Design of an expression system to enhance MBP-mediated crystallization

Jin, Tengchuan; Chuenchor, W.; Jiang, Jiansheng; Cheng, Jinbo; Li, Yajuan; Kang, Fang; Huang, M.; Smith, Patrick; Xiao, Tsan Sam

doi:10.1038/srep40991

Cited by 42 publications

(30 citation statements)

References 58 publications

(79 reference statements)

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“…To overcome the aggregation of DR3 DD due to its high hydrophobicity, we used an N‐terminal maltose‐binding protein (MBP) as a crystallization tag. This technique has been used successfully in the structural determination of several DD folds . To increase the success rate of MBP‐facilitated crystallization, we used a series of expression vectors with a set of rigid helical linkers and harvested both human and mouse MBP–DR3 DD crystal structures with one‐amino‐acid difference in the linkers between MBP tag and DR3 DD (Fig.…”

Section: Resultsmentioning

confidence: 99%

Crystal structure and activation mechanism of DR3 death domain

Yin

et al. 2019

The FEBS Journal

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Death receptor 3 (DR3) (a.k.a. tumor necrosis factor receptor superfamily 25) plays a key role in the immune system by activating nuclear factor kappa‐light‐chain‐enhancer of activated B cells signaling pathway. Here we present the crystal structures of human and mouse DR3 intracellular death domain (DD) at 2.7 and 2.5 Å resolutions, respectively. The mouse DR3 DD adopts a classical six‐helix bundle structure while human DR3 DD displays an extended fold. Though there is one‐amino‐acid difference in the linker between maltose‐binding protein (MBP) tag and DR3 DD, according to our self‐interaction analysis, the hydrophobic interface discovered in MBP–hDR3 DD crystal structure is responsible for both hDR3 DD and mDR3 DD homotypic interaction. Furthermore, our biochemical analysis indicates that the sequence variation between human and mouse DR3 DD does not affect its structure and function. Small‐angle X‐ray scattering analysis shows the averaged solution structures of both human and mouse MBP‐DR3 DD are the combination of different conformations with different proportion. Through switching to the open conformation, DR3 DD could improve the interaction with downstream element TNFR‐associated death domain (TRADD). Here we propose an activation‐dependent structural rearrangement model: the DD region is folded as the six‐helix bundles in the resting state, while upon extracellular ligand engagement, it switches to the open conformation, which facilitates its self‐association and the recruitment of TRADD. Our results provide detailed insights into the architecture of DR3 DD and the molecular mechanism of activation. Databases All refined structure coordinates as well as the corresponding structure factors have been deposited in the PDB under the accession codes http://www.rcsb.org/pdb/search/structidSearch.do?structureId=5YGS, http://www.rcsb.org/pdb/search/structidSearch.do?structureId=5YEV, http://www.rcsb.org/pdb/search/structidSearch.do?structureId=5YGP, http://www.rcsb.org/pdb/search/structidSearch.do?structureId=5ZNY, http://www.rcsb.org/pdb/search/structidSearch.do?structureId=5ZNZ.

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Section: Resultsmentioning

confidence: 99%

Crystal structure and activation mechanism of DR3 death domain

Yin

et al. 2019

The FEBS Journal

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“…In order to crystallize CAMP factor with a maltose-binding protein (MBP) tag, the CAMP-coding region was re-cloned into pET-30a vector with a non-cleavable N-terminal MBP tag. The MBP tag was designed with the following surface entropy-reducing mutations to enhance crystallization: D82A/K83A/E172A/N173A/K239A (Moon et al, 2010;Jin, Perry et al, 2013;Jin et al, 2017). A series of fusion constructs was created in which the N-terminal residue of the CAMP-factor reading frame was varied systematically in two-residue increments from Ala29 to Met50 in order to increase the chance of fusion-protein crystallization.…”

Section: Protein Expression and Purificationmentioning

confidence: 99%

“…Inspired by the successful use of MBP as a crystallization chaperone for crystallizing challenging targets by our group and several others (Potter et al, 2008;Moon et al, 2010;Jin et al, 2017), we decided to try the MBP-fusion tag to solve the phase problem of CAMP crystals. The use of a crystallization tag to aid target-protein crystallization usually requires a reasonable structural model.…”

Section: Mbp Fusion Tag-aided Crystallizationmentioning

confidence: 99%

Structure determination of the CAMP factor ofStreptococcus agalactiaewith the aid of an MBP tag and insights into membrane-surface attachment

