ABSTRACT Membrane association is governed by the thermodynamics of amino acid partitioning between water and the lipid bilayer. Here, we quantified amino acid side-chain insertion energetics in a 1-palmitoyl-2-oleoyl- sn -glycero-3-phosphocholine (POPC) bilayer using unbiased molecular dynamics simulations. Equilibrium depth distributions of 28 analogs, including multiple protonation states, were converted into potentials of mean force (PMFs) by Boltzmann inversion. The resulting PMFs reproduced the main features of bilayer partitioning. Hydrophobic analogs favored the bilayer core, aromatic analogs were stabilized in interfacial regions, and polar or charged analogs remained unfavorable in the hydrophobic interior. A diglycine analog representing the peptide backbone behaved similarly to uncharged polar residues. Depth-dependent pK a profiles and orientational analyses further showed how protonation equilibria and aromatic-ring alignment influence insertion energetics. Agreement with experimental hydrophobicity scales supports the robustness of the approach. These results provide an efficient and internally consistent framework for characterizing bilayer insertion energetics and establish a reference for future studies in more complex lipid environments. Abstract Figure SIGNIFICANCE Membrane-associated proteins represent a large fraction of the proteome and include many major drug targets, yet quantitative understanding of their interactions with lipid bilayers remains limited. Here, we present an unbiased molecular dynamics framework for systematically determining amino acid side-chain insertion free energies in a model bilayer. By deriving potentials of mean force directly from equilibrium depth distributions, this approach enables internally consistent comparisons across residue classes and protonation states without biasing restraints. The resulting free-energy profiles reproduce established hydrophobicity trends and show how protonation equilibria and aromatic-ring orientation modulate bilayer partitioning. This scalable strategy provides a quantitative reference for residue-level membrane thermodynamics and establishes a foundation for extending insertion energetics to more diverse lipid compositions and more complex membrane-associated systems.

One of the front-line drug classes used to treat invasive fungal infections is echinocandins, which target the fungal-specific beta-glucan synthase (Fks). Treatment failure due to resistance often coincides with mutations in 3 protein regions defined as hotspots. Unfortunately, the scarcity of the mutational data reported, combined with the large size and membrane-embedded nature of the enzyme hinder any effort to characterize genotype-phenotype links. Recent advances in solving the structure of Fks bring us one step closer to reliable predictions of the binding modes of each echinocandin. To help with that endeavor, we used molecular dynamics simulations to develop a membrane-embedded model of Fks that captures key structural and environmental features. Our results show that the 3 hotspots shape a single solvent-exposed binding cavity, hinting at the orientation and positioning of echinocandins. This structural framework is integrated with deep-mutational scanning to comprehensively assess the impact of mutations across the 3 hotspots in the model yeast Saccharomyces cerevisiae. We elucidate several key molecular bases of resistance to the 3 most widely used echinocandins; anidulafungin, caspofungin and micafungin and provide clues to better understand intrinsic resistance of critical fungal pathogens.

Tyrosine decarboxylases (TyDCs) are pyridoxal 5'-phosphate-dependent enzymes that catalyze the conversion of l-tyrosine into tyramine, a key entry point for Amaryllidaceae alkaloid biosynthesis. From transcriptome data of Narcissus pseudonarcissus 'King Alfred', two full-length TyDC isoforms, NpTyDC1 and NpTyDC2, were identified. In this study, both genes were cloned, heterologously expressed in Escherichia coli, and purified for functional characterization. Fluorescent protein tagging indicated nucleocytoplasmic localization of both isoforms. Enzymatic assays coupled with HPLC-MS/MS demonstrated that NpTyDC1 exhibits decarboxylase activity with a strong preference for l-tyrosine, producing tyramine as the predominant product. In contrast, NpTYDC2 showed no detectable tyrosine decarboxylase activity under the conditions tested, and no tryptophan decarboxylation despite structural features associated with indole decarboxylases but displayed minor activity toward l-phenylalanine. Collectively, these findings establish NpTyDC1 as a strong candidate for directing tyrosine into the Amaryllidaceae alkaloid biosynthesis. By integrating computational predictions with biochemical validation, this study provides new insight into the TyDC functional diversity in Narcissus.

