Initial Binding Research Articles

The hapten rigidity improves antibody performances in immunoassay for rifamycins: Immunovalidation and molecular mechanism

The immunogenicity of haptens determines the performance of the resultant antibody for small molecules. Rigidity is one of the basic physicochemical properties of haptens. However, few studies have investigated the effect of hapten rigidity on the strength of an immune response and overall antibody performance. Herein, we introduce three molecular descriptors that quantify hapten rigidity. By using of these descriptors, four rifamycin haptens with varied rigidity were designed. The structural and physicochemical feasibility of the designed haptens was then assessed by computational chemistry. Immunization demonstrated that the strength of induced immune responses, i.e., the titer and affinity of antiserum, was significantly increased with increased rigidity of haptens. Furthermore, molecular dynamic simulations demonstrated conformation constraint of rigid haptens contributed to the initial binding and activation of naïve B cells. Finally, a highly sensitive indirect competitive enzyme-linked immunosorbent assay was developed for detection of rifaximin, with an IC50 of 1.1 μg/L in buffer and a limit of detection of 0.2–11.3 μg/L in raw milk, river water, and soil samples. This work provides new insights into the effect of hapten rigidity on immunogenicity and offers new hapten design strategies for antibody discovery and vaccine development of small molecules.

Tertiary Plasticity Drives the Efficiency of Enterocin 7B Interactions with Lipid Membranes.

The ability of antimicrobial peptides to efficiently kill their bacterial targets depends on the efficiency of their binding to the microbial membrane. In the case of enterocins, there is a three-part interaction: initial binding, unpacking of helices on the membrane surface, and permeation of the lipid bilayer. Helical unpacking is driven by disruption of the peptide hydrophobic core when in contact with membranes. Enterocin 7B is a leaderless enterocin antimicrobial peptide produced from Enterococcus faecalis that functions alone, or with its cognate partner enterocin 7A, to efficiently kill a wide variety of Gram-stain positive bacteria. To better characterize the role that tertiary structural plasticity plays in the ability of enterocin 7B to interact with the membranes, a series of arginine single-site mutants were constructed that destabilize the hydrophobic core to varying degrees. A series of experimental measures of structure, stability, and function, including CD spectra, far UV CD melting profiles, minimal inhibitory concentrations analysis, and release kinetics of calcein, show that decreased stabilization of the hydrophobic core is correlated with increased efficiency of a peptide to permeate membranes and in killing bacteria. Finally, using the computational technique of adaptive steered molecular dynamics, we found that the atomistic/energetic landscape of peptide mechanical unfolding leads to free energy differences between the wild type and its mutants, whose trends correlate well with our experiment.

Genetic analysis of translation initiation in bacteria: An initiator tRNA-centric view.

Translation of messenger RNA (mRNA) in bacteria occurs in the steps of initiation, elongation, termination, and ribosome recycling. The initiation step comprises multiple stages and uses a special transfer RNA (tRNA) called initiator tRNA (i-tRNA), which is first aminoacylated and then formylated using methionine and N10 -formyl-tetrahydrofolate (N10 -fTHF), respectively. Both methionine and N10 -fTHF are produced via one-carbon metabolism, linking translation initiation with active cellular metabolism. The fidelity of i-tRNA binding to the ribosomal peptidyl-site (P-site) is attributed to the structural features in its acceptor stem, and the highly conserved three consecutive G-C base pairs (3GC pairs) in the anticodon stem. The acceptor stem region is important in formylation of the amino acid attached to i-tRNA and in its initial binding to the P-site. And, the 3GC pairs are crucial in transiting the i-tRNA through various stages of initiation. We utilized the feature of 3GC pairs to investigate the nuanced layers of scrutiny that ensure fidelity of translation initiation through i-tRNA abundance and its interactions with the components of the translation apparatus. We discuss the importance of i-tRNA in the final stages of ribosome maturation, as also the roles of the Shine-Dalgarno sequence, ribosome heterogeneity, initiation factors, ribosome recycling factor, and coevolution of the translation apparatus in orchestrating a delicate balance between the fidelity of initiation and/or its leakiness to generate proteome plasticity in cells to confer growth fitness advantages in response to the dynamic nutritional states.

The amphipathic design in helical antimicrobial peptides.

