Search

Background: Amyloids are ordered, insoluble protein aggregates, characterized by a cross-β sheet quaternary structure in which molecules in a β-strand conformation are stacked along the filament axis via intermolecular interactions. While amyloids are typically associated with pathological conditions, functional amyloids have also been identified and are present in a wide variety of organisms ranging from bacteria to humans. The cytoplasmic polyadenylation element-binding (CPEB) prion-like protein is an mRNA-binding translation regulator, whose neuronal isoforms undergo activity-dependent aggregation, a process that has emerged as a plausible biochemical substrate for memory maintenance. CPEB aggregation is driven by prion-like domains (PLD) that are divergent in sequence across species, and it remains unknown whether such divergent PLDs follow a similar aggregating assembly pathway. Here, we describe the amyloid-like features of the neuronal Aplysia CPEB (ApCPEB) PLD and compare them to those of the Drosophila ortholog, Orb2 PLD., Results: Using in vitro single-molecule and bulk biophysical methods, we find transient oligomers and mature amyloid-like filaments that suggest similarities in the late stages of the assembly pathway for both ApCPEB and Orb2 PLDs. However, while prior to aggregation the Orb2 PLD monomer remains mainly as a random coil in solution, ApCPEB PLD adopts a diversity of conformations comprising α-helical structures that evolve to coiled-coil species, indicating structural differences at the beginning of their amyloid assembly pathways., Conclusion: Our results indicate that divergent PLDs of CPEB proteins from different species retain the ability to form a generic amyloid-like fold through different assembly mechanisms.

Microbial pathogens, such as Trypanosoma brucei, have an enormous impact on global health and economic systems. Protein kinase A of T. brucei is an attractive drug target as it is an essential enzyme which differs significantly from its human homolog. The hinge region of this protein's regulatory domain is vital for enzymatic function, but its conformation is unknown. Here, the secondary structure of this region has been characterized using NMR and CD spectroscopies. More specifically, three overlapping peptides corresponding to residues T187-I211, G198-Y223 and V220-S245 called peptide 1, peptide 2 and peptide 3, respectively, were studied. The peptide 1 and peptide 2 are chiefly unfolded; only low populations (<10%) of α-helix were detected under the conditions studied. In contrast, the peptide 3 contains a long α-helix whose population is significantly higher; namely, 36% under the conditions studied. Utilizing the dihedral φ and ψ angles calculated on the basis of the NMR data, the conformation of the peptide 3 was calculated and revealed an α-helix spanning residues E230-N241. This α-helix showed amphiphilicity and reversible unfolding and refolding upon heating and cooling. Most fascinating, however, is its capacity to inhibit the activity of the catalytic domain of Trypanosoma equiperdum protein kinase A even though it is quite distinct from the canonical inhibitor motif. Based on this property, we advance that peptoids based on the peptide 3 α-helix could be novel leads for developing anti-trypanosomal therapeutics., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Activation of the cannabinoid CB1 receptor induces different cellular signaling cascades through coupling to different effector proteins (G-proteins and β-arrestins), triggering numerous therapeutic effects. Conformational changes and rearrangements at the intracellular domain of this GPCR receptor that accompany ligand binding dictate the signaling pathways. The GPCR-binding interface for G proteins has been extensively studied, whereas β-arrestin/GPCR complexes are still poorly understood. To gain knowledge in this direction, we designed peptides that mimic the motifs involved in the putative interacting region: β-arrestin1 finger loop and the transmembrane helix 7-helix 8 (TMH7-H8) elbow located at the intracellular side of the CB1 receptor. According to circular dichroism and NMR data, these peptides form a native-like, helical conformation and interact with each other in aqueous solution, in the presence of trifluoroethanol, and using zwitterionic detergent micelles as membrane mimics. These results increase our understanding of the binding mode of β-arrestin and CB1 receptor and validate minimalist approaches to structurally comprehend complex protein systems.

Fusarium oxysporum is a highly destructive plant pathogen and an emerging pathogen of humans. Like other ascomycete fungi, F. oxysporum secretes α-pheromone, a small peptide that functions both as a chemoattractant and as a quorum-sensing signal. Three of the ten amino acid residues of α-pheromone are tryptophan, an amino acid whose sidechain has high affinity for lipid bilayers, suggesting a possible interaction with biological membranes. Here we tested the effect of different lipid environments on α-pheromone structure and function. Using spectroscopic and calorimetric approaches, we show that this peptide interacts with negatively charged model phospholipid vesicles. Fluorescence emission spectroscopy and nuclear magnetic resonance (NMR) measurements revealed a key role of the positively charged groups and Trp residues. Furthermore, NMR-based calculation of the 3D structure in the presence of micelles, formed by lipid surfactants, suggests that α-pheromone can establish an intramolecular disulfide bond between the two cysteine residues during interaction with membranes, but not in the absence of lipid mimetics. Remarkably, this oxidized version of α-pheromone lacks biological activity as a chemoattractant and quorum-sensing molecule. These results suggest the presence of a previously unidentified redox regulated control of α-pheromone activity at the surface of the plasma membrane that could influence the interaction with its cognate G-protein coupled receptor., (Copyright © 2020 Partida-Hanon, Maestro-López, Vitale, Turrà, Di Pietro, Martínez-del-Pozo and Bruix.)

Microbial electrosynthesis is an emerging green technology that explores the capability of a particular group of microorganisms to drive their metabolism toward the production of hydrogen or value-added chemicals from electrons supplied by electrode surfaces. The cytochrome PccH showed the largest increase in transcription when electrons are supplied to Geobacter sulfurreducens biofilms. Gene knock-out experiments have shown that the electron transfer toward G. sulfurreducens cells was completely inhibited by the deletion of the gene encoding for cytochrome PccH. This identifies a crucial role for this protein in G. sulfurreducens microbial electrosynthesis mechanisms, which are currently unknown. In this work, we present the backbone ( 1 H, 13 C and 15 N) and heme assignment for PccH in the oxidized state. The data obtained paves the way to identify and structurally map the molecular interaction regions between the cytochrome PccH and its physiological redox partners.

Endolysins, the cell wall lytic enzymes encoded by bacteriophages to release the phage progeny, are among the top alternatives to fight against multiresistant pathogenic bacteria; one of the current biggest challenges to global health. Their narrow range of susceptible bacteria relies, primarily, on targeting specific cell-wall receptors through specialized modules. The cell wall-binding domain of Cpl-7 endolysin, made of three CW&#95;7 repeats, accounts for its extended-range of substrates. Using as model system the cell wall-binding domain of Cpl-7, here we describe the molecular basis for the bacterial cell wall recognition by the CW&#95;7 motif, which is widely represented in sequences of cell wall hydrolases. We report the crystal and solution structure of the full-length domain, identify N-acetyl-D-glucosaminyl-(β1,4)-N-acetylmuramyl-L-alanyl-D-isoglutamine (GMDP) as the peptidoglycan (PG) target recognized by the CW&#95;7 motifs, and characterize feasible GMDP-CW&#95;7 contacts. Our data suggest that Cpl-7 cell wall-binding domain might simultaneously bind to three PG chains, and also highlight the potential use of CW&#95;7-containing lysins as novel anti-infectives.

