Proteins[JOURNAL]
Sun
200 papers
The Curious Case of CysE: Diversity and Distribution of Serine Acetyltransferases in Bacteria.
Oldham KEA, Williamson AK, Grout EK … +1 more , Hicks JL
Proteins · 2026 May · PMID 41400183 · Full text
Serine acetyltransferase (CysE) is a member of the left-handed β-helix family of acetyltransferases that catalyze the rate limiting step in de novo cysteine biosynthesis. There are two isoforms of CysE that differ in len...
Serine acetyltransferase (CysE) is a member of the left-handed β-helix family of acetyltransferases that catalyze the rate limiting step in de novo cysteine biosynthesis. There are two isoforms of CysE that differ in length, with the shorter isoform lacking approximately 76 amino acids at the N-terminus of the protein from the serine acetyltransferase (SATase) domain. Here, we analyze the distribution and diversity of CysE isoforms across the bacterial kingdom. The isoforms can be classified into two discrete groups, with the truncated isoform prevalent in Gram-positive bacteria and the full-length isoform prevalent in Proteobacteria. Moreover, we demonstrate that the truncation is discrete with the loss of four N-terminal α-helices conserved for the truncated isoform. Using predictive modeling, we show that this truncation likely weakens the CysE trimer interface, potentially resulting in a trimeric assembly instead of the canonical CysE hexamer. This expands our understanding of CysE enzymes and their distribution across bacterial species, an important consideration given the increasing interest in targeting CysE enzymes for potential antimicrobials.
AmyloPick: A New Feature Selection Method and Proper Evaluation for Amyloid Hexapeptides and Aggregation-Prone Regions Prediction.
Stapor K, Lewandowska A, Fabian P … +1 more , Roterman-Konieczna I
Proteins · 2026 May · PMID 41399172 · Publisher ↗
Given the critical importance of preventing protein aggregation in neurodegenerative diseases, aggregation prediction tools are essential. Amyloid predictors would facilitate the understanding and exploitation of the amy...
Given the critical importance of preventing protein aggregation in neurodegenerative diseases, aggregation prediction tools are essential. Amyloid predictors would facilitate the understanding and exploitation of the amyloid state of proteins, providing an alternative to costly and slow laboratory tests. In recent years, hexapeptides have become a model for studying amyloid formation. Hexapeptides can also be used to identify aggregation-prone regions in proteins, particularly those involved in amyloid formation. While numerous computational methods using sophisticated feature sets and architectures have been developed for classifying hexapeptides and predicting amyloidogenic regions in proteins, predictive performance remains limited; for instance, BAP achieves only 84% accuracy. Here, we designed a novel feature selection method for hexapeptides, resulting in an easy to interpret four-feature representation called the AmyloPick model. A classifier based on this representation outperforms existing state-of-the-art methods. When extended to detect aggregation-prone regions (APRs) in full proteins, it performs comparably to established tools. A key contribution of this study is the statistical methodology that enables a rigorous performance assessment and direct comparison with other classifiers. This is particularly important because differing methodologies in the literature often hinder the comparability of the proposed methods. Our AmyloPick classifier significantly outperformed the state-of-the-art Budapest Amyloid Predictor (BAP) across all metrics, particularly in adjusted geometric mean (AGM) (0.7808 vs. 0.7649 for BAP) and accuracy (0.8089 vs. 0.7955 for BAP). For APR identification, APR-AmyloPick was comparable to ANuPP overall but significantly outperformed it in the metric. We have also developed a web server for the AmyloPick classifier.
Deciphering Glutathione S-Transferase P1 Inhibition Mechanisms for Overcoming Cancer Chemoresistance: Insights From Computational Analysis.
Aherkou M, Hakmi M, Bouricha EM … +2 more , Belyamani L, Ibrahimi A
Proteins · 2026 May · PMID 41397937 · Publisher ↗
Glutathione S-transferase P1 (GSTP1) plays a crucial role in detoxifying cytotoxic agents and contributes to cancer chemoresistance. Due to its key role in tumor progression and its impact on treatment efficacy, GSTP1 ha...
