Biochemical Journal

Alkaline ceramidase 2 (ACER2) catalyzes the hydrolysis of ceramides into sphingosine and free fatty acids, playing crucial roles in regulating various biological processes, including but not limited to cell proliferation, survival, and the DNA damage response. However, a pharmacological tool that regulates its catalytic and signaling functions remains to be developed. In this study, N-[(1S,2R)-1-hydroxy-1-phenylpropan-2-yl]dodecane-1-sulfonamide (KPB82) was identified as a potent and selective ACER2 inhibitor. In vitro enzymatic assays showed that KPB82 inhibited ACER2 with an IC50 of 190 nM, while displaying minimal activity against acid and neutral ceramidases (IC50> 100 {micro}M) and no significant inhibition of alkaline ceramidase 1 or alkaline ceramidase 3. Enzymatic kinetic studies revealed that KPB82 is a competitive inhibitor of ACER2. The three-dimensional structure of ACER2 was modeled in the AlphaFold3 server, revealing key Zn2+-coordinating histidine residues and a substrate-binding pocket. Molecular docking studies demonstrated that KPB82 binds ACER2 within this pocket, forming hydrogen bonds with the catalytically important amino residues, Asp93 and His215, and interacting with several hydrophobic residues. Lactate dehydrogenase release assays revealed that KPB82 significantly reduced cytotoxicity in HeLa cells overexpressing ACER2, as well as in HCT116 cells, RT4 cells, and human umbilical vein endothelial cells treated with the DNA-damaging agent doxorubicin. Lipidomic analysis showed that KPB82 inhibited sphingosine production in response to ACER2 overexpression or doxorubicin treatment. Collectively, these findings demonstrate that KPB82 is a potent and selective ACER2 inhibitor capable of suppressing ACER2-dependent biological processes and highlight its potential as a tool for studying ceramide metabolism and ACER2-mediated signaling pathways.

Human Ca2+/calmodulin (CaM)-dependent protein kinase II alpha (CaMKII) is a highly abundant and conserved protein in the brain. It is a multifunctional serine/threonine kinase that phosphorylates cellular substrates across subcellular compartments, activating signaling pathways essential for long-term memory storage. However, a detailed understanding of protein structure and enzyme-substrate interaction mechanisms require a method to produce active, stable, and pure proteins at scale. Since phosphorylation can alter catalytic activity and substrate affinity, examining the non-phosphorylated state enables an unbiased characterization of basal enzymatic function. Therefore, producing the non-phosphorylated form of the kinase is important for biochemical and structural studies aimed at understanding kinase activation mechanisms. We describe a detailed protocol for the expression and high-yield purification of full-length, non-phosphorylated human CaMKII in bacterial cells. CaMKII was co-expressed with {lambda}-phosphatase to maintain it in a non-phosphorylated state. Protein expression was induced by autoinduction at low temperature to enhance the stability and solubility of CaMKII. Recombinant CaMKII was purified by Ni-NTA affinity chromatography, followed by gel filtration chromatography. Although CaMKII has been considered difficult to purify in its non-phosphorylated form, we obtained it in high yield, and the protein was predominantly non-phosphorylated. Our results show that combining co-expression with autoinduction improves both the solubility and yield of the homogeneous non-phosphorylated form of CaMKII, which could be suitable for subsequent biochemical and structural studies.

Azetidine-2-carboxylic acid (Aze) is a toxic, non-proteogenic amino acid produced in restricted plants. It can enter the human food chain through direct or indirect dietary exposure and has been linked to neurological disorders due to its ability to misincorporate for its structural analog proline (Pro) during translation and lead to protein misfolding. Although several microorganisms have evolved mechanisms to detoxify Aze, these pathways have not previously been exploited to engineer Aze tolerance in plants. Here, we heterologously expressed a bacterial Aze hydrolase (HAS) in Arabidopsis thaliana to establish a metabolic strategy for Aze detoxification in plants. Transgenic plants exhibited markedly enhanced tolerance to Aze, as demonstrated by improved root growth, cotyledon development, and survival compared with wild-type plants under Aze stress. We further showed that HAS localized to the cytosol, where it converted Aze into the non-toxic metabolite 2-hydroxy-4-aminobutyrate (HAB). HAB formation was confirmed both in planta and in vitro, demonstrating that HAS remained catalytically active in plant cells and effectively detoxified Aze. Enzyme kinetics revealed that HAS has a high affinity for Aze and is stereospecific for L-Aze, with no activity toward D-Aze. Multi-omics analysis including transcriptomics and misincorporation proteomics show that the transgenic lines misincorporate Aze significantly less compared to wild-type leading to enhanced Aze tolerance. Our findings establish bacterial HAS as an effective metabolic detoxification system that confers enhanced Aze tolerance in plants. This study provides a promising strategy for engineering plant resistance to toxic non-proteogenic amino acids.

