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Apolipoprotein E (APOE) genotype determines Alzheimer's disease (AD) susceptibility, with the APOE ε4 allele being an established risk factor for late‐onset AD. The ApoE lipidation status has been reported to impact amyloid‐beta (Aβ) peptide metabolism. The details of how lipidation affects ApoE behavior remain to be elucidated. In this study, we prepared lipid‐free and lipid‐bound ApoE particles, mimicking the high‐density lipoprotein particles found in vivo, for all three isoforms (ApoE2, ApoE3, and ApoE4) and biophysically characterized them. We find that lipid‐free ApoE in solution has the tendency to aggregate in vitro in an isoform‐dependent manner under near‐physiological conditions and that aggregation is impeded by lipidation of ApoE.

Src Homology 2 and 3 (SH2 and SH3) are two key protein interaction modules involved in regulating the activity of many proteins such as tyrosine kinases and phosphatases by respective recognition of phosphotyrosine and proline-rich regions. In the Src family kinases, the inactive state of the protein is the direct result of the interaction of the SH2 and the SH3 domain with intra-molecular regions, leading to a closed structure incompetent with substrate modification. Here, we report the 1H, 15N and 13C backbone- and side-chain chemical shift assignments of the partially deuterated Fyn SH3-SH2 domain and structural differences between tandem and single domains. The BMRB accession number is 27165.

Most Alzheimer's disease (AD) cases are late-onset and characterized by the aggregation and deposition of the amyloid-beta (Aβ) peptide in extracellular plaques in the brain. However, a few rare and hereditary Aβ mutations, such as the Italian Glu22-to-Lys (E22K) mutation, guarantee the development of early-onset familial AD. This type of AD is associated with a younger age at disease onset, increased β-amyloid accumulation, and Aβ deposition in cerebral blood vessel walls, giving rise to cerebral amyloid angiopathy (CAA). It remains largely unknown how the Italian mutation results in the clinical phenotype that is characteristic of CAA. We therefore investigated how this single point mutation may affect the aggregation of Aβ1-42 in vitro and structurally characterized the resulting fibrils using a biophysical approach. This paper reports that wild-type and Italian-mutant Aβ both form fibrils characterized by the cross-β architecture, but with distinct β-sheet organizations, resulting in differences in thioflavin T fluorescence and solvent accessibility. E22K Aβ1-42 oligomers and fibrils both display an antiparallel β-sheet structure, in comparison with the parallel β-sheet structure of wild-type fibrils, characteristic of most amyloid fibrils described in the literature. Moreover, we demonstrate structural plasticity for Italian-mutant Aβ fibrils in a pH-dependent manner, in terms of their underlying β-sheet arrangement. These findings are of interest in the ongoing debate that (1) antiparallel β-sheet structure might represent a signature for toxicity, which could explain the higher toxicity reported for the Italian mutant, and that (2) fibril polymorphism might underlie differences in disease pathology and clinical manifestation., SCOPUS: ar.j, info:eu-repo/semantics/published

Design of a new catalytic function in proteins, apart from its inherent practical value, is important for fundamental understanding of enzymatic activity. Using a computationally inexpensive, minimalistic approach that focuses on introducing a single highly reactive residue into proteins to achieve catalysis we converted a 74-residue-long C-terminal domain of calmodulin into an efficient esterase. The catalytic efficiency of the resulting stereoselective, allosterically regulated catalyst, nicknamed AlleyCatE, is higher than that of any previously reported de novo designed esterases. The simplicity of our design protocol should complement and expand the capabilities of current state-of-art approaches to protein design. These results show that even a small nonenzymatic protein can efficiently attain catalytic activities in various reactions (Kemp elimination, ester hydrolysis, retroaldol reaction) as a result of a single mutation. In other words, proteins can be just one mutation away from becoming entry points for subsequent evolution.

Src homology 2 domains are interaction modules dedicated to the recognition of phosphotyrosine sites incorporated in numerous proteins found in intracellular signaling pathways. Here we provide for the first time structural insight into the dimerization of Fyn SH2 both in solution and in crystalline conditions, providing novel crystal structures of both the dimer and peptide‐bound structures of Fyn SH2. Using nuclear magnetic resonance chemical shift analysis, we show how the peptide is able to eradicate the dimerization, leading to monomeric SH2 in its bound state. Furthermore, we show that Fyn SH2's dimer form differs from other SH2 dimers reported earlier. Interestingly, the Fyn dimer can be used to construct a completed dimer model of Fyn without any steric clashes. Together these results extend our understanding of SH2 dimerization, giving structural details, on one hand, and suggesting a possible physiological relevance of such behavior, on the other hand.

Uropathogenic Escherichia coli cause urinary tract infections by adhering to mannosylated receptors on the human urothelium via the carbohydrate-binding domain of the FimH adhesin (FimHL). Numerous α-d-mannopyranosides, including α-d-heptyl mannose (HM), inhibit this process by interacting with FimHL. To establish the molecular basis of the high-affinity HM binding, we solved the solution structure of the apo form and the crystal structure of the FimHL-HM complex. NMR relaxation analysis revealed that protein dynamics were not affected by the sugar binding, yet HM addition promoted protein dimerization, which was further confirmed by small-angle X-ray scattering. Finally, to address the role of Y48, part of the "tyrosine gate" believed to govern the affinity and specificity of mannoside binding, we characterized the FimHL Y48A mutant, whose conformational, dynamical, and HM binding properties were found to be very similar to those of the wild-type protein.

Here we describe paramagnetic NMR analysis of the low- and high-spin forms of yeast cytochrome c peroxidase (CcP), a 34 kDa heme enzyme involved in hydroperoxide reduction in mitochondria. Starting from the assigned NMR spectra of a low-spin CN-bound CcP and using a strategy based on paramagnetic pseudocontact shifts, we have obtained backbone resonance assignments for the diamagnetic, iron-free protein and the high-spin, resting-state enzyme. The derived chemical shifts were further used to determine low- and high-spin magnetic susceptibility tensors and the zero-field splitting constant (D) for the high-spin CcP. The D value indicates that the latter contains a hexacoordinate heme species with a weak field ligand, such as water, in the axial position. Being one of the very few high-spin heme proteins analyzed in this fashion, the resting state CcP expands our knowledge of the heme coordination chemistry in biological systems.

