RESUMEN
Piscidins are histidine-enriched antimicrobial peptides that interact with lipid bilayers as amphipathic α-helices. Their activity at acidic and basic pH in vivo makes them promising templates for biomedical applications. This study focuses on p1 and p3, both 22-residue-long piscidins with 68% sequence identity. They share three histidines (H3, H4, and H11), but p1, which is significantly more permeabilizing, has a fourth histidine (H17). This study investigates how variations in amphipathic character associated with histidines affect the permeabilization properties of p1 and p3. First, we show that the permeabilization ability of p3, but not p1, is strongly inhibited at pH 6.0 when the conserved histidines are partially charged and H17 is predominantly neutral. Second, our neutron diffraction measurements performed at low water content and neutral pH indicate that the average conformation of p1 is highly tilted, with its C-terminus extending into the opposite leaflet. In contrast, p3 is surface bound with its N-terminal end tilted toward the bilayer interior. The deeper membrane insertion of p1 correlates with its behavior at full hydration: an enhanced ability to tilt, bury its histidines and C-terminus, induce membrane thinning and defects, and alter membrane conductance and viscoelastic properties. Furthermore, its pH-resiliency relates to the neutral state favored by H17. Overall, these results provide mechanistic insights into how differences in the histidine content and amphipathicity of peptides can elicit different directionality of membrane insertion and pH-dependent permeabilization. This work features complementary methods, including dye leakage assays, NMR-monitored titrations, X-ray and neutron diffraction, oriented CD, molecular dynamics, electrochemical impedance spectroscopy, surface plasmon resonance, and quartz crystal microbalance with dissipation.
Asunto(s)
Péptidos Catiónicos Antimicrobianos/metabolismo , Histidina/química , Membrana Dobles de Lípidos/metabolismo , Tensoactivos/metabolismo , Secuencia de Aminoácidos , Animales , Péptidos Catiónicos Antimicrobianos/química , Proteínas de Peces/química , Proteínas de Peces/metabolismo , Peces , Fluoresceínas/metabolismo , Colorantes Fluorescentes/metabolismo , Concentración de Iones de Hidrógeno , Membrana Dobles de Lípidos/química , Simulación de Dinámica Molecular , Permeabilidad/efectos de los fármacos , Fosfatidilcolinas/química , Fosfatidilgliceroles/química , Tensoactivos/químicaRESUMEN
Apolipoprotein B (apoB) is a large amphipathic protein that is the structural scaffold for the formation of several classes of lipoproteins involved in lipid transport throughout the body. The goal of the present study was to identify specific domains in the apoB sequence that contribute to its lipid binding properties. A sequence analysis algorithm was developed to identify stretches of hydrophobic amino acids devoid of charged amino acids, which are referred to as hydrophobic cluster domains (HCDs). This analysis identified 78 HCDs in apoB with hydrophobic stretches ranging from 6 to 26 residues. Each HCD was analyzed in silico for secondary structure and lipid binding properties, and a subset was synthesized for experimental evaluation. One HCD peptide, B38, showed high affinity binding to both isolated HDL and LDL, and could exchange between lipoproteins. All-atom molecular dynamics simulations indicate that B38 inserts 3.7Å below the phosphate plane of the bilayer. B38 forms an unusual α-helix with a broad hydrophobic face and polar serine and threonine residues on the opposite face. Based on this structure, we hypothesized that B38 could efflux cholesterol from cells. B38 showed a 12-fold greater activity than the 5A peptide, a bihelical Class A amphipathic helix (EC50 of 0.2658 vs. 3.188µM; p<0.0001), in promoting cholesterol efflux from ABCA1 expressing BHK-1 cells. In conclusion, we have identified novel domains within apoB that contribute to its lipid biding properties. Additionally, we have discovered a unique amphipathic helix design for efficient ABCA1-specific cholesterol efflux.
