RESUMEN
We present the high-resolution structure of stem-loop 4 of the 5'-untranslated region (5_SL4) of the severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2) genome solved by solution state nuclear magnetic resonance spectroscopy. 5_SL4 adopts an extended rod-like structure with a single flexible looped-out nucleotide and two mixed tandem mismatches, each composed of a Gâ¢U wobble base pair and a pyrimidineâ¢pyrimidine mismatch, which are incorporated into the stem-loop structure. Both the tandem mismatches and the looped-out residue destabilize the stem-loop structure locally. Their distribution along the 5_SL4 stem-loop suggests a role of these non-canonical elements in retaining functionally important structural plasticity in particular with regard to the accessibility of the start codon of an upstream open reading frame located in the RNA's apical loop. The apical loop-although mostly flexible-harbors residual structural features suggesting an additional role in molecular recognition processes. 5_SL4 is highly conserved among the different variants of SARS-CoV-2 and can be targeted by small molecule ligands, which it binds with intermediate affinity in the vicinity of the non-canonical elements within the stem-loop structure.
Asunto(s)
COVID-19 , SARS-CoV-2 , Humanos , Secuencia de Bases , COVID-19/virología , Espectroscopía de Resonancia Magnética , Conformación de Ácido Nucleico , ARN Viral/química , SARS-CoV-2/química , SARS-CoV-2/genéticaRESUMEN
Antimicrobial peptides (AMPs) are major components of the innate immune defense. Accumulating evidence suggests that the antibacterial activity of many AMPs is dependent on the formation of amyloid-like fibrils. To identify novel fibril forming AMPs, we generated a spleen-derived peptide library and screened it for the presence of amyloidogenic peptides. This approach led to the identification of a C-terminal 32-mer fragment of alpha-hemoglobin, termed HBA(111-142). The non-fibrillar peptide has membranolytic activity against various bacterial species, while the HBA(111-142) fibrils aggregated bacteria to promote their phagocytotic clearance. Further, HBA(111-142) fibrils selectively inhibited measles and herpes viruses (HSV-1, HSV-2, HCMV), but not SARS-CoV-2, ZIKV and IAV. HBA(111-142) is released from its precursor by ubiquitous aspartic proteases under acidic conditions characteristic at sites of infection and inflammation. Thus, HBA(111-142) is an amyloidogenic AMP that may specifically be generated from a highly abundant precursor during bacterial or viral infection and may play an important role in innate antimicrobial immune responses.
Asunto(s)
COVID-19 , Infección por el Virus Zika , Virus Zika , Humanos , Péptidos , Amiloide/química , Antibacterianos/farmacología , HemoglobinasRESUMEN
Both experimental and theoretical structure determinations of RNAs have remained challenging due to the intrinsic dynamics of RNAs. We report here an integrated nuclear magnetic resonance/molecular dynamics (NMR/MD) structure determination approach to describe the dynamic structure of the CUUG tetraloop. We show that the tetraloop undergoes substantial dynamics, leading to averaging of the experimental data. These dynamics are particularly linked to the temperature-dependent presence of a hydrogen bond within the tetraloop. Interpreting the NMR data by a single structure represents the low-temperature structure well but fails to capture all conformational states occurring at a higher temperature. We integrate MD simulations, starting from structures of CUUG tetraloops within the Protein Data Bank, with an extensive set of NMR data, and provide a structural ensemble that describes the dynamic nature of the tetraloop and the experimental NMR data well. We thus show that one of the most stable and frequently found RNA tetraloops displays substantial dynamics, warranting such an integrated structural approach.
