Department of Molecular Biology and Nanobiotechnology, National Institute of Chemistry, Ljubljana, Slovenia.
Theory Department, National Institute of Chemistry, Ljubljana, Slovenia.
Front Immunol. 2023 Jul 19;14:1181020. doi: 10.3389/fimmu.2023.1181020. eCollection 2023.
Perforin is a pore-forming protein that plays a crucial role in the immune system by clearing virus-infected or tumor cells. It is released from cytotoxic granules of immune cells and forms pores in targeted lipid membranes to deliver apoptosis-inducing granzymes. It is a very cytotoxic protein and is therefore adapted not to act in producing cells. Its activity is regulated by the requirement for calcium ions for optimal activity. However, the exact affinity of perforin for calcium ions has not yet been determined. We conducted a molecular dynamics simulation in the absence or presence of calcium ions that showed that binding of at least three calcium ions is required for stable perforin binding to the lipid membrane. Biophysical studies using surface plasmon resonance and microscale thermophoresis were then performed to estimate the binding affinities of native human and recombinant mouse perforin for calcium ions. Both approaches showed that mouse perforin has a several fold higher affinity for calcium ions than that of human perforin. This was attributed to a particular residue, tryptophan at position 488 in mouse perforin, which is replaced by arginine in human perforin. This represents an additional mechanism to control the activity of human perforin.
穿孔素是一种形成孔的蛋白，通过清除病毒感染或肿瘤细胞，在免疫系统中发挥着关键作用。它从免疫细胞的细胞毒性颗粒中释放出来，并在靶脂质膜中形成孔，以传递凋亡诱导的颗粒酶。它是一种非常细胞毒性的蛋白，因此不适应在产生细胞中发挥作用。其活性受到钙离子对最佳活性的要求的调节。然而，穿孔素与钙离子的确切亲和力尚未确定。我们进行了分子动力学模拟，结果表明，至少需要结合三个钙离子，才能使穿孔素稳定地与脂质膜结合。然后使用表面等离子体共振和微尺度热泳进行了生物物理研究，以估计天然人源和重组鼠源穿孔素与钙离子的结合亲和力。这两种方法都表明，鼠源穿孔素对钙离子的亲和力比人源穿孔素高几个数量级。这归因于一个特定的残基，即鼠源穿孔素第 488 位的色氨酸，而人源穿孔素的第 488 位被精氨酸取代。这代表了控制人源穿孔素活性的另一种机制。