Amphipathicity is a critical characteristic of helical antimicrobial peptides (AMPs). The hydrophilic region, primarily composed of cationic residues, plays a pivotal role in the initial binding to negatively charged components on bacterial membranes through electrostatic interactions. Subsequently, the hydrophobic region interacts with hydrophobic components, inducing membrane perturbation, ultimately leading to cell death, or inhibiting intracellular function. Due to the extensive diversity of natural and synthetic AMPs with regard to the design of amphipathicity, it is complicated to study the structure-activity relationships. Therefore, this work aims to categorize the common amphipathic design and investigate their impact on the biological properties of AMPs. Besides, the connection between current structural modification approaches and amphipathic styles was also discussed.

Molecular insights into ferric-siderophore transport by the putative TonB-dependent transporter in Mycobacterium tuberculosis

Iron acquisition is critical to the virulence of Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis. To acquire iron within the host, Mtb secretes siderophores that chelate iron with high affinity. Siderophores scavenge iron from host cells using TonB-dependent transporters like FecA. We investigated molecular mechanisms of FecA-mediated ferric-siderophore transport in Mtb. Molecular docking and molecular dynamics simulations revealed a series of interactions between ferric siderophores and FecA. The initial binding occurs at a pocket located on the extracellular surface of FecA. The ligand then migrates deeper through the transport tunnel to a subsequent binding site, aided by conformational changes in FecA that expand the tunnel diameter. We observed the key roles of precise positioning of extracellular loops in the outer membrane barrel and plug domains in the optimal ligand binding and transport. Transport of ferric–siderophore complex into Mtb follows an induced fit model, with ligand interaction eliciting 2–10 Å shifts in the barrel and plug regions. By revealing the conformational dynamics enabling iron import, these findings provide molecular-level insights into a metal ion uptake mechanism in Mtb. Iron acquisition is essential for Mtb pathogenesis, so this work may inform novel therapeutic strategies that disrupt siderophore uptake pathways. Communicated by Ramaswamy H. Sarma

Fluorescently Modified NDM-1: A Versatile Drug Sensor for Rapid In Vitro β-Lactam Antibiotic and Inhibitor Screening.

We successfully developed a fluorescent drug sensor from clinically relevant New Delhi metallo-β-lactamase-1 (NDM-1). The F70 residue was chosen to be replaced with a cysteine for conjugation with thiol-reactive fluorescein-5-maleimide to form fluorescent F70Cf, where "f" refers to fluorescein-5-maleimide. Our proteolytic studies of unlabeled F70C and labeled F70Cf monitored by electrospray ionization-mass spectrometry (ESI-MS) revealed that fluorescein-5-maleimide was specifically linked to C70 in 1:1 mole ratio (F70C:fluorophore). Our drug sensor (F70Cf) can detect the β-lactam antibiotics cefotaxime and cephalothin by giving stronger fluorescence in the initial binding phase and then declining fluorescence signals as a result of the hydrolysis of the antibiotics into acid products. F70Cf can also detect non-β-lactam inhibitors (e.g., l-captopril, d-captopril, dl-thiorphan, and thanatin). In all cases, F70Cf exhibits stronger fluorescence due to inhibitor binding and subsequently sustained fluorescence signals in a later stage. Native ESI-MS results show that F70Cf can bind to all four inhibitors. Moreover, our drug sensor is compatible with a high-throughput microplate reader and has the capability to perform in vitro drug screening.

Unraveling the atomic mechanisms underlying glyphosate insensitivity in EPSPS: implications of distal mutations

5-enolpyruvyl shikimate-3-phosphate synthase (EPSPS), as an indispensable enzyme in the shikimate pathway, is the specific target of grasser killer glyphosate (GPJ). GPJ is a competitive inhibitor of phosphoenolpyruvate (PEP), which is the natural substrate of EPSPS. A novel Ls-EPSPS gene variant discovered from Liliaceae, named ELs-EPSPS, includes five distal mutations, E112V, D142N, T351S, D425G, and R496G, endowing high GPJ insensitivity. However, the implicit molecular mechanism of the enhanced tolerance/insensitivity of GPJ in ELs-EPSPS is not fully understood. Herein, we try to interpret the hidden molecular mechanism using computational methods. Computational results reveal the enhanced flexibility of apo EPSPS upon mutations. The enhanced affinity of the initial binding substrate shikimate-3-phosphate (S3P), and the higher probability of second ligands PEP/GPJ entering the pocket are observed in the ELs-EPSPS-S3P system. Docking and MD results further confirmed the decreased GPJ-induced EPSPS inhibition upon mutations. And, the alterations of K98 and R179 side-chain orientations upon mutations are detrimental to GPJ binding at the active site. Additionally, the oscillation of side chain K98, in charge of PEP location, improves the proximity effect for substrates in the dual-substrate systems upon mutations. Our results clarify that the enhanced GPJ tolerance of EPSPS is achieved from decreased competitive inhibition of GPJ at the atomic perspective, and this finding further contributes to the cultivation of EPSPS genes with higher GPJ tolerance/insensitivity and a mighty renovation for developing glyphosate-resistant crops. Communicated by Ramaswamy H. Sarma

BRD4354 Is a Potent Covalent Inhibitor against the SARS-CoV-2 Main Protease.

