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The four WW domains of human Nedd4-1 (neuronal precursor cell expressed developmentally downregulated gene 4-1) interact with the PPxY (PY) motifs of the human epithelial Na(+) channel (hENaC) subunits, with the third WW domain (WW3*) showing the highest affinity. We have shown previously that the α-hENaC PY motif binding interface of WW3* undergoes conformational exchange on the millisecond time scale, indicating that conformational sampling plays a role in peptide recognition. To further understand this role, the structure and dynamics of hNedd4-1 WW3* were investigated. The nuclear Overhauser effect-derived structure of apo-WW3* resembles the domain in complex with the α-hENaC peptide, although particular side chain conformations change upon peptide binding, which was further investigated by molecular dynamics simulations. Model-free analysis of the (15)N nuclear magnetic resonance spin relaxation data showed that the apo and peptide-bound states of WW3* have similar backbone picosecond to nanosecond time scale dynamics. However, apo-WW3* exhibits pronounced chemical exchange on the millisecond time scale that is quenched upon peptide binding. (1)HN and (15)N Carr-Purcell-Meiboom-Gill (CPMG) relaxation dispersion experiments at various temperatures revealed that apo-WW3* exists in an equilibrium between the natively folded peptide binding-competent state and a random coil-like denatured state. The thermodynamics of the folding equilibrium was determined by fitting a thermal denaturation profile monitored by circular dichroism spectroscopy in combination with the CPMG data, leading to the conclusion that the unfolded state is populated to ∼ 20% at 37 °C. These results show that the binding of the hNedd4-1 WW3* domain to α-hENaC is coupled to the folding equilibrium.

Hyperpolarization activated and cyclic nucleotide-gated (HCN) ion channels as well as cyclic nucleotide-gated (CNG) ion channels are essential for the regulation of cardiac cells, neuronal excitability, and signaling in sensory cells. Both classes are composed of four subunits. Each subunit comprises a transmembrane region, intracellular N- and C-termini, and a C-terminal cyclic nucleotide-binding domain (CNBD). Binding of cyclic nucleotides to the CNBD promotes opening of both CNG and HCN channels. In case of CNG channels, binding of cyclic nucleotides to the CNBD is sufficient to open the channel. In contrast, HCN channels open upon membrane hyperpolarization and their activity is modulated by binding of cyclic nucleotides shifting the activation potential to more positive values. Although several high-resolution structures of CNBDs from HCN and CNG channels are available, the gating mechanism for murine HCN2 channel, which leads to the opening of the channel pore, is still poorly understood. As part of a structural investigation, here, we report the complete backbone and side chain resonance assignments of the murine HCN2 CNBD with part of the C-linker.

