RESUMO
Antibiotics that use novel mechanisms are needed to combat antimicrobial resistance1-3. Teixobactin4 represents a new class of antibiotics with a unique chemical scaffold and lack of detectable resistance. Teixobactin targets lipid II, a precursor of peptidoglycan5. Here we unravel the mechanism of teixobactin at the atomic level using a combination of solid-state NMR, microscopy, in vivo assays and molecular dynamics simulations. The unique enduracididine C-terminal headgroup of teixobactin specifically binds to the pyrophosphate-sugar moiety of lipid II, whereas the N terminus coordinates the pyrophosphate of another lipid II molecule. This configuration favours the formation of a ß-sheet of teixobactins bound to the target, creating a supramolecular fibrillar structure. Specific binding to the conserved pyrophosphate-sugar moiety accounts for the lack of resistance to teixobactin4. The supramolecular structure compromises membrane integrity. Atomic force microscopy and molecular dynamics simulations show that the supramolecular structure displaces phospholipids, thinning the membrane. The long hydrophobic tails of lipid II concentrated within the supramolecular structure apparently contribute to membrane disruption. Teixobactin hijacks lipid II to help destroy the membrane. Known membrane-acting antibiotics also damage human cells, producing undesirable side effects. Teixobactin damages only membranes that contain lipid II, which is absent in eukaryotes, elegantly resolving the toxicity problem. The two-pronged action against cell wall synthesis and cytoplasmic membrane produces a highly effective compound targeting the bacterial cell envelope. Structural knowledge of the mechanism of teixobactin will enable the rational design of improved drug candidates.
Assuntos
Antibacterianos , Bactérias , Membrana Celular , Depsipeptídeos , Viabilidade Microbiana , Antibacterianos/química , Antibacterianos/farmacologia , Bactérias/citologia , Bactérias/efeitos dos fármacos , Membrana Celular/efeitos dos fármacos , Parede Celular/efeitos dos fármacos , Parede Celular/metabolismo , Depsipeptídeos/química , Depsipeptídeos/farmacologia , Difosfatos/química , Farmacorresistência Bacteriana/efeitos dos fármacos , Humanos , Lipídeos/química , Testes de Sensibilidade Microbiana , Viabilidade Microbiana/efeitos dos fármacos , Microscopia de Força Atômica , Simulação de Dinâmica Molecular , Ressonância Magnética Nuclear Biomolecular , Estrutura Secundária de Proteína , Pirrolidinas/química , Açúcares/químicaRESUMO
Helical structures in proteins include not only α-helices but also 310 and π helices. These secondary structures differ in the registry of the CâO···H-N hydrogen bonds, which are i to i + 4 for α-helices, i to i + 3 for 310 helices, and i to i + 5 for π-helices. The standard NMR observable of protein secondary structures are chemical shifts, which are, however, insensitive to the precise type of helices. Here, we introduce a three-dimensional (3D) 1H-detected experiment that measures and assigns CO-HN cross-peaks to distinguish the different types of hydrogen-bonded helices. This hCOhNH experiment combines efficient cross-polarization from CO to HN with 13C, 15N, and 1H chemical shift correlation to detect the relative proximities of the COi-Hi+jN spin pairs. We demonstrate this experiment on the membrane-bound transmembrane domain of the SARS-CoV-2 envelope (E) protein (ETM). We show that the C-terminal five residues of ETM form a 310-helix, whereas the rest of the transmembrane domain have COi-Hi+4N hydrogen bonds that are characteristic of α-helices. This result confirms the recent high-resolution solid-state NMR structure of the open state of ETM, which was solved in the absence of explicit hydrogen-bonding restraints. This C-terminal 310 helix may facilitate proton and calcium conduction across the hydrophobic gate of the channel. This hCOhNH experiment is generally applicable and can be used to distinguish not only different types of helices but also different types of ß-strands and other hydrogen-bonded conformations in proteins.
