PKA type IIalpha holoenzyme reveals a combinatorial strategy for isoform diversity

The catalytic (C) subunit of cyclic adenosine monophosphate (cAMP)-dependent protein kinase (PKA) is inhibited by two classes of regulatory subunits, RI and RII. The RII subunits are substrates as well as inhibitors and do not require adenosine triphosphate (ATP) to form holoenzyme, which distinguishes them from RI subunits. To understand the molecular basis for isoform diversity, we solved the crystal structure of an RIIalpha holoenzyme and compared it to the RIalpha holoenzyme. Unphosphorylated RIIalpha(90-400), a deletion mutant, undergoes major conformational changes as both of the cAMP-binding domains wrap around the C subunit's large lobe. The hallmark of this conformational reorganization is the helix switch in domain A. The C subunit is in an open conformation, and its carboxyl-terminal tail is disordered. This structure demonstrates the conserved and isoform-specific features of RI and RII and the importance of ATP, and also provides a new paradigm for designing isoform-specific activators or antagonists for PKA.     

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.     

Structure of the nucleotide activation switch in glycogen phosphorylase a

Adenosine monophosphate is required for the activation of glycogen phosphorylase b and for release of the inhibition of phosphorylase a by glucose. Two molecules of adenosine monophosphate (AMP) bind to symmetry related sites at the subunit interface of the phosphorylase dimer. Adenosine triphosphate (ATP) binds to the same site, but does not promote catalytic activity. The structure of glucose-inhibited phosphorylase a bound to AMP and also of the complex formed with glucose and ATP is described. Crystallographic refinement of these complexes reveals that structural changes are associated with AMP but not ATP binding. The origin of these effects can be traced to different effector binding modes exhibited by AMP and ATP, respectively. The conformational changes associated with AMP binding traverse multiple paths in the enzyme and link the effector and catalytic sites.     

Structure of a peptide inhibitor bound to the catalytic subunit of cyclic adenosine monophosphate-dependent protein kinase

The structure of a 20-amino acid peptide inhibitor bound to the catalytic subunit of cyclic AMP-dependent protein kinase, and its interactions with the enzyme, are described. The x-ray crystal structure of the complex is the basis of the analysis. The peptide inhibitor, derived from a naturally occurring heat-stable protein kinase inhibitor, contains an amphipathic helix that is followed by a turn and an extended conformation. The extended region occupies the cleft between the two lobes of the enzyme and contains a five-residue consensus recognition sequence common to all substrates and peptide inhibitors of the catalytic subunit. The helical portion of the peptide binds to a hydrophobic groove and conveys high affinity binding. Loops from both domains converge at the active site and contribute to a network of conserved residues at the sites of magnesium adenosine triphosphate binding and catalysis. Amino acids associated with peptide recognition, nonconserved, extend over a large surface area.     

Domain separation in the activation of glycogen phosphorylase a

The crystal structure of glycogen phosphorylase a complexed with its substrates, orthophosphate and maltopentaose, has been determined and refined at a resolution of 2.8 angstroms. With oligosaccaride bound at the glycogen storage site, the phosphate ion binds at the catalytic site and causes the regulatory and catalytic domains to separate with the loss of stabilizing interactions between them. Homotropic cooperativity between the active sites of the allosteric dimer results from rearrangements in isologous contacts between symmetry-related helices in the subunit interface. The conformational changes in the core of the interface are correlated with those observed on covalent activation by phosphorylation at Ser14 (phosphorylase b----a).     

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.     