Glutathione S-transferase P1 (GSTP1) plays a crucial role in detoxifying cytotoxic agents and contributes to cancer chemoresistance. Due to its key role in tumor progression and its impact on treatment efficacy, GSTP1 has emerged as a promising therapeutic target for anticancer therapies. Ethacrynic acid (EA) is a known GSTP1 inhibitor; however, the specific molecular mechanisms behind its inhibitory action remain unclear. To clarify the effects of EA and its glutathione conjugate (EA-GSH) on the GSTP1 dimer, we conducted a comparative molecular dynamics (MD) study of four enzymatic states: apo (unbound), holo (GSH-bound), the GSTP1-EA and GSTP1-EA-GSH complexes, to analyze both interchain and ligand-enzyme interactions. Our results showed that GSTP1 flexibility depends on the movement of the α2 helix, which appears essential for accommodating substrates. Ligand binding made the enzyme more rigid, and EA disrupted dynamic coordination within the dimer by altering secondary-structure elements, potentially impairing enzymatic activity. Additionally, EA influenced dimerization by reducing binding energy at the dimer interface, possibly interfering with GSTP1's nonenzymatic role in apoptosis signaling. Energy analysis demonstrated that while GSH conjugation enhanced EA's binding affinity through favorable electrostatic interactions, it also imposed a significant energetic penalty due to increased solvent exposure. These findings highlight the need to optimize the lipophilic/hydrophilic balance of future GSTP1 inhibitors to match the physicochemical properties of the binding pocket. Overall, this study offers a deeper understanding of the molecular mechanisms behind GSTP1 inhibition and provides a structural basis for designing targeted therapies to overcome cancer chemoresistance.
Distinct Substrate and Intermediate Recognition via Mutation Effects on Mycobacterium tuberculosis Methionyl-tRNA Synthetase.
Thakur S, Mehra R
Proteins · 2026 May · PMID 41392593 · Publisher ↗
Tuberculosis kills millions worldwide. Drug-resistance demands exploring new targets against this illness. Methionyl-tRNA synthetase (MetRS) is a crucial target in Mycobacterium tuberculosis (Mtb) that participates in th...
Tuberculosis kills millions worldwide. Drug-resistance demands exploring new targets against this illness. Methionyl-tRNA synthetase (MetRS) is a crucial target in Mycobacterium tuberculosis (Mtb) that participates in the initiation and elongation of translation and represents a protein of evolutionary interest. To elucidate the structure-function relationships of MetRS, we performed detailed sequence analyses and molecular dynamics simulations of Mtb MetRS in the substrate-bound (methionine and ATP) and intermediate (methionyl-AMP) states, for both the wild-type and three single-mutant forms (H21A, K54A, and E130A). Eight systems (two wild-type and six mutants) were simulated for 36 μs. Differential dynamics and binding effects of the substrate versus intermediate states were identified, along with the molecular reasons for the loss of activity in mutants. The wild-type substrate state was more stable than the intermediate state. In contrast, the mutants were more unstable in the substrate state but incorporated stability into the intermediate state protein. These findings suggest that methionyl-AMP, being a reaction intermediate, exhibits a short residence time at the protein's active site, while the substrate state shows a longer residence time of methionine and ATP. The increased instability of mutants in the substrate state indicates disruption of the pyrophosphate-ATP exchange by altering substrate-protein interactions. Once the intermediate is formed, the mutations have minimal or no effect. These observations are consistent with experimental data. In brief, our study finds the molecular basis for the distinct substrate and intermediate recognition by Mtb MetRS and establishes a mechanism for the loss of activity in the mutants.
On the Variation of Structural Divergence Among Residues in Enzyme Evolution.
Echave J, Carpentier M
Proteins · 2026 May · PMID 41387199 · Publisher ↗
Structural divergence varies among protein residues, yet this variation has been largely overlooked compared with the well-studied case of sequence rate variation. Here we show that, in families of functionally conserved...
Structural divergence varies among protein residues, yet this variation has been largely overlooked compared with the well-studied case of sequence rate variation. Here we show that, in families of functionally conserved homologous enzymes, structural divergence increases with both residue flexibility and distance from the active site. Although these properties are correlated, modeling reveals that the pattern arises from two independent types of evolutionary constraints: non-functional and functional. The balance between these constraints varies widely across enzyme families, from non-functional to functional dominance. As functional constraints strengthen, structural divergence patterns are reshaped, becoming increasingly distinct from flexibility patterns and breaking the commonly assumed correspondence between evolutionary and dynamical structural ensembles. Active sites are more structurally conserved than average, but this conservation stems not only from functional constraints. Because active sites typically lie in rigid regions where non-functional constraints are high, both constraint types contribute comparably on average, with dominance shifting from one to the other depending on active-site rigidity. Together, these findings revise two long-standing assumptions: that evolutionary structural variation universally mirrors protein dynamics and that active-site conservation reflects functional requirements alone. Both depend on the balance between non-functional and functional constraints that shape enzyme structural evolution.
