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Interpretation of the colossal number of genetic variants identified from sequencing applications is one of the major bottlenecks in clinical genetics, with the inference of the effect of amino acidsubstituting missense variations on protein structure and function being especially challenging. Here we characterize the three-dimensional (3D) amino acid positions affected in pathogenic and population variants from 1,330 disease-associated genes using over 14,000 experimentally solved human protein structures. By measuring the statistical burden of variations (i.e., point mutations) from all genes on 40 3D protein features, accounting for the structural, chemical, and functional context of the variations’ positions, we identify features that are generally associated with pathogenic and population missense variants. We then perform the same amino acid-level analysis individually for 24 protein functional classes, which reveals unique characteristics of the positions of the altered amino acids: We observe up to 46% divergence of the class-specific features from the general characteristics obtained by the analysis on all genes, which is consistent with the structural diversity of essential regions across different protein classes. We demonstrate that the function-specific 3D features of the variants match the readouts of mutagenesis experiments for BRCA1 and PTEN, and positively correlate with an independent set of clinically interpreted pathogenic and benign missense variants. Finally, we make our results available through a web server to foster accessibility and downstream research. Our findings represent a crucial step toward translational genetics, from highlighting the impact of mutations on protein structure to rationalizing the variants’ pathogenicity in terms of the perturbed molecular mechanisms.

Moonlighting proteins—single polypeptides performing multiple, often unrelated functions—are increasingly recognized as key players in human disease and microbial pathogenesis, making their identification crucial for understanding disease mechanisms and developing targeted therapies. This study addresses the unresolved question of how such multifunctionality evolves, focusing on two potential structural mechanisms: Non-Orthologous Gene Displacement/Non-Homologous Isofunctional Enzymes (NOGD/NHIE), where evolutionarily unrelated proteins perform the same function, and Fold-Switching Proteins (FSP), which adopt alternative secondary structures to switch functions without sequence changes. We analyzed the overlap between known human moonlighting proteins (from MultitaskProtDB-II) and curated datasets of NOGD/NHIE (Non-Orthologous Gene Displacement/Non-Homologous Isofunctional Enzymes) and fold-switching proteins (FSPs), using Fisher’s exact test for statistical validation. Moonlighting proteins showed extraordinary enrichment for NOGD/NHIE (19.89% vs. 0.39% in non-moonlighting proteins; odds ratio = 63.1, p < 2.2 × 10−16) and strong enrichment for FSPs (6.99% vs. 0.26%; odds ratio = 28.7, p = 1.13 × 10−14), corresponding to ~51-fold and ~27-fold higher risks, respectively. These findings establish intrinsic structural plasticity—whether through evolutionary replacement (NOGD/NHIE) or conformational switching (FSP)—as a central mechanism enabling functional moonlighting in the human proteome. The results suggest that such plasticity facilitates functional innovation while preserving sequence integrity, and that both NOGD/NHIE and FSP features may serve as predictive signatures for identifying novel moonlighting proteins, particularly those with implications for disease mechanisms and therapeutic targeting.

Membrane proteins often possess critical structural features, such as transmembrane domains (TMs), N-glycosylation, and disulfide bonds (SS bonds), which are essential to their structure and function. Here, we extend the study of the motifs carrying N-glycosylation, i.e. the sequons, and the Cys residues supporting the SS bonds, to the whole human proteome with a particular focus on the Cys positions in human proteins with respect to those of sequons and TMs. As the least abundant amino acid residue in protein sequences, the positions of Cys residues in proteins are not random but rather selected through evolution. We discovered that the frequency of Cys residues in proteins is length dependent, and the frequency of CC gaps formed between adjacent Cys residues can be used as a classifier to distinguish proteins with special structures and functions, such as keratin-associated proteins (KAPs), extracellular proteins with EGF-like domains, and nuclear proteins with zinc finger C2H2 domains. Most importantly, by comparing the positions of Cys residues to those of sequons and TMs, we discovered that these structural features can form dense clusters in highly repeated and mutually exclusive modalities in protein sequences. The evolutionary advantages of such complementarity among the three structural features are discussed, particularly in light of structural dynamics in proteins that are lacking from computational predictions. The discoveries made here highlight the sequence-structure-function axis in biological organisms that can be utilized in future protein engineering toward synthetic biology.

HMGB3 protein belongs to the group of HMGB proteins from the superfamily of nuclear proteins with high electrophoretic mobility. HMGB proteins play an active part in almost all cellular processes associated with DNA—repair, replication, recombination, and transcription—and, additionally, can act as cytokines during infectious processes, inflammatory responses, and injuries. Although the structure and functions of HMGB1 and HMGB2 proteins have been intensively studied for decades, very little attention has been paid to HMGB3 until recently. In this review, we summarize the currently available data on the molecular structure, post-translational modifications, and biological functions of HMGB3, as well as the possible role of the ubiquitin–proteasome system-dependent HMGB3 degradation in tumor development.

Amino acid networks (AANs) are undirected networks consisting of amino acid residues and their interactions in three-dimensional protein structures. The analysis of AANs provides novel insight into protein science, and several common amino acid network properties have revealed diverse classes of proteins. In this review, we first summarize methods for the construction and characterization of AANs. We then compare software tools for the construction and analysis of AANs. Finally, we review the application of AANs for understanding protein structure and function, including the identification of functional residues, the prediction of protein folding, analyzing protein stability and protein–protein interactions, and for understanding communication within and between proteins.