Allosteric modulation enables precise control of protein activity but remains difficult to harness for selective inhibitor design. Traditional high-throughput screening for allosteric modulators is still costly and time-consuming, underscoring the need for predictive computational approaches. Here, we combined network and shortest-path analyses to predict interprotomer communication nodes that regulate the pro-apoptotic activity of human galectin-7 (GAL-7). We identify a minimal electrostatic network (R20-R22-D103) as a key allosteric node controlling dimer stability and signal transmission between the two distant glycan binding sites. Our predictions guided the engineering of four variants (R20A, R22A, D103A, and R20A-R22A), all of which impaired GAL-7-induced apoptosis in human T cells. Biophysical and structural analyses confirmed that disrupting the R20-D103 interaction weakens interprotomer communication and destabilizes the dimer, while compensatory edges partially restore connectivity. These results demonstrate that residue-network fingerprinting enables predictive mapping of global communication pathways and reveal R20, R22, and D103 as key allosteric determinants of GAL-7 function. The integrative framework introduced here can be extended to identify and exploit allosteric communication pathways in other homodimeric proteins, offering a generalizable strategy for rational modulator design.

Galanthamine, a therapeutic Amaryllidaceae alkaloid produced exclusively by species within the Amaryllidoideae subfamily, is a key treatment for early-stage symptoms of Alzheimer′s disease. Elucidating its biosynthetic pathway is essential for strategies aimed at enhancing production through metabolic engineering. Galantamine derives from the metabolic precursor 4′-O-methylnorbelladine, which undergoes cytochrome P450-mediated para-ortho′ C-C phenol coupling to yield nornarwedine. Two competing terminal routes have been proposed: (i) reduction of nornarwedine to norgalanthamine, followed by N-methylation, or (ii) N-methylation of nornarwedine to narwedine prior to reduction. Here, we identify three N-methyltransferase (NMT) candidates from Leucojum aestivum: LaNMT, related to coclaurine NMTs, and two γ-tocopherol methyltransferases (TMT) homologs, LaTMT1 and LaTMT2. Subcellular localization studies revealed distinct compartmentalization, with LaNMT targeted to the ER-cytosol, LaTMT1 to plastids, and LaTMT2 to the Golgi apparatus. In vitro enzyme assays demonstrated that LaTMT2 methylates both nornarwedine and norgalanthamine, with a kinetic preference for nornarwedine. Agroinfiltration for transient expression in Nicotiana benthamiana further confirmed LaTMT2 as a catalytically efficient and substrate-promiscuous enzyme that supports both terminal routes. These findings identify LaTMT2 as a key branch-enabling N-methyltransferase that reconcile long-standing models of galanthamine biosynthesis and provides a strategic target for metabolic engineering strategies to enhance galanthamine production. ### Competing Interest Statement The authors have declared no competing interest. Canada Research Chair, CRC-2023-00353

The molecular interactions between a fungal membrane model and SJGAP, a 32-amino-acid antimicrobial peptide (AMP) derived from skipjack tuna GAPDH, as well as four analogs, were investigated using molecular dynamics simulations and Fourier transform infrared (FTIR) spectroscopy. In a previous study, Analog 7, modified by replacing three alanine residues with leucine residues, exhibited unique antifungal activity without any antibacterial effect. This contrasts with other analogs, which showed both antifungal and antibacterial effects. In this study, Analog 7 displayed the strongest interactions with the membrane's hydrophobic core, inserting more deeply and causing significantly greater membrane deformation and thinning compared with the other analogs. Its presence caused significant membrane deformation, evident from the displacement of both the phosphate groups and terminal methyls of the lipids. Notably, Analog 7 was the only analog to induce a marked depletion of ergosterol around the peptide insertion site. FTIR spectroscopy experiments further confirmed the distinctive impact of Analog 7 on a fungal membrane model. The combined results from molecular dynamics simulations and spectroscopy emphasize the critical role of leucine substitutions in Analog 7, particularly at residues 18 and 19 within the central α helix, in promoting membrane thinning and inducing ergosterol depletion, suggesting increased membrane permeabilization, which could explain its previously reported antifungal specificity. This study provides the first insights into the molecular interactions between a GAPDH-derived AMP and a fungal membrane model, offering valuable information about its antifungal mechanism of action.