Src homology 2 (SH2) domains are key modulators in various signaling pathways allowing the recognition of phosphotyrosine sites of different proteins. Despite the fact that SH2 domains acquire their biological functions in a monomeric state, a multitude of reports have shown their tendency to dimerize. Here, we provide a technical description on how to isolate and characterize by gel filtration, circular dichroism (CD), and nuclear magnetic resonance (NMR) each conformational state of p59(fyn) SH2 domain.

To enhance our understanding of the potential therapeutic utility of insulin-degrading enzyme (IDE) in Alzheimer's disease (AD), we studied in vitro IDE-mediated degradation of different amyloid-beta (Aβ) peptide aggregation states. Our findings show that IDE activity is driven by the dynamic equilibrium between Aβ monomers and higher ordered aggregates. We identify Met(35)-Val(36) as a novel IDE cleavage site in the Aβ sequence and show that Aβ fragments resulting from IDE cleavage form non-toxic amorphous aggregates. These findings need to be taken into account in therapeutic strategies designed to increase Aβ clearance in AD patients by modulating IDE activity.

Sugar binding by a cell surface ∼29 kDa lectin (RSL) from the bacterium Ralstonia solanacearum was characterized by NMR spectroscopy. The complexes formed with four monosaccharides and four fucosides were studied. Complete resonance assignments and backbone dynamics were determined for RSL in the sugar-free form and when bound to l-fucose or d-mannose. RSL was found to interact with both the α- and the β-anomer of l-fucose and the "fucose like" sugars d-arabinose and l-galactose. Peak splitting was observed for some resonances of the binding site residues. The assignment of the split signals to the α- or β-anomer was confirmed by comparison with the spectra of RSL bound to methyl-α-l-fucoside or methyl-β-l-fucoside. The backbone dynamics of RSL were sensitive to the presence of ligand, with the protein adopting a more compact structure upon binding to l-fucose. Taking advantage of tryptophan residues in the binding sites, we show that the indole resonance is an excellent reporter on ligand binding. Each sugar resulted in a distinct signature of chemical shift perturbations, suggesting that tryptophan signals are a sufficient probe of sugar binding.

Summary To survive hostile conditions, the bacterial pathogen Mycobacterium tuberculosis produces millimolar concentrations of mycothiol as a redox buffer against oxidative stress. The reductases that couple the reducing power of mycothiol to redox active proteins in the cell are not known. We report a novel mycothiol-dependent reductase (mycoredoxin-1) with a CGYC catalytic motif. With mycoredoxin-1 and mycothiol deletion strains of Mycobacterium smegmatis, we show that mycoredoxin-1 and mycothiol are involved in the protection against oxidative stress. Mycoredoxin-1 acts as an oxidoreductase exclusively linked to the mycothiol electron transfer pathway and it can reduce S-mycothiolated mixed disulphides. Moreover, we solved the solution structures of oxidized and reduced mycoredoxin-1, revealing a thioredoxin fold with a putative mycothiol-binding site. With HSQC snapshots during electron transport, we visualize the reduction of oxidized mycoredoxin-1 as a function of time and find that mycoredoxin-1 gets S-mycothiolated on its N-terminal nucleophilic cysteine. Mycoredoxin-1 has a redox potential of −218 mV and hydrogen bonding with neighbouring residues lowers the pKa of its N-terminal nucleophilic cysteine. Determination of the oxidized and reduced structures of mycoredoxin-1, better understanding of mycothiol-dependent reactions in general, will likely give new insights in how M. tuberculosis survives oxidative stress in human macrophages.

SH2 domains are widespread protein-binding modules that recognize phosphotyrosines and play central roles in intracellular signalling pathways. The SH2 domain of the human protein tyrosine kinase Fyn has been expressed, purified and crystallized in the unbound state and in complex with a high-affinity phosphotyrosine peptide. X-ray data were collected to a resolution of 2.00 Å for the unbound form and 1.40 Å for the protein in complex with the phosphotyrosine peptide.

Cytochrome c (Cc) is a soluble electron carrier protein, transferring reducing equivalents between Cc reductase and Cc oxidase in eukaryotes. In this work, we assessed the structural differences between reduced and oxidized Cc in solution by paramagnetic NMR spectroscopy. First, we have obtained nearly-complete backbone NMR resonance assignments for iso-1-yeast Cc and horse Cc in both oxidation states. These were further used to derive pseudocontact shifts (PCSs) arising from the paramagnetic haem group. Then, an extensive dataset comprising over 450 measured PCSs and high-resolution X-ray and solution NMR structures of both proteins were used to define the anisotropic magnetic susceptibility tensor, Δχ. For most nuclei, the PCSs back-calculated from the Δχ tensor are in excellent agreement with the experimental PCS values. However, several contiguous stretches-clustered around G41, N52, and A81-exhibit large deviations both in yeast and horse Cc. This behaviour is indicative of redox-dependent structural changes, the extent of which is likely conserved in the protein family. We propose that the observed discrepancies arise from the changes in protein dynamics and discuss possible functional implications.

Protein-protein interactions occur with a wide range of affinities from tight complexes characterized by femtomolar dissociation constants to weak, and more transient, complexes of millimolar affinity. Many of the weak and transiently formed protein-protein complexes have escaped characterization due to the difficulties in obtaining experimental parameters that report on the complexes alone without contributions from the unbound, free proteins. Here, we review recent developments for characterizing the structures of weak protein-protein complexes using nuclear magnetic resonance spectroscopy with special emphasis on the utility of residual dipolar couplings.

