The multifunctional nature of human flavoproteins is critically linked to their ability to populate multiple conformational states. Ligand binding, post-translational modifications and disease-associated mutations can reshape this functional landscape, although the structure-function relationships of these effects are not well understood. Herein, we characterized the structural and functional consequences of two mutations (the cancer-associated P187S and the phosphomimetic S82D) on different ligation states which are relevant to flavin binding, intracellular stability and catalysis of the disease-associated NQO1 flavoprotein. We found that these mutations affected the stability locally and their effects propagated differently through the protein structure depending both on the nature of the mutation and the ligand bound, showing directional preference from the mutated site and leading to specific phenotypic manifestations in different functional traits (FAD binding, catalysis and inhibition, intracellular stability and pharmacological response to ligands). Our study thus supports that pleitropic effects of disease-causing mutations and phosphorylation events on human flavoproteins may be caused by long-range structural propagation of stability effects to different functional sites that depend on the ligation-state and site-specific perturbations. Our approach can be of general application to investigate these pleiotropic effects at the flavoproteome scale in the absence of high-resolution structural models.

Center for Rare Diseases Hospital Universitario de Canarias Universidad de la Laguna 38320 Tenerife Spain

Departamento de Bioquímica y Biología Molecular y Celular Facultad de Ciencias Instituto de Biocomputación y Física de Sistemas Complejos Universidad de Zaragoza 50009 Zaragoza Spain

Departamento de Química Física Unidad de Excelencia en Química Aplicada a Biomedicina y Medioambiente e Instituto de Biotecnología Universidad de Granada Av Fuentenueva s n 18071 Granada Spain

Departamento de Química Física Universidad de Granada Av Fuentenueva s n 18071 Granada Spain

Department of Biotechnology Bhupat and Jyoti Mehta School of Biosciences Indian Institute of Technology Madras Chennai 600036 India

Departmento de Bioquímica y Biología Molecular 1 Facultad de Ciencias y Centro de Investigaciones Biomédicas Universidad de Granada Granada Spain

Institute of Microbiology Academy of Sciences of the Czech Republic Videnska 1083 Prague 4 142 20 Czech Republic

Institute of Microbiology Academy of Sciences of the Czech Republic Videnska 1083 Prague 4 142 20 Czech Republic; Department of Biochemistry Faculty of Science Charles University Hlavova 2030 8 Prague 2 128 43 Czech Republic

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