Asunto(s)
Apolipoproteínas B/química , Apolipoproteínas B/metabolismo , Lípidos/química , Estructura Secundaria de Proteína/fisiología , Transportador 1 de Casete de Unión a ATP/química , Transportador 1 de Casete de Unión a ATP/metabolismo , Aminoácidos/química , Aminoácidos/metabolismo , Sitios de Unión/fisiología , Células Cultivadas , HDL-Colesterol/química , HDL-Colesterol/metabolismo , LDL-Colesterol/química , LDL-Colesterol/metabolismo , Humanos , Interacciones Hidrofóbicas e Hidrofílicas , Péptidos/química , Péptidos/metabolismo , Unión Proteica/fisiologíaRESUMEN
An all-atom molecular dynamics simulation of the archetype barrel-stave alamethicin (alm) pore in a 1,2-dioleoyl-sn-glycero-3-phosphocholine bilayer at 313 K indicates that â¼7 µs is required for equilibration of a preformed 6-peptide pore; the pore remains stable for the duration of the remaining 7 µs of the trajectory, and the structure factors agree well with experiment. A 5 µs simulation of 10 surface-bound alm peptides shows significant peptide unfolding and some unbinding, but no insertion. Simulations at 363 and 413 K with a -0.2 V electric field yield peptide insertion in 1 µs. Insertion is initiated by the folding of residues 3-11 into an α-helix, and mediated by membrane water or by previously inserted peptides. The stability of five alm pore peptides at 413 K with a -0.2 V electric field demonstrates a significant preference for a transmembrane orientation. Hence, and in contrast to the cationic antimicrobial peptide described in the following article, alm shows a strong preference for the inserted over the surface-bound state.
Asunto(s)
Alameticina/química , Antibacterianos/química , Membrana Dobles de Lípidos/química , Alameticina/metabolismo , Animales , Antibacterianos/metabolismo , Péptidos Catiónicos Antimicrobianos/química , Campos Electromagnéticos , Proteínas de Peces/química , Peces , Proteínas Fúngicas/química , Proteínas Fúngicas/metabolismo , Glicerilfosforilcolina/análogos & derivados , Glicerilfosforilcolina/química , Interacciones Hidrofóbicas e Hidrofílicas , Simulación de Dinámica Molecular , Fosfatidilcolinas , Unión Proteica , Conformación Proteica en Hélice alfa , Pliegue de Proteína , Trichoderma , ViscosidadRESUMEN
Antimicrobial peptides (AMPs) that disrupt bacterial membranes are promising therapeutics against the growing number of antibiotic-resistant bacteria. The mechanism of membrane disruption by the AMP piscidin 1 was examined with multimicrosecond all-atom molecular dynamics simulations and solid-state NMR spectroscopy. The primary simulation was initialized with 20 peptides in four barrel-stave pores in a fully hydrated 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine/1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoglycerol bilayer. The four pores relaxed to toroidal by 200 ns, only one porelike structure containing two transmembrane helices remained at 26 µs, and none of the 18 peptides released to the surface reinserted to form pores. The simulation was repeated at 413 K with an applied electric field and all peptides were surface-bound by 200 ns. Trajectories of surface-bound piscidin with and without applied fields at 313 and 413 K and totaling 6 µs show transient distortions of the bilayer/water interface (consistent with (31)P NMR), but no insertion to transmembrane or pore states. (15)N chemical shifts confirm a fully surface-bound conformation. Taken together, the simulation and experimental results imply that transient defects rather than stable pores are responsible for membrane disruption by piscidin 1, and likely other AMPs.