TACC3 is a centrosomal adaptor protein that plays important roles during mitotic spindle assembly. It interacts with chTOG/XMAP215, which catalyzes the addition of tubulin dimers during microtubule growth. A 3D coiled-coil model for this interaction is available but the structural details are not well described. To characterize this interaction at atomic resolution, we have designed a simplified version of the system based on small peptides. Four different peptides have been studied by circular dichroism and nuclear magnetic resonance both singly and in all possible combinations; namely, five peptide pairs and two trios. In cosolvents, all single peptides tend to adopt helical conformations resembling those of the full-length protein. However, neither the single peptides nor pairs of peptides form coiled coils. We show that the simultaneous presence of all preformed helices is a prerequisite for binding. The simplest 3D model for the interaction, based on the NMR results, is proposed. Interestingly, the peptide's structure remains unaffected by mutations at essential positions for TACC3 activity. This suggests that the lack of interaction of this TACC3 mutant with XMAP does not correlate with changes in the protein structure and that specific interactions are likely responsible for the interaction and stability of the complex., (© 2017 Wiley Periodicals, Inc.)

Gene knock-out studies on Geobacter sulfurreducens cells showed that the outer membrane-associated monoheme cytochrome OmcF is involved in respiratory pathways leading to the extracellular reduction of Fe(III) and U(VI). In addition, microarray analysis of an OmcF-deficient mutant revealed that many of the genes with decreased transcript level were those whose expression is up-regulated in cells grown with a graphite electrode as electron acceptor, suggesting that OmcF also regulates the electron transfer to electrode surfaces and the concomitant electricity production by G. sulfurreducens in microbial fuel cells. 15 N, 13 C-labeled OmcF was produced and NMR spectroscopy was used to determine the solution structure of the protein in the fully reduced state and the pH-dependent conformational changes. In addition, 15 N relaxation NMR experiments were used to characterize the overall and internal backbone dynamics of OmcF. The structure obtained is well-defined, with an average pairwise root mean square deviation of 0.37Å for the backbone atoms and 0.98Å for all heavy atoms. For the first time a solution structure and the protein motions were determined for an outer membrane cytochrome from G. sulfurreducens, which constitutes an important step to understand the extracellular electron transfer mechanism in Geobacter cells., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Protein engineering studies often suggest the emergence of completely new enzyme functionalities to be highly improbable. However, enzymes likely catalysed many different reactions already in the last universal common ancestor. Mechanisms for the emergence of completely new active sites must therefore either plausibly exist or at least have existed at the primordial protein stage. Here, we use resurrected Precambrian proteins as scaffolds for protein engineering and demonstrate that a new active site can be generated through a single hydrophobic-to-ionizable amino acid replacement that generates a partially buried group with perturbed physico-chemical properties. We provide experimental and computational evidence that conformational flexibility can assist the emergence and subsequent evolution of new active sites by improving substrate and transition-state binding, through the sampling of many potentially productive conformations. Our results suggest a mechanism for the emergence of primordial enzymes and highlight the potential of ancestral reconstruction as a tool for protein engineering.

Apoptin is a nonstructural protein encoded by one of the three open reading frames of the chicken anemia virus genome. It has attracted a great deal of interest due to its ability to induce apoptosis in multiple transformed and malignant mammalian cell lines without affecting primary and non-transformed cells. However, the use of Apoptin as an anticancer drug is restricted by its strong tendency to aggregate. A number of methods to overcome this problem have been proposed, including transduction techniques to deliver the Apoptin gene into tumor cells, but all such methods have certain drawbacks. Here we describe that a truncated variant of Apoptin, lacking residues 1 to 43, is a soluble, non-aggregating protein that maintains most of the biological properties of wild-type Apoptin when transfected into cells. We show that the cytotoxic effect of this variant is also present when it is added exogenously to cancer cells, but not to normal cells. In addition to the interest this protein has attracted as a promising therapeutic strategy, it is also an excellent model to study the structural properties of Apoptin and how they relate to its mechanism of action.

Despite the growing number of carbohydrate-binding modules (CBMs) that are being uncovered, information on the structural determinants for the sugar-binding regions at atomic resolution is scarce. It is widely accepted that aromatic and H-bonding interactions govern these processes, and reported simulations and theoretical calculations are valuable tools to quantify and understand these interactions. We present here a computational model derived from experimental data that provide a unique atomistic picture of an uncharacterized binding mode of laminarin to the CBM family 43. The present study, which is among the first describing an isolated CBM with the bound carbohydrate, is complemented with quantum mechanical calculations. This allows us to attribute certain experimental observations (binding affinities) to key interactions (H-bonds and aromatic stacking), on the basis of NMR-driven docking structure., (Copyright © 2017 Elsevier Inc. All rights reserved.)

During sexual development ascomycete fungi produce two types of peptide pheromones termed a and α. The α pheromone from the budding yeast Saccharomyces cerevisiae , a 13-residue peptide that elicits cell cycle arrest and chemotropic growth, has served as paradigm for the interaction of small peptides with their cognate G protein-coupled receptors. However, no structural information is currently available for α pheromones from filamentous ascomycetes, which are significantly shorter and share almost no sequence similarity with the S. cerevisiae homolog. High resolution structure of synthetic α-pheromone from the plant pathogenic ascomycete Fusarium oxysporum revealed the presence of a central β-turn resembling that of its yeast counterpart. Disruption of the-fold by d-alanine substitution of the conserved central Gly 6 -Gln 7 residues or by random sequence scrambling demonstrated a crucial role for this structural determinant in chemoattractant activity. Unexpectedly, the growth inhibitory effect of F. oxysporum α-pheromone was independent of the cognate G protein-coupled receptors Ste2 and of the central β-turn but instead required two conserved Trp 1 -Cys 2 residues at the N terminus. These results indicate that, despite their reduced size, fungal α-pheromones contain discrete functional regions with a defined secondary structure that regulate diverse biological processes such as polarity reorientation and cell division., (© 2017 by The American Society for Biochemistry and Molecular Biology, Inc.)

The periplasmic triheme cytochrome PpcA from Geobacter sulfurreducens is highly abundant; it is the likely reservoir of electrons to the outer surface to assist the reduction of extracellular terminal acceptors; these include insoluble metal oxides in natural habitats and electrode surfaces from which electricity can be harvested. A detailed thermodynamic characterization of PpcA showed that it has an important redox-Bohr effect that might implicate the protein in e - /H + coupling mechanisms to sustain cellular growth. This functional mechanism requires control of both the redox state and the protonation state. In the present study, isotope-labeled PpcA was produced and the three-dimensional structure of PpcA in the oxidized form was determined by NMR. This is the first solution structure of a G. sulfurreducens cytochrome in the oxidized state. The comparison of oxidized and reduced structures revealed that the heme I axial ligand geometry changed and there were other significant changes in the segments near heme I. The pH-linked conformational rearrangements observed in the vicinity of the redox-Bohr center, both in the oxidized and reduced structures, constitute the structural basis for the differences observed in the pK a values of the redox-Bohr center, providing insights into the e - /H + coupling molecular mechanisms driven by PpcA in G. sulfurreducens., (© 2017 The Author(s); published by Portland Press Limited on behalf of the Biochemical Society.)