BACKGROUND:Antibiotic resistance is a major issue affecting all spheres of human activity, including agriculture. One significant example is the Avian Pathogenic Escherichia coli (APEC), a bacterium that infects poultry and leads to substantial economic losses in the farming industry. As antibiotics lose efficacity, bacteriophages (phages) -viruses that specifically target bacteria-are emerging as a promising alternative to antibiotics for treating and preventing bacterial infections. However, bacteria can develop resistance to phages through various mechanisms. Studying the coevolution between a phage and its host bacterium is important to gain insight into the phage's potential as a therapeutic agent. This study investigates the evolutionary responses of an APEC strain and a laboratory E. coli strain to a commercial phage originally isolated from APEC. RESULTS:In most cases, phage resistance resulted in a significant increase in mucoidy. Genomic analysis revealed that this resistance consistently correlated with amino acid changes, particularly in proteins involved in colanic acid production, such as YrfF. Further investigation of a mutation found in the YrfF protein demonstrated that this mutation altered the protein's structure and its interaction with the membrane. Transcriptomic analysis confirmed that the genes involved in colanic acid production were significantly overexpressed. Although the strains possessed a CRISPR-Cas system, it did not contribute to phage resistance. CONCLUSIONS:This study suggests that specific amino acid changes in key proteins may be a mechanism employed by E. coli, including APEC, to defend against phage infections.

Amaryllidaceae alkaloids are a diverse group of alkaloids exclusively reported from the Amaryllidaceae plant family. In planta, their biosynthesis is still not fully characterized; however, a labeling study established 4'-O-methylnorbelladine as the key intermediate compound of the pathway. Previous reports have characterized O-methyltransferases from several Amaryllidaceae species. Nevertheless, the formation of the different O-methylnorbelladine derivatives (3'-O-methylnorbelladine, 4'-O-methylnorbelladine, and 3'4'-O-dimethylnorbelladine), the role, and the preferred substrates of O-methyltransferases are not clearly understood. In this study, we performed the biochemical characterization of an O-methyltransferase candidate from Narcissus papyraceus (NpOMT) in vitro and in vivo, following biotransformation of norbelladine in Nicotiana benthamiana having transient expression of NpOMT. Docking analysis was further used to investigate substrate preferences, as well as key interacting residues of NpOMT. Our study shows that NpOMT methylates norbelladine preferentially at the 4'-OH position in vitro and in planta. Interestingly, NpOMT also catalyzed the synthesis of 3',4'-O-dimethylnorbelladine from norbelladine and 4'-O-methylnorbelladine during in vitro enzymatic assay. Furthermore, we show that NpOMT methylates 3,4-dihydroxybenzylaldehyde and caffeic acid in a nonregiospecific manner to produce meta/para monomethylated products. This study reveals a novel catalytic potential of an Amaryllidaceae O-methyltransferase and its ability to regioselectively methylate norbelladine in the heterologous host N. benthamiana.

One of the front-line drug classes used to treat invasive fungal infections is echinocandins, which target the fungal-specific beta-glucan synthase (Fks). Treatment failure due to resistance often coincides with mutations in two protein regions known as hotspots. The biophysical bases by which such mutations confer resistance and cross-resistance among echinocandins are largely unknown. Here, we use deep-mutational scanning to quantify the resistance level of 660 mutations in the hotspots of two homologous Fks. We detail the constraints acting on drug binding and explain the resistance specificity for some mutations using the drug-protein interactions from our molecular models. Our findings will enable DNA sequence-based predictions of resistance to this important drug family and the improvement of future molecules that could overcome current resistance mutations. One sentence summary Disruption of specific interactions between echinocandins and key residues from their target lead to drug-specific resistance. ### Competing Interest Statement The authors have declared no competing interest.

