The alpha subunit of the GTP binding protein Gk opens atrial potassium channels

Guanine nucleotide binding (G) proteins (subunit composition alpha beta gamma) dissociate on activation with guanosine triphosphate (GTP) analogs and magnesium to give alpha-guanine nucleotide complexes and free beta gamma subunits. Whether the opening of potassium channels by the recently described Gk in isolated membrane patches from mammalian atrial myocytes was mediated by the alpha k subunit or beta gamma dimer was tested. The alpha k subunit was found to be active, while the beta gamma dimer was inactive in stimulating potassium channel activity. Thus, Gk resembles Gs, the stimulatory regulatory component of adenylyl cyclase, and transducin, the regulatory component of the visual system, in that it regulates its effector function--the activity of the ligand-gated potassium channel--through its guanine nucleotide binding subunit.     

The high-affinity E. coli methionine ABC transporter: structure and allosteric regulation

The crystal structure of the high-affinity Escherichia coli MetNI methionine uptake transporter, a member of the adenosine triphosphate (ATP)-binding cassette (ABC) family, has been solved to 3.7 angstrom resolution. The overall architecture of MetNI reveals two copies of the adenosine triphosphatase (ATPase) MetN in complex with two copies of the transmembrane domain MetI, with the transporter adopting an inward-facing conformation exhibiting widely separated nucleotide binding domains. Each MetI subunit is organized around a core of five transmembrane helices that correspond to a subset of the helices observed in the larger membrane-spanning subunits of the molybdate (ModBC) and maltose (MalFGK) ABC transporters. In addition to the conserved nucleotide binding domain of the ABC family, MetN contains a carboxyl-terminal extension with a ferredoxin-like fold previously assigned to a conserved family of regulatory ligand-binding domains. These domains separate the nucleotide binding domains and would interfere with their association required for ATP binding and hydrolysis. Methionine binds to the dimerized carboxyl-terminal domain and is shown to inhibit ATPase activity. These observations are consistent with an allosteric regulatory mechanism operating at the level of transport activity, where increased intracellular levels of the transported ligand stabilize an inward-facing, ATPase-inactive state of MetNI to inhibit further ligand translocation into the cell.     

Micelles protect membrane complexes from solution to vacuum

The ability to maintain interactions between soluble protein subunits in the gas phase of a mass spectrometer gives critical insight into the stoichiometry and interaction networks of protein complexes. Conversely, for membrane protein complexes in micelles, the transition into the gas phase usually leads to the disruption of interactions, particularly between cytoplasmic and membrane subunits, and a mass spectrum dominated by large aggregates of detergent molecules. We show that by applying nanoelectrospray to a micellar solution of a membrane protein complex, the heteromeric adenosine 5'-triphosphate (ATP)-binding cassette transporter BtuC2D2, we can maintain the complex intact in the gas phase of a mass spectrometer. Dissociation of either transmembrane (BtuC) or cytoplasmic (BtuD) subunits uncovers modifications to the transmembrane subunits and cooperative binding of ATP. By protecting a membrane protein complex within a n-dodecyl-beta-d-maltoside micelle, we demonstrated a powerful strategy that will enable the subunit stoichiometry and ligand-binding properties of membrane complexes to be determined directly, by precise determination of the masses of intact complexes and dissociated subunits.     

A mutation in the catalytic subunit of cAMP-dependent protein kinase that disrupts regulation

A mutant catalytic subunit of adenosine 3',5'-monophosphate (cAMP)-dependent protein kinase has been isolated from Saccharomyces cerevisiae that is no longer subject to regulation yet retains its catalytic activity. Biochemical analysis of the mutant subunit indicates a 100-fold decreased affinity for the regulatory subunit. The mutant catalytic subunit exhibits approximately a threefold increase in Michaelis constant for adenosine triphosphate and peptide cosubstrates, and is essentially unchanged in its catalytic rate. The nucleotide sequence of the mutant gene contains a single nucleotide change resulting in a threonine-to-alanine substitution at amino acid 241. This residue is conserved in other serine-threonine protein kinases. These results identify this threonine as an important contact between catalytic and regulatory subunits but only a minor contact in substrate recognition.     

