RESUMO
Protein-ligand complex formation is fundamental to biological function. A central question is whether proteins spontaneously adopt binding-competent conformations to which ligands bind conformational selection (CS) or whether ligands induce the binding-competent conformation induced fit (IF). Here, we resolve the CS and IF binding pathways by characterizing protein conformational dynamics over a wide range of ligand concentrations using NMR relaxation dispersion. We determined the relative flux through the two pathways using a four-state binding model that includes both CS and IF. Experiments conducted without ligand show that galectin-3 exchanges between the ground-state conformation and a high-energy conformation similar to the ligand-bound conformation, demonstrating that CS is a plausible pathway. Near-identical crystal structures of the apo and ligand-bound states suggest that the high-energy conformation in solution corresponds to the apo crystal structure. Stepwise additions of the ligand lactose induce progressive changes in the relaxation dispersions that we fit collectively to the four-state model, yielding all microscopic rate constants and binding affinities. The ligand affinity is higher for the bound-like conformation than for the ground state, as expected for CS. Nonetheless, the IF pathway contributes greater than 70% of the total flux even at low ligand concentrations. The higher flux through the IF pathway is explained by considerably higher rates of exchange between the two protein conformations in the ligand-associated state. Thus, the ligand acts to decrease the activation barrier between protein conformations in a manner reciprocal to enzymatic transition-state stabilization of reactions involving ligand transformation.
Assuntos
Proteínas , Modelos Moleculares , Ligantes , Ligação Proteica , Proteínas/química , Conformação ProteicaRESUMO
Structural characterization of protein-ligand binding interfaces at atomic resolution is essential for improving the design of specific and potent inhibitors. Herein, we explored fast 19F- and 1H-detected magic angle spinning NMR spectroscopy to investigate the interaction between two fluorinated ligand diastereomers with the microcrystalline galectin-3 carbohydrate recognition domain. The detailed environment around the fluorine atoms was mapped by 2D 13C-19F and 1H-19F dipolar correlation experiments and permitted characterization of the binding interface. Our results demonstrate that 19F MAS NMR is a powerful tool for detailed characterization of protein-ligand interfaces and protein interactions at the atomic level.
RESUMO
Histidine is a key amino-acid residue in proteins with unique properties engendered by its imidazole side chain that can exist in three different states: two different neutral tautomeric forms and a protonated, positively charged one with a pKa value close to physiological pH. Commonly, two or all three states coexist and interchange rapidly, enabling histidine to act as both donor and acceptor of hydrogen bonds, coordinate metal ions, and engage in acid/base catalysis. Understanding the exchange dynamics among the three states is critical for assessing histidine's mechanistic role in catalysis, where the rate of proton exchange and interconversion among tautomers might be rate limiting for turnover. Here, we determine the exchange kinetics of histidine residues with pKa values representative of the accessible range from 5 to 9 by measuring pH-dependent 15N, 13C, and 1H transverse relaxation rate constants for 5 nuclei in each imidazole. Proton exchange between the imidazole and the solvent is mediated by hydronium ions at acidic and neutral pH, whereas hydroxide mediated exchange becomes the dominant mechanism at basic pH. Proton transfer is very fast and reaches the diffusion limit for pKa values near neutral pH. We identify a direct pathway between the two tautomeric forms, likely mediated by a bridging water molecule or, in the case of high pH, hydroxide ion. For histidines with pKa 7, we determine all rate constants (lifetimes) involving protonation over the entire pH range. Our approach should enable critical insights into enzymatic acid/base catalyzed reactions involving histidines in proteins.