Asunto(s)
Simulación de Dinámica Molecular , ARN , ARN/química , Conformación de Ácido Nucleico , Espectroscopía de Resonancia Magnética , TemperaturaRESUMEN
The ribosomal S1 protein (rS1) is indispensable for translation initiation in Gram-negative bacteria. rS1 is a multidomain protein that acts as an RNA chaperone and ensures that mRNAs can bind the ribosome in a single-stranded conformation, which could be related to fast recognition. Although many ribosome structures were solved in recent years, a high-resolution structure of a two-domain mRNA-binding competent rS1 construct is not yet available. Here, we present the NMR solution structure of the minimal mRNA-binding fragment of Vibrio Vulnificus rS1 containing the domains D3 and D4. Both domains are homologues and adapt an oligonucleotide-binding fold (OB fold) motif. NMR titration experiments reveal that recognition of miscellaneous mRNAs occurs via a continuous interaction surface to one side of these structurally linked domains. Using a novel paramagnetic relaxation enhancement (PRE) approach and exploring different spin-labeling positions within RNA, we were able to track the location and determine the orientation of the RNA in the rS1-D34 bound form. Our investigations show that paramagnetically labeled RNAs, spiked into unmodified RNA, can be used as a molecular ruler to provide structural information on protein-RNA complexes. The dynamic interaction occurs on a defined binding groove spanning both domains with identical ß2-ß3-ß5 interfaces. Evidently, the 3'-ends of the cis-acting RNAs are positioned in the direction of the N-terminus of the rS1 protein, thus towards the 30S binding site and adopt a conformation required for translation initiation.
Asunto(s)
Proteínas Bacterianas/química , ARN Mensajero/química , Proteínas Ribosómicas/química , Vibrio vulnificus/química , Modelos Moleculares , Resonancia Magnética Nuclear Biomolecular , Unión Proteica , Biosíntesis de Proteínas , Dominios Proteicos , RiboswitchRESUMEN
Past sequencing campaigns overlooked small proteins as they seemed to be irrelevant due to their small size. However, their occurrence is widespread, and there is growing evidence that these small proteins are in fact functionally very important in organisms found in all kingdoms of life. Within a global proteome analysis for small proteins of the archaeal model organism Haloferax volcanii, the HVO_2922 protein has been identified. It is differentially expressed in response to changes in iron and salt concentrations, thus suggesting that its expression is stress-regulated. The protein is conserved among Haloarchaea and contains an uncharacterized domain of unknown function (DUF1508, UPF0339 family protein). We elucidated the NMR solution structure, which shows that the isolated protein forms a symmetrical dimer. The dimerization is found to be concentration-dependent and essential for protein stability and most likely for its functionality, as mutagenesis at the dimer interface leads to a decrease in stability and protein aggregation.
Asunto(s)
Proteínas Arqueales/química , Haloferax volcanii/química , Termodinámica , Proteínas Arqueales/metabolismo , Haloferax volcanii/metabolismo , Modelos Moleculares , Resonancia Magnética Nuclear Biomolecular , Conformación Proteica , Estabilidad Proteica , SolucionesRESUMEN
Proteins encoded by small open reading frames (sORFs) have a widespread occurrence in diverse microorganisms and can be of high functional importance. However, due to annotation biases and their technically challenging direct detection, these small proteins have been overlooked for a long time and were only recently rediscovered. The currently rapidly growing number of such proteins requires efficient methods to investigate their structure-function relationship. Herein, a method is presented for fast determination of the conformational properties of small proteins. Their small size makes them perfectly amenable for solution-state NMR spectroscopy. NMR spectroscopy can provide detailed information about their conformational states (folded, partially folded, and unstructured). In the context of the priority program on small proteins funded by the German research foundation (SPP2002), 27 small proteins from 9 different bacterial and archaeal organisms have been investigated. It is found that most of these small proteins are unstructured or partially folded. Bioinformatics tools predict that some of these unstructured proteins can potentially fold upon complex formation. A protocol for fast NMR spectroscopy structure elucidation is described for the small proteins that adopt a persistently folded structure by implementation of new NMR technologies, including automated resonance assignment and nonuniform sampling in combination with targeted acquisition.
Asunto(s)
Archaea/metabolismo , Proteínas Arqueales/química , Bacterias/metabolismo , Proteínas Bacterianas/química , Biología Computacional/métodos , Resonancia Magnética Nuclear Biomolecular/métodos , Pliegue de Proteína , Sistemas de Lectura Abierta , Conformación ProteicaRESUMEN
G-protein-coupled receptors (GPCRs) are the most important signal transducers in higher eukaryotes. Despite considerable progress, the molecular basis of subtype-specific ligand selectivity, especially for peptide receptors, remains unknown. Here, by integrating DNP-enhanced solid-state NMR spectroscopy with advanced molecular modeling and docking, the mechanism of the subtype selectivity of human bradykinin receptors for their peptide agonists has been resolved. The conserved middle segments of the bound peptides show distinct conformations that result in different presentations of their N and C termini toward their receptors. Analysis of the peptide-receptor interfaces reveals that the charged N-terminal residues of the peptides are mainly selected through electrostatic interactions, whereas the C-terminal segments are recognized via both conformations and interactions. The detailed molecular picture obtained by this approach opens a new gateway for exploring the complex conformational and chemical space of peptides and peptide analogs for designing GPCR subtype-selective biochemical tools and drugs.
