Interaction of asymmetric ABCC9-encoded nucleotide binding domains determines KATP channel SUR2A catalytic activity

Nucleotide binding domains (NBDs) secure ATP-binding cassette (ABC) transporter function. Distinct from traditional ABC transporters, ABCC9-encoded sulfonylurea receptors (SUR2A) form, with Kir6.2 potassium channels, ATP-sensitive K+ (K ATP) channel complexes. SUR2A contains ATPase activity harbored within NBD2 and, to a lesser degree, NBD1, with catalytically driven conformations exerting determinate linkage on the Kir6.2 channel pore. While homodomain interactions typify NBDs of conventional ABC transporters, heterodomain NBD interactions and their functional consequence have not been resolved for the atypical SUR2A protein. Here, nanoscale protein topography mapped assembly of monodisperse purified recombinant SUR2A NBD1/NBD2 domains, precharacterized by dynamic light scattering. Heterodomain interaction produced conformational rearrangements inferred by secondary structural change in circular dichroism, and validated by atomic force and transmission electron microscopy. Physical engagement of NBD1 with NBD2 translated into enhanced intrinsic ATPase activity. Molecular modeling delineated a complemental asymmetry of NBD1/NBD2 ATP-binding sites. Mutation in the predicted catalytic base residue, D834E of NBD1, altered NBD1 ATPase activity disrupting potentiation of catalytic behavior in the NBD1/NBD2 interactome. Thus, NBD1/NBD2 assembly, resolved by a panel of proteomic approaches, provides a molecular substrate that determines the optimal catalytic activity in SUR2A, establishing a paradigm for the structure-function relationship within the K ATP channel complex.     

Quaternary structure of flavorubredoxin as revealed by synchrotron radiation small-angle X-ray scattering

Flavodiiron proteins (FDP) are modular enzymes which function as NO and/or O(2) reductases. Although the majority is composed of two structural domains, the homolog found in Escherichia coli, flavorubredoxin, possesses an extra C-terminal module consisting of a linker and a rubredoxin (Rd) domain necessary for interprotein redox processes. In order to investigate the location of the Rd domain with respect to the flavodiiron structural core, small-angle X-ray scattering was used to construct low-resolution structural models of flavorubredoxin. Scattering patterns from the Rd domain, the FDP core, and full-length flavorubredoxin were collected. The latter two species were found to be tetrameric in solution. Ab initio shape reconstruction and rigid-body modeling indicate a peripheral location for the Rd domains, which appear to have weak contacts with the FDP core. This finding suggests that Rd behaves as an independent domain and is freely available to participate in redox reactions with protein partners.     

Regulation of KATP channel expression and activity by the SUR1 nucleotide binding fold 1

ATP-sensitive K(+) (K(ATP)) channels are oligomeric complexes of pore-forming Kir6 subunits and regulatory Sulfonylurea Receptor (SUR) subunits. SUR, an ATP-Binding Cassette (ABC) transporter, confers Mg-nucleotide stimulation to the channel via nucleotide interactions with its two cytoplasmic domains (Nucleotide Binding Folds 1 and 2; NBF1 and NBF2). Regulation of K(ATP) channel expression is a complex process involving subunit assembly in the ER, SUR glycosylation in the Golgi, and trafficking to the plasma membrane. Dysregulation can occur at different steps of the pathway, as revealed by disease-causing mutations. Here, we have addressed the role of SUR1 NBF1 in gating and expression of reconstituted channels. Deletion of NBF1 severely impairs channel expression and abolishes MgADP stimulation. Total SUR1 protein levels are decreased, suggestive of increased protein degradation, but they are not rescued by treatment with sulfonylureas or the proteasomal inhibitor MG-132. Similar effects of NBF1 deletion are observed in recombinant K(ATP) channels obtained by "splitting" SUR1 into two separate polypeptides (a N-terminal "half" and a C-terminal "half"). Interestingly, the location of the "splitting point" in the vicinity of NBF1 has marked effects on the MgADP stimulation of resulting channels. Finally, ablation of the ER retention motif upstream of NBF1 (in either "split" or full-length SUR1) does not rescue expression of channels lacking NBF1. These results indicate that, in addition to NBF1 being required for MgADP stimulation of the channel, it plays an important role in the regulation of channel expression that is independent of the ER retention checkpoint and the proteasomal degradation pathway.     