Assuntos
Histidina , Prótons , Histidina/química , Concentração de Íons de Hidrogênio , Cinética , Ressonância Magnética Nuclear BiomolecularRESUMO
Protein-ligand-exchange kinetics determines the duration of biochemical signals and consequently plays an important role in drug design. Binding studies commonly require solubilization of designed ligands in solvents such as dimethyl sulfoxide (DMSO), resulting in residual amounts of DMSO following titration of solubilized ligands into aqueous protein samples. Therefore, it is critical to establish whether DMSO influences protein-ligand binding. Here, we address the general and indirect effect of DMSO on protein-ligand binding caused by solvent viscosity, which is strongly dependent on the relative concentrations of DMSO and water. As a model system, we studied the binding of a drug-like ligand to the carbohydrate recognition domain of galectin-3 in the presence of variable amounts of DMSO. We used isothermal titration calorimetry to characterize binding thermodynamics and 15N NMR relaxation to monitor kinetics. The binding enthalpy is not affected, but we observe a subtle trend of increasingly unfavorable entropy of binding, and consequently decreased affinity, with increasing DMSO concentration. The increasing concentration of DMSO results in a reduced association rate of binding, while the dissociation rate is less affected. The observed association rate is inversely proportional to the viscosity of the DMSO-water mixture, as expected from theory, but significantly reduced from the diffusion-controlled limit. By comparing the viscosity dependence of the observed association rate with that of the theoretical diffusion-controlled association rate, we estimate the success rate of productive complex formation following an initial encounter of proteins and ligands, showing that only one out of several hundred binding "attempts" are successful.
Assuntos
Dimetil Sulfóxido , Proteínas , Solventes/química , Dimetil Sulfóxido/química , Ligantes , Viscosidade , Proteínas/química , Água/química , CinéticaRESUMO
The nucleation of Alzheimer-associated Aß peptide monomers can be catalyzed by preexisting Aß fibrils. This leads to autocatalytic amplification of aggregate mass and underlies self-replication and generation of toxic oligomers associated with several neurodegenerative diseases. However, the nature of the interactions between the monomeric species and the fibrils during this key process, and indeed the ultrastructural localization of the interaction sites have remained elusive. Here we used NMR and optical spectroscopy to identify conditions that enable the capture of transient species during the aggregation and secondary nucleation of the Aß42 peptide. Cryo-electron microscopy (cryo-EM) images show that new aggregates protrude from the entire length of the progenitor fibril. These protrusions are morphologically distinct from the well-ordered fibrils dominating at the end of the aggregation process. The data provide direct evidence that self-replication through secondary nucleation occurs along the sides of fibrils, which become heavily decorated under the current solution conditions (14 µM Aß42, 20 mM sodium phosphate, 200 µM EDTA, pH 6.8).
Assuntos
Peptídeos beta-Amiloides/metabolismo , Peptídeos beta-Amiloides/ultraestrutura , Fragmentos de Peptídeos/metabolismo , Fragmentos de Peptídeos/ultraestrutura , Doença de Alzheimer/patologia , Amiloide/metabolismo , Amiloide/ultraestrutura , Peptídeos beta-Amiloides/química , Benzotiazóis/química , Benzotiazóis/metabolismo , Microscopia Crioeletrônica , Humanos , Concentração de Íons de Hidrogênio , Cinética , Espectroscopia de Ressonância Magnética , Fragmentos de Peptídeos/química , Imagem com Lapso de TempoRESUMO
Plant hemoglobins, often referred to as phytoglobins, play important roles in abiotic stress tolerance. Several essential small physiological metabolites can be bound to these heme proteins. In addition, phytoglobins can catalyze a range of different oxidative reactions in vivo. These proteins are often oligomeric, but the degree and relevance of subunit interactions are largely unknown. In this study, we delineate which residues are involved in dimer formation of a sugar beet phytoglobin type 1.2 (BvPgb1.2) using NMR relaxation experiments. E. coli cells harboring a phytoglobin expression vector were cultivated in isotope-labeled (2H, 13C and 15N) M9 medium. The triple-labeled protein was purified to homogeneity using two chromatographic steps. Two forms of BvPgb1.2 were examined, the oxy-form and the more stable cyanide-form. Using three-dimensional triple-resonance NMR experiments, sequence-specific assignments for CN-bound BvPgb1.2 were achieved for 137 backbone amide cross-peaks in the 1H-15N TROSY spectrum, which amounts to 83% of the total number of 165 expected cross-peaks. A large proportion of the non-assigned residues are located in α-helixes G and H, which are proposed to be involved in protein dimerization. Such knowledge around dimer formation will be instrumental for developing a better understanding of phytoglobins' roles in planta.