Cryo-EM Analysis in CASP16.
Mulvaney T, Kryshtafovych A, Topf M
Proteins · 2026 Jan · PMID 41380080 · Full text
Since CASP13, experimentalists have been encouraged to provide their cryo-EM data along with the derived atomic models to the CASP organizers to aid assessment. In CASP16, 38 cryo-EM datasets were provided for assessment...
Since CASP13, experimentalists have been encouraged to provide their cryo-EM data along with the derived atomic models to the CASP organizers to aid assessment. In CASP16, 38 cryo-EM datasets were provided for assessment, which represented most cryo-EM targets. The corresponding targets typically comprised a single derived atomic structure; however, that model may be only one of several valid conformations. Flexibility often manifests as low-resolution regions in a cryo-EM reconstruction, particularly in RNA but often also in protein complexes. We show that local resolution in the reconstruction correlates well with the root-mean-square fluctuations (RMSF) of residues of accurate CASP predictions. The correlation between the local resolution and pLDDT was less clear, especially when mobile domains were present. When the resolution allowed, assessment of features such as sidechains, using our variant of SMOC with local fragment alignment, indicated that even high-quality predictions have room for improvement; on the other hand, some predictions fitted the density better in specific regions, indicating modeling discrepancies in the target. In one extreme case, a submitted target had regions of low-resolution that limited unambiguous model building. In such cases, part of the target structure is essentially a prediction itself, with implications for the assessment. Experimental data remain essential for model-free assessment of predictions and offer unique analyses such as comparisons to local resolution and thus flexibility.
Structure and Activity of a β-Mannosidase Involved in Core N-Glycan Degradation.
Zea G, Landry J, Winchester A … +2 more , Sharma S, Higgins MA
Proteins · 2026 May · PMID 41376108 · Publisher ↗
N-glycans are structurally complex carbohydrates commonly found on eukaryotic glycoproteins, where they play essential roles in protein folding, stability, and cellular signaling. Some bacteria have evolved specialized d...
N-glycans are structurally complex carbohydrates commonly found on eukaryotic glycoproteins, where they play essential roles in protein folding, stability, and cellular signaling. Some bacteria have evolved specialized degradation pathways to access N-glycans as nutrient sources, terminating in enzymes that cleave the conserved core Manβ1-4GlcNAc disaccharide. Members of glycoside hydrolase family 5 subfamily 18 (GH5_18) have recently been identified to catalyze this reaction. Here, we report the biochemical and structural characterization of MoGH5_18, which is encoded within a gene cluster consisting of other genes likely involved in N-glycan degradation. Biochemical assays show that MoGH5_18 hydrolyzes Manβ1-4GlcNAc but not Manβ1-4Man, consistent with substrate specificity observed in other GH5_18s. We solved the crystal structure of MoGH5_18 to 1.92 Å resolution, revealing a canonical (β/α) 8 TIM-barrel fold, dimeric architecture, and a conserved active site architecture. These findings demonstrate that MoGH5_18, despite sequence divergence, retains the structural and functional hallmarks of GH5_18 enzymes and further illustrate the power of SSN-guided approaches to uncover conserved enzymatic mechanisms within diverse glycan degradation pathways.
Structure and Mechanism of PhdC, a Prenylated-Flavin Maturase.
Whittall DR, Box HG, Payne KAP … +2 more , Marshall SA, Leys D
Proteins · 2026 May · PMID 41363046 · Full text
Prenylated flavin mononucleotide (prFMN) is a modified flavin cofactor required by the UbiD family of (de)carboxylase enzymes. While the reduced prFMNH form is produced by the flavin prenyltransferase UbiX, the correspon...
