Glucose-6-phosphate dehydrogenase (G6PD) deficiency is the most common blood disorder, presenting multiple symptoms, including hemolytic anemia. It affects 400 million people worldwide, with more than 160 single mutations reported in G6PD. The most severe mutations (about 70) are classified as class I, leading to more than 90% loss of activity of the wild-type G6PD. The crystal structure of G6PD reveals these mutations are located away from the active site, concentrating around the noncatalytic NADP⁺-binding site and the dimer interface. However, the molecular mechanisms of class I mutant dysfunction have remained elusive, hindering the development of efficient therapies. To resolve this, we performed integral structural characterization of five G6PD mutants, including four class I mutants, associated with the noncatalytic NADP⁺ and dimerization, using crystallography, small-angle X-ray scattering (SAXS), cryogenic electron microscopy (cryo-EM), and biophysical analyses. Comparisons with the structure and properties of the wild-type enzyme, together with molecular dynamics simulations, bring forward a universal mechanism for this severe G6PD deficiency due to the class I mutations. We highlight the role of the noncatalytic NADP⁺-binding site that is crucial for stabilization and ordering two β-strands in the dimer interface, which together communicate these distant structural aberrations to the active site through a network of additional interactions. This understanding elucidates potential paths for drug development targeting G6PD deficiency.

葡萄糖-6-磷酸脱氢酶 (G6PD) 缺乏症是最常见的血液疾病，呈现多种症状，包括溶血性贫血。它影响着全球 4 亿人，在 G6PD 中报告了 160 多个单一突变。最严重的突变（约 70 个）被归类为 I 类，导致野生型 G6PD 活性丧失 90% 以上。G6PD 的晶体结构显示这些突变远离活性位点，集中在非催化 NADP ⁺-结合位点和二聚体界面。然而，I类突变功能障碍的分子机制仍然难以捉摸，阻碍了有效疗法的发展。为了解决这个问题，我们使用晶体学、小角 X 射线散射 (SAXS)、低温电子显微镜 (cryo- ^EM ) 和生物物理分析。与野生型酶的结构和性质的比较，以及分子动力学模拟，为这种由于 I 类突变引起的严重 G6PD 缺陷提出了普遍的机制。我们强调非催化 NADP ^+的作用-结合位点对于稳定和有序化二聚体界面中的两条 β 链至关重要，它们通过额外的相互作用网络将这些遥远的结构异常传递给活性位点。这种理解阐明了针对 G6PD 缺乏症的药物开发的潜在途径。