Fehlfaltung, Aggregation und Amyloidfibrillenbil- dung des Proteins a-Synuclein (a-syn) sind an der Pathogenese der Parkinson-Krankheit und weiterer Synucleinopathien be- teiligt. Die Entwicklung der Amyloid-Toxizitt steht in Zu- sammenhang mit der Bildung partiell gefalteter Aggregations- intermediate. Hier beschreiben wir die Entwicklung einer Klasse gegen a-syn gerichteter Bindeproteine, die wir als b- Wrapine (b-Wrap-Proteine) bezeichnen. Die NMR-Struktur eines a-syn:b-Wrapin-Komplexes zeigt eine b-Haarnadel-Kon- formation von a-syn im Sequenzbereich a-syn(37-54). Sub- stcchiometrische Mengen des Bindeproteins inhibieren die a- syn-Aggregation und -Toxizitt, das b-Wrapin greift folglich in die Bildung der Aggregationskeime ein. Die Ablagerung spezifischer Peptide und Proteine in Amy- loid-Fibrillen liegt einer Vielzahl neurodegenerativer Erkran- kungen zugrunde. Monomere und oligomere Fehlfaltungsin- termediate sind wichtige Komponenten des Aggregationsme- chanismus. (1) Die Stabilisierung partiell gefalteter Zustnde amyloidogener Proteine kcnnte ein Ansatzpunkt fr die Arzneimittelentwicklung sein. (1d) Das Protein a-Synuclein (a-syn)ist der hauptschliche Bestandteil der intrazellulren neuronalen Ablagerungen, die Parkinson und weitere Synucleinopathien pathologisch kenn- zeichnen. (2) Genetische Studien zeigen einen Zusammenhang zwischen krankheitsassoziierter Mutation oder Multiplikati- on des fr a-syn codierenden Gens und verstrkter Aggre- gationsneigung und weisen somit auf eine Rolle der a-syn- Aggregation in der Pathogenese hin. Das a-syn-Protein zeichnet sich durch eine hohe konformative Flexibilitt aus. Freies monomeres a-syn ist intrinsisch ungeordnet, jedoch gehen einige Regionen des Proteins intramolekulare lang- reichweitige Wechselwirkungen ein. (3) An synthetische oder biologische Membranen gebunden nimmt a-syn a-helikale Konformation an. (4) In fibrillren Aggregaten bildet die zentrale Sequenzregion mehrere b-Strnge, die sich zu inter- molekularen parallelen b-Faltblttern organisieren, whrend die N-terminalen und C-terminalen Regionen entfaltet blei- ben. (5) Im Unterschied zur parallelen Anordnung der b- Strnge in Fibrillen (5b, 6) wurde eine antiparallele b-Faltblatt- Struktur in Oligomeren nachgewiesen. (7)

The misfolding and aggregation of the protein α-synuclein (α-syn), which results in the formation of amyloid fibrils, is involved in the pathogenesis of Parkinson's disease and other synucleinopathies. The emergence of amyloid toxicity is associated with the formation of partially folded aggregation intermediates. Here, we engineered a class of binding proteins termed β-wrapins (β-wrap proteins) with affinity for α-synuclein (α-syn). The NMR structure of an α-syn:β-wrapin complex reveals a β-hairpin of α-syn comprising the sequence region α-syn(37-54). The β-wrapin inhibits α-syn aggregation and toxicity at substoichiometric concentrations, demonstrating that it interferes with the nucleation of aggregation.

Summary Type 1 secretion systems (T1SS) catalyze the one step protein transport across the membranes of Gram-negative bacteria and are composed of an outer membrane protein, a membrane fusion protein and an ABC transporter. The ABC transporter consists of the canonical nucleotide binding and transmembrane domains. For the toxin hemolysin A (HlyA), the ABC transporter HlyB carries an additional, N-terminal domain sharing about 40% homology to C39 peptidases, but this "C39-like domain" (CLD) is suggested to feature another, yet unknown function. Our functional and structural analysis demonstrates that the CLD is essential for secretion and that it specifically interacts with the unfolded state of HlyA. We determined the nuclear magnetic resonance structure of the CLD as well as the substrate-binding region within the CLD. This mode of action, represents a mechanism within T1SS and answers the question, how a large and unfolded substrate is protected inside the cells during secretion.

The aggregation of the Aβ plays a fundamental role in the pathology of AD. Recently, N-terminally modified Aβ species, pE-Aβ, have been described as major constituents of Aβ deposits in the brains of AD patients. pE-Aβ has an increased aggregation propensity and shows increased toxicity compared with Aβ1-40 and Aβ1-42. In the present work, high-resolution NMR spectroscopy was performed to study pE-Aβ3-40 in aqueous TFE-containing solution. Two-dimensional TOCSY and NOESY experiments were performed. On the basis of NOE and chemical shift data, pE-Aβ3-40 was shown to contain two helical regions formed by residues 14-22 and 30-36. This is similar as previously described for Aβ1-40. However, the secondary chemical shift data indicate decreased helical propensity in pE-Aβ3-40 when compared with Aβ1-40 under exactly the same conditions. This is in agreement with the observation that pE-Aβ3-40 shows a drastically increased tendency to form β-sheet-rich structures under more physiologic conditions. Structural studies of pE-Aβ are crucial for better understanding the structural basis of amyloid fibril formation in the brain during development of AD, especially because an increasing number of reports indicate a decisive role of pE-Aβ for the pathogenesis of AD.