Asunto(s)
Péptidos Catiónicos Antimicrobianos/química , Proteínas de Peces/química , Animales , Péptidos Catiónicos Antimicrobianos/metabolismo , Proteínas de Peces/metabolismo , Peces , Membrana Dobles de Lípidos/química , Simulación de Dinámica Molecular , Resonancia Magnética Nuclear Biomolecular , Fosfatidilcolinas/química , Fosfatidilgliceroles/química , Conformación Proteica en Hélice alfa , Estabilidad Proteica , Propiedades de Superficie , Agua/químicaRESUMEN
The initial steps of membrane disruption by antimicrobial peptides (AMPs) involve binding to bacterial membranes in a surface-bound (S) orientation. To evaluate the effects of lipid composition on the S state, molecular dynamics simulations of the AMPs piscidin 1 (p1) and piscidin 3 (p3) were carried out in four different bilayers: 3:1 DMPC/DMPG, 3:1 POPC/POPG, 1:1 POPE/POPG, and 4:1 POPC/cholesterol. In all cases, the addition of 1:40 piscidin caused thinning of the bilayer, though thinning was least for DMPC/DMPG. The peptides also insert most deeply into DMPC/DMPG, spanning the region from the bilayer midplane to the headgroups, and thereby only mildly disrupting the acyl chains. In contrast, the peptides insert less deeply in the palmitoyl-oleoyl containing membranes, do not reach the midplane, and substantially disrupt the chains, i.e., the neighboring acyl chains bend under the peptide, forming a basket-like conformation. Curvature free energy derivatives calculated from the simulation pressure profiles reveal that the peptides generate positive curvature in membranes with palmitoyl and oleoyl chains but negative curvature in those with myristoyl chains. Curvature inductions predicted with a continuum elastic model follow the same trends, though the effect is weaker, and a small negative curvature induction is obtained in POPC/POPG. These results do not directly speak to the relative stability of the inserted (I) states or ease of pore formation, which requires the free energy pathway between the S and I states. Nevertheless, they do highlight the importance of lipid composition and acyl chain packing.
Asunto(s)
Péptidos Catiónicos Antimicrobianos/química , Proteínas de Peces/química , Dimiristoilfosfatidilcolina/química , Simulación de Dinámica Molecular , Fosfatidilcolinas/química , Fosfatidiletanolaminas/química , Fosfatidilgliceroles/química , Estructura Secundaria de Proteína , TermodinámicaRESUMEN
A module for fast determination of reduction potentials, E°, of redox-active proteins has been implemented in the CHARMM INterface and Graphics (CHARMMing) web portal (www.charmming.org). The free energy of reduction, which is proportional to E°, is composed of an intrinsic contribution due to the redox site and an environmental contribution due to the protein and solvent. Here, the intrinsic contribution is selected from a library of pre-calculated density functional theory values for each type of redox site and redox couple, while the environmental contribution is calculated from a crystal structure of the protein using Poisson-Boltzmann continuum electrostatics. An accompanying lesson demonstrates a calculation of E°. In this lesson, an ionizable residue in a [4Fe-4S]-protein that causes a pH-dependent E° is identified, and the E° of a mutant that would test the identification is predicted. This demonstration is valuable to both computational chemistry students and researchers interested in predicting sequence determinants of E° for mutagenesis.
Asunto(s)
Biología Computacional/educación , Biología Computacional/métodos , Transporte de Electrón , Internet , Proteínas/química , Proteínas/metabolismo , Oxidación-Reducción , TermodinámicaRESUMEN
While antimicrobial peptides (AMPs) have been widely investigated as potential therapeutics, high-resolution structures obtained under biologically relevant conditions are lacking. Here, the high-resolution structures of the homologous 22-residue long AMPs piscidin 1 (p1) and piscidin 3 (p3) are determined in fluid-phase 3:1 phosphatidylcholine/phosphatidylglycerol (PC/PG) and 1:1 phosphatidylethanolamine/phosphatidylglycerol (PE/PG) bilayers to identify molecular features important for membrane destabilization in bacterial cell membrane mimics. Structural refinement of (1)H-(15)N dipolar couplings and (15)N chemical shifts measured by oriented sample solid-state NMR and all-atom molecular dynamics (MD) simulations provide structural and orientational information of high precision and accuracy about these interfacially bound α-helical peptides. The tilt of the helical axis, τ, is between 83° and 93° with respect to the bilayer normal for all systems and analysis methods. The average azimuthal rotation, ρ, is 235°, which results in burial of hydrophobic residues in the bilayer. The refined NMR and MD structures reveal a slight kink at G13 that delineates two helical segments characterized by a small difference in their τ angles (<10°) and significant difference in their ρ angles (~25°). Remarkably, the kink, at the end of a G(X)4G motif highly conserved among members of the piscidin family, allows p1 and p3 to adopt ρ angles that maximize their hydrophobic moments. Two structural features differentiate the more potent p1 from p3: p1 has a larger ρ angle and less N-terminal fraying. The peptides have comparable depths of insertion in PC/PG, but p3 is 1.2 Å more deeply inserted than p1 in PE/PG. In contrast to the ideal α-helical structures typically assumed in mechanistic models of AMPs, p1 and p3 adopt disrupted α-helical backbones that correct for differences in the amphipathicity of their N- and C-ends, and their centers of mass lie ~1.2-3.6 Å below the plane defined by the C2 atoms of the lipid acyl chains.