Maintenance of membrane homeostasis is essential for bacterial viability, yet the molecular functions of many membrane proteins involved in this process remain poorly understood. The widely distributed DedA superfamily of integral membrane proteins has been implicated in membrane homeostasis, with deletion of DedA genes resulting in sensitivities to temperature, pH and a range of antimicrobial compounds. Several bacterial DedA proteins have been linked to lipid transport, providing a potential connection between function and phenotype. However, a lack of direct functional and structural data means that the precise role of DedA proteins in bacterial membrane homeostasis remains unclear. Here, using analytical ultracentrifugation (AUC), we show that the Escherichia coli DedA protein YqjA exists predominantly as a dimer and, by combining structural modelling with site-specific cysteine crosslinking, we have identified the most likely dimer interface. Modelling of dimeric YqjA in the presence of lipids predicts an interfacial lipid-binding site located in close proximity to several conserved, functionally important residues. Consistent with lipid binding, addition of lipid substantially increased the thermal stability of YqjA. Furthermore, mutation of residues associated with the predicted binding site impaired YqjA function. Together, these findings provide new structural and functional insights into the DedA family and support a role for lipid binding in the activity of YqjA.

Calcium (Ca2+) is a universal second messenger that orchestrates plant growth, development, and stress responses. Plant calcineurin B-like (CBL) proteins sense and relay Ca2+; signals in response to pathogens to activate downstream defense signalling pathways. Early blight disease, caused by the necrotrophic fungal pathogen Alternaria solani, affects tomato crops and severely limits their productivity. In the present study, we found that the tolerant tomato cultivar (Arka Rakshak) restricts A. solani infection and disease progression compared to the susceptible cultivar (PKM-1). Comparative transcriptome and qRT-PCR analysis of A. solani-infected tomato leaves showed that SlCBL1-2 (Solyc08g007160.3), a Ca2+; sensor, is up-regulated in the tolerant cultivar, both relative to its own uninfected control and the infected susceptible cultivar, whereas it was not significantly induced in the uninfected susceptible cultivar. Protein sequence analysis indicates that SlCBL1-2 contains four EF-hand motifs, an N-terminal N-myristoylation/S-acylation module and a C-terminal FPSF motif, and it is predicted to interact with several CBL-interacting serine/threonine protein kinases (CIPKs). However, the evolutionary analysis, conformational transitions upon binding to Ca2+, and the Ca2+-binding affinity of SlCBL1-2 have not been previously characterized. Molecular Dynamics (MD) simulation of the AlphaFold structure of SlCBL1-2 indicates a well-folded structure and binding of two Ca2+ ions at EF-hands 3 and 4. Further, far-UV circular dichroism and tryptophan fluorescence spectroscopy of recombinant SlCBL1-2 indicate a natively folded structure in the absence of Ca2+ and undergo minimal secondary and tertiary structural changes upon Ca2+-binding, in contrast to several Ca2+-binding proteins. Of note, SlCBL1-2 binds Ca2+ with apparent KD values of 526 {+/-} 113 {micro}M and 28.7 {+/-} 9.47 {micro}M, as evidenced by isothermal titration calorimetry. Together, our study provides an experimental description of the Ca2+-binding properties of tomato CBL protein and defines the molecular basis by which pathogen-induced SlCBL1-2 may act as a Ca2+ sensor in defense signalling.