Apoptin is a 121 residue protein which forms large, soluble aggregates and possesses an exceptionally selectively cytotoxic action on cancer cells. In the accompanying paper, we described the design, production and initial characterization of an Apoptin truncated variant called H 6 -ApopΔProΔLeu. Whereas both the variant and wild type protein possess similar selective cytotoxicity against cancer cells following transfection, only the variant is cytotoxic when added externally. Remarkably, as observed by gel filtration chromatography and dynamic light scattering, H 6 -ApopΔProΔLeu lacks the tendency of wild type Apoptin to form large aggregates, which greatly facilitated the study of its biological properties. Here, we characterize the conformation and dynamics of H 6 -ApopΔProΔLeu. Using a battery of 2D, 3D and (4,2)D NMR spectra, the essentially complete 1 H, 13 C and 15 N resonance assignments of H 6 -ApopΔProΔLeu were obtained. The analysis of these data shows that the variant is an intrinsically disordered protein, which lacks a preferred conformation. This conclusion is corroborated by a lack of protection against proteolytic cleavage and hydrogen/deuterium exchange. Moreover, the CD spectra are dominated by random coil contributions. Finally, 1 H- 15 N NOE ratios are low, which indicates flexibility on the ps-ns time scale. Interestingly, H 6 -ApopΔProΔLeu's intrinsically disordered ensemble is not significantly altered by the redox state of its Cys residues or by Thr phosphorylation, which has been proposed to play a key role in Apoptin's selective cytotoxicity. These results serve to better comprehend Apoptin's remarkably selective anticancer action and provide a framework for the future design of improved Apoptin variants., (Copyright © 2016 Elsevier Inc. All rights reserved.)

The human pathogen Streptococcus pneumoniae is decorated with a special class of surface-proteins known as choline-binding proteins (CBPs) attached to phosphorylcholine (PCho) moieties from cell-wall teichoic acids. By a combination of X-ray crystallography, NMR, molecular dynamics techniques and in vivo virulence and phagocytosis studies, we provide structural information of choline-binding protein L (CbpL) and demonstrate its impact on pneumococcal pathogenesis and immune evasion. CbpL is a very elongated three-module protein composed of (i) an Excalibur Ca 2+ -binding domain -reported in this work for the very first time-, (ii) an unprecedented anchorage module showing alternate disposition of canonical and non-canonical choline-binding sites that allows vine-like binding of fully-PCho-substituted teichoic acids (with two choline moieties per unit), and (iii) a Ltp&#95;Lipoprotein domain. Our structural and infection assays indicate an important role of the whole multimodular protein allowing both to locate CbpL at specific places on the cell wall and to interact with host components in order to facilitate pneumococcal lung infection and transmigration from nasopharynx to the lungs and blood. CbpL implication in both resistance against killing by phagocytes and pneumococcal pathogenesis further postulate this surface-protein as relevant among the pathogenic arsenal of the pneumococcus.

The mechanism by which the HIV-1 MPER epitope is recognized by the potent neutralizing antibody 10E8 at membrane interfaces remains poorly understood. To solve this problem, we have optimized a 10E8 peptide epitope and analyzed the structure and binding activities of the antibody in membrane and membrane-like environments. The X-ray crystal structure of the Fab-peptide complex in detergents revealed for the first time that the epitope of 10E8 comprises a continuous helix spanning the gp41 MPER/transmembrane domain junction (MPER-N-TMD; Env residues 671-687). The MPER-N-TMD helix projects beyond the tip of the heavy-chain complementarity determining region 3 loop, indicating that the antibody sits parallel to the plane of the membrane in binding the native epitope. Biophysical, biochemical and mutational analyses demonstrated that strengthening the affinity of 10E8 for the TMD helix in a membrane environment, correlated with its neutralizing potency. Our research clarifies the molecular mechanisms underlying broad neutralization of HIV-1 by 10E8, and the structure of its natural epitope. The conclusions of our research will guide future vaccine-design strategies targeting MPER.

Acute infection by Gram-negative pathogens can induce an exacerbated immune response that leads to lethal septic shock syndrome. Bacterial lipopolysaccharide (LPS) is a major pathogen-associated molecular pattern molecule that can initiate massive and lethal immune system stimulation. Therefore, the development of new and effective LPS-neutralizing agents is a top priority. The eosinophil cationic protein (ECP) is an antimicrobial protein secreted in response to infection, with a remarkable affinity for LPS. In the present study, we demonstrate that ECP is able to neutralize bacterial LPS and inhibit tumor necrosis factor-α production in human macrophages. We also characterized ECP neutralizing activity using progressively truncated LPS mutants, and conclude that the polysaccharide moiety and lipid A portions are required for LPS-mediated neutralization. In addition, we mapped the structural determinants required for the ECP-LPS interaction by nuclear magnetic resonance. Our results show that ECP is able to neutralize LPS and therefore opens a new route for developing novel therapeutic agents based on the ECP structural scaffolding., (© 2016 Federation of European Biochemical Societies.)

A family of triheme cytochromes from Geobacter sulfurreducens plays an important role in extracellular electron transfer. In addition to their role in electron transfer pathways, two members of this family (PpcA and PpcD) were also found to be able to couple e - /H + transfer through the redox Bohr effect observed in the physiological pH range, a feature not observed for cytochromes PpcB and PpcE. In attempting to understand the molecular control of the redox Bohr effect in this family of cytochromes, which is highly homologous both in amino acid sequence and structures, it was observed that residue 6 is a conserved leucine in PpcA and PpcD, whereas in the other two characterized members (PpcB and PpcE) the equivalent residue is a phenylalanine. To determine the role of this residue located close to the redox Bohr center, we replaced Leu 6 in PpcA with Phe and determined the redox properties of the mutant, as well as its solution structure in the fully reduced state. In contrast with the native form, the mutant PpcAL6F is not able to couple the e - /H + pathway. We carried out the reverse mutation in PpcB and PpcE (i.e., replacing Phe 6 in these two proteins by leucine) and the mutated proteins showed an increased redox Bohr effect. The results clearly establish the role of residue 6 in the control of the redox Bohr effect in this family of cytochromes, a feature that could enable the rational design of G. sulfurreducens strains that carry mutant cytochromes with an optimal redox Bohr effect that would be suitable for various biotechnological applications.

It has been suggested that DYNLT1, a dynein light chain known to bind to various cellular and viral proteins, can function both as a molecular clamp and as a microtubule-cargo adapter. Recent data have shown that the DYNLT1 homodimer binds to two dynein intermediate chains to subsequently link cargo proteins such as the guanine nucleotide exchange factor Lfc or the small GTPases RagA and Rab3D. Although over 20 DYNLT1-interacting proteins have been reported, the exact sequence requirements that enable their association to the canonical binding groove or to the secondary site within the DYNLT1 surface are unknown. We describe herein the sequence recognition properties of the hydrophobic groove of DYNLT1 known to accommodate dynein intermediate chain. Using a pepscan approach, we have substituted each amino acid within the interacting peptide for all 20 natural amino acids and identified novel binding sequences. Our data led us to propose activin receptor IIB as a novel DYNLT1 ligand and suggest that DYNLT1 functions as a molecular dimerization engine bringing together two receptor monomers in the cytoplasmic side of the membrane. In addition, we provide evidence regarding a dual binding mode adopted by certain interacting partners such as Lfc or the parathyroid hormone receptor. Finally, we have used NMR spectroscopy to obtain the solution structure of human DYNLT1 forming a complex with dynein intermediate chain of ∼74 kDa; it is the first mammalian structure available., (© 2016 by The American Society for Biochemistry and Molecular Biology, Inc.)