The GABA type A receptor (GABAAR) belongs to the family of pentameric ligand-gated ion channels and plays a key role in inhibition in adult mammalian brains. Dysfunction of this macromolecule may lead to epilepsy, anxiety disorders, autism, depression, and schizophrenia. GABAAR is also a target for multiple physiologically and clinically relevant modulators, such as benzodiazepines (BDZs), general anesthetics, and neurosteroids. The first GABAAR structure appeared in 2014, but the past years have brought a particularly abundant surge in structural data for these receptors with various ligands and modulators. Although the open conformation remains elusive, this novel information has pushed the structure–function studies to an unprecedented level. Electrophysiology, mutagenesis, photolabeling, and in silico simulations, guided by novel structural information, shed new light on the molecular mechanisms of receptor functioning. The main goal of this review is to present the current knowledge of GABAAR functional and structural properties. The review begins with an outline of the functional and structural studies of GABAAR, accompanied by some methodological considerations, especially biophysical methods, enabling the reader to follow how major breakthroughs in characterizing GABAAR features have been achieved. The main section provides a comprehensive analysis of the functional significance of specific structural elements in GABAARs. We additionally summarize the current knowledge on the binding sites for major GABAAR modulators, referring to the molecular underpinnings of their action. The final chapter of the review moves beyond examining GABAAR as an isolated macromolecule and describes the interactions of the receptor with other proteins in a broader context of inhibitory plasticity. In the final section, we propose a general conclusion that agonist binding to the orthosteric binding sites appears to rely on local interactions, whereas conformational transitions of bound macromolecule (gating) and allosteric modulation seem to reflect more global phenomena involving vast portions of the macromolecule.

Evolution shapes protein sequences for their functions. Here, we studied the moonlighting functions of the N-linked sequon NXS/T, where X is not P, in human nucleocytosolic proteins. By comparing membrane and secreted proteins in which sequons are well known for N-glycosylation, we discovered that cyto-sequons can participate in nucleic acid binding, particularly in zinc finger proteins. Our global studies further discovered that sequon occurrence is largely proportional to protein length. The contribution of sequons to protein functions, including both N-glycosylation and nucleic acid binding, can be regulated through their density as well as the biased usage between NXS and NXT. In proteins where other PTMs or structural features are rich, such as phosphorylation, transmembrane ɑ-helices, and disulfide bridges, sequon occurrence is scarce. The information acquired here should help understand the relationship between protein sequence and function and assist future protein design and engineering.

Higher concentration of protons in the mitochondrial intermembrane space compared to the matrix results in an electrochemical potential causing the back flux of protons to the matrix. This proton transport can take place through ATP synthase complex (leading to formation of ATP) or can occur via proton transporters of the mitochondrial carrier superfamily and/or membrane lipids. Some mitochondrial proton transporters, such as uncoupling proteins (UCPs), transport protons as their general regulating function; while others are symporters or antiporters, which use the proton gradient as a driving force to co-transport other substrates across the mitochondrial inner membrane (such as phosphate carrier, a symporter; or aspartate/glutamate transporter, an antiporter). Passage (or leakage) of protons across the inner membrane to matrix from any route other than ATP synthase negatively impacts ATP synthesis. The focus of this review is on regulated proton transport by UCPs. Recent findings on the structure and function of UCPs, and the related research methodologies, are also critically reviewed. Due to structural similarity of members of the mitochondrial carrier superfamily, several of the known structural features are potentially expandable to all members. Overall, this report provides a brief, yet comprehensive, overview of the current knowledge in the field.

The human RAD52 protein, which forms an oligomeric ring structure, is involved in DNA double‐strand break repair. The N‐terminal half of RAD52 is primarily responsible for self‐oligomerisation and DNA binding. Crystallographic studies have revealed the detailed structure of the N‐terminal half. However, only low‐resolution structures have been reported for the full‐length protein, and thus the structural role of the C‐terminal half in self‐oligomerisation has remained elusive. In this study, we determined the solution structure of the human RAD52 protein by cryo‐electron microscopy (cryo‐EM), at an average resolution of 3.5 Å. The structure revealed an undecameric ring that is nearly identical to the crystal structures of the N‐terminal half. The cryo‐EM map for the C‐terminal half was poorly defined, indicating that the region is intrinsically disordered. The present cryo‐EM structure provides important insights into the mechanistic roles played by the N‐terminal and C‐terminal halves of RAD52 during DNA double‐strand break repair. The cryo‐EM structure of the human DNA repair protein RAD52 was determined at near‐atomic resolution, which revealed that the full‐length protein oligomerises into an undecameric ring. The N‐terminal half is responsible for the oligomerisation. By contrast, the C‐terminal half was not visible in the structure, which is consistent with the predicted intrinsic disorder of the region.

Several food-derived molecules, including proteins and peptides, can show bioactivities toward the promotion of well-being and disease prevention in humans. There is still a lack of information about the potential effects on immune and inflammatory responses in mammalian cells following the ingestion of seed storage proteins. This study, for the first time, describes the potential immunomodulation capacity of chenopodin, the major protein component of quinoa seeds. After characterizing the molecular features of the purified protein, we were able to separate two different forms of chenopodin, indicated as LcC (Low charge Chenopodin, 30% of total chenopodin) and HcC (High charge Chenopodin, 70% of total chenopodin). The biological effects of LcC and HcC were investigated by measuring NF-κB activation and IL-8 expression studies in undifferentiated Caco-2 cells. Inflammation was elicited using IL-1β. The results indicate that LcC and HcC show potential anti-inflammatory activities in an intestinal cell model, and that the proteins can act differently, depending on their structural features. Furthermore, the molecular mechanisms of action and the structural/functional relationships of the protein at the basis of the observed bioactivity were investigated using in silico analyses and structural predictions.