Latent tgf-beta structure and activation

Protein Expr. Zou, Z. Type 1 transforming growth factor beta: amplified expression and secretion of mature and precursor polypeptides in Chinese hamster ovary cells. Brunner, A. Site-directed mutagenesis of glycosylation sites in the transforming growth factor-beta 1 TGF beta 1 and TGF beta 2 precursors and of cysteine residues within mature TGF beta 1: effects on secretion and bioactivity.

Heras, B. Post-crystallization treatments for improving diffraction quality of protein crystals. Acta Crystallogr. D 61 , — Otwinowski, Z. Processing of X-ray diffraction data collected in oscillation mode.

Methods Enzymol. Kabsch, W. Adams, P. D 58 , — Vagin, A. D 66 , 22—25 Bailey, S. The CCP4 suite: programs for protein crystallography. D 50 , — Cowtan, K. Recent developments in classical density modification. D 66 , — Emsley, P. Coot: model-building tools for molecular graphics. D 60 , — Davis, I. MolProbity: all-atom contacts and structure validation for proteins and nucleic acids. Nucleic Acids Res.

Takagi, J. Global conformational rearrangements in integrin extracellular domains in outside-in and inside-out signaling. Cell , — Chen, X. Download references. We thank P. You can also search for this author in PubMed Google Scholar. Correspondence to Timothy A. The file contains Supplementary Figures with legends and Supplementary Table 1. PDF kb. Reprints and Permissions. Shi, M. Download citation. Received : 20 December Accepted : 27 April Published : 15 June Issue Date : 16 June Anyone you share the following link with will be able to read this content:.

Sorry, a shareable link is not currently available for this article. More Filters. Frontiers in Immunology. Highly Influenced. View 3 excerpts, cites methods and background. Structural biology: Growth factor rattled out of its cage.

View 9 excerpts, cites background and methods. Matrix biology : journal of the International Society for Matrix Biology. Growth factors. View 19 excerpts, cites background. Structure and activation of pro-activin A. Nature communications. Biochimica et biophysica acta. Reviews on cancer. Journal of biochemistry. The flow-through with two column volumes of TBS pH 7. Hits were optimized in well plates using hanging-drop vapour diffusion. Crystals are summarized in Supplementary Table 1.

There are two complexes per asymmetric unit, with a Matthews coefficient of 2. After replacement with the same medium, cells were cultured for 4 d. Furthermore, a heavy-atom derivative was obtained by soaking crystals in mother liquor containing 0.

Statistics are in Supplementary Table 1. Electron density maps from Se-Met phasing, calculated after fourfold non-crystallographic symmetry NCS averaging, clearly defined the orientation of each monomer. The prodo-main was built into the map manually. These steps were cycled twice. Model building in COOT 43 was based on multi-crystal, multi-domain averaged electron density maps and 2 F o — F c maps.

The sequence-to-structure register was confirmed using Se anomalous maps. Two residues from the 3C protease site remain at the N terminus after cleavage.

The structures include residues 0—62, 70—, —, — and one N -acetylglucosamine NAG residue chain A ; residues 1—62, 70—, —, —, and two NAG residues chain B ; residues 1—62, 68—, —, — and three NAG residues chain C and residues 0—62, 69—, —, — and two NAG residues chain D. All structure figures were generated using Pymol DeLano Scientific. In its absence, Leu may mediate hydrophobic interactions within the bowtie. Site-directed mutagenesis was performed using QuikChange Stratagene.

All mutations were confirmed by DNA sequencing. The cells were then cultured in FreeStyle serum-free medium Invitrogen for 3 d.

After transfer to polyvinylidene difluoride membranes Millipore , biotin was detected using streptavidin-horseradish peroxidase with the ECL-plus western blotting kit GE Healthcare.

Transformed mink lung epithelial cells TMLCs stably transfected with a luciferase construct under plasminogen activator inhibitor promoter 1 ref. Rifkin New York University. Cells stably transfected with empty vector were used as a control. After 16—24 h, each well was used to seed 3 wells of a well plate with about 15, cells, which were co-cultured with 15, TMLCs in ml DMEM with 0.

Peak fractions corresponding to the purified proteins or complexes were subjected to negative-stain electron microscopy. Particle selection, alignment, classification and averaging were conducted as previously described We thank P. Author Contributions M. Full Methods and any associated references are available in the online version of the paper at www.

Supplementary Information is linked to the online version of the paper at www. National Center for Biotechnology Information , U. Author manuscript; available in PMC Jan Springer 1. Find articles by Minlong Shi. Find articles by Jianghai Zhu. Find articles by Rui Wang. Find articles by Xing Chen. Find articles by Lizhi Mi. Find articles by Thomas Walz. Timothy A. Find articles by Timothy A. Author information Copyright and License information Disclaimer.

Correspondence and requests for materials should be addressed to T. Copyright notice. Reprints and permissions information is available at www. The authors declare no competing financial interests. Readers are welcome to comment on the online version of this article at www. The publisher's final edited version of this article is available at Nature. See other articles in PMC that cite the published article. Associated Data Supplementary Materials Supporting.

Open in a separate window. Figure 1. Figure 4. Figure 2. Figure 3. Supplementary Material Supporting Click here to view. Acknowledgements We thank P. Footnotes Author Contributions M.

References 1. Derynck R, Miyazono K. Derynck R, Miyazono K, editors. Cold Spring Harbor Laboratory Press; Cell Biol. Ramirez F, Sakai LY. Biogenesis and function of fibrillin assemblies. Cell Tissue Res.