RESUMO
Enzymes are known to sample various conformations, many of which are critical for their biological function. However, structural characterizations of enzymes predominantly focus on the most populated conformation. As a result, single-point mutations often produce structures that are similar or essentially identical to those of the wild-type enzyme despite large changes in enzymatic activity. Here, we show for mutants of a histone deacetylase enzyme (HDAC8) that reduced enzymatic activities, reduced inhibitor affinities, and reduced residence times are all captured by the rate constants between intrinsically sampled conformations that, in turn, can be obtained independently by solution NMR spectroscopy. Thus, for the HDAC8 enzyme, the dynamic sampling of conformations dictates both enzymatic activity and inhibitor potency. Our analysis also dissects the functional role of the conformations sampled, where specific conformations distinct from those in available structures are responsible for substrate and inhibitor binding, catalysis, and product dissociation. Precise structures alone often do not adequately explain the effect of missense mutations on enzymatic activity and drug potency. Our findings not only assign functional roles to several conformational states of HDAC8 but they also underscore the paramount role of dynamics, which will have general implications for characterizing missense mutations and designing inhibitors.
Assuntos
Mutação de Sentido Incorreto , Conformação Proteica , Ressonância Magnética Nuclear Biomolecular/métodos , CatáliseRESUMO
Intrinsically disordered proteins (IDPs) are highly dynamic biomolecules that rapidly interconvert among many structural conformations. These dynamic biomolecules are involved in cancers, neurodegeneration, cardiovascular illnesses, and viral infections. Despite their enormous therapeutic potential, IDPs have generally been considered undruggable because of their lack of classical long-lived binding pockets for small molecules. Currently, only a few instances are known where small molecules have been observed to interact with IDPs, and this situation is further exacerbated by the limited sensitivity of experimental techniques to detect such binding events. Here, using experimental nuclear magnetic resonance (NMR) spectroscopy 19F transverse spin-relaxation measurements, we discovered that a small molecule, 5-fluoroindole, interacts with the disordered domains of non-structural protein 5A from hepatitis C virus with a Kd of 260 ± 110 µM. Our analysis also allowed us to determine the rotational correlation times (τc) for the free and bound states of 5-fluoroindole. In the free state, we observed a rotational correlation time of 27.0 ± 1.3 ps, whereas in the bound state, τc only increased to 46 ± 10 ps. Our findings imply that it is possible for small molecules to engage with IDPs in exceptionally dynamic ways, in sharp contrast to the rigid binding modes typically exhibited when small molecules bind to well-defined binding pockets within structured proteins.
Assuntos
Proteínas Intrinsicamente Desordenadas , Ressonância Magnética Nuclear Biomolecular/métodos , Proteínas Intrinsicamente Desordenadas/química , Espectroscopia de Ressonância Magnética , Conformação ProteicaRESUMO
Cyclisation of peptides by forming thioether (lanthionine), disulfide (cystine) or methylene thioacetal bridges between side chains is established as an important tool to stabilise a given structure, enhance metabolic stability and optimise both potency and selectivity. However, a systematic comparative study of the effects of differing bridging modalities on peptide conformation has not previously been carried out. In this paper, we have used the NMR deconvolution algorithm, NAMFIS, to determine the conformational ensembles, in aqueous solution, of three cyclic analogues of angiotensin(1-7), incorporating either disulfide, or non-reducible thioether or methylene thioacetal bridges. We demonstrate that the major solution conformations are conserved between the different bridged peptides, but the distribution of conformations differs appreciably. This suggests that subtle differences in ring size and bridging structure can be exploited to fine-tune the conformational properties of cyclic peptides, which may modulate their bioactivities.
RESUMO
Boron is absent in proteins, yet is a micronutrient. It possesses unique bonding that could expand biological function including modes of Lewis acidity not available to typical elements of life. Here we show that post-translational Cß-Bγ bond formation provides mild, direct, site-selective access to the minimally sized residue boronoalanine (Bal) in proteins. Precise anchoring of boron within complex biomolecular systems allows dative bond-mediated, site-dependent protein Lewis acid-base-pairing (LABP) by Bal. Dynamic protein-LABP creates tunable inter- and intramolecular ligand-host interactions, while reactive protein-LABP reveals reactively accessible sites through migratory boron-to-oxygen Cß-Oγ covalent bond formation. These modes of dative bonding can also generate de novo function, such as control of thermo- and proteolytic stability in a target protein, or observation of transient structural features via chemical exchange. These results indicate that controlled insertion of boron facilitates stability modulation, structure determination, de novo binding activities and redox-responsive 'mutation'.