Interactions between SJGAP (skipjack tuna GAPDH-related antimicrobial peptide) and four analogs thereof with model bacterial membranes were studied using Fourier-transform infrared spectroscopy (FTIR) and molecular dynamics (MD) simulations. MD trajectory analyses showed that the N-terminal segment of the peptide analogs has many contacts with the polar heads of membrane phospholipids, while the central α helix interacts strongly with the hydrophobic core of the membranes. The peptides also had a marked influence on the wave numbers associated with the phase transition of phospholipids organized as liposomes in both the interface and aliphatic chain regions of the infrared spectra, supporting the interactions observed in the MD trajectories. In addition, interesting links were found between peptide interactions with the aliphatic chains of membrane phospholipids, as determined by FTIR and from the MD trajectories, and the membrane permeabilization capacity of these peptide analogs, as previously demonstrated. To summarize, the combined experimental and computational efforts have provided insights into crucial aspects of the interactions between the investigated peptides and bacterial membranes. This work thus makes an original contribution to our understanding of the molecular interactions underlying the antimicrobial activity of these GAPDH-related antimicrobial peptides from Scombridae.

The outer membrane (OM) of gram-negative bacteria serves as a vital organelle that is densely populated with OM proteins (OMPs) and plays pivotal roles in cellular functions and virulence. The assembly and insertion of these OMPs into the OM represent a fundamental process requiring specialized molecular chaperones. One example is the translocation and assembly module (TAM), which functions as a transenvelope chaperone promoting the folding of specific autotransporters, adhesins, and secretion systems. The catalytic unit of TAM, TamA, comprises a catalytic β-barrel domain anchored within the OM and three periplasmic polypeptide-transport-associated (POTRA) domains that recruit the TamB subunit. The latter acts as a periplasmic ladder that facilitates the transport of unfolded OMPs across the periplasm. In addition to their role in recruiting the auxiliary protein TamB, our data demonstrate that the POTRA domains mediate interactions with the inner surface of the OM, ultimately modulating the membrane properties. Through the integration of X-ray crystallography, molecular dynamic simulations, and biomolecular interaction methodologies, we located the membrane-binding site on the first and second POTRA domains. Our data highlight a binding preference for phosphatidylglycerol, a minor lipid constituent present in the OM, which has been previously reported to facilitate OMP assembly. In the context of the densely OMP-populated membrane, this association may serve as a mechanism to secure lipid accessibility for nascent OMPs through steric interactions with existing OMPs, in addition to creating favorable conditions for OMP biogenesis.

Pneumocystis jirovecii is a fungal pathogen that causes pneumocystis pneumonia, a disease that mainly affects immunocompromised individuals. This fungus has historically been hard to study because of our inability to grow it in vitro . One of the main drug targets in P. jirovecii is its dihydrofolate reductase (PjDHFR). Here, by using functional complementation of the baker’s yeast ortholog, we show that PjDHFR can be inhibited by the antifolate methotrexate in a dose-dependent manner. Using deep mutational scanning of PjDHFR, we identify mutations conferring resistance to methotrexate. Thirty-one sites spanning the protein have at least one mutation that leads to resistance, for a total of 355 high-confidence resistance mutations. Most resistance-inducing mutations are found inside the active site, and many are structurally equivalent to mutations known to lead to resistance to different antifolates in other organisms. Some sites show specific resistance mutations, where only a single substitution confers resistance, whereas others are more permissive, as several substitutions at these sites confer resistance. Surprisingly, one of the permissive sites (F199) is without direct contact to either ligand or cofactor, suggesting that it acts through an allosteric mechanism. Modeling changes in binding energy between F199 mutants and drug shows that most mutations destabilize interactions between the protein and the drug. This evidence points towards a more important role of this position in resistance than previously estimated and highlights potential unknown allosteric mechanisms of resistance to antifolate in DHFRs. Our results offer unprecedented resources for the interpretation of mutation effects in the main drug target of an uncultivable fungal pathogen.