Quaternary structure of Azospirillum brasilense NADPH-dependent glutamate synthase in solution as revealed by synchrotron radiation x-ray scattering

Azospirillum brasilense glutamate synthase (GltS) is the prototype of bacterial NADPH-dependent enzymes, a class of complex iron-sulfur flavoproteins essential in ammonia assimilation processes. The catalytically active GltS alpha beta holoenzyme and its isolated alpha and beta subunits (162 and 52 kDa, respectively) were analyzed using synchrotron radiation x-ray solution scattering. The GltS alpha subunit and alpha beta holoenzyme were found to be tetrameric in solution, whereas the beta subunit was a mixture of monomers and dimers. Ab initio low resolution shapes restored from the scattering data suggested that the arrangement of alpha subunits in the (alpha beta)4 holoenzyme is similar to that in the tetrameric alpha 4 complex and that beta subunits occupy the periphery of the holoenzyme. The structure of alpha 4 was further modeled using the available crystallographic coordinates of the monomeric alpha subunit assuming P222 symmetry. To model the entire alpha beta holoenzyme, a putative alpha beta protomer was constructed from the coordinates of the alpha subunit and those of the N-terminal region of porcine dihydropyrimidine dehydrogenase, which is similar to the beta subunit. Rigid body refinement yielded a model of GltS with an arrangement of alpha subunits similar to that in alpha 4, but displaying contacts also between beta subunits belonging to adjacent protomers. The holoenzyme model allows for independent catalytic activity of the alpha beta protomers, which is consistent with the available biochemical evidence.     

Activation of the KATP channel by Mg-nucleotide interaction with SUR1

The mechanism of adenosine triphosphate (ATP)-sensitive potassium (K_ATP) channel activation by Mg-nucleotides was studied using a mutation (G334D) in the Kir6.2 subunit of the channel that renders K_ATP channels insensitive to nucleotide inhibition and has no apparent effect on their gating. K_ATP channels carrying this mutation (Kir6.2-G334D/SUR1 channels) were activated by MgATP and MgADP with an EC₅₀ of 112 and 8 µM, respectively. This activation was largely suppressed by mutation of the Walker A lysines in the nucleotide-binding domains of SUR1: the remaining small (∼10%), slowly developing component of MgATP activation was fully inhibited by the lipid kinase inhibitor {"type":"entrez-nucleotide","attrs":{"text":"LY294002","term_id":"1257998346","term_text":"LY294002"}}LY294002. The EC₅₀ for activation of Kir6.2-G334D/SUR1 currents by MgADP was lower than that for MgATP, and the time course of activation was faster. The poorly hydrolyzable analogue MgATPγS also activated Kir6.2-G334D/SUR1. AMPPCP both failed to activate Kir6.2-G334D/SUR1 and to prevent its activation by MgATP. Maximal stimulatory concentrations of MgATP (10 mM) and MgADP (1 mM) exerted identical effects on the single-channel kinetics: they dramatically elevated the open probability (P_O > 0.8), increased the mean open time and the mean burst duration, reduced the frequency and number of interburst closed states, and eliminated the short burst states. By comparing our results with those obtained for wild-type K_ATP channels, we conclude that the MgADP sensitivity of the wild-type K_ATP channel can be described quantitatively by a combination of inhibition at Kir6.2 (measured for wild-type channels in the absence of Mg²⁺) and activation via SUR1 (determined for Kir6.2-G334D/SUR1 channels). However, this is not the case for the effects of MgATP.     

PKA phosphorylation of SUR2B subunit underscores vascular KATP channel activation by beta-adrenergic receptors

ATP-sensitive K(+) (K(ATP)) channels are activated by several vasodilating hormones and neurotransmitters through the PKA pathway. Here, we show that phosphorylation at Ser1387 of the SUR2B subunit is critical for the channel activation. Experiments were performed in human embryonic kidney (HEK) 293 cells expressing the cloned Kir6.1/SUR2B channel. In whole cell patch, the Kir6.1/SUR2B channel activity was stimulated by isoproterenol via activation of beta(2) receptors. This effect was blocked in the presence of inhibitors for adenylyl cyclase or PKA. Similar channel activation was seen by exposing inside-out patches to the catalytic subunit of PKA. Because none of the previously suggested PKA phosphorylation sites accounted for the channel activation, we performed systematic mutational analysis on Kir6.1 and SUR2B. Two serine residues (Ser1351, Ser1387) located in the NBD2 of SUR2B were critical for the channel activation. In vitro phosphorylation experiments showed that Ser1387 but not Ser1351 was phosphorylated by PKA. The PKA-dependent activation of cell-endogenous K(ATP) channels was observed in acutely dissociated mesenteric smooth myocytes and isolated mesenteric artery rings, where activation of these channels contributed significantly to the isoproterenol-induced vasodilation. Taken together, these results indicate that the Kir6.1/SUR2B channel is a target of beta(2) receptors and that the channel activation relies on PKA phosphorylation of SUR2B at Ser1387.     

