Solution Structure of the Chick TGFβ Type II Receptor Ligand-binding Domain

Marlow, Michael S.; Brown, Christopher B.; Barnett, Joey V.; Krezel, Andrzej M.

doi:10.1016/s0022-2836(03)00023-8

Cited by 9 publications

(8 citation statements)

References 38 publications

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“…The four that have been characterized structurally thus far, ActRIIa, 23 ActRIIb, 21,24 TbRII, [25][26][27] and BMPRIa, 19 have been shown to adopt a three finger toxin fold and to share four structurally conserved disulfide bonds. TbRI-ED, which has a predicted extracellular domain 101 residues in length, 11 is expected to adopt a similar structure as its ten cysteine residues are positionally conserved relative to those in BMPRIa ( Figure 1).…”

Section: Resultsmentioning

confidence: 99%

“…BMPRIa-ED 28,29 and ActRIIb-ED 21 were expressed as thioredoxin fusions that remained soluble and yielded active protein upon purification. TbRII-ED has been expressed both fused to thioredoxin 25 and alone. 30,31 The fusion protein remained soluble and yielded active protein, 25 while TbRII-ED expressed alone was insoluble but could be refolded to yield active protein.…”

Section: Resultsmentioning

confidence: 99%

“…TbRII-ED has been expressed both fused to thioredoxin 25 and alone. 30,31 The fusion protein remained soluble and yielded active protein, 25 while TbRII-ED expressed alone was insoluble but could be refolded to yield active protein. 30,31 To prepare TbRI-ED, we tested the two approaches described above.…”

Section: Resultsmentioning

confidence: 99%

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Assembly of TβRI:TβRII:TGFβ Ternary Complex in vitro with Receptor Extracellular Domains is Cooperative and Isoform-dependent

Zúñiga

Groppe

Cui

et al. 2005

Journal of Molecular Biology

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Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

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Assembly of TβRI:TβRII:TGFβ Ternary Complex in vitro with Receptor Extracellular Domains is Cooperative and Isoform-dependent

Zúñiga

Groppe

Cui

et al. 2005

Journal of Molecular Biology

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“…25 kDa) and receptor ectodomains (11 – 16 kDa) has enabled studies of both using NMR [20,55,57,58,64,79,105–107] and X-ray crystallography [19,21,22,96,98,102,108–110]. The strategies used to obtain the NMR solution structures and X-ray crystal structures of the signaling proteins and receptor ectodomains more or less follow the standard methodologies.…”

Section: Methodsmentioning

confidence: 99%

Production, Isolation, and Structural Analysis of Ligands and Receptors of the TGF-β Superfamily

Huang

Hinck²

2016

Methods in Molecular Biology

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The ability to understand the molecular mechanisms by which secreted signaling proteins of the TGF-β superfamily assemble their cell surface receptors into complexes to initiate downstream signaling is dependent upon the ability to determine atomic-resolution structures of the signaling proteins, the ectodomains of the receptors, and the complexes that they form. The structures determined to date have revealed major differences in the overall architecture of the signaling complexes formed by the TGF-βs and BMPs, which has provided insights as to how they have evolved to fulfill their distinct functions. Such studies, have however, only been applied to a few members of the TGF-β superfamily, which is largely due to the difficulty of obtaining milligram-scale quantities of highly homogenous preparations of the disulfide-rich signaling proteins and receptor ectodomains of the superfamily. Here we describe methods used to produce signaling proteins and receptor ectodomains of the TGF-β superfamily using bacterial and mammalian expression systems and procedures to purify them to homogeneity.

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“…Our attempts to follow reported protocols for folding TBRIII-ECD from inclusion bodies [11,12,31] afforded highly variable yields of active protein; often less than 1 mg of protein was recovered from 50 mg of solubilized inclusion bodies. Although a thioredoxin fusion enhanced the solubility of chicken Trx-TBRII-ECD [32,33], production of the corresponding human Trx-TBRII-ECD fusion resulted in soluble but nonfunctional protein that failed to bind TGF-β1. A protocol for folding Trx-TBRII-ECD on Ni–NTA agarose was previously reported [13]; however, it employs a high concentration of arginine at pH 8.0, which interferes with protein binding to Ni–NTA agarose [34].…”

Section: Resultsmentioning

confidence: 99%

The non-detergent sulfobetaine-201 acts as a pharmacological chaperone to promote folding and crystallization of the type II TGF-β receptor extracellular domain

Wangkanont

Forest

Kiessling

2015

Protein Expression and Purification

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The roles of the extracellular domain of type II TGF-β receptor (TBRII-ECD) in physiological processes ranging from development to cancer to wound healing render it an attractive target for exploration with chemical tools. For such applications, large amounts of active soluble protein are needed, but the yields of TBRII-ECD we obtained with current folding protocols were variable. To expedite the identification of alternative folding conditions, we developed an on-plate screen. This assay indicated that effective folding additives included the non-detergent sulfobetaine-201 (NDSB-201). Although NDSB-201 can facilitate protein folding, the mode by which it does so is poorly understood. We postulated that specific interactions between NDSB-201 and TBRII-ECD might be responsible. Analysis by X-ray crystallography indicates that the TBRII-ECD possesses a binding pocket for NDSB-201. The pyridinium group of the additive stacks with a phenylalanine side chain in the binding site. The ability of NDSB-201 to occupy a pocket on the protein provides a molecular mechanism for the additive’s ability to minimize TBRII-ECD aggregation and stabilize the folded state. NDSB-201 also accelerates TBRII-ECD crystallization, suggesting it may serve as a useful crystallization additive for proteins refolded with it. Our results also suggest there is a site on TBRII-ECD that could be targeted by small-molecule modulators.

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Solution Structure of the Chick TGFβ Type II Receptor Ligand-binding Domain

Cited by 9 publications

References 38 publications

Assembly of TβRI:TβRII:TGFβ Ternary Complex in vitro with Receptor Extracellular Domains is Cooperative and Isoform-dependent

Assembly of TβRI:TβRII:TGFβ Ternary Complex in vitro with Receptor Extracellular Domains is Cooperative and Isoform-dependent

Production, Isolation, and Structural Analysis of Ligands and Receptors of the TGF-β Superfamily

The non-detergent sulfobetaine-201 acts as a pharmacological chaperone to promote folding and crystallization of the type II TGF-β receptor extracellular domain

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