TEM-1 β-lactamase is a highly efficient enzyme that is involved in bacterial resistance against β-lactam antibiotics such as penicillin. It is also a robust scaffold protein which can be engineered by molecular-evolution techniques to bind a variety of targets. One such β-lactamase variant (BlaKr) has been constructed to bind kanamycin (kan) and other aminoglycoside antibiotics, which are neither substrates nor ligands of native β-lactamases. In addition to recognizing kan, BlaKr activity is up-regulated by its binding via an activation mechanism which is not yet understood at the molecular level. In order to fill this gap, determination of the structure of the BlaKr-kan complex was embarked upon. A crystallization condition for BlaKr-kan was identified using high-throughput screening, and crystal growth was further optimized using streak-seeding and hanging-drop methods. The crystals belonged to the orthorhombic space group P2(1)2(1)2(1), with unit-cell parameters a = 47.01, b = 72.33, c = 74.62 Å, and diffracted to 1.67 Å resolution using synchrotron radiation. The X-ray structure of BlaKr with its ligand kanamycin should provide the molecular-level details necessary for understanding the activation mechanism of the engineered enzyme.

MazF proteins are ribonucleases that cleave mRNA with high sequence-specificity as part of bacterial stress response and that are neutralized by the action of the corresponding antitoxin MazE. Prolonged activation of the toxin MazF leads to cell death. Several mazEF modules from gram-negative bacteria have been characterized in terms of catalytic activity, auto-regulation mechanism and structure, but less is known about their distant relatives found in gram-positive organisms. Currently, no solution NMR structure is available for any wild-type MazF toxin. Here we report the (1)H, (15)N and (13)C backbone and side-chain chemical shift assignments of this toxin from the pathogen bacterium Staphylococcus aureus. The BMRB accession number is 17288.

Rop is a four-helix bundle protein composed of two identical helix-loop-helix monomers. Protein folding monitored by stopped-flow fluorescence or CD exhibits biphasic kinetics when folding to low final denaturant concentrations. As the final concentration of denaturant is increased, the amplitude of the fast phase decreases, until at the highest concentrations the kinetics appear monophasic. We propose that the fast phase represents the formation of an intermediate. Here, we use real-time NMR to detect the formation of this intermediate and to characterize its structural features.

Alzheimer's disease (AD) is a progressive neurodegenerative brain disorder that involves a plethora of molecular pathways. In the context of therapeutic treatment and biomarker profiling, the amyloid-beta (A beta) peptide constitutes an interesting research avenue that involves interactions within a complex mixture of A beta alloforms and other disease-modifying factors. Here, we explore the potential of an ecosystem paradigm as a novel way to consider AD and A beta dynamics in particular. We discuss the example that the complexity of the A beta network not only exhibits interesting parallels with the functioning of complex systems such as ecosystems but that this analogy can also provide novel insights into the neurobiological phenomena in AD and serve as a communication tool. We propose that combining network medicine with general ecosystem management principles could be a new and holistic approach to understand AD pathology and design novel therapies. (C) 2016 The Authors. Published by Elsevier Inc.

Vacuolar (H+)-ATPase (V-ATPase) is a proton pump present in several compartments of eukaryotic cells to regulate physiological processes. From biochemical studies it is known that the interaction between arginine 735 present in the seventh transmembrane (TM7) segment from subunit a and specific glutamic acid residues in the subunit c assembly plays an essential role in proton translocation. To provide more detailed structural information about this protein domain, a peptide resembling TM7 (denoted peptide MTM7) from Saccharomyces cerevisiae (yeast) V-ATPase was synthesized and dissolved in two membrane-mimicking solvents: DMSO and SDS. For the first time the secondary structure of the putative TM7 segment from subunit a is obtained by the combined use of CD and NMR spectroscopy. SDS micelles reveal an alpha-helical conformation for peptide MTM7 and in DMSO three alpha-helical regions are identified by 2D 1H-NMR. Based on these conformational findings a new structural model is proposed for the putative TM7 in its natural environment. It is composed of 32 amino acid residues that span the membrane in an alpha-helical conformation. It starts at the cytoplasmic side at residue T719 and ends at the luminal side at residue W751. Both the luminal and cytoplasmatic regions of TM7 are stabilized by the neighboring hydrophobic transmembrane segments of subunit a and the subunit c assembly from V-ATPase.

The N-terminally truncated variant of photoactive yellow protein (Delta25-PYP) undergoes a very similar photocycle as the corresponding wild-type protein (WT-PYP), although the lifetime of its light-illuminated (pB) state is much longer. This has allowed determination of the structure of both its dark- (pG) as well as its pB-state in solution by nuclear magnetic resonance (NMR) spectroscopy. The pG structure shows a well-defined fold, similar to WT-PYP and the X-ray structure of the pG state of Delta25-PYP. In the long-lived photocycle intermediate pB, the central beta sheet is still intact, as well as a small part of one alpha helix. The remainder of pB is unfolded and highly flexible, as evidenced by results from proton-deuterium exchange and NMR relaxation studies. Thus, the partially unfolded nature of the presumed signaling state of PYP in solution, as suggested previously, has now been structurally demonstrated.

For the purpose of equilibrium and kinetic folding-unfolding studies, the SH3 domain of alpha-spectrin (spc-SH3) has long been considered a classic two-state folding protein. In this work we have indeed observed that the thermal unfolding curves of spc-SH3 measured at pH 3.0 by differential scanning calorimetry, circular dichroism, and NMR follow apparently the two-state model when each unfolding profile is considered individually. Nevertheless, we have found that protein concentration has a marked effect upon the thermal unfolding profiles. This effect cannot be properly explained in terms of the two-state unfolding model and can only be interpreted in terms of the accumulation of intermediate associated states in equilibrium with the monomeric native and unfolded states. By chemical cross-linking and pulsed-field gradient NMR diffusion experiments we have been able to confirm the existence of associated states formed during spc-SH3 unfolding. A three-state model, in which a dimeric intermediate state is assumed to be significantly populated, provides the simplest interpretation of the whole set of thermal unfolding data and affords a satisfactory explanation for the concentration effects observed. Whereas at low concentrations the population of the associated intermediate state is negligible and the unfolding process consequently takes place in a two-state fashion, at concentrations above approximately 0.5 mM the population of the intermediate state becomes significant at temperatures between 45 degrees C and 80 degrees C and reaches up to 50% at the largest concentration investigated. The thermodynamic properties of the intermediate state implied by this analysis fall in between those of the unfolded state and the native ones, indicating a considerably disordered conformation, which appears to be stabilized by oligomerization.