Assuntos
Proteínas , Prótons , Ligação de Hidrogênio , Proteínas/química , Estrutura Secundária de Proteína , Espectroscopia de Ressonância Magnética , Conformação ProteicaRESUMO
The envelope (E) protein of SARS-CoV-2 is the smallest of the three structural membrane proteins of the virus. E mediates budding of the progeny virus in the endoplasmic reticulum Golgi intermediate compartment of the cell. It also conducts ions, and this channel activity is associated with the pathogenicity of SARS-CoV-2. The structural basis for these functions is still poorly understood. Biochemical studies of E in detergent micelles found a variety of oligomeric states, but recent 19F solid-state NMR data indicated that the transmembrane domain (ETM, residues 8-38) forms pentamers in lipid bilayers. Hexamethylene amiloride (HMA), an E inhibitor, binds the pentameric ETM at the lipid-exposed helix-helix interface. Here, we investigate the oligomeric structure and drug interaction of an ectodomain-containing E construct, ENTM (residues 1-41). Unexpectedly, 19F spin diffusion NMR data reveal that ENTM adopts an average oligomeric state of dimers instead of pentamers in lipid bilayers. A new amiloride inhibitor, AV-352, shows stronger inhibitory activity than HMA in virus-like particle assays. Distance measurements between 13C-labeled protein and a trifluoromethyl group of AV-352 indicate that the drug binds ENTM with a higher stoichiometry than ETM. We measured protein-drug contacts using a sensitivity-enhanced two-dimensional 13C-19F distance NMR technique. The results indicate that AV-352 binds the C-terminal half of the TM domain, similar to the binding region of HMA. These data provide evidence for the existence of multiple oligomeric states of E in lipid bilayers, which may carry out distinct functions and may be differentially targeted by antiviral drugs.
RESUMO
Internuclear distances represent one of the main structural constraints in molecular structure determination using solid-state NMR spectroscopy, complementing chemical shifts and orientational restraints. Although a large number of magic-angle-spinning (MAS) NMR techniques have been available for distance measurements, traditional 13C and 15N NMR experiments are inherently limited to distances of a few angstroms due to the low gyromagnetic ratios of these nuclei. Recent development of fast MAS triple-resonance 19F and 1H NMR probes has stimulated the design of MAS NMR experiments that measure distances in the 1-2 nm range with high sensitivity. This review describes the principles and applications of these multiplexed multidimensional correlation distance NMR experiments, with an emphasis on 19F- and 1H-based distance experiments. Representative applications of these long-distance NMR methods to biological macromolecules as well as small molecules are reviewed.
Assuntos
Imageamento por Ressonância Magnética , Proteínas , Espectroscopia de Ressonância Magnética , Proteínas/químicaRESUMO
The SARS-CoV-2 envelope (E) protein is a viroporin associated with the acute respiratory symptoms of COVID-19. E forms cation-selective ion channels that assemble in the lipid membrane of the endoplasmic reticulum Golgi intermediate compartment. The channel activity of E is linked to the inflammatory response of the host cell to the virus. Like many viroporins, E is thought to oligomerize with a well-defined stoichiometry. However, attempts to determine the E stoichiometry have led to inconclusive results and suggested mixtures of oligomers whose exact nature might vary with the detergent used. Here, we employ 19F solid-state nuclear magnetic resonance and the centerband-only detection of exchange (CODEX) technique to determine the oligomeric number of E's transmembrane domain (ETM) in lipid bilayers. The CODEX equilibrium value, which corresponds to the inverse of the oligomeric number, indicates that ETM assembles into pentamers in lipid bilayers, without any detectable fraction of low-molecular-weight oligomers. Unexpectedly, at high peptide concentrations and in the presence of the lipid phosphatidylinositol, the CODEX data indicate that more than five 19F spins are within a detectable distance of about 2 nm, suggesting that the ETM pentamers cluster in the lipid bilayer. Monte Carlo simulations that take into account peptide-peptide and peptide-lipid interactions yielded pentamer clusters that reproduced the CODEX data. This supramolecular organization is likely important for E-mediated virus assembly and budding and for the channel function of the protein.
