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

Garcia, Adriana Ann; Mathews, Irimpan I.; Horikoshi, Naoki; Matsui, Tsutomu; Kaur, Manat; Wakatsuki, Soichi; Mochly-Rosen, Daria

doi:10.1016/j.jbc.2022.101610

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

Journal Article · 19 January 2022 · Journal of Biological Chemistry

DOI: https://doi.org/10.1016/j.jbc.2022.101610 · OSTI ID:1872111

Garcia, Adriana Ann ^[1]; Mathews, Irimpan I. ^[2];

^[3]; Matsui, Tsutomu ^[2]; Kaur, Manat ^[1]; Wakatsuki, Soichi ^[4]; Mochly-Rosen, Daria ^[1]

Stanford Univ., CA (United States)
SLAC National Accelerator Lab., Menlo Park, CA (United States)
Univ. of Tsukuba (Japan); SLAC National Accelerator Lab., Menlo Park, CA (United States); Stanford Univ., CA (United States)
SLAC National Accelerator Lab., Menlo Park, CA (United States); Stanford Univ., CA (United States)

Glucose-6-phosphate dehydrogenase (G6PD) deficiency is a genetic trait that can cause hemolytic anemia. To date, over 150 nonsynonymous mutations have been identified in G6PD, with pathogenic mutations clustering near the dimer and/or tetramer interface and the allosteric NADP⁺-binding site. Recently, our lab identified a small molecule that activates G6PD variants by stabilizing the allosteric NADP⁺ and dimer complex, suggesting therapeutics that target these regions may improve structural defects. Here, we elucidated the connection between allosteric NADP⁺ binding, oligomerization, and pathogenicity to determine whether oligomer stabilization can be used as a therapeutic strategy for G6PD deficiency (G6PD^def). We first solved the crystal structure for G6PD^K403Q, a mutant that mimics the physiological acetylation of wild-type G6PD in erythrocytes and demonstrated that loss of allosteric NADP⁺ binding induces conformational changes in the dimer. These structural changes prevent tetramerization, are unique to Class I variants (the most severe form of G6PD^def), and cause the deactivation and destabilization of G6PD. We also introduced nonnative cysteines at the oligomer interfaces and found that the tetramer complex is more catalytically active and stable than the dimer. Furthermore, stabilizing the dimer and tetramer improved protein stability in clinical variants, regardless of clinical classification, with tetramerization also improving the activity of G6PD^K403Q and Class I variants. These findings were validated using enzyme activity and thermostability assays, analytical size-exclusion chromatography (SEC), and SEC coupled with small-angle X-ray scattering (SEC-SAXS). Taken together, our findings suggest a potential therapeutic strategy for G6PD^def and provide a foundation for future drug discovery efforts.

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Research Organization:: SLAC National Accelerator Laboratory (SLAC), Menlo Park, CA (United States)

Sponsoring Organization:: USDOE Office of Science (SC), Basic Energy Sciences (BES); National Institutes of Health (NIH); National Science Foundation (NSF); National Institute of General Medical Sciences

Grant/Contract Number:: AC02-76SF00515

OSTI ID:: 1872111

Journal Information:: Journal of Biological Chemistry, Journal Name: Journal of Biological Chemistry Journal Issue: 3 Vol. 298; ISSN 0021-9258

Publisher:: American Society for Biochemistry and Molecular BiologyCopyright Statement

Country of Publication:: United States

Language:: English

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

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