The high-resolution 3D structure of the octapeptide hormone angiotensin II (AII) in aqueous solution has been obtained by simulated annealing calculations, using high-resolution NMR-derived restraints. After final refinement in explicit water, a family of 13 structures was obtained with a backbone RMSD of 0.73 +/- 0.23 A. AII adopts a fairly compact folded structure, with its C-terminus and N-terminus approaching to within approximately 7.2 A of each other. The side chains of Arg2, Tyr4, Ile5 and His6 are oriented on one side of a plane defined by the peptide backbone, and the Val3 and Pro7 are pointing in opposite directions. The stabilization of the folded conformation can be explained by the stacking of the Val3 side chain with the Pro7 ring and by a hydrophobic cluster formed by the Tyr4, Ile5 and His6 side chains. Comparison between the NMR-derived structure of AII in aqueous solution and the refined crystal structure of the complex of AII with a high-affinity mAb (Fab131) [Garcia, K.C., Ronco, P.M., Verroust, P.J., Brunger, A.T., Amzel, L.M. (1992) Science257, 502-507] provides important quantitative information on two common structural features: (a) a U-shaped structure of the Tyr4-Ile5-His6-Pro7 sequence, which is the most immunogenic epitope of the peptide, with the Asp1 side chain oriented towards the interior of the turn approaching the C-terminus; (b) an Asx-turn-like motif with the side chain aspartate carboxyl group hydrogen-bonded to the main chain NH group of Arg2. It can be concluded that small rearrangements of the epitope 4-7 in the solution structure of AII are required by a mean value of 0.76 +/- 0.03 A for structure alignment and approximately 1.27 +/- 0.02 A for sequence alignment with the X-ray structure of AII bound to the mAb Fab131. These data are interpreted in terms of a biological "nucleus" conformation of the hormone in solution, which requires a limited number of structural rearrangements for receptor-antigen recognition and binding.

Mersacidin belongs to the type B lantibiotics (lanthionine-containing antibiotics) that contain post-translationally modified amino acids and cyclic ring structures. It targets the cell wall precursor lipid II and thereby inhibits cell wall synthesis. In light of the emerging antibiotics resistance problem, the understanding of the antibacterial activity on a structural basis provides a key to circumvent this issue. Here we present solution NMR studies of mersacidin-lipid II interaction in dodecylphosphocholine (DPC) micelles. Distinct solution structures of mersacidin were determined in three different states: in water/methanol solution and in DPC micelles with and without lipid II. The structures in various sample conditions reveal remarkable conformational changes in which the junction between Ala-12 and Abu-13 (where Abu is aminobutyric acid) effectively serves as the hinge for the opening and closure of the ring structures. The DPC micelle-bound form resembles the previously determined NMR and x-ray crystal structures of mersacidin in pure methanol but substantially deviates from the other two states in our current report. The structural changes delineate the large chemical shift perturbations observed during the course of a two-step (15)N-(1)H heteronuclear single quantum coherence titration. They also modulate the surface charge distribution of mersacidin suggesting that electrostatics play a central role in the mersacidin-lipid II interaction. The observed conformational adaptability of mersacidin might be a general feature of lipid II-interacting antibiotics/peptides.

The long-lived light-induced intermediate (pB) of the E46Q mutant (glutamic acid is replaced by glutamine at position 46) of photoactive yellow protein (PYP) has been investigated by NMR spectroscopy. The ground state of this mutant is very similar to that of wild-type PYP (WT), whereas the pB state, formed upon illumination, appears to be much more structured in E46Q than in WT. The differences are most striking in the N-terminal domain of the protein. In WT, the side-chain carboxylic group of E46 is known to donate its proton to the chromophore upon illumination. The absence of the carboxylic group near the chromophore in the E46Q mutant prohibits the formation of a negative charge at this position upon formation of pB. This prevents the partial unfolding of the mutant, as evidenced from NMR chemical shift comparison and proton/deuterium (H/D) exchange studies.

oxin-antitoxin (TA) modules are pairs of genes essential for bacterial regulation upon environmental stresses. The mazEF module encodes the MazF toxin and its cognate MazE antitoxin. The highly dynamic MazE possesses an N-terminal DNA binding domain through which it can negatively regulate its own promoter. Despite being one of the first TA systems studied, transcriptional regulation of Escherichia coli mazEF remains poorly understood. This paper presents the solution structure of C-terminal truncated E. coli MazE and a MazE-DNA model with a DNA palindrome sequence about 10 bp upstream of the mazEF promoter. The work has led to a transcription regulator-DNA model, which has remained elusive thus far in the E. coli toxin-antitoxin family. Multiple complementary techniques including NMR, SAXS and ITC show that the long intrinsically disordered C-termini in MazE, required for MazF neutralization, does not affect the interactions between the antitoxin and its operator. Rather, the MazE C-terminus plays an important role in the MazF binding, which was found to increase the MazE affinity for the palindromic single site operator.

The Staphylococcus aureus genome contains three toxin-antitoxin modules, including one mazEF module, SamazEF. Using an on-column separation protocol we are able to obtain large amounts of wild-type SaMazF toxin. The protein is well-folded and highly resistant against thermal unfolding but aggregates at elevated temperatures. Crystallographic and nuclear magnetic resonance (NMR) solution studies show a well-defined dimer. Differences in structure and dynamics between the X-ray and NMR structural ensembles are found in three loop regions, two of which undergo motions that are of functional relevance. The same segments also show functionally relevant dynamics in the distantly related CcdB family despite divergence of function. NMR chemical shift mapping and analysis of residue conservation in the MazF family suggests a conserved mode for the inhibition of MazF by MazE., info:eu-repo/semantics/published