Assuntos
Proteínas do Envelope de Coronavírus , Bicamadas Lipídicas , SARS-CoV-2 , Bicamadas Lipídicas/química , Domínios Proteicos , Proteínas Viroporinas , Proteínas do Envelope de Coronavírus/químicaRESUMO
The envelope (E) protein of the SARS-CoV-2 virus is a membrane-bound viroporin that conducts cations across the endoplasmic reticulum Golgi intermediate compartment (ERGIC) membrane of the host cell to cause virus pathogenicity. The structure of the closed state of the E transmembrane (TM) domain, ETM, was recently determined using solid-state NMR spectroscopy. However, how the channel pore opens to mediate cation transport is unclear. Here, we use 13C and 19F solid-state NMR spectroscopy to investigate the conformation and dynamics of ETM at acidic pH and in the presence of calcium ions, which mimic the ERGIC and lysosomal environment experienced by the E protein in the cell. Acidic pH and calcium ions increased the conformational disorder of the N- and C-terminal residues and also increased the water accessibility of the protein, indicating that the pore lumen has become more spacious. ETM contains three regularly spaced phenylalanine (Phe) residues in the center of the peptide. 19F NMR spectra of para-fluorinated Phe20 and Phe26 indicate that both residues exhibit two sidechain conformations, which coexist within each channel. These two Phe conformations differ in their water accessibility, lipid contact, and dynamics. Channel opening by acidic pH and Ca2+ increases the population of the dynamic lipid-facing conformation. These results suggest an intricate aromatic network that regulates the opening of the ETM channel pore. This aromatic network may be a target for E inhibitors against SARS-CoV-2 and related coronaviruses.
Assuntos
COVID-19 , Cálcio , Cálcio/metabolismo , Humanos , Concentração de Íons de Hidrogênio , Íons , Lipídeos , Conformação Proteica , SARS-CoV-2 , ÁguaRESUMO
Specific interactions with phospholipids are often critical for the function of proteins or drugs, but studying these interactions at high resolution remains difficult, especially in complex membranes that mimic biological conditions. In principle, molecular interactions with phospholipids could be directly probed by solid-state NMR (ssNMR). However, due to the challenge to detect specific lipids in mixed liposomes and limited spectral sensitivity, ssNMR studies of specific lipids in complex membranes are scarce. Here, by using purified biological 13 C,15 N-labeled phospholipids, we show that we can selectively detect traces of specific lipids in complex membranes. In combination with 1 H-detected ssNMR, we show that our approach provides unprecedented high-resolution insights into the mechanisms of drugs that target specific lipids. This broadly applicable approach opens new opportunities for the molecular characterization of specific lipid interactions with proteins or drugs in complex fluid membranes.
Assuntos
Lipossomos , Proteínas , Ressonância Magnética Nuclear Biomolecular/métodos , Proteínas/química , Espectroscopia de Ressonância Magnética , Lipossomos/química , Fosfolipídeos , Bicamadas Lipídicas/químicaRESUMO
The alarming rise of antimicrobial resistance (AMR) imposes severe burdens on healthcare systems and the economy worldwide, urgently calling for the development of new antibiotics. Antimicrobial peptides could be ideal templates for next-generation antibiotics, due to their low propensity to cause resistance. An especially promising branch of antimicrobial peptides target lipidâ II, the precursor of the bacterial peptidoglycan network. To develop these peptides into clinically applicable compounds, detailed information on their pharmacologically relevant modes of action is of critical importance. Here we review the binding modes of a selection of peptides that target lipidâ II and highlight shortcomings in our molecular understanding that, at least partly, relate to the widespread use of artificial membrane mimics for structural studies of membrane-active antibiotics. In particular, with the example of the antimicrobial peptide nisin, we showcase how the native cellular membrane environment can be critical for understanding of the physiologically relevant binding mode.
