RESUMO
We describe complex formation between a designed pentameric ß-propeller and the anionic macrocycle sulfonato-calix[8]arene (sclx8), as characterized by X-ray crystallography and NMR spectroscopy. Two crystal structures and 15N HSQC experiments reveal a single calixarene binding site in the concave pocket of the ß-propeller toroid. Despite the symmetry mismatch between the pentameric protein and the octameric macrocycle, they form a high affinity multivalent complex, with the largest protein-calixarene interface observed to date. This system provides a platform for investigating multivalency.
Assuntos
Calixarenos , Calixarenos/química , Lectinas , Cristalografia por Raios X , Espectroscopia de Ressonância Magnética , Sítios de LigaçãoRESUMO
Understanding the fine structural details of inhibitor binding at the active site of metalloenzymes can have a profound impact on the rational drug design targeted to this broad class of biomolecules. Structural techniques such as NMR, cryo-EM, and X-ray crystallography can provide bond lengths and angles, but the uncertainties in these measurements can be as large as the range of values that have been observed for these quantities in all the published structures. This uncertainty is far too large to allow for reliable calculations at the quantum chemical (QC) levels for developing precise structure-activity relationships or for improving the energetic considerations in protein-inhibitor studies. Therefore, the need arises to rely upon computational methods to refine the active site structures well beyond the resolution obtained with routine application of structural methods. In a recent paper, we have shown that it is possible to refine the active site of cobalt(II)-substituted MMP12, a metalloprotein that is a relevant drug target, by matching to the experimental pseudocontact shifts (PCS) those calculated using multireference ab initio QC methods. The computational cost of this methodology becomes a significant bottleneck when the starting structure is not sufficiently close to the final one, which is often the case with biomolecular structures. To tackle this problem, we have developed an approach based on a neural network (NN) and a support vector regression (SVR) and applied it to the refinement of the active site structure of oxalate-inhibited human carbonic anhydrase 2 (hCAII), another prototypical metalloprotein target. The refined structure gives a remarkably good agreement between the QC-calculated and the experimental PCS. This study not only contributes to the knowledge of CAII but also demonstrates the utility of combining machine learning (ML) algorithms with QC calculations, offering a promising avenue for investigating other drug targets and complex biological systems in general.
Assuntos
Domínio Catalítico , Aprendizado de Máquina , Metaloproteínas , Teoria Quântica , Metaloproteínas/química , Humanos , Modelos Moleculares , Metaloproteinase 12 da Matriz/química , Metaloproteinase 12 da Matriz/metabolismoRESUMO
The assessment of the higher-order structure (HOS) by NMR is a powerful methodology to characterize the structural features of biologics. Forced oxidative stress studies are used to investigate the stability profile, to develop pharmaceutical formulations and analytical methods. Here, the effects of forced oxidative stress by H2O2 on the monoclonal antibody Abituzumab have been characterized by a multianalytical approach combining NMR spectroscopy, mass spectrometry, differential scanning calorimetry, surface plasmon resonance, computational tools, and bioassays. This integrated strategy has provided qualitative and semiquantitative characterization of the samples and information at residue level of the effects that oxidation has on the HOS of Abituzumab, correlating them to the loss of the biological activity.
Assuntos
Anticorpos Monoclonais , Peróxido de Hidrogênio , Fluxo de Trabalho , Anticorpos Monoclonais/química , Espectroscopia de Ressonância MagnéticaRESUMO
The calcium release activated calcium channel is activated by the endoplasmic reticulum-resident calcium sensor protein STIM1. On activation, STIM1 C terminus changes from an inactive, tight to an active, extended conformation. A coiled-coil clamp involving the CC1 and CC3 domains is essential in controlling STIM1 activation, with CC1 as the key entity. The nuclear magnetic resonance-derived solution structure of the CC1 domain represents a three-helix bundle stabilized by interhelical contacts, which are absent in the Stormorken disease-related STIM1 R304W mutant. Two interhelical sites between the CC1α1 and CC1α2 helices are key in controlling STIM1 activation, affecting the balance between tight and extended conformations. Nuclear magnetic resonance-directed mutations within these interhelical interactions restore the physiological, store-dependent activation behavior of the gain-of-function STIM1 R304W mutant. This study reveals the functional impact of interhelical interactions within the CC1 domain for modifying the CC1-CC3 clamp strength to control the activation of STIM1.
