Human frataxin is an iron‐binding protein involved in the mitochondrial iron–sulfur (Fe–S) clusters assembly, a process fundamental for the functional activity of mitochondrial proteins. Decreased level of frataxin expression is associated with the neurodegenerative disease Friedreich ataxia. Defective function of frataxin may cause defects in mitochondria, leading to increased tumorigenesis. Tumor‐initiating cells show higher iron uptake, a decrease in iron storage and a reduced Fe–S clusters synthesis and utilization. In this study, we selected, from COSMIC database, the somatic human frataxin missense variants found in cancer tissues p.D104G, p.A107V, p.F109L, p.Y123S, p.S161I, p.W173C, p.S181F, and p.S202F to analyze the effect of the single amino acid substitutions on frataxin structure, function, and stability. The spectral properties, the thermodynamic and the kinetic stability, as well as the molecular dynamics of the frataxin missense variants found in cancer tissues point to local changes confined to the environment of the mutated residues. The global fold of the variants is not altered by the amino acid substitutions; however, some of the variants show a decreased stability and a decreased functional activity in comparison with that of the wild‐type protein.

Large scale genome sequencing allowed the identification of a massive number of genetic variations, whose impact on human health is still unknown. In this review we analyze, by an in silico-based strategy, the impact of missense variants on cancer-related genes, whose effect on protein stability and function was experimentally determined. We collected a set of 164 variants from 11 proteins to analyze the impact of missense mutations at structural and functional levels, and to assess the performance of state-of-the-art methods (FoldX and Meta-SNP) for predicting protein stability change and pathogenicity. The result of our analysis shows that a combination of experimental data on protein stability and in silico pathogenicity predictions allowed the identification of a subset of variants with a high probability of having a deleterious phenotypic effect, as confirmed by the significant enrichment of the subset in variants annotated in the COSMIC database as putative cancer-driving variants. Our analysis suggests that the integration of experimental and computational approaches may contribute to evaluate the risk for complex disorders and develop more effective treatment strategies.

The extracellular-signal-regulated kinase 2 (ERK2), a mitogen-activated protein kinase (MAPK) located downstream of the Ras-Raf-MEK-ERK signal transduction cascade, is involved in the regulation of a large variety of cellular processes. The ERK2, activated by phosphorylation, is the principal effector of a central signaling cascade that converts extracellular stimuli into cells. Deregulation of the ERK2 signaling pathway is related to many human diseases, including cancer. This study reports a comprehensive biophysical analysis of structural, function, and stability data of pure, recombinant human non-phosphorylated (NP-) and phosphorylated (P-) ERK2 wild-type and missense variants in the common docking site (CD-site) found in cancer tissues. Because the CD-site is involved in interaction with protein substrates and regulators, a biophysical characterization of missense variants adds information about the impact of point mutations on the ERK2 structure–function relationship. Most of the P-ERK2 variants in the CD-site display a reduced catalytic efficiency, and for the P-ERK2 D321E, D321N, D321V and E322K, changes in thermodynamic stability are observed. The thermal stability of NP-ERK2 and P-ERK2 D321E, D321G, and E322K is decreased with respect to the wild-type. In general, a single residue mutation in the CD-site may lead to structural local changes that reflects in alterations in the global ERK2 stability and catalysis.

Bromodomains (BRDs) are small protein interaction modules of about 110 amino acids that selectively recognize acetylated lysine in histones and other proteins. These domains have been identified in a variety of multi-domain proteins involved in transcriptional regulation or chromatin remodeling in eukaryotic cells. BRD inhibition is considered an attractive therapeutic approach in epigenetic disorders, particularly in oncology. Here, we present a Φ value analysis to investigate the folding pathway of the second domain of BRD2 (BRD2(2)). Using an extensive mutational analysis based on 25 site-directed mutants, we provide structural information on both the intermediate and late transition state of BRD2(2). The data reveal that the C-terminal region represents part of the initial folding nucleus, while the N-terminal region of the domain consolidates its structure only later in the folding process. Furthermore, only a small number of native-like interactions have been identified, suggesting the presence of a non-compact, partially folded state with scarce native-like characteristics. Taken together, these results indicate that, in BRD2(2), a hierarchical mechanism of protein folding can be described with non-native interactions that play a significant role in folding.

This work studies the stability of wild-type frataxin and some of its variants found in cancer tissues upon Co2+ binding. Although the physiologically involved metal ion in the frataxin enzymatic activity is Fe2+, as it is customarily done, Co2+ is most often used in experiments because Fe2+ is extremely unstable owing to the fast oxidation reaction Fe2+ → Fe3+. Protein stability is monitored following the conformational changes induced by Co2+ binding as measured by circular dichroism, fluorescence spectroscopy, and melting temperature measurements. The stability ranking among the wild-type frataxin and its variants obtained in this way is confirmed by a detailed comparative analysis of the XAS spectra of the metal-protein complex at the Co K-edge. In particular, a fit to the EXAFS region of the spectrum allows positively identifying the frataxin acidic ridge as the most likely location of the metal-binding sites. Furthermore, we can explain the surprising feature emerging from a detailed analysis of the XANES region of the spectrum, showing that the longer 81-210 frataxin fragment has a smaller propensity for Co2+ binding than the shorter 90-210 one. This fact is explained by the peculiar role of the N-terminal disordered tail in modulating the protein ability to interact with the metal.