Cyclic nucleotide-sensitive ion channels, known as HCN and CNG channels, are activated by binding of ligands to a domain (CNBD) located on the cytoplasmic side of the channel. The underlying mechanisms are not well understood. To elucidate the gating mechanism, structures of both the ligand-free and -bound CNBD are required. Several crystal structures of the CNBD from HCN2 and a bacterial CNG channel (MloK1) have been solved. However, for HCN2, the cAMP-free and -bound state did not reveal substantial structural rearrangements. For MloK1, structural information for the cAMP-free state has only been gained from mutant CNBDs. Moreover, in the crystal, the CNBD molecules form an interface between dimers, proposed to be important for allosteric channel gating. Here, we have determined the solution structure by NMR spectroscopy of the cAMP-free wild-type CNBD of MloK1. A comparison of the solution structure of cAMP-free and -bound states reveals large conformational rearrangement on ligand binding. The two structures provide insights on a unique set of conformational events that accompany gating within the ligand-binding site.

Cyclic nucleotide-sensitive ion channels, known as HCN and CNG channels play crucial roles in neuronal excitability and signal transduction of sensory cells. These channels are activated by binding of cyclic nucleotides to their intracellular cyclic nucleotide-binding domain (CNBD). A comparison of the structures of wildtype ligand-free and ligand-bound CNBD is essential to elucidate the mechanism underlying nucleotide-dependent activation of CNBDs. We recently reported the solution structure of the Mesorhizobium loti K1 (MloK1) channel CNBD in complex with cAMP. We have now extended these studies and achieved nearly complete assignments of (1)H, (13)C and (15)N resonances of the nucleotide-free CNBD. A completely new assignment of the nucleotide-free wildtype CNBD was necessary due to the sizable chemical shift differences as compared to the cAMP bound CNBD and the slow exchange behaviour between both forms. Scattering of these chemical shift differences over the complete CNBD suggests that nucleotide binding induces significant overall conformational changes.

Substrate-binding proteins or extracellular solute receptors (ESRs) are components of both ABC (ATP binding cassette) and TRAP-T (tripartite ATP-independent periplasmic transporter). The TRAP-T system UehABC from Silicibacter pomeroyi DSS-3 imports the compatible solutes ectoine and 5-hydroxyectoine as nutrients. UehA, the ESR of the UehABC operon, binds both ectoine and 5-hydroxyectoine with high affinity (K(d) values of 1.4+/-0.1 and 1.1+/-0.1 microM, respectively) and delivers them to the TRAP-T complex. The crystal structure of UehA in complex with ectoine was determined at 2.9-A resolution and revealed an overall fold common for all ESR proteins from TRAP systems determined so far. A comparison of the recently described structure of TeaA from Halomonas elongata and an ectoine-binding protein (EhuB) from an ABC transporter revealed a conserved ligand binding mode that involves both directed and cation-pi interactions. Furthermore, a comparison with other known TRAP-T ESRs revealed a helix that might act as a selectivity filter imposing restraints on the ESRs that fine-tune ligand recognition and binding and finally might determine the selection of the cognate substrate.

Autophagy is a fundamental homeostatic process in eukaryotic organisms, fulfilling essential roles in development and adaptation to stress. Among other factors, formation of autophagosomes critically depends on proteins of the Atg8 (autophagy-related protein 8) family, which are reversibly conjugated to membrane lipids. We have applied X-ray crystallography, nuclear magnetic resonance spectroscopy, and molecular dynamics simulations to study the conformational dynamics of Atg8-type proteins, using GATE-16 (Golgi-associated ATPase enhancer of 16 kDa), also known as GABARAPL2, as a model system. This combination of complementary approaches provides new insight into a structural transition centered on the C-terminus, which is crucial for the biological activity of these proteins.