Prenylated flavin mononucleotide (prFMN) is a modified flavin cofactor required by the UbiD family of (de)carboxylase enzymes. While the reduced prFMNH form is produced by the flavin prenyltransferase UbiX, the corresponding two-electron oxidized prFMN form is required to support UbiD catalysis. Thus, oxidative maturation of prFMNH is required, which can be catalyzed by UbiD. However, heterologous (over)expression of UbiDs frequently leads to the accumulation of the stable but non-active one-electron oxidized purple prFMN species. A dedicated prFMN maturase enzyme (PhdC) from Mycolicibacterium fortuitum was recently identified as capable of catalyzing the oxidative maturation of prFMN to prFMN, thereby enabling an effective supply of active cofactor to the associated phenazine-1-carboxylate (de)carboxylase PhdA. We report the crystal structure of PhdC in complex with flavin, revealing it is a distant member of the class I HpaC-like family of short-chain dimeric flavin reductases and demonstrate catalytic conversion of the prFMN species to prFMN in the presence of oxygen or ferricyanide. Co-expression of PhdC or a distant homologue from Priestia megaterium (YclD) with the canonical UbiD from Escherichia coli leads to activation of the latter, similar in effect to co-expression with the prFMNH-binding chaperone LpdD. Conserved Glu residues in the PhdC active site suggest catalysis occurs through C1' proton-abstraction coupled oxidation. This study thus provides both structural and mechanistic insight into the function of PhdC, adding to the expanding repertoire of prFMN-binding proteins associated with the widespread UbiDX system.
Biochemical, Biophysical, and Mechanistic Insights on a Putative Oxidoreductase From Mycobacterium tuberculosis That Shares Structural Homology With Tetracycline Destructase.
Shiraz M, Biswas D, Priyanka G … +2 more , Prabhu NP, Akif M
Proteins · 2026 Apr · PMID 41351180 · Publisher ↗
Mycobacterium tuberculosis (Mtb) is the causative agent of tuberculosis. The emergence of Mtb's multidrug-resistant and extremely drug-resistant strains has imposed a great challenge for TB treatment. Hence, there is alw...
Mycobacterium tuberculosis (Mtb) is the causative agent of tuberculosis. The emergence of Mtb's multidrug-resistant and extremely drug-resistant strains has imposed a great challenge for TB treatment. Hence, there is always a demand to explore new targets that may be crucial for the survival and pathogenicity of the bacilli. Oxidoreductases are a class of enzymes that transfer electrons in various biological pathways and reactions, at the expense of cellular NADPH/NADH. Here, we analyzed oxidoreductases from the H37Rv proteome and identified two uncharacterized putative oxidoreductases, Rv1260 and Rv1714. These putative oxidoreductases showed conservation among pathogenic and opportunistic mycobacterial species and were predicted to be virulence factors essential for the pathogen's survival. The 3D structural model and amino acid sequence of one of the oxidoreductases, Rv1260, showed similarities with tetracycline destructase, a flavin-dependent monooxygenase. Thin-layer chromatography and UV-visible spectroscopic experiments confirmed the presence of the FAD molecule in a bound form with the recombinant protein. Fluorescence quenching studies demonstrated a comparatively better affinity of NADPH than NADH with the protein. The protein also displayed efficient binding with chlortetracycline. Molecular dynamics simulations were employed to gain insights into the substrate binding and conformational changes in the protein. Moreover, the importance of the substrate binding region, the C-terminal helix, and the FAD binding cavity, located near the isoalloxazine ring, was highlighted. Overall, the study provides biochemical, biophysical, and mechanistic insights into one of the putative Mtb oxidoreductases. Based on our data, we propose that this protein may perform monooxygenation functions under specific redox conditions and contribute to the redox processes in Mtb.
Computational Analyses of Bifurcated Inter-Protein Interactions in Protein-Protein Assemblies Reveal Their Pivotal Role in Conferring Stability.
Bheemireddy S, Menon R, Chavali S … +2 more , Sowdhamini R, Srinivasan N
Proteins · 2026 Apr · PMID 41315005 · Publisher ↗
Majority of the proteome is constituted by oligomers and their function is governed by underlying protein-protein interactions. Interfacial residues, namely residues right at the interface of two protein chains, are know...
Majority of the proteome is constituted by oligomers and their function is governed by underlying protein-protein interactions. Interfacial residues, namely residues right at the interface of two protein chains, are known to confer stability and specificity in dimers. However, other interactions play a significant role in the formation and maintenance of oligomers in protein assemblies as well. Inter-protein bifurcated interactions are those where one residue simultaneously interacts with two residues belonging to two neighboring protein chains. The characteristic features of such higher-order interactions remain largely unexplored and unknown. In this study, we focused on residues specifically involved in bifurcated interactions (referred as RBI). We examine the bifurcated inter-protein interactions by assembling a dataset of protein assemblies of known 3D structures. We have characterized the type of interactions and the residues involved in the interactions using parameters like energy contributions and conservation scores. We find that the residues participating in bifurcated inter-protein interactions contribute more to the stability of the complex than other interfacial residues. Furthermore, we have presented examples where mutation of a residue involved in a bifurcated interaction results in detrimental outcomes. This study highlights the significance of inter-protein bifurcated interactions that contribute to the stability of multiple interfaces in protein oligomers and hence contribute to the expansion of the understanding of protein assemblies.