Chimeric alpha 2-,beta 2-adrenergic receptors: delineation of domains involved in effector coupling and ligand binding specificity

The alpha 2 and beta 2 adrenergic receptors, both of which are activated by epinephrine, but which can be differentiated by selective drugs, have opposite effects (inhibitory and stimulatory) on the adenylyl cyclase system. The two receptors are homologous with each other, rhodopsin, and other receptors coupled to guanine nucleotide regulatory proteins and they contain seven hydrophobic domains, which may represent transmembrane spanning segments. The function of specific structural domains of these receptors was determined after construction and expression of a series of chimeric alpha 2-,beta 2-adrenergic receptor genes. The specificity for coupling to the stimulatory guanine nucleotide regulatory protein lies within a region extending from the amino terminus of the fifth hydrophobic domain to the carboxyl terminus of the sixth. Major determinants of alpha 2- and beta 2-adrenergic receptor agonist and antagonist ligand binding specificity are contained within the seventh membrane spanning domain. Chimeric receptors should prove useful for elucidating the structural basis of receptor function.     

A monoclonal antibody to the alpha subunit of Gk blocks muscarinic activation of atrial K+ channels

The activated heterotrimeric guanine nucleotide binding (G) protein Gk, at subpicomolar concentrations, mimics muscarinic stimulation of a specific atrial potassium current. Reconstitution studies have implicated the alpha and beta gamma subunits as mediators, but subunit coupling by the endogenous G protein has not been analyzed. To study this process, a monoclonal antibody (4A) that binds to alpha k but not to beta gamma was applied to the solution bathing an inside-out patch of atrial membrane; the antibody blocked carbachol-activated currents irreversibly. The state of the endogenous Gk determined its susceptibility to block by the antibody. When agonist was absent or when activation by muscarinic stimulation was interrupted by withdrawal of guanosine triphosphate (GTP) in the presence or absence of guanosine diphosphate (GDP), the effects of the antibody did not persist. Thus, monoclonal antibody 4A blocked muscarinic activation of potassium channels by binding to the activated G protein in its holomeric form or by binding to the dissociated alpha subunit.     

The MerR metalloregulatory protein binds mercuric ion as a tricoordinate, metal-bridged dimer

Bacterial MerR proteins are dimeric DNA-binding proteins that mediate the Hg(II)-dependent induction of mercury resistance operons. Site-directed mutagenesis of the Bacillus sp. RC607 MerR protein reveals that three of four Cys residues per monomer are required for Hg(II) binding at the single high-affinity binding site. Inactive mutant homodimers can exchange subunits to form heterodimers active for Hg(II) binding. Studies of a heterodimer retaining only three of eight cysteine residues per dimer reveal that Cys79 in one subunit and Cys114 and Cys123 in the second subunit are necessary and sufficient for high-affinity Hg(II) binding in an asymmetric, subunit bridging coordination complex.     

Hepatitis C virus IRES RNA-induced changes in the conformation of the 40s ribosomal subunit

Initiation of protein synthesis in eukaryotes requires recruitment of the 40S ribosomal subunit to the messenger RNA (mRNA). In most cases, this depends on recognition of a modified nucleotide cap on the 5' end of the mRNA. However, an alternate pathway uses a structured RNA element in the 5' untranslated region of the messenger or viral RNA called an internal ribosomal entry site (IRES). Here, we present a cryo-electron microscopy map of the hepatitis C virus (HCV) IRES bound to the 40S ribosomal subunit at about 20 A resolution. IRES binding induces a pronounced conformational change in the 40S subunit and closes the mRNA binding cleft, suggesting a mechanism for IRES-mediated positioning of mRNA in the ribosomal decoding center.     

Crystal structure of the catalytic subunit of cyclic adenosine monophosphate-dependent protein kinase

The crystal structure of the catalytic subunit of cyclic adenosine monophosphate-dependent protein kinase complexed with a 20-amino acid substrate analog inhibitor has been solved and partially refined at 2.7 A resolution to an R factor of 0.212. The magnesium adenosine triphosphate (MgATP) binding site was located by difference Fourier synthesis. The enzyme structure is bilobal with a deep cleft between the lobes. The cleft is filled by MgATP and a portion of the inhibitor peptide. The smaller lobe, consisting mostly of amino-terminal sequence, is associated with nucleotide binding, and its largely antiparallel beta sheet architecture constitutes an unusual nucleotide binding motif. The larger lobe is dominated by helical structure with a single beta sheet at the domain interface. This lobe is primarily involved in peptide binding and catalysis. Residues 40 through 280 constitute a conserved catalytic core that is shared by more than 100 protein kinases. Most of the invariant amino acids in this conserved catalytic core are clustered at the sites of nucleotide binding and catalysis.     