Assuntos
Beta vulgaris , Beta vulgaris/metabolismo , Escherichia coli/metabolismo , Hemoglobinas/metabolismo , Espectroscopia de Ressonância Magnética , Conformação Proteica , Proteínas de Plantas/químicaRESUMO
The bromodomain and extra-terminal (BET) protein BRD4 regulates gene expression via recruitment of transcriptional regulatory complexes to acetylated chromatin. Like other BET proteins, BRD4 contains two bromodomains, BD1 and BD2, that can interact cooperatively with target proteins and designed ligands, with important implications for drug discovery. Here, we used nuclear magnetic resonance (NMR) spectroscopy to study the dynamics and interactions of the isolated bromodomains, as well as the tandem construct including both domains and the intervening linker, and investigated the effects of binding a tetra-acetylated peptide corresponding to the tail of histone 4. The peptide affinity is lower for both domains in the tandem construct than for the isolated domains. Using 15N spin relaxation, we determined the global rotational correlation times and residue-specific order parameters for BD1 and BD2. Isolated BD1 is monomeric in the apo state but apparently dimerizes upon binding the tetra-acetylated peptide. Isolated BD2 partially dimerizes in both the apo and peptide-bound states. The backbone order parameters reveal marked differences between BD1 and BD2, primarily in the acetyl-lysine binding site where the ZA loop is more flexible in BD2. Peptide binding reduces the order parameters of the ZA loop in BD1 and the ZA and BC loops in BD2. The AB loop, located distally from the binding site, shows variable dynamics that reflect the different dimerization propensities of the domains. These results provide a basis for understanding target recognition by BRD4.
Assuntos
Histonas , Proteínas Nucleares , Histonas/metabolismo , Proteínas Nucleares/metabolismo , Fatores de Transcrição/química , Sítios de Ligação , Peptídeos/metabolismo , Proteínas de Ciclo Celular/metabolismoRESUMO
Aromatic side chains are attractive probes of protein dynamic, since they are often key residues in enzyme active sites and protein binding sites. Dynamic processes on microsecond to millisecond timescales can be studied by relaxation dispersion experiments that attenuate conformational exchange contributions to the transverse relaxation rate by varying the refocusing frequency of applied radio-frequency fields implemented as either CPMG pulse trains or continuous spin-lock periods. Here we present an aromatic 1H R1ρ relaxation dispersion experiment enabling studies of two to three times faster exchange processes than achievable by existing experiments for aromatic side chains. We show that site-specific isotope labeling schemes generating isolated 1H-13C spin pairs with vicinal 2H-12C moieties are necessary to avoid anomalous relaxation dispersion profiles caused by Hartmann-Hahn matching due to the 3JHH couplings and limited chemical shift differences among 1H spins in phenylalanine, tyrosine and the six-ring moiety of tryptophan. This labeling pattern is sufficient in that remote protons do not cause additional complications. We validated the approach by measuring ring-flip kinetics in the small protein GB1. The determined rate constants, kflip, agree well with previous results from 13C R1ρ relaxation dispersion experiments, and yield 1H chemical shift differences between the two sides of the ring in good agreement with values measured under slow-exchange conditions. The aromatic1H R1ρ relaxation dispersion experiment in combination with the site-selective 1H-13C/2H-12C labeling scheme enable measurement of exchange rates up to kex = 2kflip = 80,000 s-1, and serve as a useful complement to previously developed 13C-based methods.
