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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.

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.

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 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.

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.