Asunto(s)
Péptidos Catiónicos Antimicrobianos/química , Proteínas de Peces/química , Membrana Dobles de Lípidos/química , Interacciones Hidrofóbicas e Hidrofílicas , Inmersión , Cristales Líquidos/química , Simulación de Dinámica Molecular , Fosfatidilcolinas/química , Fosfatidilgliceroles/química , Estructura Secundaria de ProteínaRESUMEN
Lyme disease (LD), caused by spirochete bacteria of the genus Borrelia burgdorferi sensu lato, remains the most common vector-borne disease in the northern hemisphere. Borrelia outer surface protein A (OspA) is an integral surface protein expressed during the tick cycle, and a validated vaccine target. There are at least 20 recognized Borrelia genospecies, that vary in OspA serotype. This study presents a new in silico sequence-based method for OspA typing using next-generation sequence data. Using a compiled database of over 400 Borrelia genomes encompassing the 4 most common disease-causing genospecies, we characterized OspA diversity in a manner that can accommodate existing and new OspA types and then defined boundaries for classification and assignment of OspA types based on the sequence similarity. To accommodate potential novel OspA types, we have developed a new nomenclature: OspA in silico type (IST). Beyond the ISTs that corresponded to existing OspA serotypes 1-8, we identified nine additional ISTs that cover new OspA variants in B. bavariensis (IST9-10), B. garinii (IST11-12), and other Borrelia genospecies (IST13-17). The IST typing scheme and associated OspA variants are available as part of the PubMLST Borrelia spp. database. Compared to traditional OspA serotyping methods, this new computational pipeline provides a more comprehensive and broadly applicable approach for characterization of OspA type and Borrelia genospecies to support vaccine development.
Asunto(s)
Antígenos de Superficie , Proteínas de la Membrana Bacteriana Externa , Lipoproteínas , Enfermedad de Lyme , Proteínas de la Membrana Bacteriana Externa/genética , Enfermedad de Lyme/microbiología , Lipoproteínas/genética , Antígenos de Superficie/genética , Borrelia burgdorferi/genética , Borrelia burgdorferi/clasificación , Simulación por Computador , Humanos , Genoma Bacteriano , Grupo Borrelia Burgdorferi/genética , Grupo Borrelia Burgdorferi/clasificación , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Serogrupo , Filogenia , Vacunas BacterianasRESUMEN
The pH dependence of the reduction potential E° for a metalloprotein indicates that the protonation state of at least one residue near the redox site changes and may be important for its activity. The responsible residue is usually identified by site-specific mutagenesis, which may be time-consuming. Here, the titration of E° for Chromatium vinosum high-potential iron-sulfur protein is predicted to be in good agreement with experiment using density functional theory and Poisson-Boltzmann calculations if only the sole histidine undergoes changes in protonation. The implementation of this approach into CHARMMing, a user-friendly web-based portal, allows users to identify residues in other proteins causing similar pH dependence.