Assuntos
Proteínas , Prótons , Sítios de Ligação , Cinética , Ressonância Magnética Nuclear BiomolecularRESUMO
Chemical shift tensors obtained from solid-state NMR spectroscopy are very sensitive reporters of structure and dynamics in proteins. While accurate 13 C and 15 N chemical shift tensors are accessible by magic angle spinning (MAS) NMR, their quantum mechanical calculations remain challenging, particularly for 15 N atoms. Here we compare experimentally determined backbone 13 Cα and 15 NH chemical shift tensors by MAS NMR with hybrid quantum mechanics/molecular mechanics/molecular dynamics (MD-QM/MM) calculations for the carbohydrate-binding domain of galectin-3. Excellent agreement between experimental and computed 15 NH chemical shift anisotropy values was obtained using the Amber ff15ipq force field when solvent dynamics was taken into account in the calculation. Our results establish important benchmark conditions for improving the accuracy of chemical shift calculations in proteins and may aid in the validation of protein structure models derived by MAS NMR.
Assuntos
Proteínas Sanguíneas/química , Galectinas/química , Isótopos de Carbono/química , Teoria da Densidade Funcional , Humanos , Modelos Químicos , Simulação de Dinâmica Molecular , Isótopos de Nitrogênio/química , Ressonância Magnética Nuclear BiomolecularRESUMO
Amyloid fibril formation is a hallmark of neurodegenerative disease caused by protein aggregation. Oligomeric protein states that arise during the process of fibril formation often coexist with mature fibrils and are known to cause cell death in disease model systems. Progress in this field depends critically on development of analytical methods that can provide information about the mechanisms and species involved in oligomerization and fibril formation. Here, we demonstrate how the powerful combination of diffusion NMR and multilinear data analysis can efficiently disentangle the number of involved species, their kinetic rates of formation or disappearance, spectral contributions, and diffusion coefficients, even without prior knowledge of the time evolution of the process or chemical shift assignments of the various species. Using this method we identify oligomeric species that form transiently during aggregation of human superoxide dismutase 1 (SOD1), which is known to form misfolded aggregates in patients with amyotrophic lateral sclerosis. Specifically, over a time course of 42 days, during which SOD1 fibrils form, we detect the disappearance of the native monomeric species, formation of a partially unfolded intermediate in the dimer to tetramer size range, subsequent formation of a distinct similarly sized species that dominates the final spectrum detected by solution NMR, and concomitant appearance of small peptide fragments.
Assuntos
Amiloide/química , Agregados Proteicos , Difusão , Humanos , Espectroscopia de Ressonância Magnética , Solubilidade , Superóxido Dismutase-1/químicaRESUMO
Understanding the driving forces underlying molecular recognition is of fundamental importance in chemistry and biology. The challenge is to unravel the binding thermodynamics into separate contributions and to interpret these in molecular terms. Entropic contributions to the free energy of binding are particularly difficult to assess in this regard. Here we pinpoint the molecular determinants underlying differences in ligand affinity to the carbohydrate recognition domain of galectin-3, using a combination of isothermal titration calorimetry, X-ray crystallography, NMR relaxation, and molecular dynamics simulations followed by conformational entropy and grid inhomogeneous solvation theory (GIST) analyses. Using a pair of diastereomeric ligands that have essentially identical chemical potential in the unbound state, we reduced the problem of dissecting the thermodynamics to a comparison of the two protein-ligand complexes. While the free energies of binding are nearly equal for the R and S diastereomers, greater differences are observed for the enthalpy and entropy, which consequently exhibit compensatory behavior, ΔΔ H°(R - S) = -5 ± 1 kJ/mol and - TΔΔ S°(R - S) = 3 ± 1 kJ/mol. NMR relaxation experiments and molecular dynamics simulations indicate that the protein in complex with the S-stereoisomer has greater conformational entropy than in the R-complex. GIST calculations reveal additional, but smaller, contributions from solvation entropy, again in favor of the S-complex. Thus, conformational entropy apparently dominates over solvation entropy in dictating the difference in the overall entropy of binding. This case highlights an interplay between conformational entropy and solvation entropy, pointing to both opportunities and challenges in drug design.