Background. Alternative oxidase (AOX) is a mitochondrial terminal oxidase that provides an alternative route for electron transfer, contributing to respiratory flexibility and redox homeostasis under stress conditions. In trypanosomatids, AOX displays a highly uneven distribution associated with extensive diversification of mitochondrial electron transport chains (ETCs). In addition to canonical AOX, many trypanosomatids encode an enigmatic AOX-like protein (AOX-L), whose evolutionary origin and functional significance remain unresolved. Results. Here, we investigated the evolutionary distribution, phylogenetic relationships, structural conservation, and functional divergence of AOX and AOX-L across euglenozoans. Comparative genomic analyses revealed a patchy distribution of both proteins among kinetoplastids, with multiple independent losses during evolution. Phylogenetic analyses demonstrated that AOX and AOX-L represent distinct evolutionary lineages, indicating independent origins rather than duplication-derived divergence. While AOX showed a conserved eukaryotic origin, AOX-L occupied a separate phylogenetic position and lacked key residues required for ubiquinol oxidation. Structural modelling revealed that AOX-L retained the characteristic AOX fold, predicted membrane association, and dimeric organization, but lacked the catalytic architecture necessary for enzymatic activity. Transcriptomic and biochemical analyses of four AOX-containing trypanosomatid species showed that AOX expression and activity correlated with mitochondrial ETC organization. Species lacking cytochrome-dependent complexes III and IV displayed substantially higher AOX expression and enzymatic activity, consistent with AOX functioning as the primary terminal oxidase in these lineages. Conversely, species retaining a canonical ETC exhibited lower AOX activity, suggesting a role in metabolic flexibility and redox regulation. Conclusions. Our study provides a comprehensive evolutionary and functional framework for alternative oxidases in trypanosomatids. We demonstrate that AOX and AOX-L are evolutionarily distinct proteins with different predicted functions: AOX maintains respiratory electron flow according to lineage-specific mitochondrial requirements, whereas AOX-L represents a structurally conserved but catalytically inactive protein family that may have acquired an alternative regulatory role. These findings highlight how mitochondrial respiratory components diversify during eukaryotic evolution and provide a basis for future investigations into the biological function of AOX-L.

The sulfur mobilization (SUF) system is the only Fe-S cluster biosynthesis pathway in Mycobacterium tuberculosis (M. tb) and Mycolicibacterium smegmatis (M. smegmatis). In this pathway, SufC protein acts as an ATPase in the SufBC2D scaffold for Fe-S cluster biogenesis. This crucial role positions SufC as a promising target for the development of antimycobacterial therapeutics. However, the SufC ATPases from Mycobacterial species are not yet characterized. This study includes cloning, expression, purification, and characterization of SufC homologs from M. tb (Rv1463) and M. smegmatis (MSMEG_3124) using biochemical, biophysical, and computational approaches. Both SufC proteins were purified using affinity and size-exclusion chromatography and observed to exist as monomers in solution. Although both proteins have the conserved domains, they exhibit different ATP-dependent kinetic efficiencies, demonstrating differences in their local environment and catalytic behavior. Furthermore, CRISPR interference targeting MSMEG_3124 results led to diminished M. smegmatis growth, highlighting the critical role of SufC in cellular viability. Modeling and virtual screening led to the identification of three candidate compounds, of which Z2911048515 and Z66741142 were selected for experimental evaluation. Circular dichroism spectroscopy analysis revealed slight differences in the spectral features of Rv1463 in the presence of Z2911048515 and Z66741142, while significant spectral variations were observed for MSMEG_3124. Importantly, Z66741142 was able to cause changes in the fluorescence spectra of both SufC protein homologs. Surprisingly, both compounds showed only limited inhibition of SufC ATPase activity, exhibited distinct modes of action, and may serve as preliminary chemical scaffolds for optimization and testing as effective inhibitors. This study presents functional diversity of mycobacterial SufC proteins across pathogenic and non-pathogenic species and provides a basis for developing a framework for further mechanistic and structure-guided investigation within the indispensable SUF pathway.

Structure-aware proteolysis prediction often ranks candidate cleavage sites using solvent accessibility from static protein structures, yet this assumption is rarely tested against native-state cleavage data. We examined this problem using AprX, a serralysin-family protease that readily hydrolyses caseins but shows lower activity toward native whey proteins. Across six bovine milk proteins, sequence compatibility and local scissile-bond accessibility did not separate caseins from whey proteins. In contrast, accessibility fell progressively in whey proteins as the required contiguous substrate window increased, reaching 0% across the working P4-P4' footprint, while all evaluable casein windows remained accessible in isolated-chain ensembles. Local remodelling showed that whey segments could still adopt AprX-compatible backbone geometry. An external thermolysin benchmark rejected a rigid whole-protein clash metric, but did not alter the contiguous-accessibility result. These findings identify footprint-scale accessibility as a distinct structural variable and show why single-residue solvent exposure can overestimate protease accessibility in folded proteins.

