AURELIA, a program for computer-aided analysis of multidimensional NMR spectra

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 ...

Section: Methodsmentioning

confidence: 99%

NMR Structure of the “Ball-and-chain” Domain of KCNMB2, the β2-Subunit of Large Conductance Ca2+- and Voltage-activated Potassium Channels

Bentrop

Beyermann

Wissmann

et al. 2001

“…Spectral assignment was performed by AURELIA (29) according to standard procedures using total correlation spectroscopy and DQF-COSY spectra for identification of spin systems and NOESY spectra for sequence-specific assignment. Amide-H ␣ coupling constants 3 J H NH␣ were obtained by fitting the corresponding DQF-COSY peaks to two antiphase Lorentzian functions.…”

Section: Methodsmentioning

confidence: 99%

NMR Structure and Functional Characteristics of the Hydrophilic N Terminus of the Potassium Channel β-Subunit Kvβ1.1

Wissmann

Baukrowitz

Kalbacher

et al. 1999

Rapid N-type inactivation of voltage-dependent potassium (Kv) channels controls membrane excitability and signal propagation in central neurons and is mediated by protein domains (inactivation gates) occluding the open channel pore from the cytoplasmic side. Inactivation domains (ID) are donated either by the pore-forming ␣-subunit or certain auxiliary ␤-subunits. Upon coexpression, Kv␤1.1 was found to endow non-inactivating members of the Kv1␣ family with fast inactivation via its unique N terminus. Here we investigated structure and functional properties of the Kv␤1.1 N terminus (amino acids 1-62, ␤N-(1-62)) using NMR spectroscopy and patch clamp recordings. ␤N-(1-62) showed all hallmarks of N-type inactivation: it inactivated non-inactivating Kv1.1 channels when applied to the cytoplasmic side as a synthetic peptide, and its interaction with the ␣-subunit was competed with tetraethylammonium and displayed an affinity in the lower micromolar range. In aequous and physiological salt solution, ␤N-(1-62) showed no well defined three-dimensional structure, it rather existed in a fast equilibrium of multiple weakly structured states. These structural and functional properties of ␤N-(1-62) closely resemble those of the "unstructured" ID from Shaker B, but differ markedly from those of the compactly folded ID of the Kv3.4 ␣-subunit.Fast N-type inactivation of voltage-gated potassium (Kv) 1 channels shapes the action potential, governs the firing rate (spiking), and controls signal propagation in central neurons (1). Biophysically, N-type inactivation has long served as the model for gating transitions in ion channels and is realized by a "ball plug-in" mechanism. In this mechanism a protein domain termed "inactivation gate" or "inactivation ball" binds to its receptor at the inner vestibule of the open channel and thereby occludes the ion pathway (2-5). Such inactivation gates have been localized in the N terminus of various Kv␣ subunits and were shown to be functional entities, i.e. they conferred rapid inactivation to "ball-less" Kv␣ subunits when applied to the cytoplasmic side of the channels as synthetic peptides (5-8). Identical to protein-harbored inactivation domains, the synthetic gates interacted with channels in the open state, blocked the pore with low voltage dependence, and were competed with the channel blocker tetraethylammonium (TEA) (9 -11). Recently, the structures of the inactivation domains (ID) from Kv1.4 and Kv3.4 were determined with NMR spectroscopy. Both IDs were found to exhibit well defined and compact folding in aequous solution (12). In contrast, the ID from Shaker B showed no unique, folded structure (13,14).Besides with Kv␣ subunits owning an N-terminal ID, fast inactivation was observed for a subset of non-inactivating Kv␣1 channels when coexpressed with certain ␤-subunits (15-17). These auxiliary subunits constitute a family of cytoplasmic proteins (subdivided into subfamilies Kv␤1, -2, and -3) that are made up of two distinct regions: a highly conserved core region that shows homolo...

“…Homonuclear two-dimensional experiments and 15 N/ 1 H-heteronuclear HSQC experiments were recorded following the Varian NMR suite with typically 2048 ϫ 512 data points. NMR spectra were converted to Bruker format and processed with XWINNMR and evaluated and plotted with Aurelia (35). The assignment strategy was based on the identification of individual resonance spin systems from homonuclear TOCSY experiments following the sequential path with homonuclear two-dimensional and 15 N-separated three-dimensional NOESY spectra similarly are described (36).…”

Section: Nmr Spectroscopy-nmr Experiments With Homonuclear Ormentioning

confidence: 99%

Biochemical Characterization of the Diaphanous Autoregulatory Interaction in the Formin Homology Protein FHOD1

Schönichen

Alexander

Gasteier

et al. 2006

Self Cite

Diaphanous related formins (DRFs) are cytoskeleton remodeling proteins that mediate specific upstream GTPase signals to regulate cellular processes such as cytokinesis, cell polarity, and organelle motility. Previous work on the Rho-interacting DRF mDia has established that the biological activity of DRFs is regulated by an autoinhibitory interaction of a C-terminal diaphanous autoregulatory domain (DAD) with the DRF N terminus. This autoinhibition is released upon competitive binding of an activated GTPase to the N terminus of the DRF. Analyzing autoregulation of the Rac1-interacting DRF FHOD1, we utilized in vitro binding studies to identify a 60-amino acid DAD at the protein C terminus that recognizes an N-terminal formin homology (FH) 3 domain. Importantly, the FH3 domain of FHOD1 does not overlap with the proposed Rac1-binding domain. The FHOD1 DAD was found to contain one functional hydrophobic autoregulatory motif, while a previously uncharacterized basic cluster that is conserved in all DRF family DADs also contributed to the FH3-DAD interaction. Simultaneous mutation of both motifs efficiently released autoinhibition of FHOD1 in NIH3T3 cells resulting in the formation of actin stress fibers and increased serum response element transcription. A second putative hydrophobic autoregulatory motif N-terminal of the DAD belongs to a unique FHOD subdomain of yet undefined function. NMR structural analysis and size exclusion chromatography experiments revealed that the FHOD1 DAD is intrinsically unstructured with a tendency for a helical conformation in the hydrophobic autoregulation motif. Together, these data suggest that in FHOD1, DAD acts as signal sequence for binding to the well folded and monomeric FH3 domain and imply an activation mechanism that differs from competitive binding of Rac1 and DAD to one interaction site.Formin proteins are involved in the regulation of many cytoskeletal processes including cytokinesis, actin cable and stress fiber formation, polarity establishment, neurite outgrowth, and intracellular trafficking (reviewed in Refs. 1 and 2). These functions are achieved by their ability to promote F-actin assembly at the filament barbed end and to move processively with the barbed end as it elongates (3-7). Formins are large proteins of typically more than 1000 amino acids that are defined by the presence of two conserved regions, namely the formin homology 1 and 2 (FH1 and FH2) 2 domains (1). Additional conserved domains such as a N-terminal GTPase-binding domain (GBD) and a C-terminal diaphanous autoregulation domain (DAD) were found to constitute a formin subfamily, the diaphanous related formins (DRFs) (8). Over the last years, DRFs have emerged as a group of proteins with the potential to bridge between G-protein signals and the cytoskeleton via their ability to bind activated small GTPases and to subsequently remodel the cytoskeleton (9 -13).At present, phylogenetic analyses of FH2 domains suggest that metazoan formins fall into seven groups, termed Dia (diaphanous), DAAM (dishevelle...