Assuntos
Entropia , Galectina 3/química , Sítios de Ligação , Cristalografia por Raios X , Galectina 3/isolamento & purificação , Ligantes , Conformação Molecular , Simulação de Dinâmica Molecular , Solubilidade , EstereoisomerismoRESUMO
Studies of protein-ligand binding often rely on dissolving the ligand in dimethyl sulfoxide (DMSO) to achieve sufficient solubility, and then titrating the ligand solution into the protein solution. As a result, the final protein-ligand solution contains small amounts of DMSO in the buffer. Here we report how the addition of DMSO impacts studies of protein conformational dynamics. We used 15 N NMR relaxation to compare the rotational diffusion correlation time (τC ) of proteins in aqueous buffer with and without DMSO. We found that τC scales with the viscosity of the water-DMSO mixture, which depends sensitively on the amount of DMSO and varies by a factor of 2 across the relevant concentration range. NMR relaxation studies of side chains dynamics are commonly interpreted using τC as a fixed parameter, obtained from backbone 15 N relaxation data acquired on a separate sample. Model-free calculations show that errors in τC , arising from mismatched DMSO concentration between samples, lead to significant errors in order parameters. Our results highlight the importance of determining τC for each sample or carefully matching the DMSO concentrations between samples.
Assuntos
Dimetil Sulfóxido/química , Ressonância Magnética Nuclear Biomolecular/métodos , Proteínas/química , Solventes/química , Viscosidade , Ligação ProteicaRESUMO
Nonuniform sampling (NUS) of multidimensional NMR data offers significant time savings while improving spectral resolution or increasing sensitivity per unit time. However, NUS has not been widely used for quantitative analysis because of the nonlinearity of most methods used to model NUS data, which leads to problems in estimating signal intensities, relaxation rate constants, and their error bounds. Here, we present an approach that avoids these limitations by combining accordion spectroscopy and NUS in the indirect dimensions of multidimensional spectra and then applying sparse exponential mode analysis, which is well suited for analyzing accordion-type relaxation data in a NUS context. By evaluating the Cramér-Rao lower bound of the variances of the estimated relaxation rate constants, we achieve a robust benchmark for the underlying reconstruction model. Furthermore, we design NUS schemes optimized with respect to the information theoretical lower bound of the error in the parameters of interest, given a specified number of sampling points. The accordion-NUS method compares favorably with conventional relaxation experiments in that it produces identical results, within error, while shortening the length of the experiment by an order of magnitude. Thus, our approach enables rapid acquisition of NMR relaxation data for optimized use of spectrometer time or accurate measurements on samples of limited lifetime.
RESUMO
FKBP12 (FK506 binding protein 12 kDa) is an important drug target. Nuclear magnetic resonance (NMR) order parameters, describing amplitudes of motion on the pico- to nanosecond time scale, can provide estimates of changes in conformational entropy upon ligand binding. Here we report backbone and methyl-axis order parameters of the apo and FK506-bound forms of FKBP12, based on 15N and 2H NMR relaxation. Binding of FK506 to FKBP12 results in localized changes in order parameters, notably for the backbone of residues E54 and I56 and the side chains of I56, I90, and I91, all positioned in the binding site. The order parameters increase slightly upon FK506 binding, indicating an unfavorable entropic contribution to binding of TΔ S = -18 ± 2 kJ/mol at 293 K. Molecular dynamics simulations indicate a change in conformational entropy, associated with all dihedral angles, of TΔ S = -26 ± 9 kJ/mol. Both these values are significant compared to the total entropy of binding determined by isothermal titration calorimetry and referenced to a reactant concentration of 1 mM ( TΔ S = -29 ± 1 kJ/mol). Our results reveal subtle differences in the response to ligand binding compared to that of the previously studied rapamycin-FKBP12 complex, despite the high degree of structural homology between the two complexes and their nearly identical ligand-FKBP12 interactions. These results highlight the delicate dependence of protein dynamics on drug interactions, which goes beyond the view provided by static structures, and reinforce the notion that protein conformational entropy can make important contributions to the free energy of ligand binding.
