GABAB receptors (GBRs) are key regulators of synaptic release but little is known about trafficking mechanisms that control their presynaptic abundance. We now show that sequence-related epitopes in APP, AJAP-1 and PIANP bind with nanomolar affinities to the N-terminal sushi-domain of presynaptic GBRs. Of the three interacting proteins, selectively the genetic loss of APP impaired GBR-mediated presynaptic inhibition and axonal GBR expression. Proteomic and functional analyses revealed that APP associates with JIP and calsyntenin proteins that link the APP/GBR complex in cargo vesicles to the axonal trafficking motor. Complex formation with GBRs stabilizes APP at the cell surface and reduces proteolysis of APP to Aβ, a component of senile plaques in Alzheimer’s disease patients. Thus, APP/GBR complex formation links presynaptic GBR trafficking to Aβ formation. Our findings support that dysfunctional axonal trafficking and reduced GBR expression in Alzheimer’s disease increases Aβ formation.

The solution structure of the dicerium(III) complex of the N-terminal domain of calmodulin (Ce2-TR1C hereafter) has been solved employing paramagnetic T1 relaxation enhancements and pseudocontact shifts introduced by the Ce3+ ions, together with conventional NOE constraints. The use of pseudocontact shift constraints constitutes the first attempt to locate metal ions within a protein structure by NMR. Like calcium(II), paramagnetic cerium(III) has been found to bind to the two metal binding sites of the TR1C fragment of calmodulin in a cooperative manner. Due to the presence of pseudocontact interactions between the Ce3+ ions and protons of the 76-residue protein, the 1H NMR spectra of the complex show resonances shifted between +22 and -9 ppm. Eighty percent of its proton resonances could be assigned through a standard approach using TOCSY/COSY and NOESY spectra and through 1D NOE difference spectra for the broad resonances of protons close to the paramagnetic ions. A family of structures was calculated by means of the torsion angle dynamics program DYANA [Güntert, P., Mumenthaler, C., & Wüthrich, K. (1996) XVIIthInternational Conference on Magnetic Resonance inBiological Systems (Abstract)] using 1012 NOEs. Longitudinal proton relaxation times helped to roughly define the position of the metal ions within the protein. A total of 381 pseudocontact shift constraints, whose evaluation and use are critically discussed, have then been added to further refine the metal coordinates within the protein frame and to improve the structure resolution. A dramatic resolution improvement of the metal coordinates together with a sizable resolution improvement in the regions close to the paramagnetic centers, where the number of NOEs is low, is observed. The good quality of the solution structure permitted a meaningful comparison with the solid-state structure of calcium-loaded calmodulin at 1.7 A resolution [Chattopadhyaya, R., Meador, W. E., Means, A. R., & Quiocho, F. A. (1992) J. Mol. Biol. 228, 1177]. The Ce2-TR1C complex is overall more compact than the Ca form.

