Phosphoglycerate kinase has been a model for the stability, folding cooperativity and catalysis of a two-domain protein. The human isoform 1 (hPGK1) is associated with cancer development and rare genetic diseases that affect several of its features. To investigate how mutations affect hPGK1 folding landscape and interaction networks, we have introduced mutations at a buried site in the N-terminal domain (F25 mutants) that either created cavities (F25L, F25V, F25A), enhanced conformational entropy (F25G) or introduced structural strain (F25W) and evaluated their effects using biophysical experimental and theoretical methods. All F25 mutants folded well, but showed reduced unfolding cooperativity, kinetic stability and altered activation energetics according to the results from thermal and chemical denaturation analyses. These alterations correlated well with the structural perturbation caused by mutations in the N-terminal domain and the destabilization caused in the interdomain interface as revealed by H/D exchange under native conditions. Importantly, experimental and theoretical analyses showed that these effects are significant even when the perturbation is mild and local. Our approach will be useful to establish the molecular basis of hPGK1 genotype-phenotype correlations due to phosphorylation events and single amino acid substitutions associated with disease.

• MeSH
• denaturace proteinů MeSH
• fosfoglycerátkinasa genetika   metabolismus   MeSH
• hydrofobní a hydrofilní interakce MeSH
• kinetika MeSH
• lidé MeSH
• sbalování proteinů * MeSH
• termodynamika MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• fosfoglycerátkinasa MeSH
• PGK1 protein, human MeSH Prohlížeč

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.

• Klíčová slova
• FAD binding, allosterism, antioxidant defense, cavity-making mutation, flavoprotein, protein core, structural perturbation,
• Publikační typ
• časopisecké články MeSH

The multifunctional nature of human flavoproteins is critically linked to their ability to populate multiple conformational states. Ligand binding, post-translational modifications and disease-associated mutations can reshape this functional landscape, although the structure-function relationships of these effects are not well understood. Herein, we characterized the structural and functional consequences of two mutations (the cancer-associated P187S and the phosphomimetic S82D) on different ligation states which are relevant to flavin binding, intracellular stability and catalysis of the disease-associated NQO1 flavoprotein. We found that these mutations affected the stability locally and their effects propagated differently through the protein structure depending both on the nature of the mutation and the ligand bound, showing directional preference from the mutated site and leading to specific phenotypic manifestations in different functional traits (FAD binding, catalysis and inhibition, intracellular stability and pharmacological response to ligands). Our study thus supports that pleitropic effects of disease-causing mutations and phosphorylation events on human flavoproteins may be caused by long-range structural propagation of stability effects to different functional sites that depend on the ligation-state and site-specific perturbations. Our approach can be of general application to investigate these pleiotropic effects at the flavoproteome scale in the absence of high-resolution structural models.

• Klíčová slova
• Disease-causing mutation, Flavoprotein, Ligand binding, Multifunctional protein, NQO1, Post-translational modification,
• MeSH
• chinony MeSH
• flavinadenindinukleotid metabolismus   MeSH
• lidé MeSH
• missense mutace * MeSH
• NAD(P)H dehydrogenasa (chinon) * genetika   metabolismus   MeSH
• NAD MeSH
• vazba proteinů MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• chinony MeSH
• flavinadenindinukleotid MeSH
• NAD(P)H dehydrogenasa (chinon) * MeSH
• NAD MeSH
• NQO1 protein, human MeSH Prohlížeč