Allosteric Communication in the Multifunctional and Redox NQO1 Protein Studied by Cavity-Making Mutations
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Antioxidant defenseFlavoproteinFAD bindingStructural perturbationProtein coreAllosterismCavity-making mutation
Pacheco-Garcia, J.L... [et al.]. Allosteric Communication in the Multifunctional and Redox NQO1 Protein Studied by Cavity-Making Mutations. Antioxidants 2022, 11, 1110. [https://doi.org/10.3390/antiox11061110]
SponsorshipERDF/Spanish Ministry of Science, Innovation and Universities-State Research Agency RTI2018-096246-B-I00; Junta de Andalucia P18-RT-2413; ERDF/Counseling of Economic transformation, Industry, Knowledge and Universities B-BIO-84-UGR20; Government of Aragon-FEDER E35_20R; Department of Science & Technology (India); Science Engineering Research Board (SERB), India MTR/2019/000392; Horizon 2020 EPIC-XS project 82383; EU/MEYS project BioCeV CZ.1.05/1.1.00/02.0109; ERDF/Counseling of Economic transformation, Industry, Knowledge and Universities, Junta de Andalucia B-BIO-84-UGR20; EU/MEYS project CIISB LM2018127; MCIN/AEI PID2019-103901GB-I00
Allosterism is a common phenomenon in protein biochemistry that allows rapid regulation of protein stability; dynamics and function. However, the mechanisms by which allosterism occurs (by mutations or post-translational modifications (PTMs)) may be complex, particularly due to long-range propagation of the perturbation across protein structures. In this work, we have investigated allosteric communication in the multifunctional, cancer-related and antioxidant protein NQO1 by mutating several fully buried leucine residues (L7, L10 and L30) to smaller residues (V, A and G) at sites in the N-terminal domain. In almost all cases, mutated residues were not close to the FAD or the active site. Mutations L -> G strongly compromised conformational stability and solubility, and L30A and L30V also notably decreased solubility. The mutation L10A, closer to the FAD binding site, severely decreased FAD binding affinity (approximate to 20 fold vs. WT) through long-range and context-dependent effects. Using a combination of experimental and computational analyses, we show that most of the effects are found in the apo state of the protein, in contrast to other common polymorphisms and PTMs previously characterized in NQO1. The integrated study presented here is a first step towards a detailed structural-functional mapping of the mutational landscape of NQO1, a multifunctional and redox signaling protein of high biomedical relevance.