Assuntos
Proteína 1A de Ligação a Tacrolimo/química , Tacrolimo/química , Cristalografia por Raios X , Entropia , Humanos , Simulação de Dinâmica Molecular , Ressonância Magnética Nuclear Biomolecular , Conformação Proteica , Domínios Proteicos , Sirolimo/química , Sirolimo/metabolismo , Tacrolimo/metabolismo , Proteína 1A de Ligação a Tacrolimo/metabolismoRESUMO
The human molecular chaperone DNAJB6, an oligomeric protein with a conserved S/T-rich region, is an efficient suppressor of amyloid fibril formation by highly aggregation-prone peptides such as the Aß and polyQ peptides associated with Alzheimer's and Huntington's disease, respectively. We previously showed that DNAJB6 can inhibit the processes through which amyloid fibrils are formed via strong interactions with aggregated forms of Aß42 that become sequestered. Here we report that the concentration-dependent capability of DNAJB6 to suppress fibril formation in thioflavin T fluorescence assays decreases progressively with an increasing number of S/T substitutions, with an almost complete loss of suppression when 18 S/T residues are substituted. The kinetics of primary nucleation in particular are affected. No detectable changes in the structure are caused by the substitutions. Also, the level of binding of DNAJB6 to Aß42 decreases with the S/T substitutions, as determined by surface plasmon resonance and microscale thermophoresis. The aggregation process monitored using nuclear magnetic resonance spectroscopy showed that DNAJB6, in contrast to a mutational variant with 18 S/T residues substituted, can keep monomeric Aß42 soluble for an extended time. The inhibition of the primary nucleation is likely to depend on hydroxyl groups in side chains of the S/T residues, and hydrogen bonding with Aß42 is one plausible molecular mechanism, although other possibilities cannot be excluded. The loss of the ability to suppress fibril formation upon S/T to A substitution was previously observed also for polyQ peptides, suggesting that the S/T residues in the DNAJB6-like chaperones have a general ability to inhibit amyloid fibril formation by different aggregation-prone peptides.
Assuntos
Peptídeos beta-Amiloides/metabolismo , Amiloide/metabolismo , Proteínas de Choque Térmico HSP40/química , Proteínas de Choque Térmico HSP40/metabolismo , Chaperonas Moleculares/química , Chaperonas Moleculares/metabolismo , Proteínas do Tecido Nervoso/química , Proteínas do Tecido Nervoso/metabolismo , Amiloide/química , Peptídeos beta-Amiloides/química , Humanos , Ligação de Hidrogênio , Modelos Biológicos , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/metabolismoRESUMO
Small heat-shock proteins (sHsps) prevent aggregation of thermosensitive client proteins in a first line of defense against cellular stress. The mechanisms by which they perform this function have been hard to define due to limited structural information; currently, there is only one high-resolution structure of a plant sHsp published, that of the cytosolic Hsp16.9. We took interest in Hsp21, a chloroplast-localized sHsp crucial for plant stress resistance, which has even longer N-terminal arms than Hsp16.9, with a functionally important and conserved methionine-rich motif. To provide a framework for investigating structure-function relationships of Hsp21 and understanding these sequence variations, we developed a structural model of Hsp21 based on homology modeling, cryo-EM, cross-linking mass spectrometry, NMR, and small-angle X-ray scattering. Our data suggest a dodecameric arrangement of two trimer-of-dimer discs stabilized by the C-terminal tails, possibly through tail-to-tail interactions between the discs, mediated through extended IXVXI motifs. Our model further suggests that six N-terminal arms are located on the outside of the dodecamer, accessible for interaction with client proteins, and distinct from previous undefined or inwardly facing arms. To test the importance of the IXVXI motif, we created the point mutant V181A, which, as expected, disrupts the Hsp21 dodecamer and decreases chaperone activity. Finally, our data emphasize that sHsp chaperone efficiency depends on oligomerization and that client interactions can occur both with and without oligomer dissociation. These results provide a generalizable workflow to explore sHsps, expand our understanding of sHsp structural motifs, and provide a testable Hsp21 structure model to inform future investigations.