Assuntos
Antibacterianos/metabolismo , Peptídeos/metabolismo , Uridina Difosfato Ácido N-Acetilmurâmico/análogos & derivados , Sequência de Aminoácidos , Antibacterianos/química , Bactérias/química , Membrana Celular/metabolismo , Peptídeos/química , Ligação Proteica , Alinhamento de Sequência , Uridina Difosfato Ácido N-Acetilmurâmico/metabolismoRESUMO
BACKGROUND: Membrane lipids play critical roles in the structure and function of membrane-embedded transporters. Salmonella typhimurium MelB (MelBSt) is a symporter coupling melibiose translocation with a cation (Na+, Li+, or H+). We present an extensive study on the effects of specific phospholipids on the structure of MelBSt and the melibiose transport catalyzed by this protein. RESULTS: Lipidomic analysis and thin-layer chromatography (TLC) experiments reveal that at least one phosphatidylethanolamine (PE) and one phosphatidylglycerol (PG) molecule associate with MelBSt at high affinities. Solid-state nuclear magnetic resonance (ssNMR) spectroscopy experiments confirmed the presence of lipid tails and glycerol backbones that co-purified with MelBSt; headgroups of PG were also observed. Studies with lipid-engineered strains, including PE-deficient, cardiolipin (CL)- and PG-deficient, or CL-deficient strains, show that lack of PE or PG, however not CL, largely inhibits both H+- and Na+-coupled melibiose active transport to different extents. Interestingly, neither the co-substrate binding (melibiose or Na+) nor MelBSt folding and stability are affected by changing lipid compositions. Remarkably, the delipidated MelBSt with only 2-3 bound lipids, regardless of the headgroup species, also exhibits unchanged melting temperature values as shown by circular dichroism spectroscopy. CONCLUSIONS: (1) Lipid tails and glycerol backbones of interacting PE and PG may contribute to the stability of the structure of MelBSt. (2) The headgroups of PE and PG, but not of CL, play important roles in melibiose transport; however, lipid headgroups do not modulate the folding and stability of MelBSt.
Assuntos
Proteínas de Bactérias/genética , Melibiose/metabolismo , Salmonella typhimurium/genética , Simportadores/genética , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Cardiolipinas/química , Cardiolipinas/metabolismo , Melibiose/química , Fosfatidiletanolaminas/química , Fosfatidilgliceróis/química , Salmonella typhimurium/metabolismo , Simportadores/química , Simportadores/metabolismoRESUMO
Stem-cell behavior is regulated by the material properties of the surrounding extracellular matrix, which has important implications for the design of tissue-engineering scaffolds. However, our understanding of the material properties of stem-cell scaffolds is limited to nanoscopic-to-macroscopic length scales. Herein, a solid-state NMR approach is presented that provides atomic-scale information on complex stem-cell substrates at near physiological conditions and at natural isotope abundance. Using self-assembled peptidic scaffolds designed for nervous-tissue regeneration, we show at atomic scale how scaffold-assembly degree, mechanics, and homogeneity correlate with favorable stem cell behavior. Integration of solid-state NMR data with molecular dynamics simulations reveals a highly ordered fibrillar structure as the most favorable stem-cell scaffold. This could improve the design of tissue-engineering scaffolds and other self-assembled biomaterials.
Assuntos
Materiais Biocompatíveis/química , Matriz Extracelular , Nanofibras/química , Células-Tronco Neurais/citologia , Medicina Regenerativa , Engenharia Tecidual/métodos , Alicerces Teciduais/química , Humanos , Microscopia de Força Atômica , Fragmentos de Peptídeos/químicaRESUMO
BACKGROUND: G-quadruplexes (G4) are found at important genome regions such as telomere ends and oncogene promoters. One prominent strategy to explore the therapeutic potential of G4 is stabilized it with specific ligands. METHODS: We report the synthesis of new phenanthroline, phenyl and quinoline acyclic bisoxazole compounds in order to explore and evaluate the targeting to c-myc and human telomeric repeat 22AG G4 using FRET-melting, CD-melting, NMR, fluorescence titrations and FID assays. RESULTS: The design strategy has led to potent compounds (Phen-1 and Phen-2) that discriminate different G4 structures (human telomeric sequences and c-myc promoter) and selectively stabilize G4 over duplex DNA. CD studies show that Phen-2 binds and induces antiparallel topologies in 22AG quadruplex and also binds c-myc promotor, increasing their Tm in about 12°C and 30°C respectively. In contrast, Phen-1 induces parallel topologies in 22AG and c-myc, with a moderate stabilization of 4°C for both sequences. Consistent with a CD melting study, Phen-2 binds strongly (K=106 to 107M-1) to c-myc and 22AG quadruplexes. CONCLUSIONS: Phen-1 and Phen-2 discriminated among various quadruplex topologies and exhibited high selectivity for quadruplexes over duplexes. Phen-2 retains antiparallel topologies for quadruplex 22AG and does not induce conformational changes on the parallel c-myc quadruplex although Phen-1 favors the parallel topology. NMR studies also showed that the Phen-2 binds to the c-myc quadruplex via end stacking. GENERAL SIGNIFICANCE: Overall, the results suggest the importance of Phen-2 as a scaffold for the fine-tuning with substituents in order to enhance binding and stabilization to G4 structures. This article is part of a Special Issue entitled "G-quadruplex" Guest Editor: Dr. Concetta Giancola and Dr. Daniela Montesarchio.