Asunto(s)
Concentración de Iones de Hidrógeno , Chromatium/química , Modelos Moleculares , Oxidación-ReducciónRESUMEN
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) continues to represent a global health emergency as a highly transmissible, airborne virus. An important coronaviral drug target for treatment of COVID-19 is the conserved main protease (Mpro). Nirmatrelvir is a potent Mpro inhibitor and the antiviral component of Paxlovid. The significant viral sequencing effort during the ongoing COVID-19 pandemic represented a unique opportunity to assess potential nirmatrelvir escape mutations from emerging variants of SARS-CoV-2. To establish the baseline mutational landscape of Mpro prior to the introduction of Mpro inhibitors, Mpro sequences and its cleavage junction regions were retrieved from ~4,892,000 high-quality SARS-CoV-2 genomes in the open-access Global Initiative on Sharing Avian Influenza Data (GISAID) database. Any mutations identified from comparison to the reference sequence (Wuhan-Hu-1) were catalogued and analyzed. Mutations at sites key to nirmatrelvir binding and protease functionality (e.g., dimerization sites) were still rare. Structural comparison of Mpro also showed conservation of key nirmatrelvir contact residues across the extended Coronaviridae family (α-, ß-, and γ-coronaviruses). Additionally, we showed that over time, the SARS-CoV-2 Mpro enzyme remained under purifying selection and was highly conserved relative to the spike protein. Now, with the emergency use authorization (EUA) of Paxlovid and its expected widespread use across the globe, it is essential to continue large-scale genomic surveillance of SARS-CoV-2 Mpro evolution. This study establishes a robust analysis framework for monitoring emergent mutations in millions of virus isolates, with the goal of identifying potential resistance to present and future SARS-CoV-2 antivirals. IMPORTANCE The recent authorization of oral severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) antivirals, such as Paxlovid, has ushered in a new era of the COVID-19 pandemic. The emergence of new variants, as well as the selective pressure imposed by antiviral drugs themselves, raises concern for potential escape mutations in key drug binding motifs. To determine the potential emergence of antiviral resistance in globally circulating isolates and its implications for the clinical response to the COVID-19 pandemic, sequencing of SARS-CoV-2 viral isolates before, during, and after the introduction of new antiviral treatments is critical. The infrastructure built herein for active genetic surveillance of Mpro evolution and emergent mutations will play an important role in assessing potential antiviral resistance as the pandemic progresses and Mpro inhibitors are introduced. We anticipate our framework to be the starting point in a larger effort for global monitoring of the SARS-CoV-2 Mpro mutational landscape.
Asunto(s)
COVID-19 , SARS-CoV-2 , Animales , Antivirales/metabolismo , Proteasas 3C de Coronavirus , Cisteína Endopeptidasas/metabolismo , Combinación de Medicamentos , Humanos , Lactamas , Leucina , Nitrilos , Pandemias , Prolina , Inhibidores de Proteasas/química , Inhibidores de Proteasas/farmacología , Ritonavir , SARS-CoV-2/genética , Proteínas no Estructurales Virales/metabolismoRESUMEN
Peptides mimicking the major protein of highdensity lipoprotein (HDL), apolipoprotein A-I (apoA-I), are promising therapeutics for cardiovascular diseases. Similar to apoA-I, their atheroprotective property is attributed to their ability to form discoidal HDL-like particles by extracting cellular cholesterol and phospholipids from lipid microdomains created by the ABCA1 transporter in a process called cholesterol efflux. The structural features of peptides that enable cholesterol efflux are not well understood. Herein, four synthetic amphipathic peptides denoted ELK, which only contain Glu, Leu, Lys, and sometimes Ala, and which have a wide range of net charges and hydrophobicities, were examined for cholesterol efflux. Experiments show that ELKs with a net neutral charge and a hydrophobic face that subtends an angle of at least 140° are optimal for cholesterol efflux. All-atom molecular dynamics simulations show that peptides that are effective in promoting cholesterol efflux stabilize HDL nanodiscs formed by these peptides by the orderly covering of the hydrophobic acyl chains on the edge of the disc. In contrast to apoA-I, which forms an anti-parallel double belt around the HDL, active peptides assemble in a mostly anti-parallel "picket fence" arrangement. These results shed light on the efflux ability of apoA-I mimetics and inform the future design of such therapeutics.
Asunto(s)
Transportador 1 de Casete de Unión a ATP/metabolismo , Apolipoproteína A-I/química , Colesterol/metabolismo , Peptidomiméticos/química , Peptidomiméticos/farmacología , Secuencia de Aminoácidos , Transporte Biológico/efectos de los fármacos , Simulación de Dinámica Molecular , Fosfolípidos/metabolismo , Conformación ProteicaRESUMEN
The periodic Saffman-Delbrück (PSD) model, an extension of the Saffman-Delbrück model developed to describe the effects of periodic boundary conditions on the diffusion constants of lipids and proteins obtained from simulation, is tested using the coarse-grained Martini and all-atom CHARMM36 (C36) force fields. Simulations of pure Martini dipalmitoylphosphatidylcholine (DPPC) bilayers and those with one embedded gramicidin A (gA) dimer or one gA monomer with sizes ranging from 512 to 2048 lipids support the PSD model. Underestimates of D∞ (the value of the diffusion constant for an infinite system) from the 512-lipid system are 35% for DPPC, 45% for the gA monomer, and 70% for the gA dimer. Simulations of all-atom DPPC and dioleoylphosphatidylcholine (DOPC) bilayers yield diffusion constants not far from experiment. However, the PSD model predicts that diffusion constants at the sizes of the simulation should underestimate experiment by approximately a factor of 3 for DPPC and 2 for DOPC. This likely implies a deficiency in the C36 force field. A Bayesian method for extrapolating diffusion constants of lipids and proteins in membranes obtained from simulation to infinite system size is provided.