The auxiliary ␤-subunit KCNMB2 (␤ 2 ) endows the noninactivating large conductance Ca 2؉ -and voltagedependent potassium (BK) channel with fast inactivation. This process is mediated by the N terminus of KCNMB2 and closely resembles the "ball-and-chain"-type inactivation observed in voltage-gated potassium channels. Here we investigated the solution structure and function of the KCNMB2 N terminus (amino acids 1-45, BK␤ 2 N) using NMR spectroscopy and patch clamp recordings. BK␤ 2 N completely inactivated BK channels when applied to the cytoplasmic side; its interaction with the BK ␣-subunit is characterized by a particularly slow dissociation rate and an affinity in the upper nanomolar range. The BK␤ 2 N structure comprises two domains connected by a flexible linker: the pore-blocking "ball domain" (formed by residues 1-17) and the "chain domain" (between residues 20 -45) linking it to the membrane segment of KCNMB2. The ball domain is made up of a flexible N terminus anchored at a well ordered loop-helix motif. The chain domain consists of a 4-turn helix with an unfolded linker at its C terminus. These structural properties explain the functional characteristics of BK␤ 2 N-mediated inactivation.Large conductance K ϩ channels (BK 1 or MaxiK channels) are key modulators of excitability in many types of cell (1, 2).They are formed from four identical ␣-subunits encoded by the Slo gene and are activated by membrane depolarization and/or increase in intracellular Ca 2ϩ concentration ([Ca 2ϩ ] i (3-8)). This dual activation is unique among the large family of K ϩ channels and provides a direct feedback mechanism to regulate Ca 2ϩ influx. In many tissues, the activation gating of BK channels is modulated by accessory ␤-subunits, a family of membrane proteins (KCNMB) closely associated with the ␣-subunit (7). Four KCNMB proteins have been identified (KCNMB1-4), and they all share a prototypic topology of two transmembrane domains with intracellular N and C termini (9 -13). Functionally, each of these KCNMB proteins distinctly changes the rates of channel activation and deactivation as well as the apparent sensitivity of the channel for Ca 2ϩ (9). In addition, one of the ␤-subunits, KCNMB2 (␤ 2 ), was found to confer rapid and complete inactivation to the BK channel complex (11, 12) in a manner similar to that observed in chromaffin cells of the adrenal gland or in hippocampal CA1 neurons (14, 15). Analysis of this KCNMB2-mediated inactivation gating showed that it closely resembled the famous ball-andchain-type inactivation of voltage-gated K ϩ channels (Kv): (i) it is determined by the N terminus of KCNMB2; (ii) it occludes the open channel pore and competes with the pore-blocking agent tetraethylammonium (11, 12); (iii) recovery from inactivation is speeded up by an increase of the extracellular K ϩ concentration (11).Moreover, the N-terminal stretch of the KCNMB2 N terminus (19 amino acids) was shown to be a functional entity, i.e. its fusion to the N terminus of KCNMB1 (␤ 1 ) conferred rapid inactivation to this ...

Small conductance Ca2؉ -activated potassium (SK) channels underlie the afterhyperpolarization that follows the action potential in many types of central neurons. SK channels are voltage-independent and gated solely by intracellular Ca 2؉ in the submicromolar range. This high affinity for Ca 2؉ results from Ca 2؉ -independent association of the SK ␣-subunit with calmodulin (CaM), a property unique among the large family of potassium channels. Here we report the solution structure of the calmodulin binding domain (CaMBD, residues 396 -487 in rat SK2) of SK channels using NMR spectroscopy. The CaMBD exhibits a helical region between residues 423-437, whereas the rest of the molecule lacks stable overall folding. Disruption of the helical domain abolishes constitutive association of CaMBD with Ca 2؉ -free CaM, and results in SK channels that are no longer gated by Ca 2؉ . The results show that the Ca 2؉ -independent CaM-CaMBD interaction, which is crucial for channel function, is at least in part determined by a region different in sequence and structure from other CaMinteracting proteins.

The solution structure of the paramagnetic seven-iron ferredoxin from Bacillus schlegelii in its oxidized form has been determined by 1H NMR. The protein, which contains 77 amino acids, is thermostable. Seventy-two residues and 79% of all theoretically expected proton resonances have been assigned. The structure has been determined through torsion angle dynamics calculations with the program DYANA, using 966 meaningful NOEs (from a total of 1305), hydrogen bond constraints, and NMR derived dihedral angle constraints for the cluster ligating cysteines, and by using crystallographic information to build up the two clusters. Afterwards, restrained energy minimization and restrained molecular dynamics were applied to each conformer of the family. The final family of 20 structures has RMSD values from the mean structure of 0.68 A for the backbone atoms and of 1.16 A for all heavy atoms. The contributions to the thermal stability of the B. schlegelii ferredoxin are discussed by comparing the present structure to that of the less stable Azotobacter vinelandii ferredoxin I which is the only other available structure of a bacterial seven-iron ferredoxin. It is proposed that the hydrophobic interactions and the hydrogen bond network linking the N-terminus and the C-terminus together and a high number of salt bridges contribute to the stability.

Cumulative inactivation of voltage-gated (Kv) K؉ channels shapes the presynaptic action potential and determines timing and strength of synaptic transmission. Kv1.4 channels exhibit rapid "ball-and-chain"-type inactivation gating. Different from all other Kv␣ subunits, Kv1.4 harbors two inactivation domains at its N terminus. Here we report the solution structure and function of this "tandem inactivation domain" using NMR spectroscopy and patch clamp recordings. Inactivation domain 1 (ID1, residues 1-38) consists of a flexible N terminus anchored at a 5-turn helix, whereas ID2 (residues 40 -50) is a 2.5-turn helix made up of small hydrophobic amino acids. Functional analysis suggests that only ID1 may work as a pore-occluding ball domain, whereas ID2 most likely acts as a "docking domain" that attaches ID1 to the cytoplasmic face of the channel. Deletion of ID2 slows inactivation considerably and largely impairs cumulative inactivation. Together, the concerted action of ID1 and ID2 may promote rapid inactivation of Kv1.4 that is crucial for the channel function in short term plasticity.