17 páginas, 12 figuras, 3 tablas, TAX1BP1 is a novel ubiquitin-binding adaptor protein involved in the negative regulation of the NF-kappaB transcription factor, which is a key player in inflammatory responses, immunity and tumorigenesis. TAX1BP1 recruits A20 to the ubiquitinated signaling proteins TRAF6 and RIP1, leading to their A20-mediated deubiquitination and the disruption of IL-1-induced and TNF-induced NF-kappaB signaling, respectively. The two zinc fingers localized at its C-terminus function as novel ubiquitin-binding domains (UBZ, ubiquitin-binding zinc finger). Here we present for the first time both the solution and crystal structures of two classical UBZ domains in tandem within the human TAX1BP1. The relative orientation of the two domains is slightly different in the X-ray structure with respect to the NMR structure, indicating some degree of conformational flexibility, which is rationalized by NMR relaxation data. The observed degree of flexibility and stability between the two UBZ domains might have consequences on the recognition mechanism of interacting partners., M.A.C. is supported by an FPU grant from the Ministerio de Educación y Cultura de España. L.B. and A.G.-P. are FWO post-doc fellows. Work in N.v.N. laboratory is supported by the VIB and the Flanders Hercules Foundation. Work in J.B. laboratory was supported by Ministerio de Ciencia e Innovación (SAF2009-10667 and SAF2012-31405) and Generalitat Valenciana ACOMP/2012/024. We thank support for data collection of ID14-1 staff at the European Synchrotron Radiation Facility.

The folding of apo-pseudoazurin, a 123-residue, predominantly beta-sheet protein with a complex Greek key topology, has been investigated using several biophysical techniques. Kinetic analysis of refolding using far- and near-ultraviolet circular dichroism (UV CD) shows that the protein folds slowly to the native state with rate constants of 0.04 and 0.03 min(-1), respectively, at pH 7.0 and at 15 degrees C. This process has an activation enthalpy of approximately 90 kJ/mole and is catalyzed by cyclophilin A, indicating that folding is limited by trans-cis proline isomerization, presumably around the Xaa-Pro 20 bond that is in the cis isomer in the native state. Before proline isomerization, an intermediate accumulates during folding. This species has a substantial signal in the far-UV CD, a nonnative signal in the near-UV CD, exposed hydrophobic surfaces (judged by 1-anilino naphthalenesulphonate binding), a noncooperative denaturation transition, and a dynamic structure (revealed by line broadening on the nuclear magnetic resonance time scale). We compare the properties of this intermediate with partially folded states of other proteins and discuss its role in folding of this complex Greek key protein.

Thermodynamic characterization of the activation domain of human procarboxypeptidase A2, ADA2h, and its helix-engineered mutants was carried out by differential scanning calorimetry. The mutants were engineered by changing residues in the exposed face of the two a helices in order to increase their stability. At neutral and alkaline pH the three mutants, a-helix 1 (M1), a-helix 2 (M2) and a-helix 1 and a-helix 2 (DM), were more stable than the wild-type domain, in the order DM, M2, M1 and wild-type. Under these conditions the CD and NMR spectra of all the variants are very similar, indicating that this increase in stability is not the result of gross structural changes. Calorimetric analysis shows that the stabilizing effect of mutating the water-exposed surfaces of the helices seems to be mainly entropic, because the mutations do not change the enthalpy or the increase in heat capacity of denaturation. The unfolding behavior of all variants changes under acidic conditions: whereas wild-type and M1 have a strong tendency to aggregate, giving rise to a b conformation upon unfolding, M2 and DM unfold reversibly, M2 being more stable than DM. CD and NMR experiments at pH 3.0 suggest that a region involving residues of the second and third b strands as well as part of a-helix 1 changes its conformation. It seems that the enhanced stability of the altered conformation of M2 and DM reduces the aggregation tendency of ADA2h at acidic pH.

The recovery of enzymatic activity during the folding of muscle acylphosphatase and two single residue mutants (proline 54 to alanine and proline 71 to alanine) from 7 M urea has been monitored and compared with the development of intrinsic fluorescence emission. Fluorescence measurements reveal the presence in the wild-type protein of a major rapid refolding phase followed by a second low amplitude slow phase. The slow phase is absent in the fluorescence trace acquired with the proline 54 to alanine mutant, suggesting the involvement of this proline residue in the fluorescence-detected slow phase of the wild-type protein. The major kinetic phase is associated with a considerable recovery of enzymatic activity, indicating that a large fraction of molecules refolds with effective two-state behavior. The use of time-resolved enzymatic activity as a probe to follow the folding process reveals, however, the presence of another exponential slow phase arising from proline 71. This slow phase is not observable by utilizing optical probes, indicating that, unlike proline 54, the cis to trans isomerization of proline 71 can take place in an intermediate possessing a native-like fold. We suggest that, although spectroscopically silent and structurally insignificant, the cis-trans interconversion of proline residues in native-like intermediates may be crucial for the generation of enzymatic activity of functional enzymes.

The transition state for folding of a small protein, muscle acylphosphatase, has been studied by measuring the rates of folding and unfolding under a variety of solvent conditions. A strong dependence of the folding rate on the concentration of urea suggests the occurrence in the transition state of a large shielding of those groups that are exposed to interaction with the denaturant in the unfolded state (mainly hydrophobic moieties and groups located on the polypeptide backbone). The heat capacity change upon moving from the unfolded state to the transition state is small and is indicative of a substantial solvent exposure of hydrophobic groups. The solvent-accessibility of such groups in the transition state has also been found to be significant by measuring the rates of folding and unfolding in the presence of sugars. These rates have also been found to be accelerated by the addition of small quantities of alcohols. Trifluoroethanol and hexafluoroisopropanol were particularly effective, suggesting that stabilisation of local hydrogen bonds lowers the energy of the transition state relative to the folded and unfolded states. Finally, a study with a competitive inhibitor of acylphosphatase has provided evidence for the complete loss of ligand binding affinity in the transition state, indicating that specific long-range interactions at the level of the active site are not yet formed at this stage of the folding reaction. A model of the transition state for acylphosphatase folding, in which β-turns and one or both α-helices are formed to a significant extent but in which the persistent long-range interactions characteristic of the folded state are largely absent, accounts for all our data. These results are broadly consistent with models of the transition states for folding of other small proteins derived from mutagenesis studies, and suggest that solvent perturbation methods can provide complementary information about the transition region of the energy surfaces for protein folding.