Variants of filamin C (FLNC) have been identified as rare genetic substrate for hypertrophic cardiomyopathy (HCM). Data on the clinical course of FLNC-related HCM are conflicting with some studies suggesting mild phenotypes whereas other studies have reported more severe outcomes. In this study, we present a novel FLNC variant (Ile1937Asn) that was identified in a large family of French-Canadian descent with excellent segregation data. FLNC-Ile1937Asn is a novel missense variant characterized by full penetrance and poor clinical outcomes. End stage heart failure requiring transplantation occurred in 43% and sudden cardiac death in 29% of affected family members. Other particular features of FLNC-Ile1937Asn include an early disease onset (mean age of 19 years) and the development of a marked atrial myopathy (severe biatrial dilatation with remodeling and multiple complex atrial arrhythmias) that was present in all gene carriers. The FLNC-Ile1937Asn variant is a novel, pathogenic mutation resulting in a severe form of HCM with full disease penetrance. The variant is associated with a high proportion of end-stage heart failure, heart transplantation, and disease-related mortality. Close follow-up and appropriate risk stratification of affected individuals at specialized heart centers is recommended.

The antimicrobial activity of SJGAP (skipjack tuna GAPDH-related antimicrobial peptide) and four chemical analogs thereof was determined under different physicochemical conditions, including different pH values, the presence of monovalent and divalent cations, and after a heating treatment. The toxicity of these five peptides was also studied with hemolytic activity assays, while their stability under human gastrointestinal conditions was evaluated using a dynamic in vitro digestion model and chromatographic and mass spectrometric analyses. The antibacterial activity of all analogs was found to be inhibited by the presence of divalent cations, while monovalent cations had a much less pronounced impact, even promoting the activity of the native SJGAP. The peptides were also more active at acidic pH values, but they did not all show the same stability following a heat treatment. SJGAP and its analogs did not show significant hemolytic activity (except for one of the analogs at a concentration equivalent to 64 times that of its minimum inhibitory concentration), and the two analogs whose digestibility was studied degraded very rapidly once they entered the stomach compartment of the digestion model. This study highlights for the first time the characteristics of antimicrobial peptides from Scombridae or homologous to GAPDH that are directly related to their potential clinical or food applications.

Mining practices, chiefly froth flotation, are being critically reassessed to replace their use of biohazardous chemical reagents in favor of biofriendly alternatives as a path toward green processes. In this regard, this study aimed at evaluating the interactions of peptides, as potential floatation collectors, with quartz using phage display and molecular dynamics (MD) simulations. Quartz-selective peptide sequences were initially identified by phage display at pH = 9 and further modeled by a robust simulation scheme combining classical MD, replica exchange MD, and steered MD calculations. Our residue-specific analyses of the peptides revealed that positively charged arginine and lysine residues were favorably attracted by the quartz surface at basic pH. The negatively charged residues at pH 9 (i.e., aspartic acid and glutamic acid) further showed affinity toward the quartz surface through electrostatic interactions with the positively charged surface-bound Na+ ions. The best-binding heptapeptide combinations, however, contained both positively and negatively charged residues in their composition. The flexibility of peptide chains was also shown to directly affect the adsorption behavior of the peptide. While attractive intrapeptide interactions were dominated by a weak peptide-quartz binding, the repulsive self-interactions in the peptides improved the binding propensity to the quartz surface. Our results showed that MD simulations are fully capable of revealing mechanistic details of peptide adsorption to inorganic surfaces and are an invaluable tool to accelerate the rational design of peptide sequences for mineral processing applications.

The structure–activity relationships and mode of action of synthesized glyceraldehyde-3-phosphate dehydrogenase (GAPDH)-related antimicrobial peptides were investigated. Including the native skipjack tuna GAPDH-related peptide (SJGAP) of 32 amino acid residues (model for the study), 8 different peptide analogs were designed and synthesized to study the impact of net charge, hydrophobicity, amphipathicity, and secondary structure on both antibacterial and antifungal activities. A net positive charge increase, by the substitution of anionic residues or C-terminal amidation, improved the antimicrobial activity of the SJGAP analogs (minimal inhibitory concentrations of 16–64 μg/mL), whereas the alpha helix content, as determined by circular dichroism, did not have a very definite impact. The hydrophobicity of the peptides was also found to be important, especially for the improvement of antifungal activity. Membrane permeabilization assays showed that the active peptides induced significant cytoplasmic membrane permeabilization in the bacteria and yeast tested, but that this permeabilization did not cause leakage of 260 nm-absorbing intracellular material. This points to a mixed mode of action involving both membrane pore formation and targeting of intracellular components. This study is the first to highlight the links between the physicochemical properties, secondary structure, antimicrobial activity, and mechanism of action of antimicrobial peptides from scombrids or homologous to GAPDH.