Assuntos
Canais de Cálcio Ativados pela Liberação de Cálcio/metabolismo , Proteínas de Neoplasias/genética , Molécula 1 de Interação Estromal/genética , Transtornos Plaquetários/genética , Clonagem Molecular , Dislexia/genética , Eritrócitos Anormais , Células HEK293 , Humanos , Ictiose/genética , Espectroscopia de Ressonância Magnética , Transtornos de Enxaqueca/genética , Miose/genética , Modelos Moleculares , Fadiga Muscular/genética , Mutação/genética , Conformação de Ácido Nucleico , Proteína ORAI1/genética , Técnicas de Patch-Clamp , Baço/anormalidadesRESUMO
Targeting immune checkpoints is a well-established strategy in cancer therapy, and antibodies blocking PD-1/PD-L1 interactions to restore the immunological activity against cancer cells have been clinically validated. High-affinity mutants of the PD-1 ectodomain have recently been proposed as an alternative to antibodies to target PD-L1 on cancer cells, shedding new light on this research area. In this dynamic scenario, the PD-1 mutant, here reported, largely expands the chemical space of nonantibody and nonsmall-molecule inhibitor therapeutics that can be used to target cancer cells overexpressing PD-L1 receptors. The polyethylene glycol moieties and the immune response-stimulating carbohydrates, used as site-selective tags, represent the proof of concept for future applications.
Assuntos
Neoplasias , Receptor de Morte Celular Programada 1 , Humanos , Receptor de Morte Celular Programada 1/genética , Receptor de Morte Celular Programada 1/química , Antígeno B7-H1 , Anticorpos , Neoplasias/tratamento farmacológico , Neoplasias/genéticaRESUMO
Several protein-drug conjugates are currently being used in cancer therapy. These conjugates rely on cytotoxic organic compounds that are covalently attached to the carrier proteins or that interact with them via non-covalent interactions. Human transthyretin (TTR), a physiological protein, has already been identified as a possible carrier protein for the delivery of cytotoxic drugs. Here we show the structure-guided development of a new stable cytotoxic molecule based on a known strong binder of TTR and a well-established anticancer drug. This example is used to demonstrate the importance of the integration of multiple biophysical and structural techniques, encompassing microscale thermophoresis, X-ray crystallography and NMR. In particular, we show that solid-state NMR has the ability to reveal effects caused by ligand binding which are more easily relatable to structural and dynamical alterations that impact the stability of macromolecular complexes.
Assuntos
Proteínas de Transporte , Imageamento por Ressonância Magnética , Humanos , Preparações Farmacêuticas , Espectroscopia de Ressonância Magnética , Proteínas de Transporte/química , Cristalografia por Raios XRESUMO
Multispecific biologics are an emerging class of drugs, in which antibodies and/or proteins designed to bind pharmacological targets are covalently linked or expressed as fusion proteins to increase both therapeutic efficacy and safety. Epitope mapping on the target proteins provides key information to improve the affinity and also to monitor the manufacturing process and drug stability. Solid-state NMR has been here used to identify the pattern of the residues of the programmed cell death ligand 1 (PD-L1) ectodomain that are involved in the interaction with a new multispecific biological drug. This is possible because the large size and the intrinsic flexibility of the complexes are not limiting factors for solid-state NMR.
Assuntos
Produtos Biológicos , Anticorpos , Mapeamento de Epitopos , Espectroscopia de Ressonância Magnética , Proteínas/químicaRESUMO
Protein solid-state NMR has evolved dramatically over the last two decades, with the development of new hardware and sample preparation methodologies. This technique is now ripe for complex applications, among which one can count bioconjugation, protein chemistry and functional biomaterials. In this review, we provide our account on this aspect of protein solid-state NMR.