Assuntos
Boro/química , Proteínas/química , Alanina/química , Sequência de Aminoácidos , Oxirredução , Ligação Proteica , Processamento de Proteína Pós-Traducional , Relação Estrutura-AtividadeRESUMO
For the past decade chemical exchange saturation transfer (CEST) experiments have been successfully applied to study exchange processes in biomolecules involving sparsely populated, transiently formed conformers. Initial implementations focused on extensive sampling of the CEST frequency domain, requiring significant measurement times. Here we show that the lengthy sampling schemes often used are not optimal and that reduced frequency sampling schedules can be developed without a priori knowledge of the exchange parameters, that only depend on the chosen B1 field, and, to a lesser extent, on the intrinsic transverse relaxation rates of ground state spins. The reduced sampling approach described here can be used synergistically with other methods for reducing measurement times such as those that excite multiple frequencies in the CEST dimension simultaneously, or make use of non-uniform sampling of indirectly detected time domains, to further decrease measurement times. The proposed approach is validated by analysis of simulated and experimental datasets.
Assuntos
Imageamento por Ressonância Magnética , Proteínas , Ressonância Magnética Nuclear Biomolecular/métodos , Proteínas/química , Imageamento por Ressonância Magnética/métodosRESUMO
Macromolecules often exchange between functional states on timescales that can be accessed with NMR spectroscopy and many NMR tools have been developed to characterise the kinetics and thermodynamics of the exchange processes, as well as the structure of the conformers that are involved. However, analysis of the NMR data that report on exchanging macromolecules often hinges on complex least-squares fitting procedures as well as human experience and intuition, which, in some cases, limits the widespread use of the methods. The applications of deep neural networks (DNNs) and artificial intelligence have increased significantly in the sciences, and recently, specifically, within the field of biomolecular NMR, where DNNs are now available for tasks such as the reconstruction of sparsely sampled spectra, peak picking, and virtual decoupling. Here we present a DNN for the analysis of chemical exchange saturation transfer (CEST) data reporting on two- or three-site chemical exchange involving sparse state lifetimes of between approximately 3-60 ms, the range most frequently observed via experiment. The work presented here focuses on the 1H CEST class of methods that are further complicated, in relation to applications to other nuclei, by anti-phase features. The developed DNNs accurately predict the chemical shifts of nuclei in the exchanging species directly from anti-phase 1HN CEST profiles, along with an uncertainty associated with the predictions. The performance of the DNN was quantitatively assessed using both synthetic and experimental anti-phase CEST profiles. The assessments show that the DNN accurately determines chemical shifts and their associated uncertainties. The DNNs developed here do not contain any parameters for the end-user to adjust and the method therefore allows for autonomous analysis of complex NMR data that report on conformational exchange.
Assuntos
Inteligência Artificial , Imageamento por Ressonância Magnética , Humanos , Espectroscopia de Ressonância Magnética/métodos , Conformação Molecular , Redes Neurais de Computação , Ressonância Magnética Nuclear Biomolecular/métodosRESUMO
Nuclear magnetic resonance (NMR) experiments are frequently complicated by the presence of homonuclear scalar couplings. For the growing body of biomolecular 13C-detected NMR methods, one-bond 13C-13C couplings significantly reduce sensitivity and resolution. The solution to this problem has typically been to perform virtual decoupling by recording multiple spectra and taking linear combinations. Here, we propose an alternative method of virtual decoupling using deep neural networks, which only requires a single spectrum and gives a significant boost in resolution while reducing the minimum effective phase cycles of the experiments by at least a factor of 2. We successfully apply this methodology to virtually decouple in-phase CON (13CO-15N) protein NMR spectra, 13C-13C correlation spectra of protein side chains, and 13Cα-detected protein 13Cα-13CO spectra where two large homonuclear couplings are present. The deep neural network approach effectively decouples spectra with a high degree of flexibility, including in cases where existing methods fail, and facilitates the use of simpler pulse sequences.