ATP-binding cassette (ABC) transporters are ubiquitous integral membrane proteins, which catalyze the translocation of molecules across biological membranes in an ATP-dependent manner. Despite the diversity in the transported substrates, they all share the same architecture, comprised of two transmembrane (TMD) and two nucleotide-binding domains (NBD). Members of the bacteriocin ABC transporter subfamily feature a special domain, belonging to the C39 (cystein protease family 39) peptidase protein family. These domains are assumed to cleave a C-terminal signal sequence from the protein or peptide substrate before or during the transport process. Although the C39 peptidase-like domain of the ABC transporter haemolysin B from E. coli shows no proteolytic activity, it is essential for the function of this transporter. In order to elucidate the contribution of the isolated C39 peptidase-like domain in the whole transport process, the backbone and side chain (1)H, (15)N and (13)C-NMR chemical shifts have been assigned.

The aggregation of the Aβ plays a fundamental role in the pathology of AD. Recently, N-terminally modified Aβ species, pE-Aβ, have been described as major constituents of Aβ deposits in the brains of AD patients. pE-Aβ has an increased aggregation propensity and shows increased toxicity compared with Aβ1-40 and Aβ1-42. In the present work, high-resolution NMR spectroscopy was performed to study pE-Aβ3-40 in aqueous TFE-containing solution. Two-dimensional TOCSY and NOESY experiments were performed. On the basis of NOE and chemical shift data, pE-Aβ3-40 was shown to contain two helical regions formed by residues 14-22 and 30-36. This is similar as previously described for Aβ1-40. However, the secondary chemical shift data indicate decreased helical propensity in pE-Aβ3-40 when compared with Aβ1-40 under exactly the same conditions. This is in agreement with the observation that pE-Aβ3-40 shows a drastically increased tendency to form β-sheet-rich structures under more physiologic conditions. Structural studies of pE-Aβ are crucial for better understanding the structural basis of amyloid fibril formation in the brain during development of AD, especially because an increasing number of reports indicate a decisive role of pE-Aβ for the pathogenesis of AD.

International audience; The ABC transporter haemolysin B (HlyB) from Escherichia coli is part of a type I secretion system that translocates a 110 kDa toxin in one step across both membranes of this Gram-negative bacterium in an ATP-dependent manner. Sequence analysis indicates that HlyB contains a C39 peptidase-like domain at its N-terminus. C39 domains are thiol-dependent peptidases that cleave their substrates after a GG motif. Interestingly, the catalytically invariant cysteine is replaced by a tyrosine in the C39-like domain of HlyB. Here, the overexpression, purification and crystallization of the isolated C39-like domain are described as a first step towards obtaining structural insights into this domain and eventually answering the question concerning the function of a degenerated C39 domain in the ABC transporter HlyB.

In the soil bacterium Bacillus subtilis , five transport systems work in concert to mediate the import of various compatible solutes that counteract the deleterious effects of increases in the osmolarity of the environment. Among these five systems, the ABC transporter OpuA, which catalyzes the import of glycine betaine and proline betaine, has been studied in detail in the past. Here, we demonstrate that OpuA is capable of importing the sulfobetaine dimethylsulfonioacetate (DMSA). Since OpuA is a classic ABC importer that relies on a substrate-binding protein priming the transporter with specificity and selectivity, we analyzed the OpuA-binding protein OpuAC by structural and mutational means with respect to DMSA binding. The determined crystal structure of OpuAC in complex with DMSA at a 2.8-Å resolution and a detailed mutational analysis of these residues revealed a hierarchy within the amino acids participating in substrate binding. This finding is different from those for other binding proteins that recognize compatible solutes. Furthermore, important principles that enable OpuAC to specifically bind various compatible solutes were uncovered.