Assuntos
Imageamento por Ressonância Magnética , Proteínas , Espectroscopia de Ressonância Magnética/métodos , Proteínas/químicaRESUMO
Biocompatible hydrogels for tissue regeneration/replacement and drug release with specific architectures can be obtained by three-dimensional bioprinting techniques. The preservation of the higher order structure of the proteins embedded in the hydrogels as drugs or modulators is critical for their biological activity. Solution nuclear magnetic resonance (NMR) experiments are currently used to investigate the higher order structure of biotherapeutics in comparability, similarity, and stability studies. However, the size of pores in the gel, protein-matrix interactions, and the size of the embedded proteins often prevent the use of this methodology. The recent advancements of solid-state NMR allow for the comparison of the higher order structure of the matrix-embedded and free isotopically enriched proteins, allowing for the evaluation of the functionality of the material in several steps of hydrogel development. Moreover, the structural information at atomic detail on the matrix-protein interactions paves the way for a structure-based design of these biomaterials.
Assuntos
Bioimpressão , Liberação Controlada de Fármacos , Hidrogéis , Impressão Tridimensional , Engenharia Tecidual , Alicerces TeciduaisRESUMO
Nuclear magnetic resonance (NMR) is an effective, commonly used experimental approach to screen small organic molecules against a protein target. A very popular method consists of monitoring the changes of the NMR chemical shifts of the protein nuclei upon addition of the small molecule to the free protein. Multidimensional NMR experiments allow the interacting residues to be mapped along the protein sequence. A significant amount of human effort goes into manually tracking the chemical shift variations, especially when many signals exhibit chemical shift changes and when many ligands are tested. Some computational approaches to automate the procedure are available, but none of them as a web server. Furthermore, some methods require the adoption of a fairly specific experimental setup, such as recording a series of spectra at increasing small molecule:protein ratios. In this work, we developed a tool requesting a minimal amount of experimental data from the user, implemented it as an open-source program, and made it available as a web application. Our tool compares two spectra, one of the free protein and one of the small molecule:protein mixture, based on the corresponding peak lists. The performance of the tool in terms of correct identification of the protein-binding regions has been evaluated on different protein targets, using experimental data from interaction studies already available in the literature. For a total of 16 systems, our tool achieved between 79% and 100% correct assignments, properly identifying the protein regions involved in the interaction.
Assuntos
Algoritmos , Proteínas , Sequência de Aminoácidos , Humanos , Ligantes , Espectroscopia de Ressonância Magnética/métodos , Ressonância Magnética Nuclear Biomolecular/métodos , Proteínas/químicaRESUMO
HMGB1 is a ubiquitous non-histone protein, which biological effects depend on its expression and subcellular location. Inside the nucleus, HMGB1 is engaged in many DNA events such as DNA repair, transcription and telomere maintenance. HMGB1 has been reported to bind preferentially to bent DNA as well as to noncanonical DNA structures like 4-way junctions and, more recently, to G-quadruplexes. These are four-stranded conformations of nucleic acids involved in important cellular processes, including telomere maintenance. In this frame, G-quadruplex recognition by specific proteins represents a key event to modulate physiological or pathological pathways. Herein, to get insights into the telomeric G-quadruplex DNA recognition by HMGB1, we performed detailed biophysical studies complemented with biological analyses. The obtained results provided information about the molecular determinants for the interaction and showed that the structural variability of human telomeric G-quadruplex DNA may have significant implications in HMGB1 recognition. The biological data identified HMGB1 as a telomere-associated protein in both telomerase-positive and -negative tumor cells and showed that HMGB1 gene silencing in such cells induces telomere DNA damage foci. Altogether, these findings provide a deeper understanding of telomeric G-quadruplex recognition by HMGB1 and suggest that this protein could actually represent a new target for cancer therapy.