Ferroptosis has attracted significant attention for its potential to treat drug resistant tumors, along with its role in neurodegeneration and ischemia. Several ferroptosis targets were identified in the past decade, yet it is also highly affected by cellular metabolism and epithelial or mesenchymal phenotypes which limits the utility of these targets across diverse cell and tissue types. To address this, we utilized chemo-resistant cancer cell lines with different metastatic potential and tissue origins and discovered a common role of cytoplasmic ion changes during ferroptosis. We observed significant changes in intracellular potassium, sodium and chloride levels upon induction of ferroptosis. Therefore, we screened several ion channel inhibitors and activators (i.e. modulators) and identified Kv7.2 and CFTR modulators influence ferroptosis execution despite the fact that these cell lines do not express these proteins. We demonstrated that clinically approved modulators of CFTR and Kv7.2 can inhibit ferroptosis which indicates that these inhibitors could be repurposed as treatments in ferroptosis-associated diseases such as neurodegeneration and kidney diseases independent of ion channel expression. These modulators also influenced potassium and chloride levels in different contexts indicating off-target effects may also lead to changes in ion levels and may serve as a ferroptosis target. Further, we demonstrated that sustained imbalance of sodium, potassium and chloride ions with ferroptosis inducer salinomycin and digoxin, a Na+/K+ ATPase inhibitor, enhanced ferroptosis. The combination of salinomycin and digoxin successfully enhanced ferroptotic cell death across both cell lines with different metabolic signatures in vitro, offering a novel ferroptosis target. This combination significantly reduced tumor growth in vivo compared with the single agent alone, offering a new therapeutic modality against drug resistant tumors.

A challenge in synthetic biology is to transfer the knowledge acquired in vitro to in vivo applications. For instance, binding affinity between peptide binding domains (PBDs) and their binding peptides is usually measured in simple buffers and in the absence of the cell machinery responsible for synthesis and proteostasis. Consequently, there is often a discrepancy between interaction quantified in vitro and in vivo. To identify strongly binding PBD-peptide pairs for in vivo applications, we aim to better understand the determinants of synthetic interaction in living yeast cells. We designed libraries of peptides expected to bind to PBDs based on data collected by phage-display. Using an in vivo protein interaction assay, we assessed the interaction strength of >6,500 PBD-peptide variants in yeast. We found that most substitutions disrupt binding, but that 3.4% increased binding strength to levels higher than expected from the phage-display data. We asked if, by using our mutational data, it was possible to optimize peptide sequences to gain even more binding strength and show additivity effect up to four substitutions. We found that PBD specificity is consistent between in vitro and in vivo assays. We hypothesized that in vivo peptide abundance could contribute to the discrepancy between the PCA and the phage-display results. We performed assays to detect in vivo peptide availability, reporting on peptide abundance, and we found that peptides with a stronger binding also show a higher availability in cells. We show that peptide availability, which seems mainly defined by peptide hydrophobicity, is key for the design of strong synthetic interactions.

Background: Positive selection shapes protein function by favoring amino acid substitutions that increase fitness. Although numerous studies have identified positively selected genes (PSGs), the structural principles underlying residue-level positive selection remain incompletely understood and previous studies have produced conflicting conclusions, particularly regarding the role of intrinsically disordered regions. In order to resolve these issues and to better understand the driving forces underlying positive selection in human proteins in general, we performed a comprehensive analysis on a manually curated, high quality collection of PSGs and positively selected residues (PSRs) as well as on a large-scale PSR dataset restricted to proteins with experimental structures. Results: Human PSRs were significantly enriched in secreted and cell membrane proteins and preferentially localized to extracellular regions, solvent-exposed surfaces, coil structures, and protein-protein interaction interfaces, whereas neither domains nor intrinsically disordered regions showed an enrichment. Spatial clustering analysis reinforced that PSRs accumulate within localized surface patches. Integration of host-pathogen interaction data highlighted that adaptive changes preferentially affect extracellular molecular recognition surfaces, recurrently appearing in certain pathogen-interacting membrane protein families and particular domain types. Additionally, PSRs showed an enrichment in the residues of human-pathogen interaction interfaces that are in direct contact with pathogenic proteins, implying that neutralizing pathogen attacks is one of the driving forces behind the adaptive evolution of human proteins. By analysing human variation data we found that PSRs are enriched in benign but depleted in pathogenic substitutions, extending previous observations that positively selected genes contain elevated levels of missense variation. Notably, PSGs of the large-scale dataset were also enriched among clinical-stage drug targets, suggesting a potential link between positive selection and pharmacological relevance. Conclusion: Together, our results provide a refined structural model of adaptive evolution in human proteins and identify extracellular exposed molecular recognition surfaces and human-pathogen interaction interfaces as recurrent hotspots of positive selection. Our results point to the direction that precise identification of human PSRs could delineate important host-pathogen interfaces that exert evolutionary selection pressure on humans and appoint novel targets with therapeutic potential.