灰葡萄孢犬尿氨酸单加氧酶基因BcKMO与病菌cAMP信号途径的关系

【目的】明确灰葡萄孢（Botrytis cinerea）犬尿氨酸单加氧酶基因BcKMO与病菌cAMP信号途径之间的关系,为进一步阐明BcKMO调控灰葡萄孢生长、发育和致病力的分子机制打下基础。【方法】利用cAMP信号途径特异性抑制剂SQ22536,检测灰葡萄孢野生型BC22、BcKMO的T-DNA插入突变体BCG183和回复菌株BCG183/BcKMO对cAMP信号途径特异性抑制剂的敏感性;分别提取灰葡萄孢野生型BC22、突变体BCG183和回复菌株BCG183/BcKMO中的cAMP,利用HPLC检测各菌株中cAMP的含量;利用real-time PCR技术检测BcKMO与cAMP信号途径中cAMP依赖的蛋白激酶（PKA）的催化亚基基因pka1和pka2、调节亚基基因pkaR、编码异源三聚体G-蛋白Gα亚基基因bcg2和bcg3在病菌不同发育阶段、组织部位以及培养条件下的表达规律;利用real-time PCR技术检测BcKMO突变体中cAMP信号途径关键基因pka1、pka2、pkaR、bcg2、bcg3的表达水平;利用real-time PCR技术检测cAMP信号途径关键基因pka1、pka2、pkaR、bcg2、bcg3的RNAi突变中BcKMO的表达水平。【结果】灰葡萄孢BcKMO的T-DNA插入突变体BCG183对cAMP信号途径特异性抑制剂SQ22536不敏感,受抑制的程度明显低于野生型BC22和回复菌株BCG183/BcKMO。BcKMO的T-DNA插入突变体BCG183中cAMP含量明显低于野生型菌株BC22和回复菌株BCG183/BcKMO。BcKMO与cAMP信号途径中cAMP依赖的蛋白激酶（PKA）的催化亚基基因pka1、编码异源三聚体G-蛋白Gα亚基基因bcg2和bcg3的表达规律基本一致,均为在7 d的菌丝和菌核中表达水平较高;此外,BcKMO和cAMP信号途径关键基因pka1、pka2、pkaR、bcg2、bcg3均为在含果糖培养基上培养的病菌中表达水平较高。BcKMO的T-DNA插入突变体BCG183中cAMP信号途径关键基因pka1、pka2、pkaR、bcg2、bcg3的表达水平均明显高于野生型BC22和回复菌株BCG183/BcKMO。pka1、bcg2的RNAi突变体中BcKMO表达水平明显高于野生型,而pka2、pkaR、bcg3的RNAi突变体中BcKMO表达水平明显低于野生型。【结论】BcKMO负调控cAMP信号途径关键基因pka1、pka2、pkaR、bcg2、bcg3的表达;cAMP信号途径关键基因pka1、bcg2负调控BcKMO的表达,而cAMP信号途径关键基因pka2、pkaR、bcg3正调控BcKMO的表达。     

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 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.     

In vivo receptor-mediated phosphorylation of a G protein in Dictyostelium

Extracellular adenosine 3',5'-monophosphate (cAMP) serves multiple roles in Dictyostelium development, acting as a chemoattractant, a cell-cell signaling molecule, and an inducer of differentiation. The Dictyostelium G-protein alpha subunit G alpha 2 appears to be the major transducer linking the surface cAMP receptor to these intracellular responses. On stimulation of cells with cAMP, G alpha 2 is phosphorylated on one or more serine residues, resulting in an alteration of its electrophoretic mobility. Phosphorylation of G alpha 2 is triggered by increased occupancy of the surface cAMP receptor and is rapid and transient, coinciding with the time course of activation of physiological responses.     

Heterodimeric GTPase core of the SRP targeting complex

Two structurally homologous guanosine triphosphatase (GTPase) domains interact directly during signal recognition particle (SRP)-mediated cotranslational targeting of proteins to the membrane. The 2.05 angstrom structure of a complex of the NG GTPase domains of Ffh and FtsY reveals a remarkably symmetric heterodimer sequestering a composite active site that contains two bound nucleotides. The structure explains the coordinate activation of the two GTPases. Conformational changes coupled to formation of their extensive interface may function allosterically to signal formation of the targeting complex to the signal-sequence binding site and the translocon. We propose that the complex represents a molecular "latch" and that its disengagement is regulated by completion of assembly of the GTPase active site.     

