Allosteric Communication in the Multifunctional and Redox NQO1 Protein Studied by Cavity-Making Mutations

Antioxidants

Eduardo Salido¹  Rogelio Palomino-Morales²  Ernesto Anoz-Carbonell³  Milagros Medina³  Angel L. Pey⁴  Juan Luis Pacheco-Garcia⁵  Athi N. Naganathan⁶  Pavla Vankova⁷  Petr Man⁸  Dmitry S. Loginov⁸ 

[1] Center for Rare Diseases (CIBERER), Hospital Universitario de Canarias, Universidad de la Laguna, 38320 Tenerife, Spain;Departamento de Bioquímica y Biología Molecular I, Facultad de Ciencias y Centro de Investigaciones Biomédicas (CIBM), Universidad de Granada, 18016 Granada, Spain;Departamento de Bioquímica y Biología Molecular y Celular, Facultad de Ciencias, Instituto de Biocomputación y Física de Sistemas Complejos (BIFI) (GBsC-CSIC Joint Unit), 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 & Jyoti Mehta School of Biosciences, Indian Institute of Technology Madras (IITM), Chennai 600036, India;Institute of Biotechnology—BioCeV, Academy of Sciences of the Czech Republic, Prumyslova 595, 252 50 Vestec, Czech Republic;Institute of Microbiology—BioCeV, Academy of Sciences of the Czech Republic, Prumyslova 595, 252 50 Vestec, Czech Republic;

关键词: antioxidant defense;    flavoprotein;    FAD binding;    structural perturbation;    protein core;    allosterism;   

DOI  :  10.3390/antiox11061110

来源: DOAJ

【 摘 要 】

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 (≈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.

【 授权许可】

Unknown