The short membrane-active peptide BP100 [KKLFKKILKYL-NH2] is known as an effective antimicrobial and cell penetrating agent. For a functional alanine scan each of the 11 amino acids was replaced with deuterated Ala-d3, one at a time. MIC assays showed that a substitution of Lys did not affect the antimicrobial activity, but it decreased when a hydrophobic residue was replaced. In most cases, a reduction in hydrophobicity led to a decrease in hemolysis, and some peptide analogues had an improved therapeutic index. Circular dichroism showed that BP100 folds as an amphiphilic α-helix in a bilayer. Its alignment was determined from (2)H NMR in oriented membranes of different composition. The azimuthal rotation angle was the same under all conditions, but the average helix tilt angle and the dynamical behavior of the peptide varied in a systematic manner. In POPC/POPG bilayers, with a negative spontaneous curvature, the peptide was found to lie flat on the bilayer surface, and with little wobble. In DMPC/DMPG, with a positive spontaneous curvature, BP100 at higher concentrations became tilted obliquely into the membrane, with the uncharged C-terminus inserted more deeply into the lipid bilayer, experiencing significant fluctuations in tilt angle. In DMPC/DMPG/lyso-MPC, with a pronounced positive spontaneous curvature, the helix tilted even further and became even more mobile. The 11-mer BP100 is obviously too short to form transmembrane pores. We conclude that BP100 operates via a carpet mechanism, whereby the C-terminus gets inserted into the hydrophobic core of the bilayer, which leads to membrane perturbation and induces transient permeability., (Copyright © 2016 Elsevier B.V. All rights reserved.)

The titin I27 module from human cardiac titin has become a standard in protein nanomechanics. A proline-scanning study of its mechanical clamp found three mechanically hypomorphic mutants and a paradoxically hypermorphic mutant (I27Y9P). Both types of mutants have been commonly used as substrates of several protein unfoldase machineries in studies relating protein mechanostability to translocation or degradation rates. Using single-molecule force spectroscopy based on atomic force microscopy, polyprotein engineering, and steered molecular dynamics simulations, we show that, unexpectedly, the mechanostability of the Y9P variant is comparable to the wild type. Furthermore, the NMR analysis of homomeric polyproteins of this variant suggests that these constructs may induce slight structural perturbations in the monomer, which may explain some minor differences in this variant's properties; namely the abolishment of the mechanical unfolding intermediate and a reduced thermal stability. Our results clarify a previously reported paradoxical result in protein nanomechanics and contribute to refining our toolbox for understanding the unfolding mechanism used by translocases and degradation machines., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Coordination polymers and metal-organic frameworks are appealing as synthetic hosts for mediating chemical reactions. Here we report the preparation of a mesoscopic metal-organic structure based on single-layer assembly of aluminium chains and organic alkylaryl spacers. The material markedly accelerates condensation reactions in water in the absence of acid or base catalyst, as well as organocatalytic Michael-type reactions that also show superior enantioselectivity when comparing with the host-free transformation. The mesoscopic phase of the solid allows for easy diffusion of products and the catalytic solid is recycled and reused. Saturation transfer difference and two-dimensional (1)H nuclear Overhauser effect NOESY NMR spectroscopy show that non-covalent interactions are operative in these host-guest systems that account for substrate activation. The mesoscopic character of the host, its hydrophobicity and chemical stability in water, launch this material as a highly attractive supramolecular catalyst to facilitate (asymmetric) transformations under more environmentally friendly conditions.

Amyloids are ordered protein aggregates that are typically associated with neurodegenerative diseases and cognitive impairment. By contrast, the amyloid-like state of the neuronal RNA binding protein Orb2 in Drosophila was recently implicated in memory consolidation, but it remains unclear what features of this functional amyloid-like protein give rise to such diametrically opposed behaviour. Here, using an array of biophysical, cell biological and behavioural assays we have characterized the structural features of Orb2 from the monomer to the amyloid state. Surprisingly, we find that Orb2 shares many structural traits with pathological amyloids, including the intermediate toxic oligomeric species, which can be sequestered in vivo in hetero-oligomers by pathological amyloids. However, unlike pathological amyloids, Orb2 rapidly forms amyloids and its toxic intermediates are extremely transient, indicating that kinetic parameters differentiate this functional amyloid from pathological amyloids. We also observed that a well-known anti-amyloidogenic peptide interferes with long-term memory in Drosophila. These results provide structural insights into how the amyloid-like state of the Orb2 protein can stabilize memory and be nontoxic. They also provide insight into how amyloid-based diseases may affect memory processes.

It has been suggested that DYNLT, a dynein light chain known to bind to various cellular and viral proteins, can function as a microtubule-cargo adaptor. Recent data showed that DYNLT links the small GTPase Rab3D to microtubules and, for this to occur, the DYNLT homodimer needs to display a binding site for dynein intermediate chain together with a binding site for the small GTPase. We have analysed in detail how RagA, another small GTPase, associates to DYNLT. After narrowing down the binding site of RagA to DYNLT we could identify that a β strand, part of the RagA G3 box involved in nucleotide binding, mediates this association. Interestingly, we show that both microtubule-associated DYNLT and cytoplasmic DYNLT are equally able to bind to the small GTPases Rab3D and RagA. Using NMR spectroscopy, we analysed the binding of dynein intermediate chain and RagA to mammalian DYNLT. Our experiments identify residues of DYNLT affected by dynein intermediate chain binding and residues affected by RagA binding, hence distinguishing the docking site for each of them. In summary, our results shed light on the mechanisms adopted by DYNLT when binding to protein cargoes that become transported alongside microtubules bound to the dynein motor., (© 2015 FEBS.)

Gene knockout studies on Geobacter sulfurreducens (Gs) cells showed that the outer membrane cytochrome OmcF is involved in respiratory pathways leading to the extracellular reduction of Fe(III) citrate and U(VI) oxide. In addition, microarray analysis of OmcF-deficient mutant versus the wild-type strain revealed that many of the genes with decreased transcript level were those whose expression is upregulated in cells grown with a graphite electrode as electron acceptor. This suggests that OmcF also regulates the electron transfer to electrode surfaces and the concomitant electrical current production by Gs in microbial fuel cells. Extracellular electron transfer processes (EET) constitute nowadays the foundations to develop biotechnological applications in biofuel production, bioremediation and bioenergy. Therefore, the structural characterization of OmcF is a fundamental step to understand the mechanisms underlying EET. Here, we report the complete assignment of the heme proton signals together with (1)H, (13)C and (15)N backbone and side chain assignments of the OmcF, excluding the hydrophobic residues of the N-terminal predicted lipid anchor.