Protein Involved in Initiation 1 Interaction With Starch Synthase 4 From Arabidopsis thaliana Induces Inhibition of Elongating Activity.
Bossu M, Osman R, Brysbaert G … +3 more , Lensink MF, Dauvillée D, Bompard C
Proteins · 2026 Apr · PMID 41277197 · Full text
Starch is the major energy storage compound in plants. It accumulates in the form of insoluble, partly crystalline granules whose number and shape are specific to each plant species. These characteristics are defined ver...
Starch is the major energy storage compound in plants. It accumulates in the form of insoluble, partly crystalline granules whose number and shape are specific to each plant species. These characteristics are defined very early in starch biosynthesis, at the initiation stage. Starch biosynthesis initiation is a complex process that relies on the coordinated action of several proteins that interact together in the so-called complex of initiation. Starch Synthase 4 (SS4) is the only initiation protein with enzymatic activity. It catalyzes the formation of glucan primers, which serve as substrates for the enzymatic machinery that synthesizes starch granules. Previous studies have highlighted the importance of interactions between SS4 and regulatory proteins in this process. Among them, Protein Involved in Initiation 1 (PII1) interacts with SS4 but its function is not yet established. In this study, we explored the structural and functional implications of PII1 on SS4's enzymatic activity. Our findings reveal that PII1 contains a long coiled-coil domain that specifically interacts with SS4, leading to modification of SS4's glucan elongation activity. Importantly, this interaction is specific to SS4 and does not affect other known synthases, suggesting a targeted regulatory mechanism probably through a dimerization domain. This work describes the structural specificities of PII1 and SS4 and reveals a possible function for PII1 in the initiation complex.
Elucidation of Critical Amino Acid Residues for Antibody and Transmembrane Antigen Interactions via Cell-Free Protein Expression-Based Approach.
Karbyshev MS, Baskakova KO, Kuzmichev PK … +6 more , Okhrimenko IS, Bocharov EV, Kolesnikova IM, Shatova OP, Shestopalov AV, Rumyantsev SA
Proteins · 2026 Apr · PMID 41272942 · Publisher ↗
The generation of antibodies against integral membrane proteins (IMPs) presents unique challenges due to IMPs' low natural abundance, reduced biosynthesis rate, limited level of extraction, and low purification yield. Th...
The generation of antibodies against integral membrane proteins (IMPs) presents unique challenges due to IMPs' low natural abundance, reduced biosynthesis rate, limited level of extraction, and low purification yield. That stems from their intricate structure defining the conformal epitope location. This study introduces a novel approach to identify crucial amino acid residues involved in the interaction between antibodies and tumor-associated transmembrane antigens based on utilizing a prokaryotic cell-free expression (CFPE) system. We considered human transmembrane prostate androgen-induced protein 1 (hTMEPA1) as the target antigen. It is a single-pass α-helical protein possessing hallmarks of a promising cancer biomarker. A several-step procedure was implemented to determine key residues of hTMEPA1. We created mimotope mAbs based on in silico analysis of immunogenic regions followed by target protein antigen production in the CFPE system and further antibody characterization. The results demonstrated an exceptional way for robust and high-throughput target protein production combined with in-depth biophysical and immunochemical characterization of therapeutic and diagnostic antibody-based molecules.
Molecular Dynamics Identify Variances Between Galectin Carbohydrate-Binding Sites That Impact the Binding Site Conformation and Ligand Binding.
Go RM, Kishor C, Malde AK … +1 more , Blanchard H
Proteins · 2026 Apr · PMID 41268632 · Publisher ↗
Galectins are a family of carbohydrate-binding proteins that aid in the progression of pathological conditions such as inflammation, bone disease, and cancers, making them attractive drug targets. Galectins share a conse...