Assuntos
Ressonância Magnética Nuclear BiomolecularRESUMO
In recent years, the transformative potential of deep neural networks (DNNs) for analysing and interpreting NMR data has clearly been recognised. However, most applications of DNNs in NMR to date either struggle to outperform existing methodologies or are limited in scope to a narrow range of data that closely resemble the data that the network was trained on. These limitations have prevented a widescale uptake of DNNs in NMR. Addressing this, we introduce FID-Net, a deep neural network architecture inspired by WaveNet, for performing analyses on time domain NMR data. We first demonstrate the effectiveness of this architecture in reconstructing non-uniformly sampled (NUS) biomolecular NMR spectra. It is shown that a single network is able to reconstruct a diverse range of 2D NUS spectra that have been obtained with arbitrary sampling schedules, with a range of sweep widths, and a variety of other acquisition parameters. The performance of the trained FID-Net in this case exceeds or matches existing methods currently used for the reconstruction of NUS NMR spectra. Secondly, we present a network based on the FID-Net architecture that can efficiently virtually decouple 13Cα-13Cß couplings in HNCA protein NMR spectra in a single shot analysis, while at the same time leaving glycine residues unmodulated. The ability for these DNNs to work effectively in a wide range of scenarios, without retraining, paves the way for their widespread usage in analysing NMR data.
Assuntos
Histona Desacetilases/química , Imageamento por Ressonância Magnética/métodos , Muramidase/química , Redes Neurais de Computação , Ressonância Magnética Nuclear Biomolecular/métodos , Domínios de Homologia de src/fisiologia , Algoritmos , Biologia Computacional/métodos , Aprendizado ProfundoRESUMO
A series of potent inhibitors of histone deacetylase-8 (HDAC8) is described that contains an α-amino amide zinc-binding unit and a substituted isoindolinyl capping group. The presence of a 2,4-dichlorophenyl unit located in the acetate-release cavity was shown to confer a gain of approx. 4.3 kJ mol-1 in binding energy compared to a phenyl group, and the isoindoline linker has approx. 5.8 kJ mol-1 greater binding energy than the corresponding tetrahydroisoquinoline ring system. In a series of 5-substituted isoindolin-2-yl inhibitors, a 5-acetylamino derivative was found to be more potent than the 5-unsubstituted lead HDAC8 inhibitor (increase in binding energy of 2.0 kJ mol-1, ascribed to additional binding interactions within the Nε-acetyl-l-lysine binding tunnel in HDAC8, including hydrogen bonding to Asp101. Tolerance of a 5-substituent (capping group) on the isoindoline ring has been demonstrated, and which in some cases confers improved enzyme inhibition, the HDAC8 substrate-binding region providing a platform for additional interactions.
Assuntos
Quelantes/química , Inibidores de Histona Desacetilases/química , Histona Desacetilases/metabolismo , Isoindóis/química , Proteínas Repressoras/metabolismo , Zinco/metabolismo , Domínio Catalítico , Quelantes/síntese química , Quelantes/metabolismo , Ensaios Enzimáticos , Inibidores de Histona Desacetilases/síntese química , Inibidores de Histona Desacetilases/metabolismo , Histona Desacetilases/química , Humanos , Isoindóis/síntese química , Isoindóis/metabolismo , Simulação de Acoplamento Molecular , Estrutura Molecular , Ligação Proteica , Proteínas Repressoras/química , Relação Estrutura-AtividadeRESUMO
The von Willebrand factor (VWF) and coagulation factor VIII (FVIII) are intricately involved in hemostasis. A tight, noncovalent complex between VWF and FVIII prolongs the half-life of FVIII in plasma, and failure to form this complex leads to rapid clearance of FVIII and bleeding diatheses such as hemophilia A and von Willebrand disease (VWD) type 2N. High-resolution insight into the complex between VWF and FVIII has so far been strikingly lacking. This is particularly the case for the flexible a3 region of FVIII, which is imperative for high-affinity binding. Here, a structural and biophysical characterization of the interaction between VWF and FVIII is presented with focus on two of the domains that have been proven pivotal for mediating the interaction, namely the a3 region of FVIII and the TIL'E' domains of VWF. Binding between the FVIII a3 region and VWF TIL'E' was here observed using NMR spectroscopy, where chemical shift changes were localized to two ß-sheet regions on the edge of TIL'E' upon FVIII a3 region binding. Isothermal titration calorimetry and NMR spectroscopy were used to characterize the interaction between FVIII and TIL'E' as well as mutants of TIL'E', which further highlights the importance of the ß-sheet region of TIL'E' for high-affinity binding. Overall, the results presented provide new insight into the role the FVIII a3 region plays for complex formation between VWF and FVIII and the ß-sheet region of TIL'E' is shown to be important for FVIII binding. Thus, the results pave the way for further high-resolution insights into this imperative complex.
