Long-range structural defects by pathogenic mutations in most severe glucose-6-phosphate dehydrogenase deficiency

Abstract

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.

Stabilization of glucose-6-phosphate dehydrogenase oligomers enhances catalytic activity and stability of clinical variants

“…Therefore, we solved the protein structure for G6PD K403Q and confirmed that major structural alterations occur in the G6PD K403Q dimer: the structural NADP + is not bound, the catalytic G6P-binding site is occluded, and there are major changes in the globular structure of the dimer. These observations explain our biochemical data; G6P occlusion hinders G6PD activity, and dimer distortion prevents salt-bridge interactions at the tetramer interface, and these findings are consistent with previous structures solved for Class I variants ( 26 ).…”

“…For the G6PD K403QdLtL tetramer, a subunit of the crystal structure of G6PD WT (PDB 6E08) was used an input model and disordered N-terminal portion of 31 residues was modeled with P22 symmetry. For G6PD K403QdLtL dimer, a subunit of the crystal structure (PDB 7SEH) was split into 3 rigid bodies and 3 disordered regions in the subunit (31,26,14 residues at the N-terminus, 407 th -432 nd , and C-terminus respectively) were reconstructed during the modeling with P2 symmetry. 20 independent runs were performed and the model with the lowest 𝜒 2 value was selected as the best model.…”

Combined effects of double mutations on catalytic activity and structural stability contribute to clinical manifestations of glucose-6-phosphate dehydrogenase deficiency

“…The structural NADP + has been known to maintain G6PD catalytic activity by stabilizing the dimeric state. The pathogenic mutations located around the structural NADP + binding site were found to be structurally less stable than the G6PD WT 35 , 36 . These mutations result in the loss of structural NADP + and the disorder of the C-terminal tail, which blocks the G6P-binding site through long-range commination and reduces the enzyme activity.…”

Case Report: Drug-Induced Immune Haemolytic Anaemia Caused by Cefoperazone-Tazobactam/ Sulbactam Combination Therapy

“…Cefoperazone-dependent antibodies were detected in the patient's plasma in the presence of cefoperazone solution; no drug-dependent antibodies were detected in the presence of tazobactam or sulbactam solution. Based on the patient's medical history and results of clinical, biochemical, and immunohaematological tests, we ruled out the possibility of haemolytic anaemia caused by G6PD deficiency (19,20) and autoimmune haemolytic anaemia (21)(22)(23)(24)(25). The condition was diagnosed as DIIHA caused by cefoperazone-dependent antibodies and the NIPA effect of tazobactam and sulbactam.…”