A G protein directly regulates mammalian cardiac calcium channels

A possible direct effect of guanine nucleotide binding (G) proteins on calcium channels was examined in membrane patches excised from guinea pig cardiac myocytes and bovine cardiac sarcolemmal vesicles incorporated into planar lipid bilayers. The guanosine triphosphate analog, GTP gamma S, prolonged the survival of excised calcium channels independently of the presence of adenosine 3',5'-monophosphate (cAMP), adenosine triphosphate, cAMP-activated protein kinase, and the protein kinase C activator tetradecanoyl phorbol acetate. A specific G protein, activated Gs, or its alpha subunit, purified from the plasma membranes of human erythrocytes, prolonged the survival of excised channels and stimulated the activity of incorporated channels. Thus, in addition to regulating calcium channels indirectly through activation of cytoplasmic kinases, G proteins can regulate calcium channels directly. Since they also directly regulate a subset of potassium channels, G proteins are now known to directly gate two classes of membrane ion channels.     

Crystal structure of a complex between the catalytic and regulatory (RIalpha) subunits of PKA

The 2.0-angstrom structure of the cyclic adenosine monophosphate (cAMP)-dependent protein kinase (PKA) catalytic subunit bound to a deletion mutant of a regulatory subunit (RIalpha) defines a previously unidentified extended interface. The complex provides a molecular mechanism for inhibition of PKA and suggests how cAMP binding leads to activation. The interface defines the large lobe of the catalytic subunit as a stable scaffold where Tyr247 in the G helix and Trp196 in the phosphorylated activation loop serve as anchor points for binding RIalpha. These residues compete with cAMP for the phosphate binding cassette in RIalpha. In contrast to the catalytic subunit, RIalpha undergoes major conformational changes when the complex is compared with cAMP-bound RIalpha. The inhibitor sequence docks to the active site, whereas the linker, also disordered in free RIalpha, folds across the extended interface. The beta barrel of cAMP binding domain A, which is the docking site for cAMP, remains largely intact in the complex, whereas the helical subdomain undergoes major reorganization.     

可结合Caco-2细胞表面蛋白的副溶血弧菌外膜蛋白的筛选与鉴定

[目的]筛选可结合Caco-2细胞表面蛋白的副溶血弧菌外膜蛋白。[方法]筛选副溶血弧菌外膜中的黏附蛋白,将Caco-2细胞表面蛋白生物素化并固定于中性卵白素树脂上,进一步利用亲和层析技术来筛选副溶血弧菌的外膜蛋白。[结果]通过LC-MS/MS质谱技术鉴定出3个候选蛋白:ATP synthase subunit alpha、ATP synthase subunit beta和outer membrane protein U。通过对这3个蛋白进行克隆基因和原核表达,成功纯化得到相应重组蛋白。通过进一步的间接免疫荧光试验,发现3种蛋白对于Caco-2细胞均有黏附作用。[结论]推测ATP synthase subunit alpha、ATP synthase subunit beta和outer membrane protein U这3种蛋白可能是潜在的黏附因子。     

The alpha subunit of the GTP binding protein activates muscarinic potassium channels of the atrium

It has been debated whether the potassium channel of the atrium is activated by the alpha subunit or by the beta gamma subunits of guanine nucleotide binding (G) proteins, which dissociate on activation with guanosine triphosphate (GTP). Therefore, the channel-activating effectiveness of these subunits on isolated guinea pig atrial cells was tested. The activated alpha K subunit from human erythrocytes activated the channel in subpicomolar concentrations. The beta gamma dimer from bovine brain activated the channel in nanomolar concentrations. These results support the view that, physiologically, the alpha subunit activates the channel.     

Subunit communication in the anthranilate synthase complex from Salmonella typhimurium

The anthranilate synthase-phosphoribosyl transferase complex of the tryptophan biosynthetic pathway in Salmonella typhimurium is an allosteric, heterotetrameric (TrpE2-TrpD2) enzyme whose multiple activities are negatively feedback-regulated by L-tryptophan. A hybrid complex containing one catalytically active, feedback-insensitive and one catalytically inactive, feedback-sensitive mutant TrpE subunit was assembled in vitro and used to investigate communication between regulatory and catalytic sites located on different subunits. The properties of the hybrid complex demonstrate that the binding of a single inhibitor molecule to one TrpE subunit is sufficient for the propagation of a conformational change that affects the active site of the companion subunit.     