Time-resolved synchrotron radiation X-ray solution scattering study of DNA melting

Time resolved x-ray solution scattering measurements were made during thermal denaturation of DNA from various sources in the temperature range of 20-90 degrees C. Preliminary results on the influence of fragment length, ionic strength, and origin of the DNA on the time course of the scattering are described. Interpretation is based on model calculations of the scattering patterns. The results indicate that, for long DNA fragments at very low ionic strength, the melting process is a continuous phenomenon over the whole temperature range. It is accompanied by a progressive decrease of the radius of gyration of the cross section and of the mass per unit length. For short fragments of 146 base pair nucleosomal core DNA, stiffening of the DNA appears to precede a sharp melting transition.     

Observation of RecA protein monomer by small angle X-ray scattering with synchrotron radiation

RecA protein is capable of forming homo-oligomers in solution. The oligomeric and monomeric states of Thermus thermophilus RecA protein were studied by small angle X-ray scattering, a direct method used to measure the overall dimensions of a macromolecule. In the presence of 3 M urea or 0.2 M lithium perchlorate, RecA dissociates from higher oligomeric states to form a hexamer with a radius of gyration (R(g)) of 52 A. The value of R(g) decreased to 36 A at a higher lithium perchlorate concentration (1.0 M). The zero angle intensity, I(0), was consistent with the identification of the former state as a hexamer and the latter as a monomer.     

Quaternary structure changes in aspartate transcarbamylase studied by X-ray solution scattering. Signal transmission following effector binding

The result of binding the effectors ATP and CTP to aspartate transcarbamylase was studied by X-ray solution scattering. Binding of substrate analogues produces a substantial change in the solution scattering curve, allowing us to monitor the proportion of the different quaternary structure states present in solution. In the initial solution this ratio was made roughly unity by adding either carbamyl phosphate and succinate, or N-(phosphonacetyl)-L-aspartate (PALA). ATP or CTP were then added, and their effect on the proportion of the different quaternary structure states was followed. When using carbamyl phosphate and succinate (weakly bound), ATP or CTP had a clear effect, as observed previously by monitoring the sedimentation rate (Changeux et al., 1968). However, when PALA (strongly bound) was used, the effect of CTP was very much smaller, and that of ATP was undetectable. This result supports the explanation by Tauc et al. (1982), that nucleotides act mostly through changing the affinity of the active sites for substrate, and only to a small extent by directly modifying the quaternary structure equilibrium in the case of CTP.     

Quaternary structure of alpha-crustacyanin from lobster as seen by small-angle X-ray scattering

The structure of alpha-crustacyanin, the blue carotenoprotein of lobster (Homarus gammarus) carapace, has been investigated for the first time using small-angle X-ray scattering. In this paper, we have determined the dimensions of this protein composed of eight heterodimeric subunits of beta-crustacyanin. Analysis of the scattering spectra and estimation of the shape of alpha-crustacyanin show that the protein fits into a cylinder with an axial length of 238 A and a radius of 47.5 A, in which the eight beta-crustacyanin molecules are probably arranged in a helical manner.     