International audience; Antitoxins from prokaryotic type II toxin-antitoxin modules are characterized by a high degree of intrinsic disorder. The description of such highly flexible proteins is challenging because they cannot be represented by a single structure. Here, we present a combination of SAXS and NMR data to describe the conformational ensemble of the PaaA2 antitoxin from the human pathogen E. coli O157. The method encompasses the use of SAXS data to filter ensembles out of a pool of conformers generated by a custom NMR structure calculation protocol and the subsequent refinement by a block jackknife procedure. The final ensemble obtained through the method is validated by an established residual dipolar coupling analysis. We show that the conformational ensemble of PaaA2 is highly compact and that the protein exists in solution as two preformed helices, connected by a flexible linker, that probably act as molecular recognition elements for toxin inhibition.

Nature Chemical Biology 9(12), 811 - 817 (2013). doi:10.1038/nchembio.1364, Fic proteins are ubiquitous in all of the domains of life and have critical roles in multiple cellular processes through AMPylation of (transfer of AMP to) target proteins. Doc from the doc-phd toxin-antitoxin module is a member of the Fic family and inhibits bacterial translation by an unknown mechanism. Here we show that, in contrast to having AMPylating activity, Doc is a new type of kinase that inhibits bacterial translation by phosphorylating the conserved threonine (Thr382) of the translation elongation factor EF-Tu, rendering EF-Tu unable to bind aminoacylated tRNAs. We provide evidence that EF-Tu phosphorylation diverged from AMPylation by antiparallel binding of the NTP relative to the catalytic residues of the conserved Fic catalytic core of Doc. The results bring insights into the mechanism and role of phosphorylation of EF-Tu in bacterial physiology as well as represent an example of the catalytic plasticity of enzymes and a mechanism for the evolution of new enzymatic activities., Published by Nature Publishing Group

Src homology 2 (SH2) domains have an important role in the regulation of protein activity and intracellular signaling processes. They are geared to bind to specific phosphotyrosine (pY) motifs, with a substrate sequence specificity depending on the three amino acids immediately C-terminal to the pY. Here we report for the first time the 1H, 15N and 13C backbone and side-chain chemical shift assignments for the C-terminal SH2 domain of the human protein tyrosine phosphatase PTPN11, both in its free and bound forms, where the ligand in the latter corresponds to a specific sequence of the human erythropoietin receptor.

17 páginas, 8 figuras, 1 tabla. Este fichero contiene 5 figuras y 3 tablas de material suplementario, The CD2AP (CD2-associated protein) and CIN85 (Cbl-interacting protein of 85 kDa) adaptor proteins each employ three Src homology 3 (SH3) domains to cluster protein partners and ensure efficient signal transduction and down-regulation of tyrosine kinase receptors. Using NMR, isothermal titration calorimetry and small-angle X-ray scattering methods, we have characterized several binding modes of the N-terminal SH3 domain (SH3A) of CD2AP and CIN85 with two natural atypical proline-rich regions in CD2 (cluster of differentiation 2) and Cbl-b (Casitas B-lineage lymphoma), and compared these data with previous studies and published crystal structures. Our experiments show that the CD2AP-SH3A domain forms a type II dimer with CD2 and both type I and type II dimeric complexes with Cbl-b. Like CD2AP, the CIN85-SH3A domain forms a type II complex with CD2, but a trimeric complex with Cbl-b, whereby the type I and II interactions take place at the same time. Together, these results explain how multiple interactions among similar SH3 domains and ligands produce a high degree of diversity in tyrosine kinase, cell adhesion or T-cell signaling pathways., This research is funded by grant FQM02838 from the Junta de Andalucía. M.A.C. is supported by an FPU grant from the Spanish Ministry of Education (MEC). A.G-P is an FWO post-doc fellow. J.L.OR is a FEBS post-doc fellow. Work in NvN laboratory is supported by the VIB and the Flanders Hercules Foundation. Work in JB laboratory was supported by Ministerio de Ciencia e Innovación (SAF2009-10667, SAF2012-31405) and Generalitat Valenciana ACOMP/2012/024.

Src homology 2 (SH2) domains have an important role in the regulation of protein activity and intracellular signaling processes. They are geared to bind to specific phosphotyrosine (pY) motifs, with a substrate sequence specificity depending on the three amino acids immediately C-terminal to the pY. Here we report for the first time the (1)H, (15)N and (13)C backbone and side-chain chemical shift assignments for the C-terminal SH2 domain of the human protein tyrosine phosphatase PTPN11, both in its free and bound forms, where the ligand in the latter corresponds to a specific sequence of the human erythropoietin receptor.

Here we present a solution NMR study of the complex between yeast cytochrome c (Cc) and cytochrome c peroxidase (CcP), a paradigm for understanding the biological electron transfer. Performed for the first time, the CcP-observed heteronuclear NMR experiments were used to probe the Cc binding in solution. Combining the Cc- and CcP-detected experiments, the binding interface on both proteins was mapped out, confirming that the X-ray structure of the complex is maintained in solution. Using NMR titrations and chemical shift perturbation analysis, we show that the interaction is independent of the CcP spin-state and is only weakly affected by the Cc redox state. Based on these findings, we argue that the complex of the ferrous Cc and the cyanide-bound CcP is a good mimic of the catalytically-active Cc-CcP compound I species. Finally, no chemical shift perturbations due to the Cc binding at the low-affinity CcP site were observed at low ionic strength. We discuss possible reasons for the absence of the effects and outline future research directions.