Galectins are a family of carbohydrate-binding proteins that aid in the progression of pathological conditions such as inflammation, bone disease, and cancers, making them attractive drug targets. Galectins share a conserved carbohydrate recognition domain providing a focus for inhibitor design incorporating modification of carbohydrate-based scaffolds that includes the addition of aromatic moieties. Some compounds exhibit nanomolar affinities but often show poor specificity toward particular galectins; thus cross-reactivity in the body is difficult to avoid and potentially detrimental for drug therapies. The low selectivity is due to the high conservation of amino acid residues among galectins that partake in ligand binding. Critically, although these amino acids are highly conserved, there are differences in the shape and physical characteristics of the binding site due to the slight variation in the surrounding amino acid residues of the more extended regions. Using molecular dynamic simulations, the effect of amino acids associated with the galectin binding site was explored. Besides the impact of large bulky side chain amino acids such as phenylalanine, tyrosine, and histidine, a series of salt-bridge interactions were identified within some galectins that influence the resulting shape of the binding site cavity, thus affecting ligand selectivity. In silico alanine point mutations disrupting these salt-bridge interactions or replacing the bulky side chain amino acid residues led to changes in the binding site conformation impacting ligand binding, as indicated by our cluster and energetic analyses. This investigation gives further insight into the galectin carbohydrate binding site that has relevance for ligand design as potential therapeutics.
Structural Modeling and Dynamics of the Full-Length Homer1 Multimer.
Kálmán ZE, Czajlik A, Maruzs B … +7 more , Farkas F, Pap I, Homonnay C, Klumpler T, Batta G, Gáspári Z, Péterfia B
Proteins · 2026 Apr · PMID 41267651 · Full text
Homer proteins are modular scaffold molecules that constitute an integral part of the protein network within the postsynaptic density. Full-length Homer1 forms a large homotetramer via a long coiled coil region, and can...
Homer proteins are modular scaffold molecules that constitute an integral part of the protein network within the postsynaptic density. Full-length Homer1 forms a large homotetramer via a long coiled coil region, and can interact with proline-rich target sequences with its globular EVH1 domain. Here we report an atomistic model of the Homer1 coiled coil region along with the NMR solution structure and backbone dynamics of its EVH1 domain, with implications for the organization of the full-length tetramer. Compared to the already available EVH1 structures, our NMR ensemble exhibits subtle differences, mostly in and around its partner binding region, suggesting the presence of ligand-induced conformational transitions. Molecular dynamics simulations of the long coiled coil reveal distinct regions with different stability and flexibility, with the N-terminal part of the coiled coil exhibiting the largest motions. Interestingly, this segment is highly conserved, pointing to the functional relevance of the observed dynamical features. Our results indicate previously unexplored aspects of the flexibility of the full-length Homer1 tetramer that might contribute to the dynamic rearrangements of the postsynaptic protein network linked to its functional transitions.
Structure of the NAD Bound Erythrose-4-Phosphate Dehydrogenase (E4PDH) Reveals the Stabilizing Effect of Polyethylene Glycol on the Quaternary Structure.
Vijayan V, Kumari A, Singh A … +5 more , Kumar A, Sharma P, Sharma S, Singh TP, Raje CI
Proteins · 2026 Apr · PMID 41230615 · Publisher ↗
Erythrose-4-phosphate dehydrogenase (E4PDH, EC 1.2.1.72) from Acinetobacter baumannii (AbE4PDH) is an essential multifunctional enzyme. E4PDH catalyzes the first step of the deoxyxylulose-5-phosphate (DXP) dependent Vita...
Erythrose-4-phosphate dehydrogenase (E4PDH, EC 1.2.1.72) from Acinetobacter baumannii (AbE4PDH) is an essential multifunctional enzyme. E4PDH catalyzes the first step of the deoxyxylulose-5-phosphate (DXP) dependent Vitamin B6 biosynthetic pathway. It utilizes nicotinamide adenine diphosphate (NAD) as a co-factor while exhibiting dual catalytic activity wherein it converts (i) D-Erythrose-4-phosphate to 4-phosphoerythronate and (ii) Glyceraldehyde-3-phosphate to Glyceraldehyde-1,3-bisphosphate. An alternate function of AbE4PDH is the capture of human transferrin (Tf) and lactoferrin (Lf) for bacterial iron acquisition. This study provides the first X-ray crystal structures of AbE4PDH at a resolution of 2.2 Å. The recombinant enzyme was crystallized under two different conditions with (i) 30% PEG-400 (Crystal/Structure 1; PDB Id-9IIL) and (ii) 1.6 M MgSO (Crystal/Structure 2; PDB Id-9IIM). The crystal structure reveals a homo-tetramer, with an NAD bound to each monomer. The structure from Crystal 2 contained only four sulfate ions which were located in the substrate binding sites. In contrast, Crystal 1 showed the presence of several polyethylene glycol (PEG) molecules located in the center of the tetramer contributing to its stability without disturbing co-factor binding. The presence of PEG molecules induced a strong conformational change in the side chain of Gln213, inducing the formation of additional intermolecular contacts and enhancing the stability of the tetramer. Overall, this novel structure has not been observed with other dehydrogenases and may be unique to E4PDH. An insight into this structure would aid in the design of potential small molecule inhibitors to target its biochemical and alternate functions.