Sulfation plays an important role in many metabolic processes such as substrate detoxification, solubilization or activation. The enzymes responsible for these reactions, sulfotransferases, occur in all domains of life and can be classified in two major, evolutionary independent groups, PAPS dependent and PAPS independent or arysulfate sulfotransferases (ASST) enzymes. Besides the fact that they play key roles in biological functions, sulfotransferases can be interesting enzymatic tools to selectively sulfate specific positions of various metabolites. To this end we need to understand the relation between sequence, structure and function. Actually, eukaryotic PAPS dependent sulfotransferases have extensively been studied, while we largely lack knowledge about the diversity of bacterial PAPS-independent ASSTs. We have conducted a multidisciplinary study of a bacterial sulfotransferase from Salmonella typhimurium, revealing the importance of active site residues for activity and guiding substrate specificity. Based on the crystal structure in complex with substrate, we used molecular docking combined with Quantum Mechanics/Molecular Mechanics (QM/MM) free energy calculations to investigate the reaction catalytic mechanism with a focus on the role of specific active site residues in substrate specificity. Our results show that the catalytic mechanism involving three conserved histidines is common with previously determined ASSTs, and highlight a specific pair of aromatic residues, Phe174 and Tyr557, that play an important role in defining the substrate specificity in ASST clade 5. These findings enhance our general understanding of how substrate specificity is attained and has the potential to guide further enzyme engineering to expand the acceptor scope of ASSTs.

Lipoate is an essential protein-bound cofactor attached to conserved lysine residues in structurally related acceptor proteins, including GcvH of the glycine cleavage system and LbpA proteins required for sulfur oxidation by the sulfur-oxidizing heterodisulfide-like (sHdr) complex. The coexistence of distinct lipoylation pathways and acceptor proteins raises the question of how the enzymes initiating lipoate attachment discriminate between homologous but functionally distinct substrates. We systematically tested lipoate:protein ligases and potential acceptor proteins from five representatives of the bacterial phyla Pseudomonadota and Aquificota using native gel-shift assays and MALDI-TOF mass spectrometry. The canonical Escherichia coli lipoate:protein ligase Lpl(AB) preferentially modified GcvH proteins but showed little or no activity toward LbpAs. In contrast, sulfur oxidation-associated lipoate:protein ligases, termed sLpl(AB), which function in the sLpl(AB)-LipS1/LipS2 lipoate assembly pathway, strongly preferred LbpA proteins and generally failed to modify GcvH. Conserved cysteine residues characteristic of LbpAs were dispensable for recognition by sLpl(AB). Instead, comparison of amino acid sequences and electrostatic surfaces revealed predominantly acidic interaction surfaces in GcvH proteins and corresponding basic regions in LbpAs. Accordingly, replacement of selected basic regions in Roseovarius mucosus LbpA2 by acidic residues reduced modification by its cognate sLpl(AB) and simultaneously enabled weak modification by E. coli Lpl(AB). These findings establish electrostatic surface properties as major determinants of substrate discrimination by lipoate ligases and show that recognition depends on distributed features of the folded acceptor protein rather than solely on the sequence surrounding the lipoylated lysine.