Assuntos
Antineoplásicos/metabolismo , DNA de Neoplasias/metabolismo , Desenho de Fármacos , Quadruplex G , Guanosina/química , Oxazóis/metabolismo , Fenantrolinas/metabolismo , Proteínas Proto-Oncogênicas c-myc/genética , Telômero/metabolismo , Antineoplásicos/síntese química , Antineoplásicos/farmacologia , Sítios de Ligação , Dicroísmo Circular , DNA de Neoplasias/química , DNA de Neoplasias/efeitos dos fármacos , Transferência Ressonante de Energia de Fluorescência , Quadruplex G/efeitos dos fármacos , Humanos , Ligantes , Espectroscopia de Ressonância Magnética , Desnaturação de Ácido Nucleico , Oxazóis/síntese química , Oxazóis/farmacologia , Fenantrolinas/síntese química , Fenantrolinas/farmacologia , Regiões Promotoras Genéticas/efeitos dos fármacos , Relação Estrutura-Atividade , Telômero/química , Telômero/efeitos dos fármacos , TemperaturaRESUMO
1H-detected solid-state NMR in combination with 1H/2D exchange steps allows for the direct identification of very strong hydrogen bonds in membrane proteins. On the example of the membrane-embedded potassium channel KcsA, we quantify the longevity of such very strong hydrogen bonds by combining time-resolved 1H-detected solid-state NMR experiments and molecular dynamics simulations. In particular, we show that the carboxyl-side chain of the highly conserved residue Glu51 is involved in ultra-strong hydrogen bonds, which are fully-water-exposed and yet stable for weeks. The astonishing stability of these hydrogen bonds is important for the structural integrity of potassium channels, which we further corroborate by computational studies.
Assuntos
Proteínas de Membrana/química , Simulação de Dinâmica Molecular , Sequência de Aminoácidos , Ligação de Hidrogênio , Conformação Proteica , Fatores de TempoRESUMO
The segregation of cellular surfaces in heterogeneous patches is considered to be a common motif in bacteria and eukaryotes that is underpinned by the observation of clustering and cooperative gating of signaling membrane proteins such as receptors or channels. Such processes could represent an important cellular strategy to shape signaling activity. Hence, structural knowledge of the arrangement of channels or receptors in supramolecular assemblies represents a crucial step towards a better understanding of signaling across membranes. We herein report on the supramolecular organization of clusters of the K+ channel KcsA in bacterial membranes, which was analyzed by a combination of DNP-enhanced solid-state NMR experiments and MD simulations. We used solid-state NMR spectroscopy to determine the channel-channel interface and to demonstrate the strong correlation between channel function and clustering, which suggests a yet unknown mechanism of communication between K+ channels.
RESUMO
1 H detection can significantly improve solid-state NMR spectral sensitivity and thereby allows studying more complex proteins. However, the common prerequisite for 1 Hâ detection is the introduction of exchangeable protons in otherwise deuterated proteins, which has thus far significantly hampered studies of partly water-inaccessible proteins, such as membrane proteins. Herein, we present an approach that enables high-resolution 1 H-detected solid-state NMR (ssNMR) studies of water-inaccessible proteins, and that even works in highly complex environments such as cellular surfaces. In particular, the method was applied to study the K+ channel KcsA in liposomes and inâ situ in native bacterial cell membranes. We used our data for a dynamic analysis, and we show that the selectivity filter, which is responsible for ion conduction and highly conserved in K+ channels, undergoes pronounced molecular motion. We expect this approach to open new avenues for biomolecular ssNMR.