Numerous organic molecules are known to inhibit the main protease (MPro) of SARS-CoV-2, the pathogen of Coronavirus Disease 2019 (COVID-19). Guided by previous research on zinc-ligand inhibitors of MPro and zinc-dependent histone deacetylases (HDACs), we identified BRD4354 as a potent inhibitor of MPro. The in vitro protease activity assays show that BRD4354 displays time-dependent inhibition against MPro with an IC50 (concentration that inhibits activity by 50%) of 0.72 ± 0.04 μM after 60 min of incubation. Inactivation follows a two-step process with an initial rapid binding step with a KI of 1.9 ± 0.5 μM followed by a second slow inactivation step, kinact,max of 0.040 ± 0.002 min-1. Native mass spectrometry studies indicate that a covalent intermediate is formed where the ortho-quinone methide fragment of BRD4354 forms a covalent bond with the catalytic cysteine C145 of MPro. Based on these data, a Michael-addition reaction mechanism between MPro C145 and BRD4354 was proposed. These results suggest that both preclinical testing of BRD4354 and structure-activity relationship studies based on BRD4354 are warranted to develop more effective anti-COVID therapeutics.

Tuning the Selectivity of Nitrate Reduction via Fine Composition Control of RuPdNP Catalysts.

Herein, aqueous nitrate (NO3 -) reduction is used to explore composition-selectivity relationships of randomly alloyed ruthenium-palladium nanoparticle catalysts to provide insights into the factors affecting selectivity during this and other industrially relevant catalytic reactions. NO3 - reduction proceeds through nitrite (NO2 -) and then nitric oxide (NO), before diverging to form either dinitrogen (N2) or ammonium (NH4 +) as final products, with N2 preferred in potable water treatment but NH4 + preferred for nitrogen recovery. It is shown that the NO3 - and NO starting feedstocks favor NH4 + formation using Ru-rich catalysts, while Pd-rich catalysts favor N2 formation. Conversely, a NO2 - starting feedstock favors NH4 + at ≈50 atomic-% Ru and selectivity decreases with higher Ru content. Mechanistic differences have been probed using density functional theory (DFT). Results show that, for NO3 - and NO feedstocks, the thermodynamics of the competing pathways for N-H and N-N formation lead to preferential NH4 +or N2 production, respectively, while Ru-rich surfaces are susceptible to poisoning by NO2 - feedstock, which displaces H atoms. This leads to a decrease in overall reduction activity and an increase in selectivity toward N2 production. Together, these results demonstrate the importance of tailoring both the reaction pathway thermodynamics and initial reactant binding energies to control overall reaction selectivity.

Addressing Suboptimal Poses in Nonequilibrium Alchemical Calculations.

Alchemical transformations can be used to quantitatively estimate absolute binding free energies at a reasonable computational cost. However, most of the approaches currently in use require knowledge of the correct (crystallographic) pose. In this paper, we present a combined Hamiltonian replica exchange nonequilibrium alchemical method that allows us to reliably calculate absolute binding free energies, even when starting from suboptimal initial binding poses. Performing a preliminary Hamiltonian replica exchange enhances the sampling of slow degrees of freedom of the ligand and the target, allowing the system to populate the correct binding pose when starting from an approximate docking pose. We apply the method on 6 ligands of the first bromodomain of the BRD4 bromodomain-containing protein. For each ligand, we start nonequilibrium alchemical transformations from both the crystallographic pose and the top-scoring docked pose that are often significantly different. We show that the method produces statistically equivalent binding free energies, making it a useful tool for computational drug discovery pipelines.