Assuntos
Quadruplex G , Proteína HMGB1/genética , Conformação de Ácido Nucleico , Telômero/genética , DNA/química , DNA/genética , Escherichia coli/genética , Proteína HMGB1/química , Humanos , Telomerase/química , Telomerase/genética , Telômero/químicaRESUMO
Mutating the side-chains of amino acids in a peptide ligand, with unnatural amino acids, aiming to mitigate its short half-life is an established approach. However, it is hypothesized that mutating specific backbone peptide bonds with bioisosters can be exploited not only to enhance the proteolytic stability of parent peptides, but also to tune its receptor subtype selectivity. Towards this end, four [Y]6 -Angiotensin II analogues are synthesized where amide bonds have been replaced by 1,4-disubstituted 1,2,3-triazole isosteres in four different backbone locations. All the analogues possessed enhanced stability in human plasma in comparison with the parent peptide, whereas only two of them achieved enhanced AT2 R/AT1 R subtype selectivity. This diversification has been studied through 2D NMR spectroscopy and unveiled a putative more structured microenvironment for the two selective ligands accompanied with increased number of NOE cross-peaks. The most potent analogue, compoundâ 2, has been explored regarding its neurotrophic potential and resulted in an enhanced neurite growth with respect to the established agent C21.
Assuntos
Angiotensina II/química , Angiotensina II/metabolismo , Mutação , Peptídeos/genética , Receptores de Angiotensina/química , Receptores de Angiotensina/metabolismo , Aminoácidos/genética , Angiotensina II/genética , Animais , Células HEK293 , Humanos , Ligantes , Peptídeos/química , Peptídeos/metabolismo , Especificidade por SubstratoRESUMO
Paramagnetic restraints have been used in biomolecular NMR for the last three decades to elucidate and refine biomolecular structures, but also to characterize protein-ligand interactions. A common technique to generate such restraints in proteins, which do not naturally contain a (paramagnetic) metal, consists in the attachment to the protein of a lanthanide-binding-tag (LBT). In order to design such LBTs, it is important to consider the efficiency and stability of the conjugation, the geometry of the complex (conformational exchanges and coordination) and the chemical inertness of the ligand. Here we describe a photo-catalyzed thiol-ene reaction for the cysteine-selective paramagnetic tagging of proteins. As a model, we designed an LBT with a vinyl-pyridine moiety which was used to attach our tag to the protein GB1 in fast and irreversible fashion. Our tag T1 yields magnetic susceptibility tensors of significant size with different lanthanides and has been characterized using NMR and relaxometry measurements.
Assuntos
Proteínas/química , Compostos de Sulfidrila/química , Catálise , Cisteína/química , Elementos da Série dos Lantanídeos/química , Ligantes , Espectroscopia de Ressonância Magnética/métodos , Ressonância Magnética Nuclear Biomolecular/métodos , Processos Fotoquímicos , Piridinas/químicaRESUMO
FlowNMR has the aim of continuously monitoring processes that occur in conditions that are not compatible with being carried out within a closed tube. However, it is sample intensive and not suitable for samples, such as proteins or living cells, that are often available in limited volumes and possibly low concentrations. We here propose a dialysis-based modification of a commercial flowNMR setup that allows for recycling the medium while confining the sample (proteins and cells) within the active volume of the tube. This approach is demonstrated in the specific cases of in-cell NMR and protein-based ligand studies.
Assuntos
Técnicas de Cultura Celular por Lotes/métodos , Reatores Biológicos , Espectroscopia de Ressonância Magnética/métodos , Técnicas de Cultura Celular por Lotes/instrumentação , Anidrase Carbônica II/antagonistas & inibidores , Anidrase Carbônica II/metabolismo , Descoberta de Drogas/métodos , Inibidores Enzimáticos/farmacologia , Células HEK293 , Humanos , Espectroscopia de Ressonância Magnética/instrumentação , Ligação Proteica , Superóxido Dismutase/antagonistas & inibidores , Superóxido Dismutase/metabolismoRESUMO
Solid state NMR (SSNMR) has earned a substantial success in the characterization of paramagnetic systems over the last decades. Nowadays, the resolution and sensitivity of solid state NMR in biological molecules has improved significantly and these advancements can be translated into the study of paramagnetic biomolecules. However, the electronic properties of different metal centers affect the quality of their SSNMR spectra differently, and not all systems turn out to be equally easy to approach by this technique. In this review we will try to give an overview of the properties of different paramagnetic centers and how they can be used to increase the chances of experimental success.