The precision with which X-ray crystallography and nuclear magnetic resonance (NMR) have provided structural models of biologically active and inactive conformations of countless proteins belies an easily overlooked dilemma. Proteins are inherently dynamic, exhibiting conformational freedom on timescales from picoseconds to seconds, implicating structural rearrangements that are essential for their biological function. Classical structural biology determines static models, that afford little insight into the underlying conformational equilibrium. The role that structural dynamics play in biological processes can only be understood by characterizing all thermally accessible protein conformations and their populations. NMR spectroscopy is uniquely sensitive to the presence of conformational dynamics in solution. Residual dipolar couplings (RDCs) measured in weakly aligned proteins, scalar couplings, and chemical shifts, probe motions occurring on timescales faster than 100 s of microseconds. These parameters therefore offer general tools to characterize protein motion on physiologically important timescales. A common approach to the dynamic interpretation of RDCs is to combine experimental restraint terms with a classical potential-energy force field to develop a conformational ensemble in agreement with experimental data. RDCs have also been exploited to characterize the conformational space sampled by the protein backbone either by fitting experimental data to determine angular excursions of internuclear bond vectors, or in comparison with different levels of accelerated molecular dynamics (AMD) to describe the most appropriate ensemble. Comparison of motions modeled using the Gaussian axial fluctuation (GAF) model, with ensembles derived from restraint-free AMD, demonstrated that such methods can provide a convergent description of protein motion.

The structure of the phosphorylated form of the histidine-containing phosphocarrier protein HPr from Escherichia coli has been solved by NMR and compared with that of unphosphorylated HPr. The structural changes that occur upon phosphorylation of His 15, monitored by changes in NOE patterns, 3JNHH,,-coupling constants, and chemical shifts, are limited to the region around the phosphorylation site. The His15 backbone torsion angles become strained upon phosphorylation. The release of this strain during the phosphoryl-transfer to Enzyme I1 facilitates the transport of carbohydrates across the membrane. From an X-ray study of Streptococcus faeculis HPr (Jia Z, Vandonselaar M, Quail JW, Delbaere LTJ, 1993, Nature 361:94-97), it was proposed that the observed torsion-angle strain at residue 16 in unphosphorylated S. fuecalis HPr has a role to play in the protein's phosphocarrier function. The model predicts that this strain is released upon phosphorylation. Our observations on E. coli HPr in solution, which shows strain only after phosphorylation, and the fact that all other HPrs studied thus far in their unphosphorylated forms show no strain either, led us to investigate the possibility that the crystal environment causes the strain in S. faecalis HPr. A 1-ns molecular dynamics simulation of S. faecalis HPr, under conditions that mimic the crystal environment, confirms the observations from the X-ray study, including the torsion-angle strain at residue 16. The strain disappeared, however, when S. faecalis HPr was simulated in a water environment, resulting in an active site configuration virtually the same as that observed in all other unphosphorylated HPrs. This indicates that the torsion-angle strain at Ala 16 in S. faecalis HPr is a result of crystal contacts or conditions and does not play a role in the phosphorylationdephosphorylation cycle.

The VAR2CSA protein has been closely associated with pregnancy-associated malaria and is recognized as the main adhesin exposed on the surface of Plasmodium falciparum-infected erythrocytes. Chondroitin sulfate A was identified as the main host receptor in the placenta. Single-domain heavy-chain camelid antibodies, more commonly called nanobodies, were selected and produced against the DBL6"-FCR3 domain of VAR2CSA. Crystals of two specific nanobodies, Nb2907 and Nb2919, identified as strong binders to DBL6e-FCR3 and the full-length VAR2CSA exposed on the surface of FCR3 P. falciparum-infected erythrocytes, were obtained. Crystals of Nb2907 diffract to 2.45 A resolution and belong to space group C2 with unit-cell parameters a = 136.1, b = 78.5, c = 103.4 A, beta = 118.8°, whereas Nb2919 crystals diffract to 2.15 A resolution and belong to space group P4(3)2(1)2 with unit-cell parameters a = b = 62.7, c = 167.2 A.

14 páginas, 6 figuras. En material suplementario: 3 figuras, 2 tablas, SH3 domains constitute a new type of ubiquitin-binding domains. We previously showed that the third SH3 domain (SH3-C) of CD2AP binds ubiquitin in an alternative orientation. We have determined the structure of the complex between first CD2AP SH3 domain and ubiquitin and performed a structural and mutational analysis to decipher the determinants of the SH3-C binding mode to ubiquitin. We found that the Phe-to-Tyr mutation in CD2AP and in the homologous CIN85 SH3-C domain does not abrogate ubiquitin binding, in contrast to previous hypothesis and our findings for the first two CD2AP SH3 domains. The similar alternative binding mode of the SH3-C domains of these related adaptor proteins is characterised by a higher affinity to C-terminal extended ubiquitin molecules. We conclude that CD2AP/CIN85 SH3-C domain interaction with ubiquitin constitutes a new ubiquitin-binding mode involved in a different cellular function and thus changes the previously established mechanism of EGF-dependent CD2AP/CIN85 mono-ubiquitination., This research was funded by grant BIO2005-04650 from the Spanish Ministry of Education and Science (MEC) and FQM-02838 from the Andalucia Regional Government. Work at J.B. laboratory was funded by the Spanish Ministry of Education and Innovation (SAF2009-10667). J.L.O.R. is supported by a Federation of European Biochemical Societies (FEBS) post-doctoral grant. S.C.A. is supported by a resettlement contract from the University of Granada. N.A.J.v.N is a group leader of the Vlaams Instituut voor Biotechnologie (VIB). The 600 MHz spectra were recorded in the Centre for Scientific Instrumentation (CIC) of the University of Granada and at the RALF Large Scale Facility in Grenoble, which is funded by the 'Access to Research Infrastructures' program of the European Union.