Ole e 9 and Fra e 9 are two allergenic β-1,3-glucanases from olive and ash tree pollens, respectively. Both proteins present a modular structure with a catalytic N-terminal domain and a carbohydrate-binding module (CBM) at the C-terminus. Despite their significant sequence resemblance, they differ in some functional properties, such as their catalytic activity and the carbohydrate-binding ability. Here, we have studied the different capability of the recombinant C-terminal domain of both allergens to bind laminarin by NMR titrations, binding assays and ultracentrifugation. We show that rCtD-Ole e 9 has a higher affinity for laminarin than rCtD-Fra e 9. The complexes have different exchange regimes on the NMR time scale in agreement with the different affinity for laminarin observed in the biochemical experiments. Utilising NMR chemical shift perturbation data, we show that only one side of the protein surface is affected by the interaction and that the binding site is located in the inter-helical region between α1 and α2, which is buttressed by aromatic side chains. The binding surface is larger in rCtD-Ole e 9 which may account for its higher affinity for laminarin relative to rCtD-Fra e 9., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Multiheme cytochromes have been implicated in Geobacter sulfurreducens extracellular electron transfer (EET). These proteins are potential targets to improve EET and enhance bioremediation and electrical current production by G. sulfurreducens. However, the functional characterization of multiheme cytochromes is particularly complex due to the co-existence of several microstates in solution, connecting the fully reduced and fully oxidized states. Over the last decade, new strategies have been developed to characterize multiheme redox proteins functionally and structurally. These strategies were used to reveal the functional mechanism of G. sulfurreducens multiheme cytochromes and also to identify key residues in these proteins for EET. In previous studies, we set the foundations for enhancement of the EET abilities of G. sulfurreducens by characterizing a family of five triheme cytochromes (PpcA-E). These periplasmic cytochromes are implicated in electron transfer between the oxidative reactions of metabolism in the cytoplasm and the reduction of extracellular terminal electron acceptors at the cell's outer surface. The results obtained suggested that PpcA can couple e(-)/H(+) transfer, a property that might contribute to the proton electrochemical gradient across the cytoplasmic membrane for metabolic energy production. The structural and functional properties of PpcA were characterized in detail and used for rational design of a family of 23 single site PpcA mutants. In this review, we summarize the functional characterization of the native and mutant proteins. Mutants that retain the mechanistic features of PpcA and adopt preferential e(-)/H(+) transfer pathways at lower reduction potential values compared to the wild-type protein were selected for in vivo studies as the best candidates to increase the electron transfer rate of G. sulfurreducens. For the first time G. sulfurreducens strains have been manipulated by the introduction of mutant forms of essential proteins with the aim to develop and improve bioelectrochemical technologies.

Cyclic N6-threonylcarbamoyladenosine ('cyclic t6A', ct(6)A) is a non-thiolated hypermodification found in transfer RNAs (tRNAs) in bacteria, protists, fungi and plants. In bacteria and yeast cells ct(6)A has been shown to enhance translation fidelity and efficiency of ANN codons by improving the faithful discrimination of aminoacylated tRNAs by the ribosome. To further the understanding of ct(6)A biology we have determined the high-resolution crystal structures of CsdL/TcdA in complex with AMP and ATP, an E1-like activating enzyme from Escherichia coli, which catalyzes the ATP-dependent dehydration of t6A to form ct(6)A. CsdL/TcdA is a dimer whose structural integrity and dimer interface depend critically on strongly bound K+ and Na+ cations. By using biochemical assays and small-angle X-ray scattering we show that CsdL/TcdA can associate with tRNA with a 1:1 stoichiometry and with the proper position and orientation for the cyclization of t6A. Furthermore, we show by nuclear magnetic resonance that CsdL/TcdA engages in transient interactions with CsdA and CsdE, which, in the latter case, involve catalytically important residues. These short-lived interactions may underpin the precise channeling of sulfur atoms from cysteine to CsdL/TcdA as previously characterized. In summary, the combination of structural, biophysical and biochemical methods applied to CsdL/TcdA has afforded a more thorough understanding of how the structure of this E1-like enzyme has been fine tuned to accomplish ct(6)A synthesis on tRNAs while providing support for the notion that CsdA and CsdE are able to functionally interact with CsdL/TcdA.

Gene knock-out studies on Geobacter sulfurreducens (Gs) cells showed that the periplasmic triheme cytochrome PpcD is involved in respiratory pathways leading to the extracellular reduction of Fe(III) and U(VI) oxides. More recently, it was also shown that the gene encoding for PpcD has higher transcript abundance when Gs cells utilize graphite electrodes as sole electron donors to reduce fumarate. This sets PpcD as the first multiheme cytochrome to be involved in Gs respiratory pathways that bridge the electron transfer between the cytoplasm and cell exterior in both directions. Nowadays, extracellular electron transfer (EET) processes are explored for several biotechnological applications, which include bioremediation, bioenergy and biofuel production. Therefore, the structural characterization of PpcD is a fundamental step to understand the mechanisms underlying EET. However, compared to non-heme proteins, the presence of numerous proton-containing groups in the redox centers presents additional challenges for protein signal assignment and structure calculation. Here, we report the complete assignment of the heme proton signals together with (1)H, (13)C and (15)N backbone and side chain assignments of the reduced form of PpcD.

Human centrosomal proteins show a significant, 3.5 fold, bias to be both unstructured and coiled-coils with respect to generic human proteins, based on results from state of the art bioinformatics tools. We hypothesize that this bias means that these proteins adopt an ensemble of disordered and partially helical conformations, with the latter becoming stabilized when these proteins form complexes. Characterization of the structural properties of 13 peptides from 10 different centrosomal proteins ranging in size from 20 to 61 residues by biophysical methods led us to confirm our hypothesis in most cases. Interestingly, the secondary structure adopted by most of these peptides becomes stabilized at acidic pH and it is concentration dependent. For two of them, PIK3R1(453-513) and BRCA1(1253-1273), we observed not only the stabilization of helical structure through self-association, but also the presence of β-structures linked to the formation of high molecular weight oligomers. These oligomers are the predominant forms detected by CD, but unobservable by liquid state NMR. BRCA1(1397-1424) and MAP3K11(396-441) populate helical structures that can also self-associate at pH3 through oligomeric species. Four peptides, derived from three proteins, namely CCNA2(103-123), BRCA1(1253-1273), BRCA1(1397-1424) and PIK3R1(453-513), can form intermolecular associations that are concomitant with alpha or beta structure stabilization. The self-phosphorylation previously described for the kinase NEK2 did not lead to any stabilization in the peptide's structure of NEK2(303-333), NEK2(341-361), and NEK2(410-430). Based on these results, obtained from a series of peptides derived from a significant number of different centrosomal proteins, we propose that conformational polymorphism, modulated by intermolecular interactions is a general property of centrosomal proteins., (Copyright © 2014 Elsevier B.V. All rights reserved.)

chTOG is a conserved microtubule polymerase that catalyses the addition of tubulin dimers to promote microtubule growth. chTOG interacts with TACC3, a member of the transforming acidic coiled-coil (TACC) family. Here we analyse their association using the Xenopus homologues, XTACC3 (TACC3) and XMAP215 (chTOG), dissecting the mechanism by which their interaction promotes microtubule elongation during spindle assembly. Using SAXS, we show that the TACC domain (TD) is an elongated structure that mediates the interaction with the C terminus of XMAP215. Our data suggest that one TD and two XMAP215 molecules associate to form a four-helix coiled-coil complex. A hybrid methods approach was used to define the precise regions of the TACC heptad repeat and the XMAP215 C terminus required for assembly and functioning of the complex. We show that XTACC3 can induce the recruitment of larger amounts of XMAP215 by increasing its local concentration, thereby promoting efficient microtubule elongation during mitosis.