Rieske Iron-Sulfur Cluster Proteins From an Anaerobic Ammonium Oxidizer Suggest Unusual Energetics in Their Parent Rieske/Cytochrome b Complexes.
Hauser D, Sode M, Andreeva EA … +2 more , Parey K, Barends TRM
Proteins · 2026 Apr · PMID 41222066 · Full text
Anaerobic ammonium-oxidizing (anammox) bacteria employ a unique, hydrazine-based pathway to obtain energy from nitrite and ammonium. These organisms possess distinct Rieske/cytochrome b complexes whose precise role in an...
Anaerobic ammonium-oxidizing (anammox) bacteria employ a unique, hydrazine-based pathway to obtain energy from nitrite and ammonium. These organisms possess distinct Rieske/cytochrome b complexes whose precise role in anammox metabolism remains unclear, but which have been proposed to include the generation of NAD(P)H. This would require energetics and structural features unusual for such complexes. Here we present crystal structures and electrochemical investigations of the Rieske subunits of two of these complexes from the anammox organism Kuenenia stuttgartiensis, Kuste4569 and Kustd1480. Both proteins display high redox potentials (> + 300 mV), which can be in part explained by their crystal structures and which fit perfectly into the energetic scheme of the proposed NAD(P)H generation mechanism. Moreover, AlphaFold3 models of the parent complexes trace out a path for the electrons required for NAD(P)H production, which includes a proposed, novel b-type heme in the membrane-bound part of the complex.
Proteome-Wide Analysis of Human Deletions.
Zhang H, Luan X, Vihinen M
Proteins · 2026 Mar · PMID 41215709 · Full text
Protein deletions are frequent among both natural and pathogenic variations. Many of them are misclassified in variation databases and the literature. Nonsense-mediated decay prevents the expression of many nucleotide de...
Protein deletions are frequent among both natural and pathogenic variations. Many of them are misclassified in variation databases and the literature. Nonsense-mediated decay prevents the expression of many nucleotide deletions. Many variants classified as protein deletions are not expressed at all. We conducted an exhaustive systematic analysis of three types of deletions: N- and C-terminal deletions, as well as internal deletions within protein sequences. In addition, we compared natural and pathogenic internal deletions. We collected an extensive dataset of reliable deletions in many proteins and then performed extensive statistical analyses to investigate properties of deletions and proteins that contain them. We studied the properties of protein deletions, including deletion length and position, amino acid composition, flanking amino acid sequence context, the functions and properties of deletion-containing proteins, the functional roles of the deleted regions, the positioning within protein domains and protein structure, as well as sequence conservation and involvement in protein-protein interaction networks. We found several statistically significant differences between the deletion types and between benign and pathogenic deletions. The obtained insight can be used, for example, for variation interpretation, prediction method development, and analysis of variation mechanisms and effects.
Enhancement Effects of α4/α6-Targeted Inhibitors on Eg5-Microtubule Interaction.
Huo Z, Li J, Qin J … +1 more , Geng Y
Proteins · 2026 Apr · PMID 41215706 · Publisher ↗
Eg5 is one kind of kinesin motor that participates in various cellular processes, especially in mitosis. The tetrameric structure of Eg5 can crosslink the antiparallel microtubules and generate forces on microtubules to...