Asunto(s)
1,2-Dipalmitoilfosfatidilcolina/química , Gramicidina/química , Membrana Dobles de Lípidos/química , Simulación de Dinámica Molecular , Difusión , Tamaño de la Partícula , Propiedades de SuperficieRESUMEN
Contributions of hydroxyethyl functions to the DNA binding affinities of substituted anthracenes are evaluated by calorimetry and spectroscopy. Isothermal titration calorimetry indicated that binding of the ligands to calf thymus DNA (5 mM Tris buffer, 50 mM NaCl, pH 7.2, 25 degrees C) is exothermic. The binding constants increased from 1.5 x 10(4) to 1.7 x 10(6) M(-1) as a function of increase in the number of hydroxyethyl functions (0-4). DNA binding was accompanied by red-shifted absorption (approximately 630 cm(-1)), strong hypochromism (>65%), positive induced-circular dichroism bands, and negative linear dichroism signals. DNA binding, in general, increased the helix stabilities to a significant extent (DeltaT(m) approximately 7 degrees C, DeltaDeltaH approximately 3 kcal/mol, DeltaDeltaS approximately 6-20 cal/K.mol). The binding constants showed a strong correlation with the number of hydroxyethyl groups present on the anthracene ring system. Analysis of the binding data using the hydrophobicity parameter (Log P) showed a poor correlation between the binding affinity and hydrophobicity. This observation was also supported by a comparison of the affinities of probes carrying N-ethyl (Kb = 0.8 x 10(5) M(-1)) versus N-hydroxyethyl side chains (Kb = 5.5 x 10(5) M(-1)). These are the very first examples of a strong quantitative correlation between the DNA binding affinity of a probe and the number of hydroxyethyl groups present on the probe. These quantitative findings are useful in the rational design of new ligands for high-affinity binding to DNA.
Asunto(s)
Antracenos/química , ADN/química , Biofisica/métodos , Calorimetría/métodos , Rastreo Diferencial de Calorimetría , Fenómenos Químicos , Química Física , Dicroismo Circular , Cinética , Modelos Químicos , Unión Proteica , Programas Informáticos , Espectrofotometría/métodos , Temperatura , TermodinámicaRESUMEN
In nitrogen fixation by Azotobacter vinelandii nitrogenase, the iron protein (FeP) binds to and subsequently transfers electrons to the molybdenum-FeP, which contains the nitrogen fixation site, along with hydrolysis of two ATPs. However, the nature of the reduced state cluster is not completely clear. While reduced FeP is generally thought to contain an [Fe4 S4 ](1+) cluster, evidence also exists for an all-ferrous [Fe4 S4 ](0) cluster. Since the former indicates a single electron is transferred per two ATPs hydrolyzed while the latter indicates two electrons could be transferred per two ATPs hydrolyzed, an all-ferrous [Fe4 S4 ](0) cluster in FeP is potenially two times more efficient. However, the 1+/0 reduction potential has been measured in the protein at both 460 and 790 mV, causing the biological significance to be questioned. Here, "density functional theory plus Poisson Boltzmann" calculations show that cluster movement relative to the protein surface observed in the crystal structures could account for both measured values. In addition, elastic network mode analysis indicates that such movement occurs in low frequency vibrations of the protein, implying protein dynamics might lead to variations in reduction potential. Furthermore, the different reductants used in the conflicting measurements of the reduction potential could be differentially affecting the protein dynamics. Moreover, even if the all-ferrous cluster is not the biologically relevant cluster, mutagenesis to stabilize the conformation with the more exposed cluster may be useful for bioengineering more efficient enzymes.