Docking and molecular dynamic simulations of Mithramycin-A and Tolfenamic acid against Sp1 and survivin

Therapeutic targeting of Sp1 transcription factor and survivin, are studied in various cancers due to their consistent overexpression. These markers result in poorer cancer prognoses and their downregulation has been investigated as an effective treatment approach. Mithramycin-A and Tolfenamic acid are two drugs with innate anti-cancer properties and are suggested to be able to target Sp1 through GC/GT DNA binding interference, however in-depth binding and mechanistic studies are lacking. Through docking analysis, we investigated Mithramycin-A and Tolfenamic acid in terms of their specific binding interactions with Sp1 and survivin. Through further molecular dynamics simulations including Root Mean Square (RMS) Fluctuation and RMS Deviation, rGYr, and H-bond analysis, we identified critical residues involved in drug interactions with each protein in question. We show Mithramycin-A as the superior binding candidate to each protein and found that it exhibited stronger binding with Sp1, and then survivin. Subsequent molecular dynamics simulations followed the same trend as initial binding energy calculations and showed crucial amino acids involved in each Mithramycin-A-protein complex. Our findings warrant further investigation into Mithramycin-A and its specific interaction with Sp1 and their downstream targets giving a better understanding of Mithramycin-A and its potential as an effective cancer treatment.

Occurrence and characterization of β-lactamase-producing bacteria in biomedical wastewater and in silico enhancement of antibiotic efficacy.

Wastewater discharged from hospitals is a recognized contributor to the dissemination of antibiotic-resistant bacteria and their associated genetic traits into the environment. This study focused on the analysis of β-lactamase-producing pathogenic bacteria within untreated biomedical wastewater originating from various hospitals in Dhaka City, Bangladesh, as well as in silico evaluation and structural activity relationship mentioned antibiotics were evaluated. In silico drug design techniques were applied to identify the relationship with how the functional group impacts the binding energy. Out of the 184 isolates obtained from well-established hospital sewage discharge points in Dhaka, 89 were identified as β-lactamase positive. These bacteria were subjected to antimicrobial susceptibility testing using the VITEK-2 assay, and their profiles of extended-spectrum beta-lactamase (ESBL) production were determined through molecular methodologies. Among the β-lactamase-positive isolates, considerable resistance was observed, particularly against ampicillin, Ceftriaxone, Cefuroxime, and Meropenem. The predominant resistant species included Escherichia coli, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter cloacae. The study identified the prevalence of ESBL-producing genes, with blaNDM-1 being the most prevalent, followed by blaOXA-1, blaSHV, blaCTX-M, and blaKPC. None of the isolates carried the blaTEM gene. In addition to characterizing these bacteria, the research explored ways to enhance the binding energy of four existing antibiotics as new inhibitors through computational studies. The findings revealed significant improvements in binding energy. Specifically, Meropenem initially exhibited a binding energy of -7.5 kcal/mol, notably increasing to -8.3 kcal/mol after modification. With an initial binding energy was only -7.9 kcal/mol, Ampicillin experienced an enhancement, reaching -8.0 kcal/mol post-modification. Similarly, Ceftriaxone, with an initial binding energy of -8.2 kcal/mol, increased to -8.5 kcal/mol following structural adjustments. Finally, Cefuroxime, initially registering a binding energy of -7.1 kcal/mol, substantially increased to -8.9 kcal/mol after modification. This finding establishes a foundation for future investigations in the development of modified antibiotics to address the issue of antibiotic resistance. It presents prospective remedies for the persistent problem of antibiotic-resistant bacteria in healthcare and the environment.

Trapping a non-cognate nucleotide upon initial binding for replication fidelity control in SARS-CoV-2 RNA dependent RNA polymerase.

The RNA dependent RNA polymerase (RdRp) in SARS-CoV-2 is a highly conserved enzyme responsible for viral genome replication/transcription. To understand how the viral RdRp achieves fidelity control during such processes, here we computationally investigate the natural non-cognate vs. cognate nucleotide addition and selectivity during viral RdRp elongation. We focus on the nucleotide substrate initial binding (RdRp active site open) to the prechemical insertion (active site closed) of the RdRp. The current studies were first carried out using microsecond ensemble equilibrium all-atom molecular dynamics (MD) simulations. Due to the slow conformational changes (from open to closed) during nucleotide insertion and selection, enhanced or umbrella sampling methods have been further employed to calculate the free energy profiles of the nucleotide insertion. Our studies find notable stability of noncognate dATP and GTP upon initial binding in the active-site open state. The results indicate that while natural cognate ATP and Remdesivir drug analogue (RDV-TP) are biased toward stabilization in the closed state to facilitate insertion, the natural non-cognate dATP and GTP can be well trapped in off-path initial binding configurations and prevented from insertion so that to be further rejected. The current work thus presents the intrinsic nucleotide selectivity of SARS-CoV-2 RdRp for natural substrate fidelity control, which should be considered in antiviral drug design.

