Design, characterization, and structure of a biologically active single-chain mutant of human IFN-gamma.
Design, characterization, and structure of a biologically active single-chain mutant of human IFN-gamma.
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DOI：
10.1006/jmbi.2000.3734
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发表时间：
2000-05
影响因子：
5.6
通讯作者：
A. Landar;B. Curry;M. Parker;R. DiGiacomo;S. Indelicato;T. L. Nagabhushan;G. Rizzi;M. Walter
中科院分区：
文献类型：
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作者：
A. Landar;B. Curry;M. Parker;R. DiGiacomo;S. Indelicato;T. L. Nagabhushan;G. Rizzi;M. Walter
已设计并表征了结合一个IFN-γ受体α链（IFN-γ R α）的人干扰素-γ（IFN-γ SC 1）的突变形式。IFN-γ SC 1是通过用7个残基的接头连接IFN-γ二聚体的两条肽链并将第一条链中的His 111变为天冬氨酸残基而得到的。等温滴定量热法显示，IFN-γ SC 1与其高亲和力受体（IFN-γ R α）形成1：1复合物，亲和力为27（+/- 9）nM。IFN-γ SC 1的晶体结构已从在pH 7.6的1.4 M柠檬酸盐溶液中生长的晶体中以2.9 A分辨率测定。IFN-γ SC 1的野生型受体结合结构域和含Asp 111结构域的比较表明，它们在结构上是等同的，但具有非常不同的静电表面电位。因此，表面电荷而不是结构变化可能是His 111->Asp结构域不能结合IFN-γ R α的原因。IFN-γ SC 1的AB环采用与在IFN-γ/IFN-γ R α复合物的晶体结构中观察到的IFN-γ的有序环相似的构象。因此，IFN-γ R α结合不会导致如先前所建议的AB环中的大的构象变化。该结构还揭示了IFN-γ SC 1的最后6个C-末端氨基酸残基（残基253-258），这些残基在任何其他报道的IFN-γ结构中均未观察到。尽管仅与一种IFN-γ R α结合，但IFN-γ SC 1在细胞增殖、MHC I类诱导和抗病毒测定中具有生物活性。这表明IFN-γ的一个结构域足以将IFN-γ R α和IFN-γ R β募集到能够引发生物活性的复合物中。目前的数据与IFN-γ二聚体的主要作用一致，即降低IFN-γ对其细胞受体的解离常数。
A mutant form of human interferon-gamma (IFN-gamma SC1) that binds one IFN-gamma receptor alpha chain (IFN-gamma R alpha) has been designed and characterized. IFN-gamma SC1 was derived by linking the two peptide chains of the IFN-gamma dimer by a seven-residue linker and changing His111 in the first chain to an aspartic acid residue. Isothermal titration calorimetry shows that IFN-gamma SC1 forms a 1:1 complex with its high-affinity receptor (IFN-gamma R alpha) with an affinity of 27(+/- 9) nM. The crystal structure of IFN-gamma SC1 has been determined at 2.9 A resolution from crystals grown in 1.4 M citrate solutions at pH 7.6. Comparison of the wild-type receptor-binding domain and the Asp111-containing domain of IFN-gamma SC1 show that they are structurally equivalent but have very different electrostatic surface potentials. As a result, surface charge rather than structural changes is likely responsible for the inability of the His111-->Asp domain of to bind IFN-gamma R alpha. The AB loops of IFN-gamma SC1 adopt conformations similar to the ordered loops of IFN-gamma observed in the crystal structure of the IFN-gamma/IFN-gamma R alpha complex. Thus, IFN-gamma R alpha binding does not result in a large conformational change in the AB loop as previously suggested. The structure also reveals the final six C-terminal amino acid residues of IFN-gamma SC1 (residues 253-258) that have not been observed in any other reported IFN-gamma structures. Despite binding to only one IFN-gamma R alpha, IFN-gamma SC1 is biologically active in cell proliferation, MHC class I induction, and anti-viral assays. This suggests that one domain of IFN-gamma is sufficient to recruit IFN-gamma R alpha and IFN-gamma R beta into a complex competent for eliciting biological activity. The current data are consistent with the main role of the IFN-gamma dimer being to decrease the dissociation constant of IFN-gamma for its cellular receptors.