Asunto(s)
Cininas/química , Receptor de Bradiquinina B1/química , Receptor de Bradiquinina B2/química , Receptores Acoplados a Proteínas G/química , Electricidad Estática , Animales , Células HEK293 , Humanos , Insectos , Ligandos , Espectroscopía de Resonancia Magnética , Modelos Moleculares , Simulación del Acoplamiento Molecular , Mutación , Péptidos/química , Unión Proteica , Dominios Proteicos , Estructura Secundaria de Proteína , Células Sf9 , Transducción de SeñalRESUMEN
Despite the great interest in glycoproteins, structural information reporting on conformation and dynamics of the sugar moieties are limited. We present a new biochemical method to express proteins with glycans that are selectively labeled with NMR-active nuclei. We report on the incorporation of 13 C-labeled mannose in the C-mannosylated UNC-5 thrombospondin repeat. The conformational landscape of the C-mannose sugar puckers attached to tryptophan residues of UNC-5 is characterized by interconversion between the canonical 1 C4 state and the B03 / 1 S3 state. This flexibility may be essential for protein folding and stabilization. We foresee that this versatile tool to produce proteins with selectively labeled C-mannose can be applied and adjusted to other systems and modifications and potentially paves a way to advance glycoprotein research by unravelling the dynamical and conformational properties of glycan structures and their interactions.
RESUMEN
The discovery that MptpA (low-molecular-weight protein tyrosine phosphatase A) from Mycobacterium tuberculosis (Mtb) has an essential role for Mtb virulence has motivated research of tyrosine-specific phosphorylation in Mtb and other pathogenic bacteria. The phosphatase activity of MptpA is regulated via phosphorylation on Tyr128 and Tyr129 Thus far, only a single tyrosine-specific kinase, protein-tyrosine kinase A (PtkA), encoded by the Rv2232 gene has been identified within the Mtb genome. MptpA undergoes phosphorylation by PtkA. PtkA is an atypical bacterial tyrosine kinase, as its sequence differs from the sequence consensus within this family. The lack of structural information on PtkA hampers the detailed characterization of the MptpA-PtkA interaction. Here, using NMR spectroscopy, we provide a detailed structural characterization of the PtkA architecture and describe its intra- and intermolecular interactions with MptpA. We found that PtkA's domain architecture differs from the conventional kinase architecture and is composed of two domains, the N-terminal highly flexible intrinsically disordered domain (IDDPtkA) and the C-terminal rigid kinase core domain (KCDPtkA). The interaction between the two domains, together with the structural model of the complex proposed in this study, reveal that the IDDPtkA is unstructured and highly dynamic, allowing for a "fly-casting-like" mechanism of transient interactions with the rigid KCDPtkA This interaction modulates the accessibility of the KCDPtkA active site. In general, the structural and functional knowledge of PtkA gained in this study is crucial for understanding the MptpA-PtkA interactions, the catalytic mechanism, and the role of the kinase-phosphatase regulatory system in Mtb virulence.
Asunto(s)
Proteínas Bacterianas/química , Mycobacterium tuberculosis/enzimología , Proteínas Tirosina Quinasas/química , Proteínas Bacterianas/metabolismo , Humanos , Enlace de Hidrógeno , Modelos Moleculares , Mycobacterium tuberculosis/química , Mycobacterium tuberculosis/metabolismo , Resonancia Magnética Nuclear Biomolecular , Fosforilación , Conformación Proteica , Proteínas Tirosina Fosfatasas/metabolismo , Proteínas Tirosina Quinasas/metabolismo , Tuberculosis/microbiologíaRESUMEN
The interaction of fibroblast growth factors (FGFs) with their fibroblast growth factor receptors (FGFRs) are important in the signaling network of cell growth and development. SSR128129E (SSR), a ligand of small molecular weight with potential anti-cancer properties, acts allosterically on the extracellular domains of FGFRs. Up to now, the structural basis of SSR binding to the D3 domain of FGFR remained elusive. This work reports the structural characterization of the interaction of SSR with one specific receptor, FGFR3, by NMR spectroscopy. This information provides a basis for rational drug design for allosteric FGFR inhibitors.
