Divergent CPEB prion-like domains reveal different assembly mechanisms for a generic amyloid-like fold.

BACKGROUND: Amyloids are ordered, insoluble protein aggregates, characterized by a cross-ß sheet quaternary structure in which molecules in a ß-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.

Disorder and partial folding in the regulatory subunit hinge region of Trypanosoma brucei protein kinase A: The C-linker portion inhibits the parasite's protein kinase A.

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.

Structural Insights into ß-arrestin/CB1 Receptor Interaction: NMR and CD Studies on Model Peptides.

Activation of the cannabinoid CB1 receptor induces different cellular signaling cascades through coupling to different effector proteins (G-proteins and ß-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 ß-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: ß-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 ß-arrestin and CB1 receptor and validate minimalist approaches to structurally comprehend complex protein systems.

Structure of Fungal α Mating Pheromone in Membrane Mimetics Suggests a Possible Role for Regulation at the Water-Membrane Interface.

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.

Backbone assignment of cytochrome PccH, a crucial protein for microbial electrosynthesis in Geobacter sulfurreducens.

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 (1H, 13C and 15N) 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.

Deciphering how Cpl-7 cell wall-binding repeats recognize the bacterial peptidoglycan.

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_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_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-(ß1,4)-N-acetylmuramyl-L-alanyl-D-isoglutamine (GMDP) as the peptidoglycan (PG) target recognized by the CW_7 motifs, and characterize feasible GMDP-CW_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_7-containing lysins as novel anti-infectives.

Structural insight into the XTACC3/XMAP215 interaction from CD and NMR studies on model peptides.

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.

De novo active sites for resurrected Precambrian enzymes.

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.

Solution structure and dynamics of the outer membrane cytochrome OmcF from Geobacter sulfurreducens.

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. 15N,13C-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, 15N 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.

Insights into protein-carbohydrate recognition: A novel binding mechanism for CBM family 43.

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.

Structure-Activity Relationship of α Mating Pheromone from the Fungal Pathogen Fusarium oxysporum.

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 ß-turn resembling that of its yeast counterpart. Disruption of the-fold by d-alanine substitution of the conserved central Gly6-Gln7 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 ß-turn but instead required two conserved Trp1-Cys2 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.

A truncated apoptin protein variant selectively kills cancer cells.

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.

Redox- and pH-linked conformational changes in triheme cytochrome PpcA from Geobacter sulfurreducens.

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 pKa values of the redox-Bohr center, providing insights into the e-/H+ coupling molecular mechanisms driven by PpcA in G. sulfurreducens.

Insights into the mechanism of Apoptin's exquisitely selective anti-tumor action from atomic level characterization of its conformation and dynamics.

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 H6-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, H6-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 H6-ApopΔProΔLeu. Using a battery of 2D, 3D and (4,2)D NMR spectra, the essentially complete 1H, 13C and 15N resonance assignments of H6-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, 1H-15N NOE ratios are low, which indicates flexibility on the ps-ns time scale. Interestingly, H6-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.

Modular Architecture and Unique Teichoic Acid Recognition Features of Choline-Binding Protein L (CbpL) Contributing to Pneumococcal Pathogenesis.

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 Ca2+-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_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.

Structural basis for broad neutralization of HIV-1 through the molecular recognition of 10E8 helical epitope at the membrane interface.

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.

Structural basis for endotoxin neutralization by the eosinophil cationic protein.

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.

Unveiling the Structural Basis That Regulates the Energy Transduction Properties within a Family of Triheme Cytochromes from Geobacter sulfurreducens.

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 Leu6 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 Phe6 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.

Molecular Basis for the Protein Recognition Specificity of the Dynein Light Chain DYNLT1/Tctex1: CHARACTERIZATION OF THE INTERACTION WITH ACTIVIN RECEPTOR IIB.

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.

The Y9P Variant of the Titin I27 Module: Structural Determinants of Its Revisited Nanomechanics.

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.