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

ABSTRACT: 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.

SUBMITTER: Pacheco-Garcia JL

PROVIDER: S-EPMC9219786 | biostudies-literature | 2022 Jun

REPOSITORIES: biostudies-literature

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

Pacheco-Garcia Juan Luis JL Loginov Dmitry S DS Anoz-Carbonell Ernesto E Vankova Pavla P Palomino-Morales Rogelio R Salido Eduardo E Man Petr P Medina Milagros M Naganathan Athi N AN Pey Angel L AL

Antioxidants (Basel, Switzerland) 20220602 6

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 ful ...[more]

PMID: 35740007

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Project description:BackgroundThe S. cerevisiae carbamylphosphate synthetase - aspartate transcarbamylase multifunctional protein catalyses the first two reactions of the pyrimidine pathway. In this organism, these two reactions are feedback inhibited by the end product UTP. In the present work, the mechanisms of these integrated inhibitions were studied.ResultsThe results obtained show that the inhibition is competitive in the case of carbamylphosphate synthetase and non-competitive in the case of aspartate transcarbamylase. They also identify the substrate whose binding is altered by this nucleotide and the step of the carbamylphosphate synthetase reaction which is inhibited. Furthermore, the structure of the domains catalyzing these two reactions were modelled in order to localize the mutations which, specifically, alter the aspartate transcarbamylase sensitivity to the feedback inhibitor UTP. Taken together, the results make it possible to propose a model for the integrated regulation of the two activities of the complex. UTP binds to a regulatory site located in the vicinity of the carbamylphosphate synthetase catalytic subsite which catalyzes the third step of this enzyme reaction. Through a local conformational change, this binding decreases, competitively, the affinity of this site for the substrate ATP. At the same time, through a long distance signal transmission process it allosterically decreases the affinity of the aspartate transcarbamylase catalytic site for the substrate aspartate.ConclusionThis investigation provides informations about the mechanisms of allosteric inhibition of the two activities of the CPSase-ATCase complex. Although many allosteric monofunctional enzymes were studied, this is the first report on integrated allosteric regulation in a multifunctional protein. The positions of the point mutations which specifically abolish the sensitivity of aspartate transcarbamylase to UTP define an interface between the carbamylphosphate synthetase and aspartate transcarbamylase domains, through which the allosteric signal for the regulation of aspartate transcarbamylase must be propagated.

Project description:The stress induced protein NQO1 can participate in a wide range of biological pathways which are dependent upon the interaction of NQO1 with protein targets. Many of the protein-protein interactions involving NQO1 have been shown to be regulated by the pyridine nucleotide redox balance. NQO1 can modify its conformation as a result of redox changes in pyridine nucleotides and sites on the C-terminal and helix seven regions of NQO1 have been identified as potential areas that may be involved in redox-dependent protein-protein interactions. Since post-translational modifications can modify the functionality of proteins, we examined whether redox-dependent conformational changes induced in NQO1 would alter lysine acetylation. Recombinant NQO1 was incubated with and without NADH then acetylated non-enzymatically by acetic anhydride or S-acetylglutathione (Ac-GSH). NQO1 acetylation was determined by immunoblot and site-specific lysine acetylation was quantified by mass spectrometry (MS). NQO1 was readily acetylated by acetic anhydride and Ac-GSH. Interestingly, despite a large number of lysine residues (9%) in NQO1 only a small subset of lysines were acetylated and the majority of these were located in or near the functional C-terminal or helix seven regions. Reduction of NQO1 by NADH prior to acetylation resulted in almost complete protection of NQO1 from lysine acetylation as confirmed by immunoblot analysis and MS. Lysines located within the redox-active C-terminus and helix seven regions were readily acetylated when NQO1 was in an oxidized conformation but were protected from acetylation when NQO1 was in the reduced conformation. To investigate regulatory mechanisms of enzymatic deacetylation, NQO1 was acetylated by Ac-GSH then exposed to purified sirtuins (SIRT 1-3) or histone deacetylase 6 (HDAC6). NQO1 could be deacetylated by all sirtuin isoforms and quantitative MS analysis performed using SIRT2 revealed very robust deacetylation of NQO1, specifically at K262 and K271 in the C-terminal region. No deacetylation of NQO1 by HDAC6 was detected. These data demonstrate that the same subset of key lysine residues in the C-terminal and helix seven regions of NQO1 undergo redox dependent acetylation and are regulated by sirtuin-mediated deacetylation.

Dataset Information

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

Publications

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

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