms plaques on specific serovars of Salmonella enterica subsp. enterica and on Cronobacter sakazakii. Analysis of phage resistant mutants suggests that the O-antigen of lipopolysaccharide is the phage receptor in both bacterial genera. The S144 genome consists of 53,628 bp and encodes 80 open reading frames (ORFs), but no tRNA genes. In total, 32 ORFs coding for structural proteins were confirmed by ESI-MS/MS analysis, whereas 45 gene products were functionally annotated within DNA metabolism, packaging, nucleotide biosynthesis and phage morphogenesis. Transmission electron microscopy showed that phage S144 is a myovirus, with a prolate head and short tail fibers. The putative S144 tail fiber structure is, overall, similar to the tail fiber of phage Mu and the C-terminus shows amino acid similarity to tail fibers of otherwise unrelated phages infecting Cronobacter. Since all phages in the Loughboroughvirus genus encode tail fibers similar to S144, we suggest that phages in this genus infect Cronobacter sakazakii and are polyvalent.

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“…TheaDomEx server [ 57 ] using fasta sequence suggested segmentation of the amino acid sequence of Alr0765 into two parts, N terminal comprising CBS domain from residue 1-127 and C terminal comprising CP12 domain of about 77 residues long. ThreaDomEx suggested typical patterns of α-β-α-β-β-α repeated two times exhibited by N terminal part (1-127 residues long) comprising CBS domain of target protein (Fig.…”

Section: Resultsmentioning

confidence: 99%

Functional Characterization of Alr0765, A Hypothetical Protein from Anabaena PCC 7120 Involved in Cellular Energy Status Sensing, Iron Acquisition and Abiotic Stress Management in E. coli Using Molecular, Biochemical and Computational Approaches

Chatterjee

Singh

Rai

et al. 2020

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Background: Cyanobacteria are excellent model to understand the basic metabolic processes taking place in response to abiotic stress. The present study involves characterization of a hypothetical protein Alr0765 of Anabaena PCC7120 comprising CBS-CP12 domain and deciphering its role in abiotic stress tolerance. Methods: Molecular cloning, heterologous expression and protein purification using affinity chromatography was performed to obtain native purified protein Alr0765. Energy sensing property of Alr0765 was inferred from its binding affinity with different ligand molecules as analyzed by FTIR and TNP-ATP binding assay. AAS and real time-PCR were applied to evaluate the iron acquisition property and cyclic voltammetry was employed to check redox sensitivity of the target protein. Transcript level under different abiotic stresses as well as spot assay, CFU count, ROS level and cellular H2O2 level were used to show potential role of Alr0765 in abiotic stress tolerance. In-silico analysis of Alr0765 included molecular function probability analysis, multiple sequence analysis, protein domain and motif finding, secondary structure analysis, protein ligand interaction, homologous modeling, model refinement and verification and molecular docking was performed with COFACTOR, PROMALS-3D, InterProScan, MEME, TheaDomEx, COACH, Swiss modeller, Modrefiner, PROCHECK, ERRAT, MolProbity, ProSA, TM-align, and Discovery studio respectively. Results: Transcript levels of alr0765 significantly increased by 20, 13, 15, 14.8, 12, 7, 6 and 2.5 fold when Anabaena PCC7120 treated with LC50 dose of heat, arsenic, cadmium, butachlor, salt, mannitol (drought), UV-B, and methyl viologen respectively, with respect to control (untreated). Heterologous expression resulted in 23KDa protein observed on the SDS-PAGE. Immunoblotting and MALDI-TOF-MS/MS followed by MASCOT search analysis confirmed the identity of the protein and ESI/MS revealed the purified protein was a dimer. Binding possibility of Alr0765 with ATP was observed with almost 6-fold increment in relative fluorescence during TNP-ATP binding assay with a ƛ max of 538 nm. FTIR spectra revealed modification in protein confirmation upon binding of Alr0765 with ATP, ADP, AMP and NADH. A 10-fold higher accumulation of iron was observed in digests of E. coli with recombinant vector after induction as compared to control affirms the iron acquisition property of protein. Moreover, generation of redox potential of 146 mV by Alr0765 suggested its probable role in maintaining redox status of the cell under environmental constraints. As per CFU count recombinant E. coli BL21 cells showed about 14.7, 7.3, 6.9, 1.9, 3, 4.9 fold higher number of colonies under heat, cadmium (CdCl2), arsenic (Na3AsO4), salt (NaCl), UV-B and drought (mannitol) respectively compared to pET21a harboring E. coli BL21 cells. Deterioration in cellular ROS level and total cellular H2O2 concentration validated stress tolerance ability of Alr0765. In-silico analysis unraveled novel findings and attested experimental findings in determining the role of Alr0765. Conclusion: Alr0765 is a novel CBS-CP12 domain protein that maintains cellular energy level and iron homeostasis provide tolerance against multiple abiotic stresses.

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ThreaDomEx: a unified platform for predicting continuous and discontinuous protein domains by multiple-threading and segment assembly

Cited by 27 publications

References 50 publications

Missing regions within the molecular architecture of human fibrin clots structurally resolved by XL-MS and integrative structural modeling

Missing regions within the molecular architecture of human fibrin clots structurally resolved by XL-MS and integrative structural modeling

Phage S144, a New Polyvalent Phage Infecting Salmonella spp. and Cronobacter sakazakii

Functional Characterization of Alr0765, A Hypothetical Protein from Anabaena PCC 7120 Involved in Cellular Energy Status Sensing, Iron Acquisition and Abiotic Stress Management in E. coli Using Molecular, Biochemical and Computational Approaches

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