Initiation factor 3 bound to the 30S ribosomal subunit in an initial step of translation.

Bacterial ribosomes require three initiation factors IF1, IF2, and IF3 during the initial steps of translation. These IFs ensure correct base pairing of the initiator tRNA anticodon with the start codon in the mRNA located at the P-site of the 30S ribosomal subunit. IF3 is one of the first IFs to bind to the 30S and plays a crucial role in the selection of the correct start codon and codon: anticodon base pairing. IF3 also prevents the premature association of the 50S subunit of ribosomes and aids in ribosome recycling. IF3 is reported to change binding sites and conformation to ensure translation initiation fidelity. A recent study suggested an initial binding of IF3 CTD away from the P-site and that IF1 and IF2 promote the movement of CTD to the P-site and concomitant movement of NTD. Hence, to visualize the position of IF3 in the absence of any other IFs, we determined cryo-EM structure of the 30S-IF3 complex. The map shows that IF3 is present in an extended conformation with CTD present at the P-site and NTD near the platform even in the absence of IF1 and IF2. Hence, IF3 CTD binds at the P-site and moves away during the accommodation of the initiator tRNA at the P-site in the later steps of translation initiation. Overall, we report the structure of 30S-IF3 which demystifies the starting binding site and conformation of IF3 on the 30S ribosomal subunit.

Computational analysis of long-range allosteric communications in CFTR.

Malfunction of the CFTR protein results in cystic fibrosis, one of the most common hereditary diseases. CFTR functions as an anion channel, the gating of which is controlled by long-range allosteric communications. Allostery also has direct bearings on CF treatment: the most effective CFTR drugs modulate its activity allosterically. Herein, we integrated Gaussian network model, transfer entropy, and anisotropic normal mode-Langevin dynamics and investigated the allosteric communications network of CFTR. The results are in remarkable agreement with experimental observations and mutational analysis and provide extensive novel insight. We identified residues that serve as pivotal allosteric sources and transducers, many of which correspond to disease-causing mutations. We find that in the ATP-free form, dynamic fluctuations of the residues that comprise the ATP-binding sites facilitate the initial binding of the nucleotide. Subsequent binding of ATP then brings to the fore and focuses on dynamic fluctuations that were present in a latent and diffuse form in the absence of ATP. We demonstrate that drugs that potentiate CFTR's conductance do so not by directly acting on the gating residues, but rather by mimicking the allosteric signal sent by the ATP-binding sites. We have also uncovered a previously undiscovered allosteric 'hotspot' located proximal to the docking site of the phosphorylated regulatory (R) domain, thereby establishing a molecular foundation for its phosphorylation-dependent excitatory role. This study unveils the molecular underpinnings of allosteric connectivity within CFTR and highlights a novel allosteric 'hotspot' that could serve as a promising target for the development of novel therapeutic interventions.

Computational analysis of long-range allosteric communications in CFTR

Malfunction of the CFTR protein results in cystic fibrosis, one of the most common hereditary diseases. CFTR functions as an anion channel, the gating of which is controlled by long-range allosteric communications. Allostery also has direct bearings on CF treatment: the most effective CFTR drugs modulate its activity allosterically. Herein, we integrated Gaussian network model, transfer entropy, and anisotropic normal mode-Langevin dynamics and investigated the allosteric communications network of CFTR. The results are in remarkable agreement with experimental observations and mutational analysis and provide extensive novel insight. We identified residues that serve as pivotal allosteric sources and transducers, many of which correspond to disease-causing mutations. We find that in the ATP-free form, dynamic fluctuations of the residues that comprise the ATP-binding sites facilitate the initial binding of the nucleotide. Subsequent binding of ATP then brings to the fore and focuses on dynamic fluctuations that were present in a latent and diffuse form in the absence of ATP. We demonstrate that drugs that potentiate CFTR’s conductance do so not by directly acting on the gating residues, but rather by mimicking the allosteric signal sent by the ATP-binding sites. We have also uncovered a previously undiscovered allosteric ‘hotspot’ located proximal to the docking site of the phosphorylated regulatory (R) domain, thereby establishing a molecular foundation for its phosphorylation-dependent excitatory role. This study unveils the molecular underpinnings of allosteric connectivity within CFTR and highlights a novel allosteric ‘hotspot’ that could serve as a promising target for the development of novel therapeutic interventions.