Assuntos
Proteínas de Arabidopsis/metabolismo , Proteínas de Choque Térmico/metabolismo , Chaperonas Moleculares/metabolismo , Proteínas de Plantas/metabolismo , Motivos de Aminoácidos , Sequência de Aminoácidos , Proteínas de Cloroplastos/metabolismo , Cloroplastos/metabolismo , Microscopia Crioeletrônica , Processamento de Imagem Assistida por Computador , Espectroscopia de Ressonância Magnética , Espectrometria de Massas , Mutação , Mutação Puntual , Ligação Proteica , Domínios Proteicos , Dobramento de Proteína , Multimerização Proteica , Proteínas Recombinantes/metabolismo , Espalhamento de Radiação , Raios XRESUMO
FKBP12, a small human enzyme, aids protein folding by catalyzing cis-trans isomerization of peptidyl-prolyl bonds, and is involved in cell signaling pathways, calcium regulation, and the immune response. The underlying molecular mechanisms are not fully understood, but it is well-known that aromatic residues in the active site and neighboring loops are important for substrate binding and catalysis. Here we report micro- to millisecond exchange dynamics of aromatic side chains in the active site region of ligand-free FKBP12, involving a minor state population of 0.5% and an exchange rate of 3600 s-1, similar to previous results for the backbone and methyl-bearing side chains. The exchange process involves tautomerization of H87. In the major state H87 is highly flexible and occupies the common HNε2 tautomer, while in the minor state it occupies the rare HNδ1 tautomer, which typically requires stabilization by specific interactions, such as hydrogen bonds. This finding suggests that the exchange process is coupled to a rearrangement of the hydrogen bond network around H87. Upon addition of the active-site inhibitor FK506 the exchange of all aromatic residues is quenched, with exception of H87. The H87 resonances are broadened beyond detection, suggesting that interconversion between tautomers prevail in the FK506-bound state. While key active-site residues undergo conformational exchange in the apo state, the exchange rate is considerably faster than the catalytic turnover, as determined herein by Michaelis-Menten type analysis of NMR line shapes and chemical shifts. We discuss alternative interpretations of this observation in terms of FKBP12 function.
Assuntos
Aminoácidos Aromáticos/química , Domínio Catalítico , Conformação Proteica , Proteína 1A de Ligação a Tacrolimo/química , Aminoácidos Aromáticos/metabolismo , Sítios de Ligação/genética , Histidina/química , Histidina/metabolismo , Humanos , Ligação de Hidrogênio , Isomerismo , Cinética , Espectroscopia de Ressonância Magnética , Modelos Moleculares , Mutação , Ligação Proteica , Tacrolimo/química , Tacrolimo/metabolismo , Proteína 1A de Ligação a Tacrolimo/genética , Proteína 1A de Ligação a Tacrolimo/metabolismoRESUMO
We have recently discovered that the ZZ zinc finger domain represents a novel small ubiquitin-like modifier (SUMO) binding motif. In this study we identify the binding epitopes in the ZZ domain of CBP (CREB-binding protein) and SUMO1 using NMR spectroscopy. The binding site on SUMO1 represents a unique epitope for SUMO interaction spatially opposite to that observed for canonical SUMO interaction motifs (SIMs). HADDOCK docking simulations using chemical shift perturbations and residual dipolar couplings was employed to obtain a structural model for the ZZ domain-SUMO1 complex. Isothermal titration calorimetry experiments support this model by showing that the mutation of key residues in the binding site abolishes binding and that SUMO1 can simultaneously and non-cooperatively bind both the ZZ domain and a canonical SIM motif. The binding dynamics of SUMO1 was further characterized using (15)N Carr-Purcell-Meiboom-Gill (CPMG) relaxation dispersions, which define the off rates for the ZZ domain and SIM motif and show that the dynamic binding process has different characteristics for the two cases. Furthermore, in the absence of bound ligands SUMO1 transiently samples a high energy conformation, which might be involved in ligand binding.