Background: Anisakiasis is a re-emerging global disease caused by consumption of raw or lightly cooked fish contaminated with L3 Anisakis larvae. This zoonotic disease is characterized by severe gastrointestinal and/or allergic symptoms which may misdiagnosed as appendicitis, gastric ulcer or other food allergies. The Anisakis allergen Ani s 5 is a protein belonging to the SXP/RAL-2 family; it is detected exclusively in nematodes. Previous studies showed that SXP/RAL-2 proteins are active antigens; however, their structure and function remain unknown. The aim of this study was to elucidate the three-dimensional structure of Ani s 5 and its main IgE and IgG4 binding regions., Methodology/principal Findings: The tertiary structure of recombinant Ani s 5 in solution was solved by nuclear magnetic resonance. Mg2+, but not Ca2+, binding was determined by band shift using SDS-PAGE. IgE and IgG4 epitopes were elucidated by microarray immunoassay and SPOTs membranes using sera from nine Anisakis allergic patients. The tertiary structure of Ani s 5 is composed of six alpha helices (H), with a Calmodulin like fold. H3 is a long, central helix that organizes the structure, with H1 and H2 packing at its N-terminus and H4 and H5 packing at its C-terminus. The orientation of H6 is undefined. Regarding epitopes recognized by IgE and IgG4 immunoglobulins, the same eleven peptides derived from Ani s 5 were bound by both IgE and IgG4. Peptides 14 (L40-K59), 26 (A76-A95) and 35 (I103-D122) were recognized by three out of nine sera., Conclusions/significance: This is the first reported 3D structure of an Anisakis allergen. Magnesium ion binding and structural resemblance to Calmodulin, suggest some putative functions for SXP/RAL-2 proteins. Furthermore, the IgE/IgG4 binding regions of Ani s 5 were identified as segments localized on its surface. These data will contribute towards a better understanding of the interactions that occur between immunoglobulins and allergens and, in turn, facilitate the design of novel diagnostic tests and immunotherapeutic strategies.

Sea anemone actinoporins constitute an optimum model to investigate mechanisms of membrane pore formation. All actinoporins of known structure show a general fold of a β-sandwich motif flanked by two α-helices. The crucial structure for pore formation seems to be the helix located at the N-terminal end. The role of several other protein regions in membrane attachment is also well established. However, not much is known about the protein residues involved in the oligomerization required for pore formation. Previous detailed analysis of the soluble three-dimensional structures of different wild-type and mutant actinoporins from Stychodactyla helianthus suggested residues which could be involved in this oligomerization. One of these stretches contains a conserved sequence compatible with an integrin-binding RGD motif. The results presented now deal with mutants affecting this motif in the well-characterized actinoporin sticholysin II. Small modifications along this three-residue sequence had profound effects on its solubility. Just a single methyl group yielded an RAD mutant version with a highly diminished haemolytic activity and altered oligomerization behaviour. The results obtained are discussed in terms of a key role for the RGD motif in maintaining the actinoporins' pore-competent state of protein oligomerization., (© 2014 FEBS.)

Here we perform a large-scale study of the structural properties and the expression of proteins that constitute the human Centrosome. Centrosomal proteins tend to be larger than generic human proteins (control set), since their genes contain in average more exons (20.3 versus 14.6). They are rich in predicted disordered regions, which cover 57% of their length, compared to 39% in the general human proteome. They also contain several regions that are dually predicted to be disordered and coiled-coil at the same time: 55 proteins (15%) contain disordered and coiled-coil fragments that cover more than 20% of their length. Helices prevail over strands in regions homologous to known structures (47% predicted helical residues against 17% predicted as strands), and even more in the whole centrosomal proteome (52% against 7%), while for control human proteins 34.5% of the residues are predicted as helical and 12.8% are predicted as strands. This difference is mainly due to residues predicted as disordered and helical (30% in centrosomal and 9.4% in control proteins), which may correspond to alpha-helix forming molecular recognition features (α-MoRFs). We performed expression assays for 120 full-length centrosomal proteins and 72 domain constructs that we have predicted to be globular. These full-length proteins are often insoluble: Only 39 out of 120 expressed proteins (32%) and 19 out of 72 domains (26%) were soluble. We built or retrieved structural models for 277 out of 361 human proteins whose centrosomal localization has been experimentally verified. We could not find any suitable structural template with more than 20% sequence identity for 84 centrosomal proteins (23%), for which around 74% of the residues are predicted to be disordered or coiled-coils. The three-dimensional models that we built are available at http://ub.cbm.uam.es/centrosome/models/index.php.

Actinoporins are water-soluble proteins with the ability to form pores upon insertion into biological membranes. They constitute a family of proteins with high degree of sequence identities but different hemolytic activities, suggesting that minor conformational arrangements result in major functional changes. A good example of this situation is the sea anemone Stichodactyla helianthus which produces two very similar actinoporins, sticholysins I (StnI) and II (StnII), but of very different hemolytic efficiency. Within this idea, given that the high resolution three-dimensional structure of StnII is already known, we have now solved that one corresponding to StnI in order to analyze the influence of particular residues on the conformation and activity of these proteins. In addition, random mutagenesis has been also used to produce five less hemolytic variants of StnI. All these mutations map to functionally relevant regions because they are probably involved in conformational changes associated with pore formation, which take place after membrane binding, and involve long-distance rearrangements of the polypeptide chain of actinoporins., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Onconase(®) FL-G zymogen is a 120 residue protein produced by circular permutation of the native Onconase(®) sequence. In this construction, the wild type N- and C-termini are linked by a 16 residue segment and new N- and C-termini are generated at wild type positions R73 and S72. This novel segment linking the native N- and C-termini is designed to obstruct Onconase's(®) active site and encloses a cleavage site for the HIV-1 protease. As a first step towards the resolution of its 3D structure and the study of its structure-function relationships, we report here the nearly complete NMR (1)H, (13)C and (15)N resonance chemical shift assignments at pH 5.2 and 35°C (BMRB deposit no 17973). The results presented here clearly show that the structure of the wild type Onconase(®) is conserved in the FL-G zymogen.

Intrinsically disordered proteins (IDPs) have recently attracted the attention of the scientific community challenging the well accepted structure-function paradigm. In the characterization of the dynamic features of proteins nuclear magnetic resonance spectroscopy (NMR) is a strategic tool of investigation. However the peculiar properties of IDPs, with the lack of a unique 3D structure and their high flexibility, have a strong impact on NMR observables (low chemical shift dispersion, efficient solvent exchange broadening) and thus on the quality of NMR spectra. Key aspects to be considered in the design of new NMR experiments optimized for the study of IDPs are discussed. A new experiment, based on direct detection of (13)C(α), is proposed.