Assuntos
Proteínas de Bactérias/química , Canais de Potássio/química , Água/química , Membrana Celular/química , Lipossomos/química , Espectroscopia de Prótons por Ressonância MagnéticaRESUMO
Antimicrobial resistance is a leading cause of mortality, calling for the development of new antibiotics. The fungal antibiotic plectasin is a eukaryotic host defence peptide that blocks bacterial cell wall synthesis. Here, using a combination of solid-state nuclear magnetic resonance, atomic force microscopy and activity assays, we show that plectasin uses a calcium-sensitive supramolecular killing mechanism. Efficient and selective binding of the target lipid II, a cell wall precursor with an irreplaceable pyrophosphate, is achieved by the oligomerization of plectasin into dense supra-structures that only form on bacterial membranes that comprise lipid II. Oligomerization and target binding of plectasin are interdependent and are enhanced by the coordination of calcium ions to plectasin's prominent anionic patch, causing allosteric changes that markedly improve the activity of the antibiotic. Structural knowledge of how host defence peptides impair cell wall synthesis will likely enable the development of superior drug candidates.
Assuntos
Cálcio , Parede Celular , Peptídeos , Uridina Difosfato Ácido N-Acetilmurâmico , Parede Celular/metabolismo , Parede Celular/efeitos dos fármacos , Parede Celular/química , Cálcio/metabolismo , Peptídeos/farmacologia , Peptídeos/metabolismo , Peptídeos/química , Uridina Difosfato Ácido N-Acetilmurâmico/análogos & derivados , Uridina Difosfato Ácido N-Acetilmurâmico/metabolismo , Uridina Difosfato Ácido N-Acetilmurâmico/química , Microscopia de Força Atômica , Antibacterianos/farmacologia , Antibacterianos/química , Espectroscopia de Ressonância Magnética , Ligação ProteicaRESUMO
The envelope (E) protein of the SARS-CoV-2 virus forms cation-conducting channels in the endoplasmic reticulum Golgi intermediate compartment (ERGIC) of infected cells. The calcium channel activity of E is associated with the inflammatory responses of COVID-19. Using solid-state NMR (ssNMR) spectroscopy, we have determined the open-state structure of E's transmembrane domain (ETM) in lipid bilayers. Compared to the closed state, open ETM has an expansive water-filled amino-terminal chamber capped by key glutamate and threonine residues, a loose phenylalanine aromatic belt in the middle, and a constricted polar carboxyl-terminal pore filled with an arginine and a threonine residue. This structure gives insights into how protons and calcium ions are selected by ETM and how they permeate across the hydrophobic gate of this viroporin.
Assuntos
COVID-19 , Proteínas Viroporinas , Humanos , Transporte de Íons , SARS-CoV-2 , TreoninaRESUMO
The SARS-CoV-2 envelope (E) protein forms a five-helix bundle in lipid bilayers whose cation-conducting activity is associated with the inflammatory response and respiratory distress symptoms of COVID-19. E channel activity is inhibited by the drug 5-(N,N-hexamethylene) amiloride (HMA). However, the binding site of HMA in E has not been determined. Here we use solid-state NMR to measure distances between HMA and the E transmembrane domain (ETM) in lipid bilayers. 13 C, 15 N-labeled HMA is combined with fluorinated or 13 C-labeled ETM. Conversely, fluorinated HMA is combined with 13 C, 15 N-labeled ETM. These orthogonal isotopic labeling patterns allow us to conduct dipolar recoupling NMR experiments to determine the HMA binding stoichiometry to ETM as well as HMA-protein distances. We find that HMA binds ETM with a stoichiometry of one drug per pentamer. Unexpectedly, the bound HMA is not centrally located within the channel pore, but lies on the lipid-facing surface in the middle of the TM domain. This result suggests that HMA may inhibit the E channel activity by interfering with the gating function of an aromatic network. These distance data are obtained under much lower drug concentrations than in previous chemical shift perturbation data, which showed the largest perturbation for N-terminal residues. This difference suggests that HMA has higher affinity for the protein-lipid interface than the channel pore. These results give insight into the inhibition mechanism of HMA for SARS-CoV-2 E.