Assuntos
Fator VIII/química , Fator VIII/metabolismo , Fator de von Willebrand/química , Fator de von Willebrand/metabolismo , Calorimetria , Espectroscopia de Ressonância Magnética , Proteínas Mutantes/química , Proteínas Mutantes/metabolismo , Mutação/genética , Ligação Proteica , Domínios Proteicos , Fator de von Willebrand/genéticaRESUMO
Non-uniform and sparse sampling of multi-dimensional NMR spectra has over the last decade become an important tool to allow for fast acquisition of multi-dimensional NMR spectra with high resolution. The success of non-uniform sampling NMR hinge on both the development of algorithms to accurately reconstruct the sparsely sampled spectra and the design of sampling schedules that maximise the information contained in the sampled data. Traditionally, the reconstruction tools and algorithms have aimed at reconstructing the full spectrum and thus 'fill out the missing points' in the time-domain spectrum, although other techniques are based on multi-dimensional decomposition and extraction of multi-dimensional shapes. Also over the last decade, machine learning, deep neural networks, and artificial intelligence have seen new applications in an enormous range of sciences, including analysis of MRI spectra. As a proof-of-principle, it is shown here that simple deep neural networks can be trained to reconstruct sparsely sampled NMR spectra. For the reconstruction of two-dimensional NMR spectra, reconstruction using a deep neural network performs as well, if not better than, the currently and widely used techniques. It is therefore anticipated that deep neural networks provide a very valuable tool for the reconstruction of sparsely sampled NMR spectra in the future to come.
Assuntos
Algoritmos , Redes Neurais de Computação , Ressonância Magnética Nuclear Biomolecular/métodos , Processamento de Imagem Assistida por Computador/métodos , Tamanho da Amostra , Aprendizado de Máquina SupervisionadoRESUMO
Methyl-TROSY based NMR experiments have over the last two decades become one of the most important means to characterise dynamics and functional mechanisms of large proteins and macromolecular machines in solution. The chemical shift assignment of methyl groups in large proteins is, however, still not trivial and it is typically performed using backbone-dependent experiments in a 'divide and conquer' approach, mutations, structure-based assignments or a combination of these. Structure-based assignment of methyl groups is an emerging strategy, which reduces the time and cost required as well as providing a method that is independent of a backbone assignment. One crucial step in available structure-based assignment protocols is linking the two prochiral methyl groups of leucine and valine residues. This has previously been achieved by recording NOESY spectra with short mixing times or by comparing NOESY spectra. Herein, we present a method based on through-bond scalar coupling transfers, a 3D-HMBC-HMQC experiment, to link the intra-residue methyl groups of leucine and valine. It is shown that the HMBC-HMQC method has several advantages over solely using NOESY spectra since a unique intra-residue cross-peak is observed. Moreover, overlap in the methyl-TROSY HMQC spectrum can easily be identified with the HMBC-HMQC experiment, thereby removing possible ambiguities in the assignment.
Assuntos
Leucina/química , Ressonância Magnética Nuclear Biomolecular/métodos , Proteínas/química , Valina/química , Metano/análogos & derivados , Metano/química , Conformação MolecularRESUMO
Natural products that target lipid II, such as the lantibiotic nisin, are strategically important in the development of new antibacterial agents to combat the rise of antimicrobial resistance. Understanding the structural factors that govern the highly selective molecular recognition of lipid II by the N-terminal region of nisin, nisin(1-12), is a crucial step in exploiting the potential of such compounds. In order to elucidate the relationships between amino acid sequence and conformation of this bicyclic peptide fragment, we have used solid-phase peptide synthesis to prepare two novel analogues of nisin(1-12) in which the dehydro residues have been replaced. We have carried out an NMR ensemble analysis of one of these analogues and of the wild-type nisin(1-12) peptide in order to compare the conformations of these two bicyclic peptides. Our analysis has shown the effects of residue mutation on ring conformation. We have also demonstrated that the individual rings of nisin(1-12) are pre-organised to an extent for binding to the pyrophosphate group of lipid II, with a high degree of flexibility exhibited in the central amide bond joining the two rings.