Asunto(s)
Antineoplásicos/química , Indolizinas/química , Inhibidores de Proteínas Quinasas/química , Receptores de Factores de Crecimiento de Fibroblastos/antagonistas & inhibidores , ortoaminobenzoatos/química , Regulación Alostérica , Diseño de Fármacos , Simulación del Acoplamiento Molecular , Unión Proteica , Receptores de Factores de Crecimiento de Fibroblastos/química , Relación Estructura-Actividad , TermodinámicaRESUMEN
Photolabile protecting groups are widely used to trigger oligonucleotide activity. The ON/OFF-amplitude is a critical parameter. An experimental setup has been developed to identify protecting group derivatives with superior caging properties. Bulky rests are attached to the cage moiety via Cu-catalyzed azide-alkyne cycloaddition post-synthetically on DNA. Interestingly, the decrease in melting temperature upon introducing o-nitrobenzyl-caged (NPBY-) and diethylaminocoumarin-cages (DEACM-) in DNA duplexes reaches a limiting value. NMR spectroscopy was used to characterize individual base-pair stabilities and determine experimental structures of a selected number of photocaged DNA molecules. The experimental structures agree well with structures predicted by MD simulations. Combined, the structural data indicate that once a sterically demanding group is added to generate a tri-substituted carbon, the sterically less demanding cage moiety points towards the neighboring nucleoside and the bulkier substituents remain in the major groove.
Asunto(s)
ADN/química , Nucleósidos/química , Alquinos/química , Azidas/química , Emparejamiento Base , Catálisis , Cobre/química , Reacción de Cicloadición , Espectroscopía de Resonancia Magnética , Simulación de Dinámica Molecular , Conformación de Ácido Nucleico , EstereoisomerismoRESUMEN
Encodable lanthanide binding tags (LBTs) have become an attractive tool in modern structural biology as they can be expressed as fusion proteins of targets of choice. Previously, we have demonstrated the feasibility of inserting encodable LBTs into loop positions of interleukin-1ß (Barthelmes et al. in J Am Chem Soc 133:808-819, 2011). Here, we investigate the differences in fast dynamics of selected loop-LBT interleukin-1ß constructs by measuring 15N nuclear spin relaxation experiments. We show that the loop-LBT does not significantly alter the dynamic motions of the host protein in the sub-τc-timescale and that the loop-LBT adopts a rigid conformation with significantly reduced dynamics compared to the terminally attached encodable LBT leading to increased paramagnetic alignment strength. We further analyze residual dipolar couplings (RDCs) obtained by loop-LBTs and additional liquid crystalline media to assess the applicability of the loop-LBT approach for RDC-based methods to determine structure and dynamics of proteins, including supra-τc dynamics. Using orthogonalized linear combinations (OLCs) of RDCs and Saupe matrices, we show that the combined use of encodable LBTs and external alignment media yields up to five linear independent alignments.
Asunto(s)
Interleucina-1beta/química , Elementos de la Serie de los Lantanoides/química , Conformación ProteicaRESUMEN
Telomeric G-quadruplexes have recently emerged as drug targets in cancer research. Herein, we present the first NMR structure of a telomeric DNA G-quadruplex that adopts the biologically relevant hybrid-2 conformation in a ligand-bound state. We solved the complex with a metalorganic gold(III) ligand that stabilizes G-quadruplexes. Analysis of the free and bound structures reveals structural changes in the capping region of the G-quadruplex. The ligand is sandwiched between one terminal G-tetrad and a flanking nucleotide. This complex structure involves a major structural rearrangement compared to the free G-quadruplex structure as observed for other G-quadruplexes in different conformations, invalidating simple docking approaches to ligand-G-quadruplex structure determination.
