Lee B L, Ciardelli T L
Department of Pharmacology and Toxicology, Dartmouth Medical School, Hanover, New Hampshire 03755, USA.
Biochem Biophys Res Commun. 1997 Apr 17;233(2):309-15. doi: 10.1006/bbrc.1997.6453.
Interleukin-2 (IL-2) is a cytokine essential for the growth and proliferation of T-cells. The purpose of this research was to study the effects of altering the "kink" caused by Pro in an alpha-helix of the protein. The Pro-47 residue was chosen because it was originally, but mistakenly, traced to the kink of an alpha-helix [1]. Pro-65 is now recognized to be situated in the middle of helix B [2]. To study the significance of this Pro for the bioactivity and overall conformation of IL-2 it was mutated to Gly and Ala. We successfully obtained 17 different mutants at position 47 and two mutants at position 65. Certain amino acid substitutions representing different categories of amino acids, namely, acidic, neutral and helix stabilizing, were chosen for more thorough investigation. The results showed that Asn-47 and Asp-47 decreased the bioactivity of these mutants by 50- and 700-fold respectively, while the Kd to its high affinity receptors was increased 180- and 90-fold respectively, compared to IL-2. The intermediate binding affinity of Asn-47 and Asp-47 was decreased 8- and 37-fold, respectively. On the other hand, Gly-47, Gly-65 and Ala-65 showed less dramatic decreases in bioactivity and high affinity binding. The intermediate binding affinity of these mutants decreased from 5- to 3-fold and low affinity binding decreased approximately 4-fold suggesting some structural and conformational changes. From these observations, we conclude that Asn-47 or Asp-47 disrupt the hydrophobic packing of the core and thus changed the overall conformation of the protein, thereby giving rise to partial agonists. Although Pro-65 lies within the helix, it may be near the surface of the protein but may not be the actual binding site and thus any conservative mutation can be better tolerated.
白细胞介素-2（IL-2）是一种对T细胞生长和增殖至关重要的细胞因子。本研究的目的是研究改变蛋白质α-螺旋中由脯氨酸（Pro）引起的“扭结”的影响。选择Pro-47残基是因为它最初被错误地追踪到α-螺旋的扭结处[1]。现在已知Pro-65位于螺旋B的中间[2]。为了研究该脯氨酸对IL-2生物活性和整体构象的重要性，将其突变为甘氨酸（Gly）和丙氨酸（Ala）。我们成功获得了17种位于47位的不同突变体和2种位于65位的突变体。选择了代表不同氨基酸类别的某些氨基酸替代，即酸性、中性和螺旋稳定型，进行更深入的研究。结果表明，与IL-2相比，Asn-47和Asp-47分别使这些突变体的生物活性降低了50倍和700倍，而它们与高亲和力受体的解离常数（Kd）分别增加了180倍和90倍。Asn-47和Asp-47的中间结合亲和力分别降低了8倍和37倍。另一方面，Gly-47、Gly-65和Ala-65的生物活性和高亲和力结合的降低没有那么显著。这些突变体的中间结合亲和力降低了5至3倍，低亲和力结合降低了约4倍，表明存在一些结构和构象变化。从这些观察结果中，我们得出结论，Asn-47或Asp-47破坏了核心的疏水堆积，从而改变了蛋白质的整体构象，进而产生了部分激动剂。虽然Pro-65位于螺旋内，但它可能靠近蛋白质表面，但可能不是实际的结合位点，因此任何保守突变都可能更易被耐受。