Assuntos
Nisina/análogos & derivados , Peptídeos/síntese química , Uridina Difosfato Ácido N-Acetilmurâmico/análogos & derivados , Sequência de Aminoácidos , Ligação de Hidrogênio , Nisina/metabolismo , Ressonância Magnética Nuclear Biomolecular , Peptídeos/química , Peptídeos/metabolismo , Conformação Proteica , Uridina Difosfato Ácido N-Acetilmurâmico/química , Uridina Difosfato Ácido N-Acetilmurâmico/metabolismoRESUMO
The rate with which labile backbone hydrogen atoms in proteins exchange with the solvent has long been used to probe protein interactions in aqueous solutions. Arginine, an essential amino acid found in many interaction interfaces, is capable of an impressive range of interactions via its guanidinium group. The hydrogen exchange rate of the guanidinium hydrogens therefore becomes an important measure to quantify side-chain interactions. Herein we present an NMR method to quantify the hydrogen exchange rates of arginine side-chain 1 Hϵ protons and thus present a method to gauge the strength of arginine side-chain interactions. The method employs 13 C-detection and the one-bond deuterium isotope shift observed for 15 Nϵ to generate two exchanging species in 1 H2 O/2 H2 O mixtures. An application to the protein T4 Lysozyme is shown, where protection factors calculated from the obtained exchange rates correlate well with the interactions observed in the crystal structure. The methodology presented provides an important step towards characterising interactions of arginine side-chains in enzymes, in phase separation, and in protein interaction interfaces in general.
RESUMO
In response to the growing threat posed by antibiotic-resistant bacterial strains, extensive research is currently focused on developing antimicrobial agents that target lipid II, a vital precursor in the biosynthesis of bacterial cell walls. The lantibiotic nisin and related peptides display unique and highly selective binding to lipid II. A key feature of the nisin-lipid II interaction is the formation of a cage-like complex between the pyrophosphate moiety of lipid II and the two thioether-bridged rings, rings A and B, at the N-terminus of nisin. To understand the important structural factors underlying this highly selective molecular recognition, we have used solid-phase peptide synthesis to prepare individual ring A and B structures from nisin, the related lantibiotic mutacin, and synthetic analogues. Through NMR studies of these rings, we have demonstrated that ring A is preorganized to adopt the correct conformation for binding lipid II in solution and that individual amino acid substitutions in ring A have little effect on the conformation. We have also analyzed the turn structures adopted by these thioether-bridged peptides and show that they do not adopt the tight α-turn or ß-turn structures typically found in proteins.
Assuntos
Antibacterianos/síntese química , Bacteriocinas/síntese química , Nisina/química , Técnicas de Síntese em Fase Sólida/métodos , Uridina Difosfato Ácido N-Acetilmurâmico/análogos & derivados , Antibacterianos/química , Antibacterianos/farmacologia , Bacteriocinas/química , Bacteriocinas/farmacologia , Nisina/análogos & derivados , Ligação Proteica , Conformação Proteica , Uridina Difosfato Ácido N-Acetilmurâmico/metabolismoRESUMO
Arginine side-chains are often key for enzyme catalysis, protein-ligand and protein-protein interactions. The importance of arginine stems from the ability of the terminal guanidinium group to form many key interactions, such as hydrogen bonds and salt bridges, as well as its perpetual positive charge. We present here an arginine 13Cζ-detected NMR experiment in which a double-quantum coherence involving the two 15Nη nuclei is evolved during the indirect chemical shift evolution period. As the precession frequency of the double-quantum coherence is insensitive to exchange of the two 15Nη; this new approach is shown to eliminate the previously deleterious line broadenings of 15Nη resonances caused by the partially restricted rotation about the Cζ-Nε bond. Consequently, sharp and well-resolved 15Nη resonances can be observed. The utility of the presented method is demonstrated on the L99A mutant of the 19 kDa protein T4 lysozyme, where the measurement of small chemical shift perturbations, such as one-bond deuterium isotope shifts, of the arginine amine 15Nη nuclei becomes possible using the double-quantum experiment.