Asunto(s)
ADN/química , G-Cuádruplex , Sustancias Macromoleculares/química , Conformación de Ácido Nucleico , Espectroscopía de Protones por Resonancia Magnética/métodos , Sitios de Unión , Oro/química , Humanos , Ligandos , Compuestos Organometálicos/química , TelómeroRESUMEN
The ability of three different bifunctional azobenzene linkers to enable the photoreversible formation of a defined intermolecular two-tetrad G-quadruplex upon UV/Vis irradiation was investigated. Circular dichroism and NMR spectroscopic data showed the formation of G-quadruplexes with K(+) â ions at room temperature in all three cases with the corresponding azobenzene linker in an Eâ conformation. However, only the para-para-substituted azobenzene derivative enables photoswitching between a nonpolymorphic, stacked, tetramolecular G-quadruplex and an unstructured state after E-Zâ isomerization.
Asunto(s)
G-Cuádruplex , Modelos Moleculares , Conformación de Ácido Nucleico , Fotoquímica , Espectroscopía de Protones por Resonancia MagnéticaRESUMEN
The energy landscapes of human telomeric G-quadruplexes are complex, and their folding pathways have remained largely unexplored. By using real-time NMR spectroscopy, we investigated the K(+)-induced folding of the human telomeric DNA sequence 5'-TTGGG(TTAGGG)3 A-3'. Three long-lived states were detected during folding: a major conformation (hybrid-1), a previously structurally uncharacterized minor conformation (hybrid-2), and a partially unfolded state. The minor hybrid-2 conformation is formed faster than the more stable hybrid-1 conformation. Equilibration of the two states is slow and proceeds via a partially unfolded intermediate state, which can be described as an ensemble of hairpin-like structures.
Asunto(s)
ADN/química , G-Cuádruplex , Telómero/química , Secuencia de Bases , Humanos , Cinética , Resonancia Magnética Nuclear BiomolecularRESUMEN
Photolabile protecting groups are a versatile tool to trigger reactions by light irradiation. In this study, we have investigated the influence of the absolute configuration of the 1-(2-nitrophenyl)ethyl (NPE) cage group on a 15-base-pair duplex DNA. Using UV melting, we determined the global stability of the unmodified and the selectively (S)- and (R)-NPE-modified DNA sequences, respectively. We observe a differently destabilizing effect for the two NPE stereoisomers on the global stability. Analysis of the temperature dependence of imino proton exchange rates measured by NMR spectroscopy reveals that this effect can be attributed to decreased base pair stabilities of the caged and the 3'-neighbouring base pair, respectively. Furthermore, our NMR based structural models of the modified duplexes provide a structural basis for the distinct effect of the (S)- and the (R)-NPE group.
Asunto(s)
Citosina/química , ADN/química , Nitrobencenos/química , Emparejamiento Base , Espectroscopía de Resonancia Magnética , Modelos Moleculares , Estructura MolecularRESUMEN
SSR128129E (SSR) is a unique small-molecule inhibitor of fibroblast growth factor receptors (FGFRs). SSR is a high-affinity allosteric binder that selectively blocks one of the two major FGFR-mediated pathways. The mechanisms of SSR activity were studied previously in much detail, allowing the identification of its binding site, located in the hydrophobic groove of the receptor D3 domain. The binding site overlaps with the position of an N-terminal helix, an element exclusive for the FGF8b growth factor, which could potentially convert SSR from an allosteric inhibitor into an orthosteric blocker for the particular FGFR/FGF8b system. In this regard, we report here on the structural and functional investigation of FGF8b/FGFR3c system and the effects imposed on it by SSR. We show that SSR is equally or more potent in inhibiting FGF8b-induced FGFR signaling compared to FGF2-induced activation. On the other hand, when studied in the context of separate extracellular domains of FGFR3c in solution with NMR spectroscopy, SSR is unable to displace the N-terminal helix of FGF8b from its binding site on FGFR3c and behaves as a weak orthosteric inhibitor. The substantial inconsistency between the results obtained with cell culture and for the individual water-soluble subdomains of the FGFR proteins points to the important role played by the cell membrane.