The current surge in defining glycobiomarkers by applying lectins rekindles interest in definition of the sugar-binding sites of lectins at high resolution. Natural complex-type N-glycans can present more than one potential binding motif, posing the question of the actual mode of interaction when interpreting, for example, lectin array data. By strategically combining N-glycan preparation with saturation-transfer difference NMR and modeling, we illustrate that epitope recognition depends on the structural context of both the sugar and the lectin (here, wheat germ agglutinin and a single hevein domain) and cannot always be predicted from simplified model systems studied in the solid state. We also monitor branch-end substitutions by this strategy and describe a three-dimensional structure that accounts for the accommodation of the α2,6-sialylated terminus of a biantennary N-glycan by viscumin. In addition, we provide a structural explanation for the role of terminal α2,6-sialylation in precluding the interaction of natural N-glycans with lectin from Maackia amurensis . The approach described is thus capable of pinpointing lectin-binding motifs in natural N-glycans and providing detailed structural explanations for lectin selectivity.

Protein-glycosaminoglycan interactions are essential in many biological processes and human diseases, yet how their recognition occurs is poorly understood. Eosinophil cationic protein (ECP) is a cytotoxic ribonuclease that interacts with glycosaminoglycans at the cell surface; this promotes the destabilization of the cellular membrane and triggers ECP's toxic activity. To understand this membrane destabilization event and the differences in the toxicity of ECP and its homologues, the high resolution solution structure of the complex between full length folded ECP and a heparin-derived trisaccharide (O-iPr-α-D-GlcNS6S-α(1-4)-L-IdoA2S-α(1-4)-D-GlcNS6S) has been solved by NMR methods and molecular dynamics simulations. The bound protein retains the tertiary structure of the free protein. The (2)S(0) conformation of the IdoA ring is preferably recognized by the protein. We have identified the precise location of the heparin binding site, dissected the specific interactions responsible for molecular recognition, and defined the structural requirements for this interaction. The structure reveals the contribution of Arg7, Gln14, and His15 in helix α1, Gln40 in strand β1, His64 in loop 4, and His128 in strand β6 in the recognition event and corroborates the previously reported participation of residues Arg34-Asn39. The participation of the catalytic triad (His15, Lys38, His128) in recognizing the heparin mimetic reveals, at atomic resolution, the mechanism of heparin's inhibition of ECP's ribonucleolytic activity. We have integrated all the available data to propose a molecular model for the membrane interaction process. The solved NMR complex provides the structural model necessary to design inhibitors to block ECP's toxicity implicated in eosinophil pathologies.

Onconase® is a highly cytotoxic amphibian homolog of Ribonuclease A. Here, we describe the construction of circularly permuted Onconase® variants by connecting the N- and C-termini of this enzyme with amino acid residues that are recognized and cleaved by the human immunodeficiency virus protease. Uncleaved circularly permuted Onconase® variants are unusually stable, non-cytotoxic and can internalize in human T-lymphocyte Jurkat cells. The structure, stability and dynamics of an intact and a cleaved circularly permuted Onconase® variant were determined by Nuclear Magnetic Resonance spectroscopy and provide valuable insight into the changes in catalytic efficiency caused by the cleavage. The understanding of the structural environment and the dynamics of the activation process represents a first step toward the development of more effective drugs for the treatment of diseases related to pathogens expressing a specific protease. By taking advantage of the protease's activity to initiate a cytotoxic cascade, this approach is thought to be less susceptible to known resistance mechanisms.

Gs (Geobacter sulfurreducens) can transfer electrons to the exterior of its cells, a property that makes it a preferential candidate for the development of biotechnological applications. Its genome encodes over 100 cytochromes and, despite their abundance and key functional roles, to date there is no structural information for these proteins in solution. The trihaem cytochrome PpcA might have a crucial role in the conversion of electronic energy into protonmotive force, a fundamental step for ATP synthesis in the presence of extracellular electron acceptors. In the present study, 15N-labelled PpcA was produced and NMR spectroscopy was used to determine its solution structure in the fully reduced state, its backbone dynamics and the pH-dependent conformational changes. The structure obtained is well defined, with an average pairwise rmsd (root mean square deviation) of 0.25 Å (1 Å=0.1 nm) for the backbone atoms and 0.99 Å for all heavy atoms, and constitutes the first solution structure of a Gs cytochrome. The redox-Bohr centre responsible for controlling the electron/proton transfer was identified, as well as the putative interacting regions between PpcA and its redox partners. The solution structure of PpcA will constitute the foundation for studies aimed at mapping out in detail these interacting regions.

The bacterium Geobacter sulfurreducens (Gs) can grow in the presence of extracellular terminal acceptors, a property that is currently explored to harvest electricity from aquatic sediments and waste organic matter into microbial fuel cells. A family composed of five triheme cytochromes (PpcA-E) was identified in Gs. These cytochromes play a crucial role by bridging the electron transfer from oxidation of cytoplasmic donors to the cell exterior and assisting the reduction of extracellular terminal acceptors. The detailed thermodynamic characterization of such proteins showed that PpcA and PpcD have an important redox-Bohr effect that might implicate these proteins in the e(-)/H(+) coupling mechanisms to sustain cellular growth. The physiological relevance of the redox-Bohr effect in these proteins was studied by determining the fractional contribution of each individual redox-microstate at different pH values. For both proteins, oxidation progresses from a particular protonated microstate to a particular deprotonated one, over specific pH ranges. The preferred e(-)/H(+) transfer pathway established by the selected microstates indicates that both proteins are functionally designed to couple e(-)/H(+) transfer at the physiological pH range for cellular growth.

Amyloidogenic neurodegenerative diseases are incurable conditions with high social impact that are typically caused by specific, largely disordered proteins. However, the underlying molecular mechanism remains elusive to established techniques. A favored hypothesis postulates that a critical conformational change in the monomer (an ideal therapeutic target) in these "neurotoxic proteins" triggers the pathogenic cascade. We use force spectroscopy and a novel methodology for unequivocal single-molecule identification to demonstrate a rich conformational polymorphism in the monomer of four representative neurotoxic proteins. This polymorphism strongly correlates with amyloidogenesis and neurotoxicity: it is absent in a fibrillization-incompetent mutant, favored by familial-disease mutations and diminished by a surprisingly promiscuous inhibitor of the critical monomeric β-conformational change, neurotoxicity, and neurodegeneration. Hence, we postulate that specific mechanostable conformers are the cause of these diseases, representing important new early-diagnostic and therapeutic targets. The demonstrated ability to inhibit the conformational heterogeneity of these proteins by a single pharmacological agent reveals common features in the monomer and suggests a common pathway to diagnose, prevent, halt, or reverse multiple neurodegenerative diseases., Competing Interests: The authors declare a competing financial interest: M.C.-V., J.O., and R.H. are co-inventors on an international patent application (No. P 201031846,PCT/ES2011/070867) covering the results contained in this article. Any potential income generated by exploitation of the patent rights and received by their employer, the CSIC, shall be shared with these authors according to Spanish law and the regulations of the CSIC.

