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Abstract：
Pacak-Zhuang syndrome is caused by mutations in the EPAS1 gene, which encodes for one of the three hypoxia-inducible factor alpha (HIFα) paralogs HIF2α and is associated with defined but varied phenotypic presentations including neuroendocrine tumors and polycythemia. However, the mechanisms underlying the complex genotype-phenotype correlations remain incompletely understood. Here, we devised a quantitative method for determining the dissociation constant (Kd) of the HIF2α peptides containing disease-associated mutations and the catalytic domain of prolyl-hydroxylase (PHD2) using microscale thermophoresis (MST) and showed that neuroendocrine-associated Class 1 HIF2α mutants have distinctly higher Kd than the exclusively polycythemia-associated Class 2 HIF2α mutants. Based on the co-crystal structure of PHD2/HIF2α peptide complex at 1.8 Å resolution, we showed that the Class 1 mutated residues are localized to the critical interface between HIF2α and PHD2, adjacent to the PHD2 active catalytic site, while Class 2 mutated residues are localized to the more flexible region of HIF2α that makes less contact with PHD2. Concordantly, Class 1 mutations were found to significantly increase HIF2α-mediated transcriptional activation in cellulo compared to Class 2 counterparts. These results reveal a structural mechanism in which the strength of the interaction between HIF2α and PHD2 is at the root of the general genotype-phenotype correlations observed in Pacak-Zhuang syndrome.
摘要：
帕卡克-庄综合征是由EPAS1基因突变引起的,它编码三种缺氧诱导因子α（HIFα）旁系同源物HIF2α之一，并与明确但变化的表型表现有关，包括神经内分泌肿瘤和红细胞增多症。然而,复杂的基因型-表型相关性的潜在机制仍未完全了解.这里,我们设计了一种定量方法，使用微尺度热电泳（MST）确定含有疾病相关突变的HIF2α肽的解离常数（Kd），并显示神经内分泌相关的1类HIF2α突变体的Kd明显高于仅与红细胞增多症相关的2类HIF2α突变体。基于PHD2/HIF2α肽复合物在1.8µ分辨率下的共晶结构，我们表明，1类突变残基位于HIF2α和PHD2之间的关键界面，邻近PHD2活性催化位点，而2类突变的残基定位于HIF2α的更柔性的区域，该区域与PHD2的接触较少。和谐地，与2类对应物相比，发现1类突变显着增加了HIF2α介导的纤维素转录激活。这些结果揭示了一种结构机制，其中HIF2α与PHD2之间的相互作用强度是在Pacak-Zhong综合征中观察到的一般基因型-表型相关性的根源。
