Biological Activity Differences between TGF-尾1 and TGF-尾3 Correlate with Differences in the Rigidity and Arrangement of Their Component Monomers
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- 作者：Tao Huang ; Seth L. Schor ; Andrew P. Hinck
- 刊名：Biochemistry
- 出版年：2014
- 出版时间：September 16, 2014
- 年：2014
- 卷：53
- 期：36
- 页码：5737-5749
- 全文大小：703K
- ISSN：1520-4995
文摘
TGF-尾1, -尾2, and -尾3 are small, secreted signaling proteins. They share 71鈥?0% sequence identity and signal through the same receptors, yet the isoform-specific null mice have distinctive phenotypes and are inviable. The replacement of the coding sequence of TGF-尾1 with TGF-尾3 and TGF-尾3 with TGF-尾1 led to only partial rescue of the mutant phenotypes, suggesting that intrinsic differences between them contribute to the requirement of each in vivo. Here, we investigated whether the previously reported differences in the flexibility of the interfacial helix and arrangement of monomers was responsible for the differences in activity by generating two chimeric proteins in which residues 54鈥?5 in the homodimer interface were swapped. Structural analysis of these using NMR and functional analysis using a dermal fibroblast migration assay showed that swapping the interfacial region swapped both the conformational preferences and activity. Conformational and activity differences were also observed between TGF-尾3 and a variant with four helix-stabilizing residues from TGF-尾1, suggesting that the observed changes were due to increased helical stability and the altered conformation, as proposed. Surface plasmon resonance analysis showed that TGF-尾1, TGF-尾3, and variants bound the type II signaling receptor, T尾RII, nearly identically, but had small differences in the dissociation rate constant for recruitment of the type I signaling receptor, T尾RI. However, the latter did not correlate with conformational preference or activity. Hence, the difference in activity arises from differences in their conformations, not their manner of receptor binding, suggesting that a matrix protein that differentially binds them might determine their distinct activities.