Assuntos
Amilorida , COVID-19 , Humanos , Amilorida/farmacologia , Amilorida/química , SARS-CoV-2 , Bicamadas Lipídicas/químicaRESUMO
The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) envelope (E) protein forms a pentameric ion channel in the lipid membrane of the endoplasmic reticulum Golgi intermediate compartment (ERGIC) of the infected cell. The cytoplasmic domain of E interacts with host proteins to cause virus pathogenicity and may also mediate virus assembly and budding. To understand the structural basis of these functions, here we investigate the conformation and dynamics of an E protein construct (residues 8-65) that encompasses the transmembrane domain and the majority of the cytoplasmic domain using solid-state NMR. 13C and 15N chemical shifts indicate that the cytoplasmic domain adopts a ß-sheet-rich conformation that contains three ß-strands separated by turns. The five subunits associate into an umbrella-shaped bundle that is attached to the transmembrane helices by a disordered loop. Water-edited NMR spectra indicate that the third ß-strand at the C terminus of the protein is well hydrated, indicating that it is at the surface of the ß-bundle. The structure of the cytoplasmic domain cannot be uniquely determined from the inter-residue correlations obtained here due to ambiguities in distinguishing intermolecular and intramolecular contacts for a compact pentameric assembly of this small domain. Instead, we present four structural topologies that are consistent with the measured inter-residue contacts. These data indicate that the cytoplasmic domain of the SARS-CoV-2 E protein has a strong propensity to adopt ß-sheet conformations when the protein is present at high concentrations in lipid bilayers. The equilibrium between the ß-strand conformation and the previously reported α-helical conformation may underlie the multiple functions of E in the host cell and in the virion.
Assuntos
SARS-CoV-2 , Humanos , Bicamadas Lipídicas/química , Modelos Moleculares , Conformação Proteica em Folha beta , SARS-CoV-2/químicaRESUMO
C-type inactivation is of great physiological importance in voltage-activated K+ channels (Kv), but its structural basis remains unresolved. Knowledge about C-type inactivation has been largely deduced from the bacterial K+ channel KcsA, whose selectivity filter constricts under inactivating conditions. However, the filter is highly sensitive to its molecular environment, which is different in Kv channels than in KcsA. In particular, a glutamic acid residue at position 71 along the pore helix in KcsA is substituted by a valine conserved in most Kv channels, suggesting that this side chain is a molecular determinant of function. Here, a combination of X-ray crystallography, solid-state NMR and MD simulations of the E71V KcsA mutant is undertaken to explore inactivation in this Kv-like construct. X-ray and ssNMR data show that the filter of the Kv-like mutant does not constrict under inactivating conditions. Rather, the filter adopts a conformation that is slightly narrowed and rigidified. On the other hand, MD simulations indicate that the constricted conformation can nonetheless be stably established in the mutant channel. Together, these findings suggest that the Kv-like KcsA mutant may be associated with different modes of C-type inactivation, showing that distinct filter environments entail distinct C-type inactivation mechanisms.
Assuntos
Proteínas de Bactérias , Canais de Potássio , Proteínas de Bactérias/metabolismo , Cristalografia por Raios X , Canais de Potássio/metabolismo , Conformação ProteicaRESUMO
Dynamic nuclear polarization (DNP) is a powerful method to enhance nuclear magnetic resonance (NMR) signal intensities, enabling unprecedented applications in life and material science. An ultimate goal is to expand the use of DNP-enhanced solid-state NMR to ultrahigh magnetic fields where optimal spectral resolution and sensitivity are integrated. Trityl-nitroxide (TN) biradicals have attracted significant interest in high-field DNP, but their application to complex (bio)molecules has so far been limited. Here we report a novel postmodification strategy for synthesis of hydrophilic TN biradicals in order to improve their use in biomolecular applications. Initially, three TN biradicals (referred to as NATriPols 1-3) with amino-acid linkers were synthesized. EPR studies showed that the α-position of the amino-acid linkers is an ideal modification site for these biradicals since their electron-electron magnetic interactions are marginally affected by the substituents at this position. On the basis of this finding, we synthesized NATriPol-4 with pyridine disulfide appended at the α-position. Postmodification of NATriPol-4 via thiol-click chemistry resulted in various TN biradicals including hydrophilic NATriPol-5 in a quantitative manner. Interestingly, DNP enhancements at 18.8 T of NATriPols for 13C,15N-proline in a glycerol/water matrix are inversely correlated with their hydrophobicity. Importantly, applications of hydrophilic NATriPol-5 and NATriPol-3 to biomolecules including a globular soluble protein and a membrane targeting peptide reveal significantly improved performance compared to TEMTriPol-1 and AMUPol. Our work provides an efficient approach for one-step synthesis of new polarizing agents with tunable physicochemical properties, thus expediting optimization of new biradicals for biomolecular applications at ultrahigh magnetic fields.