Protein structure homology modeling using SWISS-MODEL workspace

Bordoli, Lorenza; Kiefer, Florian; Arnold, Konstantin; Benkert, Pascal; Battey, James N. D.; Schwede, Torsten

doi:10.1038/nprot.2008.197

Cited by 1,131 publications

(839 citation statements)

References 74 publications

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“…22,23 Protein modeling 24 using AtCPS as the template showed SmCPS belongs to the class II synthase and the active site DXDD motif for H + -initiated cyclization is located between the βγ domains in the N-terminus (Supporting Information Figure S7). Protein modeling using taxadiene synthase as the template showed the active site of SmKSL is located at the α domain in the Cterminus (Supporting Information Figure S7).…”

Section: ■ Resultsmentioning

confidence: 99%

Modular Pathway Engineering of Diterpenoid Synthases and the Mevalonic Acid Pathway for Miltiradiene Production

Zhou

Gao²,

Rong³

et al. 2012

J. Am. Chem. Soc.

333

243

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Microbial production can be advantageous over the extraction of phytoterpenoids from natural plant sources, but it remains challenging to rationally and rapidly access efficient pathway variants. Previous engineering attempts mainly focused on the mevalonic acid (MVA) or methyl-Derythritol phosphate (MEP) pathways responsible for the generation of precursors for terpenoids biosynthesis, and potential interactions between diterpenoids synthases were unexplored. Miltiradiene, the product of the stepwise conversion of (E,E,E)-geranylgeranyl diphosphate (GGPP) catalyzed by diterpene synthases SmCPS and SmKSL, has recently been identified as the precursor to tanshionones, a group of abietane-type norditerpenoids rich in the Chinese medicinal herb Salvia miltiorrhiza. Here, we present the modular pathway engineering (MOPE) strategy and its application for rapid assembling synthetic miltiradiene pathways in the yeast Saccharomyces cerevisiae. We predicted and analyzed the molecular interactions between SmCPS and SmKSL, and engineered their active sites into close proximity for enhanced metabolic flux channeling to miltiradiene biosynthesis by constructing protein fusions. We show that the fusion of SmCPS and SmKSL, as well as the fusion of BTS1 (GGPP synthase) and ERG20 (farnesyl diphosphate synthase), led to significantly improved miltiradiene production and reduced byproduct accumulation. The MOPE strategy facilitated a comprehensive evaluation of pathway variants involving multiple genes, and, as a result, our best pathway with the diploid strain YJ2X reached miltiradiene titer of 365 mg/L in a 15-L bioreactor culture. These results suggest that terpenoids synthases and the precursor supplying enzymes should be engineered systematically to enable an efficient microbial production of phytoterpenoids.

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Section: ■ Resultsmentioning

confidence: 99%

Modular Pathway Engineering of Diterpenoid Synthases and the Mevalonic Acid Pathway for Miltiradiene Production

Zhou

Gao²,

Rong³

et al. 2012

J. Am. Chem. Soc.

333

243

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“…A three dimensional homology model has been obtained from Swiss Model [16]. The homology model was built on the basis of an alignment of the amino-acid sequence of mvWFA2 with a similar protein of known structure.…”

Section: Nmr Spectroscopy and Structure Predictionmentioning

confidence: 99%

Insight into interactions of the von-Willebrand-factor-A-like domain 2 with the FNIII-like domain 9 of collagen VII by NMR and SPR

2011

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Edited by Gianni CesareniKeywords: vWFA Domain interaction Skin structure NMR a b s t r a c t Type VII collagen as component of anchoring fibrils plays an important role in skin architecture, however, no detailed structural information is available. Here, we describe the recombinant expression, isotope labeling, and 1 H, 15 N, 13 C chemical shift assignment of a subdomain of the murine type VII collagen -the von-Willebrand-factor-A-like domain 2 (mvWFA2). vWFA2 interacts with type I collagen and plays a central role in certain skin blistering diseases. Based on these assignments a secondary structure prediction was performed showing a properly folded protein. An interaction of mvWFA2 with its neighboring domain mFNIII-9 was characterized with NMR spectroscopy and SPR.Structured summary of protein interactions: mFNIII-9 and mvWFA2 bind by nuclear magnetic resonance (View interaction) mvWFA2 binds to mFNIII-9 by surface plasmon resonance (View interaction)

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“…Using the published structure of human TIMP‐2 (accession code: 1BR9) as a template, predicted structural models were produced using SWISS‐MODEL 49, 50 for TIMP‐2, TIMP‐3, NT2/CT3 and NT3/CT2 fusions (sequence of rat origin). Structures were analysed using reported methods 36, 37, 46 and predictions of protein surface hydrophobicity (Fig.…”

Section: Resultsmentioning

confidence: 99%

“…Structural models of TIMP ‐2 and TIMP ‐3 created using SWISS ‐ MODEL 49, 50 and processed through an algorithm developed by the Warwicker group (The University of Manchester). Predictions of the (A–B) hydrophobicity (nonpolar vs. polar regions) and (C–D) electrostatic potential (positive vs. negative charge) of protein surfaces were generated for TIMP ‐2 (A,C) and TIMP ‐3 (B,D).…”

Section: Resultsmentioning

confidence: 99%

“…Predicted structural models of TIMP‐2, TIMP‐3, TIMP‐4 and TIMP fusion/mutant sequences used in this study were generated using SWISS‐MODEL 49, 50, where the published structure of human TIMP‐2 (accession code: 1BR9) was used as a template. Published structures were also analysed for ARTN (accession code: 2GYZ) and PAI‐1 (accession code: 3LW2).…”

Section: Methodsmentioning

confidence: 99%

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A protein chimera strategy supports production of a model “difficult‐to‐express” recombinant target

et al. 2018

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Due in part to the needs of the biopharmaceutical industry, there has been an increased drive to generate high quality recombinant proteins in large amounts. However, achieving high yields can be a challenge as the novelty and increased complexity of new targets often makes them ‘difficult‐to‐express’. This study aimed to define the molecular features that restrict the production of a model ‘difficult‐to‐express’ recombinant protein, Tissue Inhibitor Metalloproteinase‐3 (TIMP‐3). Building from experimental data, computational approaches were used to rationalize the redesign of this recombinant target to generate a chimera with enhanced secretion. The results highlight the importance of early identification of unfavourable sequence attributes, enabling the generation of engineered protein forms that bypass ‘secretory’ bottlenecks and result in efficient recombinant protein production.

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Protein structure homology modeling using SWISS-MODEL workspace

Cited by 1,131 publications

References 74 publications

Modular Pathway Engineering of Diterpenoid Synthases and the Mevalonic Acid Pathway for Miltiradiene Production

Modular Pathway Engineering of Diterpenoid Synthases and the Mevalonic Acid Pathway for Miltiradiene Production

Insight into interactions of the von-Willebrand-factor-A-like domain 2 with the FNIII-like domain 9 of collagen VII by NMR and SPR

A protein chimera strategy supports production of a model “difficult‐to‐express” recombinant target

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