The design of allosteric modulators to control protein function is a key objective in drug discovery programs. Altering functionally essential allosteric residue networks provides unique protein family subtype specificity, minimizes unwanted off-target effects, and helps avert resistance acquisition typically plaguing drugs that target orthosteric sites. In this work, we used protein engineering and dimer interface mutations to positively and negatively modulate the immunosuppressive activity of the proapoptotic human galectin-7 (GAL-7). Using the PoPMuSiC and BeAtMuSiC algorithms, mutational sites and residue identity were computationally probed and predicted to either alter or stabilize the GAL-7 dimer interface. By designing a covalent disulfide bridge between protomers to control homodimer strength and stability, we demonstrate the importance of dimer interface perturbations on the allosteric network bridging the two opposite glycan-binding sites on GAL-7, resulting in control of induced apoptosis in Jurkat T cells. Molecular investigation of G16X GAL-7 variants using X-ray crystallography, biophysical, and computational characterization illuminates residues involved in dimer stability and allosteric communication, along with discrete long-range dynamic behaviors involving loops 1, 3, and 5. We show that perturbing the protein-protein interface between GAL-7 protomers can modulate its biological function, even when the overall structure and ligand-binding affinity remains unaltered. This study highlights new avenues for the design of galectin-specific modulators influencing both glycan-dependent and glycan-independent interactions.

The interaction between the 20 natural amino acids and the surfaces of sphalerite, pyrite, and chalcopyrite was examined in the light of density functional theory (DFT) simulations. The electronic properties of the active functional groups and the equilibrium geometries of the sorbed amino acid species were characterized as a function the 65 species resulting from their (de)protonations. Covalent bonds are predicted between the surface metallic atoms and the carboxyl groups and, though to a lesser extent, with the side and main amino groups. Chemisorption as a determining factor in the uptake of amino acid species was assessed in terms of sorption affinities for the mineral surfaces. The largest affinities result from the carboxyl groups of the deprotonated species at intermediate pH, whereas the fully protonated amino acids in acidic media predict the lowest affinities. Affinity of amino acids towards the mineral surfaces followed the trend: pyrite >> chalcopyrite > sphalerite with speciation-dependent decreasing sequence: similar to 2.5 < pH < similar to 9.5; pH > similar to 9.5; pH < similar to 2.5. By targeting the most suitable amino acid building blocks in bio-sourced collectors, either high collecting powers or superior selectivities can be aimed at depending on pH for improved separation of sulphidic minerals in flotation processes.

Mammalian acetylcholinesterase (AChE) is well-studied, being important in both cholinergic brain synapses and the peripheral nervous systems and also a key drug target for many diseases. In contrast, little is known about the structures and molecular mechanism of prokaryotic acetylcholinesterases. We report here the structural and biochemical characterization of ChoE, a putative bacterial acetylcholinesterase fromPseudomonas aeruginosa. Analysis of WT and mutant strains indicated that ChoE is indispensable forP. aeruginosagrowth with acetylcholine as the sole carbon and nitrogen source. The crystal structure of ChoE at 1.35 ? resolution revealed that this enzyme adopts a typical fold of the SGNH hydrolase family. Although ChoE and eukaryotic AChEs catalyze the same reaction, their overall structures bear no similarities constituting an interesting example of convergent evolution. Among Ser-38, Asp-285, and His-288 of the catalytic triad residues, only Asp-285 was not essential for ChoE activity. Combined with kinetic analyses of WT and mutant proteins, multiple crystal structures of ChoE complexed with substrates, products, or reaction intermediate revealed the structural determinants for substrate recognition, snapshots of the various catalytic steps, and the molecular basis of substrate inhibition at high substrate concentrations. Our results indicate that substrate inhibition in ChoE is due to acetate release being blocked by the binding of a substrate molecule in a nonproductive mode. Because of the distinct overall folds and significant differences of the active site between ChoE and eukaryotic AChEs, these structures will serve as a prototype for other prokaryotic acetylcholinesterases.