The solution structure of the phosphorylated form of the histidine-containing phosphocarrier protein, HPr, from Escherichia coli has been determined by NMR in combination with restrained molecular dynamics simulations. The structure of phospho-HPr (P-HPr) results from a molecular dynamics simulation in water, using time-dependent distance restraints to attain agreement with the measured NOEs. Experimental restraints were identified from both three-dimensional H-1-H-1-N-15 HSQC-NOESY and two-dimensional H-1-(1)HNOESY spectra, and compared with those of the unphosphorylated form. Structural changes upon phosphorylation of HPr are Limited to the active site, as evidenced by changes in chemical shifts, in (3)J(NHH alpha)-coupling constants and NOE patterns. Chemical shift changes were obtained mainly for protons that were positioned close to the phosphoryl group attached to the Hisl5 imidazole ring. Differences could be detected in the intensity of the NOEs involving the side-chain protons of Hisl5 and Pro18, resulting from a change in the relative position of the two rings. In addition, a small change could be detected in the three-bond T-coupling between the amide proton and the H-alpha proton of Thr16 and Arg17 upon phosphorylation, in agreement with the changes of the phi torsion angle of these two residues obtained from time-averaged restrained molecular dynamics simulations in water. The proposed role of the torsion-angle strain at residue 16 in the mechanism of Streptococcus faecalis HPr is not supported by these results. In contrast, phosphorylation seems to introduce torsion angle strain at residue Hisl5. This strain could facilitate the transfer of the phosphoryl group to the A-domain at enzyme II. The phospho-histidine is not stabilised by hydrogen bends to the side-chain group of Argl7; instead stable hydrogen bonds are formed between the phosphate group and the backbone amide.protons of ThrlG and Argl7, which show the largest changes in chemical shift up on phosphorylation, and a hydrogen bond involving the side-chain O-y proton of Thr16.HPr accepts the phosphoryl group from enzyme I and donates it subsequently to the A domain of various enzyme II species. The binding site for EI on HPr resembles that of the A domain of the mannitol-specific enzyme II, as canb e concludedfrom the changes onthe amideprotonandnitrogenchemical shifts observed via heteromolecular single-quantum coherence spectroscopy.

Toxin-antitoxin (TA) modules in bacteria are involved in pathogenesis, antibiotic stress response, persister formation and programmed cell death. The toxin Doc, from the phd/doc module, blocks protein synthesis by targeting the translation machinery. Despite a large wealth of biophysical and biochemical data on the regulatory aspects of the operon transcription and role of Doc co-activator and co-repressor, little is still know on the molecular basis of Doc toxicity. Structural information about this toxin is only available for its inhibited state bound to the antitoxin Phd. Here we report the (1)H, (15)N and (13)C backbone and side chain chemical shift assignments of the toxin Doc from of bacteriophage P1 (the model protein from this family of TA modules) in its free state. The BMRB accession number is 18899.

Chemokines and their receptors play fundamental roles in many physiological and pathological processes such as leukocyte trafficking, inflammation, cancer and HIV-1 infection. Chemokine-receptor interactions are particularly intricate and therefore require precise orchestration. The flexible N-terminal domain of human chemokine receptors has regularly been demonstrated to hold a crucial role in the initial recognition and selective binding of the receptor ligands. The length and the amino acid sequences of the N-termini vary considerably among different receptors but they all show a high content of negatively charged residues and are subject to post-translational modifications such as O-sulfation and N- or O-glycosylation. In addition, a conserved cysteine that is most likely engaged in a receptor-stabilizing disulfide bond delimits two functionally distinct parts in the N-terminus, characterized by specific molecular signatures. Structural analyses have shown that the N-terminus of chemokine receptors recognizes a groove on the chemokine surface and that this interaction is stabilized by high-affinity binding to a conserved sulfotyrosine-binding pocket. Altogether, these data provide new insights on the chemokine-receptor molecular interplay and identify the receptor N-terminus-binding site as a new target for the development of therapeutic molecules. This review presents and discusses the diversity and function of human chemokine receptor N-terminal domains and provides a comprehensive annotated inventory of their sequences, laying special emphasis on the presence of post-translational modifications and functional features. Finally, it identifies new molecular signatures and proposes a computational model for the positioning and the conformation of the CXCR4 N-terminus grafted on the first chemokine receptor X-ray structure.

An efficient way to rapidly generate protein-ligand costructures based on solution-NMR using sparse NOE data combined with selective isotope labeling is presented. A docked model of the 27 kDa N-terminal ATPase domain of Hsp90 bound to a small molecule ligand was generated using only 21 intermolecular NOEs, which uniquely defined both the binding site and the orientation of the ligand. The approach can prove valuable for the early stages of fragment-based drug discovery.

To survive hostile conditions, the bacterial pathogen Mycobacterium tuberculosis produces millimolar concentrations of mycothiol as a redox buffer against oxidative stress. The reductases that couple the reducing power of mycothiol to redox active proteins in the cell are not known. We report a novel mycothiol-dependent reductase (mycoredoxin-1) with a CGYC catalytic motif. With mycoredoxin-1 and mycothiol deletion strains of Mycobacterium smegmatis, we show that mycoredoxin-1 and mycothiol are involved in the protection against oxidative stress. Mycoredoxin-1 acts as an oxidoreductase exclusively linked to the mycothiol electron transfer pathway and it can reduce S-mycothiolated mixed disulphides. Moreover, we solved the solution structures of oxidized and reduced mycoredoxin-1, revealing a thioredoxin fold with a putative mycothiol-binding site. With HSQC snapshots during electron transport, we visualize the reduction of oxidized mycoredoxin-1 as a function of time and find that mycoredoxin-1 gets S-mycothiolated on its N-terminal nucleophilic cysteine. Mycoredoxin-1 has a redox potential of -218 mV and hydrogen bonding with neighbouring residues lowers the pKa of its N-terminal nucleophilic cysteine. Determination of the oxidized and reduced structures of mycoredoxin-1, better understanding of mycothiol-dependent reactions in general, will likely give new insights in how M. tuberculosis survives oxidative stress in human macrophages.

A complete understanding of the relationship between biological activity and molecular conformation requires an understanding of the thermally accessible potential energy landscape. An extensive set of experimental NMR residual dipolar couplings (RDCs) has been used to determine the conformational behavior of CD2AP SH3C on multiple timescales, using the Gaussian Axial Fluctuation model, and comparison to restraint-free accelerated molecular dynamics simulation. These robust analyses provide a comprehensive description of conformational fluctuations on picosecond to millisecond timescales. While the β-sheets show negligible slow motions, larger amplitude slow dynamics are found in the n-SRC and RT loops that mediate physiological interactions.