Department of Cell and Molecular Biology, Uppsala University, Uppsala, Sweden.
Department of Chemistry, Uppsala University, Uppsala, Sweden.
PLoS One. 2023 Mar 23;18(3):e0282741. doi: 10.1371/journal.pone.0282741. eCollection 2023.
The interaction between human Growth Hormone (hGH) and hGH Receptor (hGHR) has basic relevance to cancer and growth disorders, and hGH is the scaffold for Pegvisomant, an anti-acromegaly therapeutic. For the latter reason, hGH has been extensively engineered by early workers to improve binding and other properties. We are particularly interested in E174 which belongs to the hGH zinc-binding triad; the substitution E174A is known to significantly increase binding, but to now no explanation has been offered. We generated this and several computationally-selected single-residue substitutions at the hGHR-binding site of hGH. We find that, while many successfully slow down dissociation of the hGH-hGHR complex once bound, they also slow down the association of hGH to hGHR. The E174A substitution induces a change in the Circular Dichroism spectrum that suggests the appearance of coiled-coiling. Here we show that E174A increases affinity of hGH against hGHR because the off-rate is slowed down more than the on-rate. For E174Y (and certain mutations at other sites) the slowdown in on-rate was greater than that of the off-rate, leading to decreased affinity. The results point to a link between structure, zinc binding, and hGHR-binding affinity in hGH.
人类生长激素 (hGH) 与 hGH 受体 (hGHR) 的相互作用与癌症和生长障碍有基本的相关性，hGH 是 Pegvisomant 的支架，Pegvisomant 是一种抗肢端肥大症的治疗药物。由于后一个原因，早期研究人员对 hGH 进行了广泛的工程改造，以提高其结合能力和其他特性。我们特别感兴趣的是 E174，它属于 hGH 锌结合三联体；已知 E174A 的取代可显著增加结合，但到目前为止还没有解释。我们在 hGH 的 hGHR 结合位点生成了这个和几个经过计算选择的单残基取代。我们发现，虽然许多取代成功地减缓了 hGH-hGHR 复合物一旦结合后的解离，但它们也减缓了 hGH 与 hGHR 的结合。E174A 取代会引起圆二色性光谱的变化，表明出现了螺旋卷曲。在这里，我们表明 E174A 增加了 hGH 对 hGHR 的亲和力，因为失活速率比激活速率慢。对于 E174Y（和其他位点的某些突变），激活速率的减慢大于失活速率的减慢，导致亲和力降低。这些结果表明 hGH 中的结构、锌结合和 hGHR 结合亲和力之间存在联系。