RESUMEN
Protein-tyrosine phosphatases (PTPs) and protein-tyrosine kinases co-regulate cellular processes. In pathogenic bacteria, they are frequently exploited to act as key virulence factors for human diseases. Mycobacterium tuberculosis, the causative organism of tuberculosis, secretes a low molecular weight PTP (LMW-PTP), MptpA, which is required for its survival upon infection of host macrophages. Although there is otherwise no sequence similarity of LMW-PTPs to other classes of PTPs, the phosphate binding loop (P-loop) CX(5)R and the loop containing a critical aspartic acid residue (D-loop), required for the catalytic activity, are well conserved. In most high molecular weight PTPs, ligand binding to the P-loop triggers a large conformational reorientation of the D-loop, in which it moves â¼10 Å, from an "open" to a "closed" conformation. Until now, there have been no ligand-free structures of LMW-PTPs described, and hence the dynamics of the D-loop have remained largely unknown for these PTPs. Here, we present a high resolution solution NMR structure of the free form of the MptpA LMW-PTP. In the absence of ligand and phosphate ions, the D-loop adopts an open conformation. Furthermore, we characterized the binding site of phosphate, a competitive inhibitor of LMW-PTPs, on MptpA and elucidated the involvement of both the P- and D-loop in phosphate binding. Notably, in LMW-PTPs, the phosphorylation status of two well conserved tyrosine residues, typically located in the D-loop, regulates the enzyme activity. PtkA, the kinase complementary to MptpA, phosphorylates these two tyrosine residues in MptpA. We characterized the MptpA-PtkA interaction by NMR spectroscopy to show that both the P- and D-loop form part of the binding interface.
Asunto(s)
Proteínas Bacterianas , Proteínas Quinasas Dependientes de AMP Cíclico , Macrófagos/enzimología , Mycobacterium tuberculosis/enzimología , Proteínas Tirosina Fosfatasas , Apoenzimas , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Cristalografía por Rayos X , Proteínas Quinasas Dependientes de AMP Cíclico/química , Proteínas Quinasas Dependientes de AMP Cíclico/genética , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Sistemas de Liberación de Medicamentos , Humanos , Macrófagos/microbiología , Mycobacterium tuberculosis/genética , Resonancia Magnética Nuclear Biomolecular , Fosforilación/genética , Estructura Secundaria de Proteína , Proteínas Tirosina Fosfatasas/química , Proteínas Tirosina Fosfatasas/genética , Proteínas Tirosina Fosfatasas/metabolismoRESUMEN
Protein kinases are highly dynamic and complex molecules. Here we present high-pressure and relaxation studies of the activated p38α mitogen-activated protein kinase (MAPK). p38α plays a central role in inflammatory diseases such as rheumatoid arthritis and is therefore a highly attractive pharmaceutical target. The combination of high pressure and NMR spectroscopy allowed for a detailed per-residue based assessment of the structural plasticity of p38α and the accessibility of low-lying excited-energy conformations throughout the kinase structure. Such information is uniquely accessible through the combination of liquid-state NMR and high pressure and is of considerable value for the drug discovery process. The interactions of p38α and DFG-in and DFG-out ligands were studied under the application of high pressure, and we demonstrate how we can alter kinase dynamics by pressure in a similar way to what has previously only been observed by ligand binding. Pressure is shown to be a mild and efficient tool for manipulation of intermediate-timescale dynamics.
Asunto(s)
Proteína Quinasa 14 Activada por Mitógenos/química , Resonancia Magnética Nuclear Biomolecular , Animales , Ratones , Proteína Quinasa 14 Activada por Mitógenos/genética , Proteína Quinasa 14 Activada por Mitógenos/metabolismo , Presión , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , TemperaturaRESUMEN
The splicing isoform b of human fibroblast growth factor 8 (FGF8b) is an important regulator of brain embryonic development. Here, we report the almost complete NMR chemical shift assignment of the backbone and aliphatic side chains of FGF8b. Obtained chemical shifts are in good agreement with the previously reported X-ray data, excluding the N-terminal gN helix, which apparently forms only in complex with the receptor. The reported data provide an NMR starting point for the investigation of FGF8b interaction with its receptors and with potential drugs or inhibitors.