Assuntos
Espectroscopia de Ressonância Magnética/métodos , Metaloproteínas/química , Metais/química , Ressonância Magnética Nuclear Biomolecular/métodos , Compostos Organometálicos/química , Cobre/química , Ferro/química , Modelos Moleculares , Níquel/química , Conformação ProteicaRESUMO
Partial symmetry, i.e., the presence of more than one molecule in the asymmetric unit of a crystal, is a relatively rare phenomenon in small-molecule crystallography, but is quite common in protein crystallography, where it is typically known as non-crystallographic symmetry (NCS). Several papers in literature propose molecular determinants such as crystal contacts, thermal factors, or TLS parameters as an explanation for the phenomenon of intrinsic asymmetry among molecules that are in principle equivalent. Nevertheless, are all of the above determinants the cause or are they rather the effect? In the general frame of the NCS often observed in crystals of biomolecules, this paper deals with nickel(II)-substituted human carbonic anhydrase(II) (hCAII) and its SAD structure determination at the nickel edge. The structure revealed two non-equivalent molecules in the asymmetric unit, the presence of a secondary nickel-binding site at the N-terminus of both molecules (which had never been found before in the nickel-substituted enzyme) and two different coordination geometries of the active site nickel (hexa-coordinated in one molecule and mainly penta-coordinated in the other). The above-mentioned standard molecular crystallographic determinants of this asymmetry are analyzed and presented in detail for this particular case. From these considerations, we speculate on the existence of a fundamental, although yet unknown, common cause for the partial symmetry that is so often encountered in X-ray structures of biomolecules.
Assuntos
Anidrase Carbônica II/química , Níquel/química , Sítios de Ligação , Cristalografia por Raios X , Humanos , Modelos Moleculares , Conformação ProteicaRESUMO
Resonance assignment and structural characterization of pharmacologically relevant proteins promise to improve understanding and safety of these proteins by rational design. However, the PEG coating that is used to evade the immune system also causes these molecules to "evade" the standard structural biology methodologies. We here demonstrate that it is possible to obtain the resonance assignment and a reliable structural model of large PEGylated proteins through an integrated approach encompassing NMR and X-ray crystallography.
Assuntos
Asparaginase , Asparaginase/química , Asparaginase/metabolismo , Materiais Revestidos Biocompatíveis , Espectroscopia de Ressonância Magnética/métodos , Polietilenoglicóis , Multimerização ProteicaRESUMO
The Human antigen R protein (HuR) is an RNA-binding protein that recognizes U/AU-rich elements in diverse RNAs through two RNA-recognition motifs, RRM1 and RRM2, and post-transcriptionally regulates the fate of target RNAs. The natural product dihydrotanshinone-I (DHTS) prevents the association of HuR and target RNAs in vitro and in cultured cells by interfering with the binding of HuR to RNA. Here, we report the structural determinants of the interaction between DHTS and HuR and the impact of DHTS on HuR binding to target mRNAs transcriptome-wide. NMR titration and Molecular Dynamics simulation identified the residues within RRM1 and RRM2 responsible for the interaction between DHTS and HuR. RNA Electromobility Shifts and Alpha Screen Assays showed that DHTS interacts with HuR through the same binding regions as target RNAs, stabilizing HuR in a locked conformation that hampers RNA binding competitively. HuR ribonucleoprotein immunoprecipitation followed by microarray (RIP-chip) analysis showed that DHTS treatment of HeLa cells paradoxically enriched HuR binding to mRNAs with longer 3'UTR and with higher density of U/AU-rich elements, suggesting that DHTS inhibits the association of HuR to weaker target mRNAs. In vivo, DHTS potently inhibited xenograft tumor growth in a HuR-dependent model without systemic toxicity.
