121158 20241125101126.0 doi 10.1111/febs.16677 sideral 131629 ART-2023-131629 eng Pacheco-García, Juan Luis Counterintuitive structural and functional effects due to naturally occurring mutations targeting the active site of the disease-associated NQO1 enzyme* 2023 Access copy available to the general public Unrestricted Our knowledge on the genetic diversity of the human genome is exponentially growing. However, our capacity to establish genotype–phenotype correlations on a large scale requires a combination of detailed experimental and computational work. This is a remarkable task in human proteins which are typically multifunctional and structurally complex. In addition, mutations often prevent the determination of mutant high-resolution structures by X-ray crystallography. We have characterized here the effects of five mutations in the active site of the disease-associated NQO1 protein, which are found either in cancer cell lines or in massive exome sequencing analysis in human population. Using a combination of H/D exchange, rapid-flow enzyme kinetics, binding energetics and conformational stability, we show that mutations in both sets may cause counterintuitive functional effects that are explained well by their effects on local stability regarding different functional features. Importantly, mutations predicted to be highly deleterious (even those affecting the same protein residue) may cause mild to catastrophic effects on protein function. These functional effects are not well explained by current predictive bioinformatic tools and evolutionary models that account for site conservation and physicochemical changes upon mutation. Our study also reinforces the notion that naturally occurring mutations not identified as disease-associated can be highly deleterious. Our approach, combining protein biophysics and structural biology tools, is readily accessible to broadly increase our understanding of genotype–phenotype correlations and to improve predictive computational tools aimed at distinguishing disease-prone against neutral missense variants in the human genome. info:eu-repo/grantAgreement/ES/DGA-FEDER/E35-20R info:eu-repo/grantAgreement/ES/ERDF-B-BIO-84-UGR20 info:eu-repo/grantAgreement/EC/H2020/731077/EU/European Network of Fourier-Transform Ion-Cyclotron-Resonance Mass Spectrometry Centers/EU_FT-ICR_MS This project has received funding from the European Union’s Horizon 2020 research and innovation program under grant agreement No H2020 731077-EU_FT-ICR_MS info:eu-repo/grantAgreement/ES/MCIU-ERDF/RTI2018-096246-B-I00 info:eu-repo/grantAgreement/ES/MICINN-AEI/PID2019-103901GB-I00 info:eu-repo/semantics/openAccess by http://creativecommons.org/licenses/by/3.0/es/ 5.5 2023 BIOCHEMISTRY & MOLECULAR BIOLOGY 58 / 313 = 0.185 2023 Q1 T1 2.003 2023 Biochemistry 2023 Q1 Molecular Biology 2023 Q1 Cell Biology 2023 Q1 11.7 2023 info:eu-repo/semantics/article info:eu-repo/semantics/publishedVersion Anoz Carbonell, Ernesto Loginov, Dmitry S. Kavan, Daniel Salido, Eduardo Man, Petr Medina, Milagros Universidad de Zaragoza (orcid)0000-0001-8743-0182 Pey, Angel L. 1002 060 Universidad de Zaragoza Dpto. Bioq.Biolog.Mol. Celular Área Bioquímica y Biolog.Mole. 290, 7 (2023), 1855-1873 FEBS J. FEBS Journal 1742-464X 4431512 http://zaguan.unizar.es/record/121158/files/texto_completo.pdf Versión publicada 2578736 http://zaguan.unizar.es/record/121158/files/texto_completo.jpg?subformat=icon icon Versión publicada oai:zaguan.unizar.es:121158 articulos driver 2024-11-22-11:57:45 ARTICLE