The protein NA14 is a key adaptor protein mediating the intermolecular interactions of microtubules and Spastin. To gain insight into its structure and function, we have expressed, purified and characterized human NA14 and some variants. NA14 is rather insoluble and tends to oligomerize and form fibrils. Successive mutation of the three Cys and two potentially exposed Leu residues (83 and 93) yielded a water-soluble quintuple variant, named 3CS-2LR. NA14 and its variants have a high helical content as determined by circular dichroism (CD). Based on nuclear magnetic resonance data of the quintuple mutant and the wild-type (wt) protein in the presence of dodecylphosphocholine micelles, the N-(M1-N13) and C-termini (K105-S119) were found to lack preferred structure. The remaining residues (14-104) participate in NA14 self-association, probably by forming a parallel coiled-coil structure. We hypothesize that Leu 83 and Leu 93 mediate interactions among NA14, Spastin and microtubules. We have also examined urea and thermal denaturation of the quintuple and other NA14 variants at different pH values by CD. The pH dependence of the conformational stability and the elevated native-state pK(a) determined for the two conserved Tyr allow us to propose that the NA14 structure may be stabilized by two Glu-COO(-) ||| HO-Tyr H-bonds, highly conserved in NA14-like proteins in other species.

LC8 dynein light chain (now termed DYNLL1 and DYNLL2 in mammals), a dimeric 89 amino acid protein, is a component of the dynein multi-protein complex. However a substantial amount of DYNLL1 is not associated to microtubules and it can thus interact with dozens of cellular and viral proteins that display well-defined, short linear motifs. Using DYNLL1 as bait in a yeast two-hybrid screen of a human heart library we identified ATMIN, an ATM kinase-interacting protein, as a DYNLL1-binding partner. Interestingly, ATMIN displays at least 18 SQ/TQ motifs in its sequence and DYNLL1 is known to bind to proteins with KXTQT motifs. Using pepscan and yeast two-hybrid techniques we show that DYNLL1 binds to multiple SQ/TQ motifs present in the carboxy-terminal domain of ATMIN. Recombinant expression and purification of the DYNLL1-binding region of ATMIN allowed us to obtain a polypeptide with an apparent molecular mass in gel filtration close to 400 kDa that could bind to DYNLL1 in vitro. The NMR data-driven modelled complexes of DYNLL1 with two selected ATMIN peptides revealed a similar mode of binding to that observed between DYNLL1 and other peptide targets. Remarkably, co-expression of mCherry-DYNLL1 and GFP-ATMIN mutually affected intracellular protein localization. In GFP-ATMIN expressing-cells DNA damage induced efficiently nuclear foci formation, which was partly impeded by the presence of mCherry-DYNLL1. Thus, our results imply a potential cellular interference between DYNLL1 and ATMIN functions., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Actinoporins constitute a group of small and basic α-pore forming toxins produced by sea anemones. They display high sequence identity and appear as multigene families. They show a singular behaviour at the water-membrane interface: In aqueous solution, actinoporins remain stably folded but, upon interaction with lipid bilayers, become integral membrane structures. These membranes contain sphingomyelin, display phase coexistence, or both. The water soluble structures of the actinoporins equinatoxin II (EqtII) and sticholysin II (StnII) are known in detail. The crystalline structure of a fragaceatoxin C (FraC) nonamer has been also determined. The three proteins fold as a β-sandwich motif flanked by two α-helices, one of them at the N-terminal end. Four regions seem to be especially important: A cluster of aromatic residues, a phosphocholine binding site, an array of basic amino acids, and the N-terminal α-helix. Initial binding of the soluble monomers to the membrane is accomplished by the cluster of aromatic amino acids, the array of basic residues, and the phosphocholine binding site. Then, the N-terminal α-helix detaches from the β-sandwich, extends, and lies parallel to the membrane. Simultaneously, oligomerization occurs. Finally, the extended N-terminal α-helix penetrates the membrane to build a toroidal pore. This model has been however recently challenged by the cryo-EM reconstruction of FraC bound to phospholipid vesicles. Actinoporins structural fold appears across all eukaryotic kingdoms in other functionally unrelated proteins. Many of these proteins neither bind to lipid membranes nor induce cell lysis. Finally, studies focusing on the therapeutic potential of actinoporins also abound., (Copyright © 2011 Elsevier B.V. All rights reserved.)

Sequence analysis of eosinophil cationic protein (ECP), a ribonuclease of broad antimicrobial activity, allowed identification of residues 1-45 as the antimicrobial domain. We have further dissected ECP(1-45) with a view to defining the minimal requirements for antimicrobial activity. Structure-based downsizing has focused on both α-helices of ECP(1-45) and yielded analogues with substantial potency against Gram-negative and -positive strains. Analogues ECP(8-36) and ECP(6-17)-Ahx-(23-36) (Ahx, 6-aminohexanoic acid) involve 36% and 40% size reduction relative to (1-45), respectively, and display a remarkably ECP-like antimicrobial profile. Both retain segments required for self-aggregation and lipolysaccharide binding, as well as the bacterial agglutination ability of parent ECP. Analogue (6-17)-Ahx-(23-36), in particular, is shown by NMR to preserve the helical traits of the native 8-16 (α1) and 33-36 (α2) regions and can be proposed as the minimal structure capable of reproducing the activity of the entire protein.

The structural determinants that are responsible for the formation of higher order associations of folded proteins remain unknown. We have investigated the role on the dimerization process of different residues of a domain-swapped dimer human pancreatic ribonuclease variant. This variant is a good model to study the dimerization and swapping processes because dimer and monomer forms interconvert, are easily isolated, and only one dimeric species is produced. Thus, simple models for the swapping process can be proposed. The dimerization (dissociation constant) and swapping propensity have been studied using different variants with changes in residues that belong to different putative molecular determinants of dimerization. Using NMR spectroscopy, we show that these mutations do not substantially alter the overall conformation and flexibility, but affect the residue level stability. Overall, the most critical residues for the swapping process are those of one subunit that interact with the hinge loop of another one-subunit residue, stabilizing it in a conformation that favors the interchange. Tyr(25), Gln(101), and Pro(19), with Asn(17), Ser(21), and Ser(23), are found to be the most significant; notably, Glu(103) and Arg(104), which were postulated to form salt bridges that would stabilize the dimer, are not critical for dimerization., (Copyright © 2011 Biophysical Society. Published by Elsevier Inc. All rights reserved.)

Actinoporins are a family of sea anemone proteins that bind to membranes and produce functional pores which result in cell lysis. Actinoporin variants with decreased lytic activity usually show a reduced affinity for membranes. However, for some of these mutant versions there is no direct correlation between the loss of binding affinity and the decrease in their overall lytic activity, suggesting that other steps in pore formation may be hampered or facilitated by the mutations. To test this hypothesis on the mechanism of pore formation by this interesting family of proteins, structural and dynamic NMR studies have been carried out on two disabled variants of the actinoporin Sticholysin II, R29Q and Y111N. It is shown that their lytic activity is not only related to their membrane affinity but also to their conformational mechanism for membrane insertion. Alterations in their activities can be explained by structural, electrostatic and dynamic differences in a cluster of aromatic moieties and the N-terminus. In addition, the dynamic properties of some segments located at the C-terminus of the R29Q variant suggest a relevant role for this region in terms of protein-protein interactions. On the basis of all these results, we propose that R29 anchors a network of electrostatic interactions crucial for the actinoporin's approach to the membrane and that Y111 induces a necessary disorder in the loop regions that bind to membranes., (© 2011 The Authors Journal compilation © 2011 FEBS.)