A synchrotron X-ray scattering characterization of purified tubulin and of its expansion induced by mild detergent binding

This report presents a synchrotron radiation X-ray scattering characterization of calf brain tubulin purified by the modified Weisenberg procedure. The results show that under nonassembly conditions (i.e., in 10 mM sodium phosphate and 0.1 mM GTP, pH 7, buffer) these preparations consist of a uniform population of molecules with a radius of gyration of 3.1 +/- 0.1 nm, which can be interpreted as arising from the native alpha-beta heterodimer. The uniformity in the population persists even at unusually high concentrations of protein. Binding of colchicine or substitution of GTP by GDP does not induce, within the statistical accuracy and resolution range of our measurements, any significant structural modification in soluble tubulin. In assembly buffer [i.e., 10 mM sodium phosphate, 6 mM magnesium chloride, 1 mM [ethylenebis(oxyethylenenitrilo)]tetraacetic acid, 1 mM GTP, and 3.4 M glycerol, pH 6.5], these preparations readily assemble into microtubules upon increasing the temperature from 4 to 37 degrees C. Binding of nondenaturing amphiphiles to soluble tubulin provides a simplified model for tubulin-membrane interactions. The X-ray scattering data show that the radius of gyration of tubulin progressively increases upon binding of the mild detergent sodium deoxycholate, reaching a maximum value of 4.3 +/- 0.1 nm at detergent saturation. The relative increase in the radius of gyration coincides within experimental error with the previously determined relative increase in the frictional coefficient [Andreu, J.M., & Mu?oz, J.A. (1986) Biochemistry 25, 5220-5230]. Analysis of these observations suggests that the effect of detergent binding is to induce an isotropic swelling of the protein structure.     

Nuclear forward scattering of synchrotron radiation by deoxymyoglobin

Nuclear forward scattering of synchrotron radiation is used to determine the quadrupole splitting and the mean square displacement of the iron atom in deoxymyoglobin in the temperature range between 50 K and 243 K. Above 200 K an abnormally fast decay of the forward scattered intensity at short times after the synchrotron flash is observed, which is caused by protein-specific motions. The results strongly support the picture that protein dynamics seen at the position of the iron can be understood by harmonic motions in the low temperature regime while in the physiological regime diffusive motions in limited space are present. The shape of the resonance broadening function is investigated. An inhomogeneous broadening with a Lorentzian distribution indicating dipole interactions results in a better agreement with the experimental data than the common Gaussian distribution. Received: 30 August 1999 / Revised version: 22 October 1999 / Accepted: 6 December 1999     

Structural stability and solution structure of chaperonin GroES heptamer studied by synchrotron small-angle X-ray scattering

The GroES protein from Escherichia coli is a well-known member of the molecular chaperones. GroES consists of seven identical 10 kDa subunits, and forms a dome-like oligomeric structure. In order to obtain information on the structural stability and unfolding-refolding mechanism of GroES protein, especially at protein concentrations (0.4-1.2 mM GroES monomer) that would mimic heat stress conditions in vivo, we have performed synchrotron small-angle X-ray scattering (SAXS) experiments. Surprisingly, in spite of the high protein concentration, reversibility in the unfolding-refolding reaction was confirmed by SAXS experiments structurally. Although the unfolding-refolding reaction showed an apparent single transition with a Cm of 1.1 M guanidium hydrochloride, a more detailed analysis of this transition demonstrated that the unfolding mechanism could be best explained by a sequential three-state model, which consists of native heptamer, dissociated monomer, and unfolded monomer. Together with our previous result that GroES unfolded completely via a partially folded monomer according to a three-state model at low protein concentration (5 microM monomer), the unfolding-refolding mechanism of GroES protein could be explained uniformly by the three-state model from low to high protein concentrations. Furthermore, to clarify an ambiguity of the native GroES structure in solution, especially mobile loop structures, we have estimated a solution structure of GroES using SAXS profiles obtained from experiments and simulation analysis. The result suggested that the native structure of GroES in solution was very similar to that seen in GroES-GroEL complex determined by crystallography.     

Quaternary structure of immunoglobulin m: A model based on small-angle X-ray scattering data

This paper presents some data on a human Waldenström immunoglobulin M.IgM_GAL based on small-angle X-ray scattering data. the IgM_GAL had molecular weight 970 000, volume 1760 nm³, radius of gyration 12 nm and maximum diameter 37 nm. The conclusions from various model calculations are discussed. A flat, star-shaped model is compatible both with X-ray scattering data and electron micrographs.     

Aluminum site structure in serum transferrin and lactoferrin revealed by synchrotron radiation X-ray spectroscopy

The Al site structure of serum transferrin and lactoferrin is investigated using X-ray absorption near edge structure (XANES) spectroscopy. Al K-edge spectra in the mono- and dialuminum forms of the proteins have been recorded for the first time. Our results show that the aluminium ion is hexa-coordinated in an octahedral-like symmetry and that the monoaluminum form, where only the C-terminal binding site is saturated, has an increased structural distortion around the metal site.     