Protein O-mannosylation in the endoplasmic reticulum (ER) regulates the maturation and function of extracellular proteins, yet the substrate spectrum and biological functions of individual O-mannosyltransferases remain incompletely understood. TMEM260 is an ER-resident O-mannosyltransferase that modifies immunoglobulin-like, plexins, transcription factors (IPT) domains. Here, we identify fibrocystin and fibrocystin-L, encoded by PKHD1 and PKHD1L1, respectively, as novel TMEM260-mediated O-mannosylated proteins. By combining screening of human proteomic datasets with mass spectrometry-based analysis of IPT domains expressed in TMEM260-deficient HEK293 cells, we demonstrate that multiple IPT domains of fibrocystin and fibrocystin-L are modified by TMEM260. Our findings expand the known substrate requirements of TMEM260 by revealing tolerance of substantial sequon variation and demonstrating serine as an alternative acceptor residue within IPT domains. In humans, loss-of-function variants in TMEM260 cause structural heart defects and renal anomalies (SHDRA) syndrome, whereas pathogenic variants in fibrocystin cause autosomal recessive polycystic kidney disease (ARPKD). Notably, patients with SHDRA and ARPKD share overlapping renal phenotypes, suggesting that impaired TMEM260 function may phenocopy aspects of fibrocystin-associated diseases. Given that fibrocystin is a substrate protein for TMEM260-mediated O-mannosylation, these findings raise the possibility that functional impairment of TMEM260 causes aberrant O-mannosylation of fibrocystin. Thereby fibrocystin function might be compromised leading to the renal phenotype observed in patients with TMEM260 mutations. To directly assess the functional relevance of this modification, we expressed recombinant fibrocystin fragments in TMEM260-deficient cells complemented with either wild-type TMEM260 or a catalytically inactive TMEM260 mutant. Further, we investigate the O-mannosylation of the threonine residue directly C-terminal to the ARPKD-associated patient mutation L1407R. Strikingly, substitution of leucine L1407 by the patient-associated arginine residue, completely abolished O-mannosylation of the adjacent threonine, demonstrating that this disease-associated mutation directly disrupts a TMEM260-dependent O-mannosylation site. Together, our findings establish fibrocystin and fibrocystin-L as TMEM260-dependent O-mannosylated proteins and reveal a potential molecular mechanism linking defective TMEM260-mediated O-mannosylation to the renal phenotype of SHDRA, which overlaps with PKHD1-associated ARPKD.

Opening of the mitochondrial permeability transition (PT) pore is an important step in necrotic cell death in various pathologic conditions such as ischemia-reperfusion injury. Methylglyoxal (MGO) is a byproduct of glycolysis that covalently modifies proteins (i.e., glycation) and is widely regarded as toxic (glycative stress), contributing to the pathology of diabetes. Paradoxically, MGO has been shown to inhibit PT pore opening, but the mechanism of this effect is poorly understood. Herein we show that glycation is elevated in various cardioprotective scenarios. Furthermore, building on a serendipitous finding of the ability of phosphate to block MGO's effect on the PT pore, we used proteomics to map potential sites mediating this effect. From this analysis, mitochondrial creatine kinase (CK) emerged as a phosphate-sensitive MGO target with links to the PT pore. Although MGO inhibited CK activity, this did not appear to be important for its effect on the PT pore. Rather, MGO modified sites on CK that are important for its membrane binding, which likely mediates the combined effects of MGO and phosphate on the PT pore. Furthermore, a consensus sequence for MGO modifications in mitochondria is reported.

MAPK-activated protein kinase 2 (MAPKAPK2, MK2) is a downstream substrate of p38 MAPK that regulates inflammatory signaling and cytokine synthesis, making it an attractive therapeutic target. Although several crystal structures of MAPKAPK2 with ATP-competitive inhibitors have supported drug discovery, structural elucidation of selective non-ATP-competitive inhibitors has remained challenging. MK-25 is a non-ATP-competitive furan-2-carboxamide MAPKAPK2 inhibitor developed at Merck to overcome the selectivity and cellular-efficacy limitations of ATP-competitive chemotypes. Here, we show through isothermal titration calorimetry that MK-25 binds the activated MAPKAPK2, consistent with state-dependent allosteric binding. To determine the binding mode of MK-25, we solved a 2.8 A resolution crystal structure of an activated MAPKAPK2 construct in ternary complex with the ATP-site inhibitor PF-3644022 and a bromo-analogue of MK-25. The ligand occupies a pocket in the C-lobe overlapping the protein-substrate recognition region and formed through rearrangements of D and G helices and the activation segment. These interactions are consistent with the structure-activity relationships reported for the furan-2-carboxamide chemotype and identify a ligandable allosteric pocket that may facilitate future structure-guided design of selective, non-ATP-competitive MAPKAPK2 inhibitors.