Heteronuclear multidimensional NMR spectroscopy was used to investigate in detail the structural and dynamical properties of a partially unfolded intermediate of the reduced high-potential iron-sulfur protein (HiPIP) from Chromatium vinosum present in 4 M guanidinium chloride solution. After an extensive assignment of 15N and 1H resonances, NOE data, proton longitudinal relaxation times, and 3JHNHalpha coupling constants as well as 15N relaxation parameters (T1, T2, T1rho, and 1H-15N NOE) were obtained and used to build a structural model of the intermediate. The Fe4S4 cluster of the HiPIP plays a decisive role in determining the resulting structure, which is random in the N-terminal half of the protein and partially organized in the loops between the cysteines bound to the cluster. Consistent with the structural data, the backbone mobility is typical of folded proteins in the regions where there are elements of structure and increases with the structural indetermination.

The solution structure of the 60-residue 1 [Fe,-S,] ferredoxin from the hyperthermophilic bacterium Thermotoga maritima was determined based on 683 distance and 35 dihedral angle restraints that were obtained from NMR data. In addition, data known from crystallographic studies of ferredoxins was used for modeling of the iron-sulfur cluster and its environment. The protein shows a globular fold very similar to the fold of the related 1 [Fe,-S,] ferredoxins from Desulfovibrio gigas and Desuljovibrio africanus, and elements of regular secondary structure similar to those in other ferredoxins were found in the 2: maritima protein. In particular, the ?: rnaritimu protein displayed a @-sheet structure made up of strands located at the very NH, and COOH termini of the protein, and an internal rx-helix. The internal @-sheet observed in the D. gigas and D. africunus ferredoxins could not be confirmed in 7: muritima ferredoxin and is thus suggested to be only weakly present or even absent in this protein. This result suggests that thermostability in ferredoxins is not necessarily correlated with the content of stable elements of regular secondary structure.Keywords; Ferredoxin; NMR ; protein structure ; iron-sulfur proteins ; Thermotoga maritima. Thermotoga maritima is a hyperthermophilic bacterium (Stetter et al., 1990) with a maximal growth temperature of 90°C (Huber et al., 1986). Together with Aquifex pyrophilus (Huber et al., 1992), 7: maritima is the only known exception to the rule that hyperthermophilic microorganisms can be classified as Archaea. In addition, as 7: maritima and A. pyrophilus represent the most original and most slowly evolving branches of bacteria, these species may have retained more characteristic properties of the presumed last common ancestor of life than most other known organisms.Ferredoxins are small iron-sulfur electron transport proteins that are involved in crucial metabolic processes such as photosynthesis, oxidative phosphorylation, or nitrogen fixation (for reviews see Beinert, 1990;Matsubara and Saeki, 1992). The function of these proteins is accomplished by Fe,S, clusters (mainly in plants and animals) or Fe,S, (Fe,S,) clusters (mainly in prokaryotes).Ferredoxin from 7: muritima (Fd,,) functions as an electron carrier for pyruvate oxidoreductase (Blarney et al., 1994). Recently, the gene encoding Fd,,,, was cloned and expressed in Escherichia coli (Darimont and Sterner, 1994). These studies Bayreuth, D-95440 Bayreuth, Germany revealed that Fd.,, contains a single Fe,S, cluster and a disulfide bond. Fd.,.,, is extremely thermostable and shows high sequence similarities to ferredoxins of hyperthermophilic Archaea, with identities of 73% and 45% between FdT,, and the proteins from Thermococcus litoralis (Fd,,) and Pyr;ococcus furiosus (Fd,,;Busse et al., 1992), respectively ( Table 1). The sequence identity to ferredoxins from mesophilic bacteria such as Desulfovibrio gigas (Fd,,;Bruschi, 1979;Travis et al., 1971) and Desulfovibrio africanus (Fd,,;Bruschi and Hatschikian, 1982) is 3...