Assuntos
Arginina/química , Guanidina/química , Ressonância Magnética Nuclear Biomolecular/métodos , Isótopos de Carbono , Muramidase/genética , Mutação de Sentido Incorreto , Isótopos de Nitrogênio , Fragmentos de Peptídeos/genéticaRESUMO
Proteins are inherently plastic molecules, whose function often critically depends on excursions between different molecular conformations (conformers). However, a rigorous understanding of the relation between a protein's structure, dynamics and function remains elusive. This is because many of the conformers on its energy landscape are only transiently formed and marginally populated (less than a few per cent of the total number of molecules), so that they cannot be individually characterized by most biophysical tools. Here we study a lysozyme mutant from phage T4 that binds hydrophobic molecules and populates an excited state transiently (about 1 ms) to about 3% at 25 °C (ref. 5). We show that such binding occurs only via the ground state, and present the atomic-level model of the 'invisible', excited state obtained using a combined strategy of relaxation-dispersion NMR (ref. 6) and CS-Rosetta model building that rationalizes this observation. The model was tested using structure-based design calculations identifying point mutants predicted to stabilize the excited state relative to the ground state. In this way a pair of mutations were introduced, inverting the relative populations of the ground and excited states and altering function. Our results suggest a mechanism for the evolution of a protein's function by changing the delicate balance between the states on its energy landscape. More generally, they show that our approach can generate and validate models of excited protein states.
Assuntos
Bacteriófago T4/enzimologia , Bacteriófago T4/genética , Modelos Moleculares , Muramidase/química , Muramidase/genética , Mutação , Evolução Molecular , Interações Hidrofóbicas e Hidrofílicas , Ligantes , Ligação Proteica , TemperaturaRESUMO
Although much of the function of von Willebrand factor (VWF) has been revealed, detailed insight into the molecular structure that enables VWF to orchestrate hemostatic processes, in particular factor VIII (FVIII) binding and stabilization in plasma, is lacking. Here, we present the high-resolution solution structure and structural dynamics of the D' region of VWF, which constitutes the major FVIII binding site. D' consists of 2 domains, trypsin-inhibitor-like (TIL') and E', of which the TIL' domain lacks extensive secondary structure, is strikingly dynamic and harbors a cluster of pathological mutations leading to decreased FVIII binding affinity (type 2N von Willebrand disease [VWD]). This indicates that the backbone malleability of TIL' is important for its biological activity. The principal FVIII binding site is localized to a flexible, positively charged region on TIL', which is supported by the rigid scaffold of the TIL' and E' domain ß sheets. Furthermore, surface-charge mapping of the TIL'E' structure reveals a potential mechanism for the electrostatically guided, high-affinity VWFâ FVIII interaction. Our findings provide novel insights into VWFâ FVIII complex formation, leading to a greater understanding of the molecular basis of the bleeding diathesis type 2N VWD.
Assuntos
Fator VIII/química , Fator de von Willebrand/química , Sítios de Ligação , Cristalografia por Raios X , Fator VIII/genética , Fator VIII/metabolismo , Humanos , Estabilidade Proteica , Estrutura Quaternária de Proteína , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Eletricidade Estática , Fator de von Willebrand/genética , Fator de von Willebrand/metabolismoRESUMO
States along the phosphoryl transfer reaction catalyzed by the nucleoside monophosphate kinase UmpK were captured and changes in the conformational heterogeneity of conserved active site arginine side-chains were quantified by NMR spin-relaxation methods. In addition to apo and ligand-bound UmpK, a transition state analog (TSA) complex was utilized to evaluate the extent to which active site conformational entropy contributes to the transition state free energy. The catalytically essential arginine side-chain guanidino groups were found to be remarkably rigid in the TSA complex, indicating that the enzyme has evolved to restrict the conformational freedom along its reaction path over the energy landscape, which in turn allows the phosphoryl transfer to occur selectively by avoiding side reactions.