Allosteric Communication in the Multifunctional and Redox NQO1 Protein Studied by Cavity-Making Mutations
Juan Luis Pacheco-Garcia,
Dmitry S. Loginov,
Ernesto Anoz-Carbonell,
Pavla Vankova,
Rogelio Palomino-Morales,
Eduardo Salido,
Petr Man,
Milagros Medina,
Athi N. Naganathan,
Angel L. Pey
Affiliations
Juan Luis Pacheco-Garcia
Departamento de Química Física, Universidad de Granada, Av. Fuentenueva s/n, 18071 Granada, Spain
Dmitry S. Loginov
Institute of Microbiology—BioCeV, Academy of Sciences of the Czech Republic, Prumyslova 595, 252 50 Vestec, Czech Republic
Ernesto Anoz-Carbonell
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
Pavla Vankova
Institute of Biotechnology—BioCeV, Academy of Sciences of the Czech Republic, Prumyslova 595, 252 50 Vestec, Czech Republic
Rogelio Palomino-Morales
Departamento de Bioquímica y Biología Molecular I, Facultad de Ciencias y Centro de Investigaciones Biomédicas (CIBM), Universidad de Granada, 18016 Granada, Spain
Eduardo Salido
Center for Rare Diseases (CIBERER), Hospital Universitario de Canarias, Universidad de la Laguna, 38320 Tenerife, Spain
Petr Man
Institute of Microbiology—BioCeV, Academy of Sciences of the Czech Republic, Prumyslova 595, 252 50 Vestec, Czech Republic
Milagros Medina
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
Athi N. Naganathan
Department of Biotechnology, Bhupat & Jyoti Mehta School of Biosciences, Indian Institute of Technology Madras (IITM), Chennai 600036, India
Angel L. Pey
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
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.