Annexin A5 derived from matrix vesicles protects against osteoporotic bone loss via mineralization

Matrix vesicles (MVs) have shown strong effects in diseases such as vascular ectopic calcification and pathological calcified osteoarthritis and in wound repair of the skeletal system due to their membranous vesicle characteristics and abundant calcium and phosphorus content. However, the role of MVs in the progression of osteoporosis is poorly understood. Here, we report that annexin A5, an important component of the matrix vesicle membrane, plays a vital role in bone matrix homeostasis in the deterioration of osteoporosis. We first identified annexin A5 from adherent MVs but not dissociative MVs of osteoblasts and found that it could be sharply decreased in the bone matrix during the occurrence of osteoporosis based on ovariectomized mice. We then confirmed its potential in mediating the mineralization of the precursor osteoblast lineage via its initial binding with collagen type I to achieve MV adhesion and the subsequent activation of cellular autophagy. Finally, we proved its protective role in resisting bone loss by applying it to osteoporotic mice. Taken together, these data revealed the importance of annexin A5, originating from adherent MVs of osteoblasts, in bone matrix remodeling of osteoporosis and provided a new strategy for the treatment and intervention of bone loss.

Shape-Induced Variations in Aromatic Thiols Adsorption on Gold Nanoparticle: A Novel Method for Accurate Evaluation of Adsorbed Molecules.

Nonspherical gold nanoparticles (GNPs) are increasingly used to enhance sensitivity and selectivity in analytical methods such as surface-enhanced Raman spectroscopy (SERS) for detecting trace biomarkers. However, there is limited research on the adsorption properties of aromatic thiols onto gold nanoparticles of different morphologies, where surface curvature varies significantly at the molecular level. In this study, we investigated the adsorption kinetics of 4-mercaptobenzoic acid, an aromatic molecule, on GNPs with different shapes using SERS. Our findings revealed significant differences in the adsorption behavior and binding site preferences of aromatic thiols on GNPs with distinct morphologies. While thiol molecules consider any surface site on nanospheres equally appealing, nanostars exhibit variations in curvature and surface energy, leading to initial binding with further repositioning from the tips of the nanostar after plasmon activation. To address these differences, we proposed a universal method to evaluate the quantity of tightly bound adsorbed molecules on GNPs independently of the particle size, shape, or concentration.

The SAP domain of Ku facilitates its efficient loading onto DNA ends.

The evolutionarily conserved DNA repair complex Ku serves as the primary sensor of free DNA ends in eukaryotic cells. Its rapid association with DNA ends is crucial for several cellular processes, including non-homologous end joining (NHEJ) DNA repair and telomere protection. In this study, we conducted a transient kinetic analysis to investigate the impact of the SAP domain on individual phases of the Ku-DNA interaction. Specifically, we examined the initial binding, the subsequent docking of Ku onto DNA, and sliding of Ku along DNA. Our findings revealed that the C-terminal SAP domain of Ku70 facilitates the initial phases of the Ku-DNA interaction but does not affect the sliding process. This suggests that the SAP domain may either establish the first interactions with DNA, or stabilize these initial interactions during loading. To assess the biological role of the SAP domain, we generated Arabidopsis plants expressing Ku lacking the SAP domain. Intriguingly, despite the decreased efficiency of the ΔSAP Ku complex in loading onto DNA, the mutant plants exhibited full proficiency in classical NHEJ and telomere maintenance. This indicates that the speed with which Ku loads onto telomeres or DNA double-strand breaks is not the decisive factor in stabilizing these DNA structures.

Charge within Nt17 peptides modulates huntingtin aggregation and initial lipid binding events

Toxic aggregation of pathogenic huntingtin protein (htt) is implicated in Huntington's disease and influenced by various factors, including the first seventeen amino acids at the N-terminus (Nt17) and the presence of lipid membranes. Nt17 has a propensity to form an amphipathic α-helix in the presence of binding partners, which promotes α-helix rich oligomer formation and facilitates htt/lipid interactions. Within Nt17 are multiple sites that are subject to post-translational modification, including acetylation and phosphorylation. Acetylation can occur at lysine 6, 9, and/or 15 while phosphorylation can occur at threonine 3, serine 13, and/or serine 16. Such modifications impact aggregation and lipid binding through the alteration of various intra- and intermolecular interactions. When incubated with htt-exon1(46Q), free Nt17 peptides containing point mutations mimicking acetylation or phosphorylation reduced fibril formation and altered oligomer morphologies. Upon exposure to lipid vesicles, changes to peptide/lipid complexation were observed and peptide-containing oligomers demonstrated reduced lipid interactions.