Quaternary structure of the chloride channel ClC-2

The chloride channel ClC-2 is thought to be essential for chloride homeostasis in neurons and critical for chloride secretion by the developing respiratory tract. In the present work, we investigated the quaternary structure of ClC-2 required to mediate chloride conduction. We found using chemical cross-linking and a novel PAGE system that tagged ClC-2 expressed in Sf9 cells exists as oligomers. Fusion of membranes from Sf9 cells expressing this protein confers double-barreled channel activity, with each pore exhibiting a unitary conductance of 32 pS. Polyhistidine-tagged ClC-2 from Sf9 cells can be purified as monomers, dimers, and tetramers. Purified, reconstituted ClC-2 monomers do not possess channel function whereas both purified ClC-2 dimers and tetramers do mediate chloride flux. In planar bilayers, reconstitution of dimeric ClC-2 leads to the appearance of a single, anion selective 32 pS pore, and tetrameric ClC-2 confers double-barreled channel activity similar to that observed in Sf9 membranes. These reconstitution studies suggest that a ClC-2 dimer is the minimum functional structure and that ClC-2 tetramers likely mediate double-barreled channel function.     

Syntaxin-1A inhibits cardiac KATP channels by its actions on nucleotide binding folds 1 and 2 of sulfonylurea receptor 2A

ATP-sensitive potassium (KATP) channels couple the metabolic status of the cell to its membrane potential to regulate a number of cell actions, including secretion (neurons and neuroendocrine cells) and muscle contractility (skeletal, cardiac, and vascular smooth muscle). KATP channels consist of regulatory sulfonylurea receptors (SUR) and pore-forming (Kir6.X) subunits. We recently reported (Pasyk, E. A., Kang, Y., Huang, X., Cui, N., Sheu, L., and Gaisano, H. Y. (2004) J. Biol. Chem. 279, 4234-4240) that syntaxin-1A (Syn-1A), known to mediate exocytotic fusion, was capable of binding the nucleotide binding folds (NBF1 and C-terminal NBF2) of SUR1 to inhibit the KATP channels in insulin-secreting pancreatic islet beta cells. This prompted us to examine whether Syn-1A might modulate cardiac SUR2A/KATP channels. Here, we show that Syn-1A is present in the plasma membrane of rat cardiac myocytes and binds the SUR2A protein (of rat brain, heart, and human embryonic kidney 293 cells expressing SUR2A/Kir6. 2) at its NBF1 and NBF2 domains to decrease KATP channel activation. Unlike islet beta cells, in which Syn-1A inhibition of the channel activity was apparently mediated only via NBF1 and not NBF2 of SUR1, both exogenous recombinant NBF1 and NBF2 of SUR2A were found to abolish the inhibitory actions of Syn-1A on K(ATP) channels in rat cardiac myocytes and HEK293 cells expressing SUR2A/Kir6.2. Together with our recent report, this study suggests that Syn-1A binds both NBFs of SUR1 and SUR2A but appears to exhibit distinct interactions with NBF2 of these SUR proteins in modulating the KATP channels in islet beta cells and cardiac myocytes.     

Small-angle X-ray scattering study of the ATP modulation of the structural features of the nucleotide binding domains of the CFTR in solution

Nucleotide binding domains (NBD1 and NBD2) of the cystic fibrosis transmembrane conductance (CFTR), the defective protein in cystic fibrosis, are responsible for controlling the gating of the chloride channel and are the putative binding site for several candidate drugs in the disease treatment. We studied the structural properties of recombinant NBD1, NBD2, and an equimolar NBD1/NBD2 mixture in solution by small-angle X-ray scattering. We demonstrated that NBD1 or NBD2 alone have an overall structure similar to that observed for crystals. Application of 2 mM ATP induces a dimerization of NBD1 but does not modify the NBD2 monomeric conformation. An equimolar mixture of NBD1/NBD2 in solution shows a dimeric conformation, and the application of ATP to the solution causes a conformational change in the NBD1/NBD2 complex into a tight heterodimer. We hypothesize that a similar conformation change occurs in situ and that transition is part of the gating mechanism. To our knowledge, this is the first direct observation of a conformational change of the NBD1/NBD2 interaction by ATP. This information may be useful to understand the physiopathology of cystic fibrosis.