Assuntos
Proteína de Ligação a CREB/química , Epitopos/química , Domínios Proteicos , Proteína SUMO-1/química , Motivos de Aminoácidos , Sequência de Aminoácidos , Sítios de Ligação/genética , Proteína de Ligação a CREB/genética , Proteína de Ligação a CREB/metabolismo , Calorimetria/métodos , Epitopos/genética , Epitopos/metabolismo , Humanos , Cinética , Ligantes , Espectroscopia de Ressonância Magnética , Modelos Moleculares , Mutação , Ligação Proteica , Proteína SUMO-1/genética , Proteína SUMO-1/metabolismo , TermodinâmicaRESUMO
Structure-based protein design offers a possibility of optimizing the overall shape of engineered binding scaffolds to match their targets better. We developed a computational approach for the structure-based design of repeat proteins that allows for adjustment of geometrical features like length, curvature, and helical twist. By combining sequence optimization of existing repeats and de novo design of capping structures, we designed leucine-rich repeats (LRRs) from the ribonuclease inhibitor (RI) family that assemble into structures with a predefined geometry. The repeat proteins were built from self-compatible LRRs that are designed to interact to form highly curved and planar assemblies. We validated the geometrical design approach by engineering a ring structure constructed from 10 self-compatible repeats. Protein design can also be used to increase our structural understanding of repeat proteins. We use our design constructs to demonstrate that buried Cys play a central role for stability and folding cooperativity in RI-type LRR proteins. The computational procedure presented here may be used to develop repeat proteins with various geometrical shapes for applications where greater control of the interface geometry is desired.
Assuntos
Biologia Computacional/métodos , Modelos Moleculares , Engenharia de Proteínas/métodos , Proteínas/química , Proteínas/metabolismo , Sequência de Aminoácidos , Cromatografia de Afinidade , Dicroísmo Circular , Dimerização , Escherichia coli , Proteínas de Repetições Ricas em Leucina , Simulação de Dinâmica Molecular , Dados de Sequência Molecular , Ligação Proteica , Conformação Proteica , Proteínas/genética , Espectroscopia de Infravermelho com Transformada de Fourier , UltracentrifugaçãoRESUMO
BACKGROUND: Peptidyl-prolyl isomerases (PPIases) catalyze cis/trans isomerization of peptidyl-prolyl bonds, which is often rate-limiting for protein folding. SlyD is a two-domain enzyme containing both a PPIase FK506-binding protein (FKBP) domain and an insert-in-flap (IF) chaperone domain. To date, the interactions of these domains with unfolded proteins have remained rather obscure, with structural information on binding to the FKBP domain being limited to complexes involving various inhibitor compounds or a chemically modified tetrapeptide. RESULTS: We have characterized the binding of 15-residue-long unmodified peptides to SlyD from Thermus thermophilus (TtSlyD) in terms of binding thermodynamics and enzyme kinetics through the use of isothermal titration calorimetry, nuclear magnetic resonance spectroscopy, and site-directed mutagenesis. We show that the affinities and enzymatic activity of TtSlyD towards these peptides are much higher than for the chemically modified tetrapeptides that are typically used for activity measurements on FKBPs. In addition, we present a series of crystal structures of TtSlyD with the inhibitor FK506 bound to the FKBP domain, and with 15-residue-long peptides bound to either one or both domains, which reveals that substrates bind in a highly adaptable fashion to the IF domain through ß-strand augmentation, and can bind to the FKBP domain as both types VIa1 and VIb-like cis-proline ß-turns. Our results furthermore provide important clues to the catalytic mechanism and support the notion of inter-domain cross talk. CONCLUSIONS: We found that 15-residue-long unmodified peptides can serve as better substrate mimics for the IF and FKBP domains than chemically modified tetrapeptides. We furthermore show how such peptides are recognized by each of these domains in TtSlyD, and propose a novel general model for the catalytic mechanism of FKBPs that involves C-terminal rotation around the peptidyl-prolyl bond mediated by stabilization of the twisted transition state in the hydrophobic binding site.