Midbody targeting of the ESCRT machinery by a noncanonical coiled coil in CEP55

The ESCRT (endosomal sorting complex required for transport) machinery is required for the scission of membrane necks in processes including the budding of HIV-1 and cytokinesis. An essential step in cytokinesis is recruitment of the ESCRT-I complex and the ESCRT-associated protein ALIX to the midbody (the structure that tethers two daughter cells) by the protein CEP55. Biochemical experiments show that peptides from ALIX and the ESCRT-I subunit TSG101 compete for binding to the ESCRT and ALIX-binding region (EABR) of CEP55. We solved the crystal structure of EABR bound to an ALIX peptide at a resolution of 2.0 angstroms. The structure shows that EABR forms an aberrant dimeric parallel coiled coil. Bulky and charged residues at the interface of the two central heptad repeats create asymmetry and a single binding site for an ALIX or TSG101 peptide. Both ALIX and ESCRT-I are required for cytokinesis, which suggests that multiple CEP55 dimers are required for function.     

Evidence for a novel thioredoxin-like catalytic property of gonadotropic hormones

It has been proposed that dithiol-disulfide interchange and oxidation-reduction reactions may play a role in hormone-induced receptor activation. Inspection of the sequences of the gonadotropic hormones revealed a homologous tetrapeptide (Cys-Gly-Pro-Cys) between the beta subunit of lutropin (LH) and the active site of thioredoxin (TD). The beta subunit of follitropin (FSH) has a similar sequence (Cys-Gly-Lys-Cys). Thioredoxin is a ubiquitous protein serving as an electron donor for ribonucleotide reductase, but it also exhibits disulfide isomerase activity. The catalytic activity of TD was assayed by its ability to reactivate reduced and denatured ribonuclease. In this assay, the purified ovine FSH and bovine LH preparations tested were approximately 60 and approximately 300 times, respectively, as active as TD on a molar basis. This heretofore unsuspected catalytic property of FSH and LH may be important in understanding their mechanism of receptor activation and signal transduction.     

Molecular mimicry regulates ABA signaling by SnRK2 kinases and PP2C phosphatases

Abscisic acid (ABA) is an essential hormone for plants to survive environmental stresses. At the center of the ABA signaling network is a subfamily of type 2C protein phosphatases (PP2Cs), which form exclusive interactions with ABA receptors and subfamily 2 Snfl-related kinase (SnRK2s). Here, we report a SnRK2-PP2C complex structure, which reveals marked similarity in PP2C recognition by SnRK2 and ABA receptors. In the complex, the kinase activation loop docks into the active site of PP2C, while the conserved ABA-sensing tryptophan of PP2C inserts into the kinase catalytic cleft, thus mimicking receptor-PP2C interactions. These structural results provide a simple mechanism that directly couples ABA binding to SnRK2 kinase activation and highlight a new paradigm of kinase-phosphatase regulation through mutual packing of their catalytic sites.     

Structural basis for the activation of cholera toxin by human ARF6-GTP

The Vibrio cholerae bacterium causes devastating diarrhea when it infects the human intestine. The key event is adenosine diphosphate (ADP)-ribosylation of the human signaling protein GSalpha, catalyzed by the cholera toxin A1 subunit (CTA1). This reaction is allosterically activated by human ADP-ribosylation factors (ARFs), a family of essential and ubiquitous G proteins. Crystal structures of a CTA1:ARF6-GTP (guanosine triphosphate) complex reveal that binding of the human activator elicits dramatic changes in CTA1 loop regions that allow nicotinamide adenine dinucleotide (NAD+) to bind to the active site. The extensive toxin:ARF-GTP interface surface mimics ARF-GTP recognition of normal cellular protein partners, which suggests that the toxin has evolved to exploit promiscuous binding properties of ARFs.