Zeng

Fan

et al. 2019

Acta Cryst Sect D Struct Biol

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CAMP factor is a unique -helical bacterial toxin that is known for its cohemolytic activity in combination with staphylococcal sphingomyelinase. It was first discovered in the human pathogen Streptococcus agalactiae (also known as group B streptococcus), but homologous genes have been found in many other Gram-positive pathogens. In this study, the efforts that led to the determination of the first structure of a CAMP-family toxin are reported. Initially, it was possible to produce crystals of the native protein which diffracted to near 2.45 Å resolution. However, a series of technical obstacles were encountered on the way to structure determination. Over a period of more than five years, many methods, including selenomethionine labeling, mutations, crystallization chaperones and heavy-atom soaking, were attempted, but these attempts resulted in limited progress. The structure was finally solved using a combination of iodine soaking and molecular replacement using the crystallization chaperone maltosebinding protein (MBP) as a search model. Analysis of native and MBP-tagged CAMP-factor structures identified a conserved interaction interface in the C-terminal domain (CTD). The positively charged surface may be critical for binding to acidic ligands. Furthermore, mutations on the interaction interface at the CTD completely abolished its co-hemolytic activities. This study provides novel insights into the mechanism of the membrane-permeabilizing activity of CAMP factor.

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“…One approach to stabilize flexible proteins has been described by Moon and coworkers and utilizes maltosebinding protein (MBP) with surface-entropy reduction (SER) mutations as an N-terminal carrier protein to enhance protein crystallization (Moon et al, 2010). MBP has been reported to enhance the solubility of proteins when expressed as an N-terminal fusion in Escherichia coli (Waugh, 2016;Jin et al, 2017) and mammalian expression systems (Reuten et al, 2016;Bokhove et al, 2016), but to date this has not been reported for insect-cell expression systems. We describe the expression of the FXIIa protease domain as a secreted MBP fusion using DES, which facilitated the crystal structure determination of MBP-FXIIa His in the active conformation in complex with the peptidomimetic inhibitor d-Phe-Pro-Arg chloromethyl ketone (PPACK).…”

Section: Introductionmentioning

confidence: 99%

Crystal structures of the recombinant β-factor XIIa protease with bound Thr-Arg and Pro-Arg substrate mimetics

Pathak

Manna

et al. 2019

Acta Cryst Sect D Struct Biol

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Coagulation factor XII (FXII) is a key initiator of the contact pathway, which contributes to inflammatory pathways. FXII circulates as a zymogen, which when auto-activated forms factor XIIa (FXIIa). Here, the production of the recombinant FXIIa protease domain (FXIIa His ) with yields of $1-2 mg per litre of insect-cell culture is reported. A second construct utilized an N-terminal maltose-binding protein (MBP) fusion (MBP-FXIIa His ). Crystal structures were determined of MBP-FXIIa His in complex with the inhibitor d-Phe-Pro-Arg chloromethyl ketone (PPACK) and of FXIIa His in isolation. The FXIIa His structure revealed that the S2 and S1 pockets were occupied by Thr and Arg residues, respectively, from an adjacent molecule in the crystal. The Thr-Arg sequence mimics the P2-P1 FXIIa cleavage-site residues present in the natural substrates prekallikrein and FXII, and Pro-Arg (from PPACK) mimics the factor XI cleavage site. A comparison of the FXIIa His structure with the available crystal structure of the zymogen-like FXII protease revealed large conformational changes centred around the S1 pocket and an alternate conformation for the 99-loop, Tyr99 and the S2 pocket. Further comparison with activated protease structures of factors IXa and Xa, which also have the Tyr99 residue, reveals that a more open form of the S2 pocket only occurs in the presence of a substrate mimetic. The FXIIa inhibitors EcTI and infestin-4 have Pro-Arg and Phe-Arg P2-P1 sequences, respectively, and the interactions that these inhibitors make with FXIIa are also described. These structural studies of FXIIa provide insight into substrate and inhibitor recognition and establish a scaffold for the structure-guided drug design of novel antithrombotic and antiinflammatory agents.

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Design of an expression system to enhance MBP-mediated crystallization

Cited by 42 publications

References 58 publications

Crystal structure and activation mechanism of DR3 death domain

Crystal structure and activation mechanism of DR3 death domain

Structure determination of the CAMP factor ofStreptococcus agalactiaewith the aid of an MBP tag and insights into membrane-surface attachment

Crystal structures of the recombinant β-factor XIIa protease with bound Thr-Arg and Pro-Arg substrate mimetics

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