Eg5 is one kind of kinesin motor that participates in various cellular processes, especially in mitosis. The tetrameric structure of Eg5 can crosslink the antiparallel microtubules and generate forces on microtubules to separate chromosomes through walking on the microtubules. Inhibition of the activity of Eg5 leads to cell cycle arrest and apoptosis. Thus, Eg5 is a potential therapeutic target in anticancer drug development. Two inhibitor-binding sites have been discovered to date. The α2/L5/α3-targeted inhibitors can inhibit the chemical cycle of Eg5. However, drug resistance to the α2/L5/α3-targeted inhibitors is found in Eg5 with point mutations. The second inhibitor-binding site is the pocket between α4 and α6 helices. The α4/α6-targeted inhibitors can inhibit the activity of Eg5 with D130V and A133D mutations. However, the molecular mechanism of inhibition of α4/α6-targeted inhibitors is still unclear. The effects of α4/α6-targeted inhibitors on the Eg5-microtubule interaction are investigated using molecular dynamics simulations. It is found that the binding of inhibitors can induce changes in the binding conformation of Eg5 on the microtubule. With inhibitors, the number of interactions formed between Eg5 and the microtubule are increased and the binding free energies are lowered. Therefore, α4/α6-targeted inhibitors can enhance the Eg5-microtubule interaction to inhibit the mechanical cycle of Eg5. It is also found that, Leu292, Leu293, and Tyr352 of Eg5 are key residues for the binding of inhibitors. These results provide molecular insight into the inhibition of α4/α6-targeted inhibitors on the activity of Eg5.
Blind Prediction of Complex Water and Ion Ensembles Around RNA in CASP16.
Kretsch RC, Posani E, Baulin EF … +48 more , Bujnicki JM, Bussi G, Cheatham TE, Chen SJ, Elofsson A, Farsani MA, Fisher ON, Gromiha MM, Gupta A, Hamada M, Harini K, Hu G, Huang D, Iwakiri J, Jain A, Kagaya Y, Kihara D, Kmiecik S, Krishnan SR, Kurisaki I, Languin-Cattoën O, Li J, Li S, Malekzadeh K, Nakamura T, Ni W, Nithin C, Palo MZ, Park JH, Pilla SP, Poblete S, Pucci F, Punuru P, Saha A, Sato K, Srivastava A, Terashi G, Tugolukova E, Verburgt J, Wuyun Q, Zerze GH, Zhang K, Zhang S, Zheng W, Zhou Y, Chiu W, Case DA, Das R
Proteins · 2026 Jan · PMID 41204761 · Full text
Biomolecules rely on water and ions for stable folding, but these interactions are often transient, dynamic, or disordered and thus hidden from experiments and evaluation challenges that represent biomolecules as single,...
Biomolecules rely on water and ions for stable folding, but these interactions are often transient, dynamic, or disordered and thus hidden from experiments and evaluation challenges that represent biomolecules as single, ordered structures. Here, we compare blindly predicted ensembles of water and ion structure to the cryo-EM densities observed around the Tetrahymena ribozyme at 2.2-2.3 Å resolution, collected through target R1260 in the CASP16 competition. Twenty-six groups participated in this solvation "cryo-ensemble" prediction challenge, submitting over 350 million atoms in total, offering the first opportunity to compare blind predictions of dynamic solvent shell ensembles to cryo-EM density. Predicted atomic ensembles were converted to density through local alignment and these densities were compared to the cryo-EM densities using Pearson correlation, Spearman correlation, mutual information, and precision-recall curves. These predictions show that an ensemble representation is able to capture information of transient or dynamic water and ions better than traditional atomic models, but there remains a large accuracy gap to the performance ceiling set by experimental uncertainty. Overall, molecular dynamics approaches best matched the cryo-EM density, with blind predictions from bussilab_plain_md, SoutheRNA, bussilab_replex, coogs2, and coogs3 outperforming the baseline molecular dynamics prediction. This study indicates that simulations of water and ions can be quantitatively evaluated with cryo-EM maps. We propose that further community-wide blind challenges can drive and evaluate progress in modeling water, ions, and other previously hidden components of biomolecular systems.
Large Extent of Convergent Evolution Towards the Double Histone Fold Revealed by Targeted Sequence and Structure Search Approach.
Miyake T, Ranaudo A, Sacco E … +1 more , Greco C
Proteins · 2026 Apr · PMID 41200785 · Publisher ↗
Histone proteins are key players in chromatin packaging. In eukaryotes, nucleosomal cores-the DNA packaging fundamental units-are formed by composition of histone dimers. The double histone fold is a protein structure wh...
Histone proteins are key players in chromatin packaging. In eukaryotes, nucleosomal cores-the DNA packaging fundamental units-are formed by composition of histone dimers. The double histone fold is a protein structure where two consecutive regions, each featuring histone fold, come together to create a histone pseudodimer. Although regarded as an uncommon fold to date, in this study we show-by protein structure and sequence analyses-that the double histone fold is widespread in eukaryotes. Perspectives of such outcome are discussed in terms of novel directions that our results may open in diverse areas, from epigenetics to the design of DNA-binding proteins.
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