Assuntos
Proteína Semelhante a ELAV 1/química , Fenantrenos/química , Fenantrenos/farmacologia , Regiões 3' não Traduzidas , Elementos Ricos em Adenilato e Uridilato , Animais , Antineoplásicos Fitogênicos/química , Antineoplásicos Fitogênicos/farmacologia , Proteína Semelhante a ELAV 1/antagonistas & inibidores , Proteína Semelhante a ELAV 1/genética , Proteína Semelhante a ELAV 1/metabolismo , Furanos , Humanos , Espectroscopia de Ressonância Magnética , Camundongos Mutantes Neurológicos , Simulação de Dinâmica Molecular , Fenantrenos/metabolismo , Mutação Puntual , Conformação Proteica , Domínios Proteicos , Quinonas , RNA Mensageiro/metabolismo , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Paramagnetic NMR data can be profitably incorporated in structural refinement protocols of metalloproteins or metal-substituted proteins, mostly as distance or angle restraints. However, they could in principle provide much more information, because the magnetic susceptibility of a paramagnetic metal ion is largely determined by its coordination sphere. This information can in turn be used to evaluate changes occurring in the coordination sphere of the metal when ligands (e.g.: inhibitors) are bound to the protein. This gives an experimental handle on the molecular structure in the vicinity of the metal which falls in the so-called blind sphere. The magnetic susceptibility anisotropy tensors of cobalt(II) and nickel(II) ions bound to human carbonic anhydrase II in free and inhibited forms have been determined. The change of the magnetic susceptibility anisotropy is directly linked to the binding mode of different ligands in the active site of the enzyme. Indication about the metal coordination sphere in the presence of an inhibitor in pharmaceutically relevant proteins could be important in the design of selective drugs with a structure-based approach.
Assuntos
Anidrase Carbônica II/metabolismo , Cobalto/metabolismo , Espectroscopia de Ressonância Magnética/estatística & dados numéricos , Metaloproteínas/metabolismo , Níquel/metabolismo , Anisotropia , Anidrase Carbônica II/química , Domínio Catalítico , Cobalto/química , Complexos de Coordenação/química , Furosemida/química , Humanos , Ligantes , Metaloproteínas/química , Modelos Teóricos , Estrutura Molecular , Níquel/química , Ligação ProteicaRESUMO
Protein glycosylation is the most complex post-translational modification process. More than 50 % of human cells proteins are glycosylated, whereas bacteria such as E. coli do not have this modification machinery. Indeed, the carbohydrate residues in natural proteins affect their folding, immunogenicity, and stability toward proteases, besides controlling biological properties and activities. It is therefore important to introduce such structural modification in bioengineered proteins lacking the presence of carbohydrate residues. This is not trivial as it requires reagents and conditions compatible with the protein's stability and reactivity. This work reports on the introduction of lactose moieties in two natural proteins, namely ubiquitin (Ub) and l-asparaginaseâ II (ANSII). The synthetic route employed is based on the sulfur(VI) fluoride exchange (SuFEx) coupling of a lactose tethered arylfluorosulfate (Lact-Ar-OSO2 F) with the ϵ-NH2 group of lysine residues of the proteins. This metal-free click SuFEx reaction relies on the properties of the fluorosulfate employed, which is easily prepared in multigram scale from available precursors and reacts chemoselectively with the ϵ-NH2 group of lysine residues under mild conditions. Thus, iterative couplings of Lact-Ar-OSO2 F to Ub and ANSII, afforded multiple glycosylations of these proteins so that up to three and four Lact-Ar-OSO2 groups were introduced in Ub and ANSII, respectively, via the formation of a sulfamoyl (OSO2 -NH) linkage.