Sequence of the alpha subunit of photoreceptor G protein: homologies between transducin, ras, and elongation factors

A bovine retinal complementary DNA clone encoding the alpha subunit of transducin (T alpha) was isolated with the use of synthetic oligodeoxynucleotides as probes, and the complete nucleotide sequence of the insert was determined. THe predicted protein sequence of 354 amino acids includes the known sequences of four tryptic peptides and sequences adjacent to the residues that undergo adenosine diphosphate ribosylation by cholera toxin and pertussis toxin. On the basis of homologies to other proteins, such as the elongation factors of protein synthesis and the ras oncogene proteins, regions are identified that are predicted to be acylated and involved in guanine nucleotide binding and hydrolysis. Amino acid sequence similarity between T alpha and ras is confined to these regions of the molecules.     

Crystal structure of a mammalian voltage-dependent Shaker family K+ channel

Voltage-dependent potassium ion (K+) channels (Kv channels) conduct K+ ions across the cell membrane in response to changes in the membrane voltage, thereby regulating neuronal excitability by modulating the shape and frequency of action potentials. Here we report the crystal structure, at a resolution of 2.9 angstroms, of a mammalian Kv channel, Kv1.2, which is a member of the Shaker K+ channel family. This structure is in complex with an oxido-reductase beta subunit of the kind that can regulate mammalian Kv channels in their native cell environment. The activation gate of the pore is open. Large side portals communicate between the pore and the cytoplasm. Electrostatic properties of the side portals and positions of the T1 domain and beta subunit are consistent with electrophysiological studies of inactivation gating and with the possibility of K+ channel regulation by the beta subunit.     

基于开放染色质的全基因组水平转录调控元件的研究方法与进展

真核生物转录调控过程是大量的顺式调控元件与反式作用因子相互作用的结果.研究发现,这一调控过程与染色质核小体的动态定位相关,调控因子的结合需要裸露的无核小体的DNA区域,即开放的染色质位点.因此,高效精确地定位基因组上的开放染色质位点为成功地发掘基因组调控元件,乃至揭示基因表达调控机制提供了重要线索和有效手段.本文对开放染色质位点的定义、主要研究方法以及功能注释进行概述,希望对在基因组水平上调控元件的发掘,尤其是在植物中的应用提供借鉴.     

Structure and allostery of the PKA RIIβ tetrameric holoenzyme

In its physiological state, cyclic adenosine monophosphate (cAMP)-dependent protein kinase (PKA) is a tetramer that contains a regulatory (R) subunit dimer and two catalytic (C) subunits. We describe here the 2.3 angstrom structure of full-length tetrameric RIIβ(2):C(2) holoenzyme. This structure showing a dimer of dimers provides a mechanistic understanding of allosteric activation by cAMP. The heterodimers are anchored together by an interface created by the β4-β5 loop in the RIIβ subunit, which docks onto the carboxyl-terminal tail of the adjacent C subunit, thereby forcing the C subunit into a fully closed conformation in the absence of nucleotide. Diffusion of magnesium adenosine triphosphate (ATP) into these crystals trapped not ATP, but the reaction products, adenosine diphosphate and the phosphorylated RIIβ subunit. This complex has implications for the dissociation-reassociation cycling of PKA. The quaternary structure of the RIIβ tetramer differs appreciably from our model of the RIα tetramer, confirming the small-angle x-ray scattering prediction that the structures of each PKA tetramer are different.     

A G protein-coupled receptor is a plasma membrane receptor for the plant hormone abscisic acid

The plant hormone abscisic acid (ABA) regulates many physiological and developmental processes in plants. The mechanism of ABA perception at the cell surface is not understood. Here, we report that a G protein-coupled receptor genetically and physically interacts with the G protein alpha subunit GPA1 to mediate all known ABA responses in Arabidopsis. Overexpressing this receptor results in an ABA-hypersensitive phenotype. This receptor binds ABA with high affinity at physiological concentration with expected kinetics and stereospecificity. The binding of ABA to the receptor leads to the dissociation of the receptor-GPA1 complex in yeast. Our results demonstrate that this G protein-coupled receptor is a plasma membrane ABA receptor.