Dysregulation of insulin-like growth factor (IGF) signaling contributes to diverse human diseases. Therapeutic strategies have largely targeted the IGF-1 receptor, but the complexity of the IGF system has limited their clinical efficacy. Pregnancy-associated plasma protein-A (PAPP-A) is a secreted metalloproteinase that regulates local IGF signaling through proteolytic cleavage of IGF-binding proteins (IGFBPs) and represents an attractive therapeutic target. Here, we report the first synthetic PAPP-A inhibitors designed to target the enzyme active site using an IGFBP-5-derived anchor sequence. Replacing the scissile bond with phosphinate or phosphonate moieties yielded potent and selective PAPP-A inhibitors. Cryo-electron microscopy revealed the molecular basis of inhibition and key active-site interactions. The compounds inhibited cleavage of IGFBP-4 and IGFBP-5, two physiological PAPP-A substrates. They suppressed IGF-1 signaling in A549 lung cancer cells, with efficacy comparable to the endogenous PAPP-A inhibitor stanniocalcin-1. They also inhibited Papp-aa-dependent IGF signaling and cell proliferation in wild-type and stc1a-/- mutant zebrafish in vivo in a dose-dependent manner. Together, these findings establish the first class of synthetic, selective PAPP-A inhibitors capable of modulating IGF signaling. They provide new chemical tools for studying and modulating pathological IGF signaling and offer new directions for developing PAPP-A-targeted therapeutic strategies.

Nitrate is an important oxyanion in bacterial physiology, with major roles as a nitrogen source for assimilation and as an alternative electron acceptor in anaerobic respiration. Accordingly, diverse nitrate-responsive regulatory systems sense nitrate availability and control the expression of genes involved in nitrate transport and metabolism. In the NreABC system, the soluble GAF-like domain protein NreA functions as a cellular nitrate sensor. To date, our molecular understanding of nitrate recognition by NreA has been derived primarily from studies of the Staphylococcus carnosus protein, with less known about homologues from other species. Here, we identified 736 NreA homologues through bioinformatic analysis of the GAF-like domain superfamily and selected five staphylococcal representatives for thermodynamic characterization by isothermal titration calorimetry. Despite conservation of the nitrate-binding pocket, these homologues resolved into high- and low-affinity groups with distinct thermodynamic profiles. Comparative structural modeling and chimeragenesis of a low-affinity homologue revealed that the C-terminal region is a non-coordinating determinant of nitrate-binding affinity. Together, these findings show that a conserved nitrate-binding pocket preserves recognition, while distal sequence variation shapes thermodynamics and tunes the nitrate concentration range over which NreA sensors can function.

Exploring the ability of metabolic enzymes to interact with diverse small molecules can provide insights into their ligand recognition and activity regulation. Glucose-6-phosphate isomerase (GPI) is an enzyme that catalyzes the second step of glycolysis, a critical node that links glucose metabolism to other cytosolic pathways, such as pentose phosphate and hexosamine biosynthetic pathways. Despite its importance in cellular metabolism, little is known about the broad ligandability of GPI. Here, we performed a structure-based virtual screening using molecular docking to identify new modulators predicted to engage with the human GPI active site. Enzyme kinetic and binding studies confirmed eleven compounds as active-site binders of GPI, exhibiting inhibitory activities and binding affinities in upper micromolar to sub-millimolar range. The binding of several ligands to GPI active site was further supported by X-ray crystallographic data. In addition, in vitro assays were performed to evaluate the modulatory effects of the GPI hits on cellular glycolysis and viability. The identified inhibitors included the mono-phosphorylated metabolite of ribavirin, which inhibited glycolytic flux and cell viability; two compounds with phosphate bioisosteres; and endogenous phosphate-containing metabolites from the pentose phosphate, hexosamine, nucleotide, and mevalonate pathways. Collectively, these findings expanded the repertoire of human GPI ligands and revealed previously unrecognized GPI-metabolite interactions that may contribute to modulation of glycolytic flux.

Although use of AI-based tools has gained ground in molecular biology research, their reliability remains a matter of debate. Here, we combined AlphaFold 3 modelling with microscale thermophoresis to investigate the interactions between glucan-binding protein C and the biofilm-inhibiting aptamer and its five derivatives. Structural modelling identified a binding motif on the aptamer and suggested a mechanism of action; however, the predictions were only partially supported by the experimental results. The AlphaFold generated confidence metrics did not correlate directly with the measured binding affinities, indicating that confidence scores can help identify plausible interaction interfaces, but they are not reliable predictors of binding strength. These results highlight the importance of integrating structural prediction with biophysical validation to dissect aptamer-protein recognition.