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.     

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.     

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.     

Structure of complex of synthetic HIV-1 protease with a substrate-based inhibitor at 2.3 A resolution

The structure of a complex between a peptide inhibitor with the sequence N-acetyl-Thr-Ile-Nle-psi[CH2-NH]-Nle-Gln-Arg.amide (Nle, norleucine) with chemically synthesized HIV-1 (human immunodeficiency virus 1) protease was determined at 2.3 A resolution (R factor of 0.176). Despite the symmetric nature of the unliganded enzyme, the asymmetric inhibitor lies in a single orientation and makes extensive interactions at the interface between the two subunits of the homodimeric protein. Compared with the unliganded enzyme, the protein molecule underwent substantial changes, particularly in an extended region corresponding to the "flaps" (residues 35 to 57 in each chain), where backbone movements as large as 7 A are observed.     

The structure of a human p110alpha/p85alpha complex elucidates the effects of oncogenic PI3Kalpha mutations

PIK3CA, one of the two most frequently mutated oncogenes in human tumors, codes for p110alpha, the catalytic subunit of a phosphatidylinositol 3-kinase, isoform alpha (PI3Kalpha, p110alpha/p85). Here, we report a 3.0 angstrom resolution structure of a complex between p110alpha and a polypeptide containing the p110alpha-binding domains of p85alpha, a protein required for its enzymatic activity. The structure shows that many of the mutations occur at residues lying at the interfaces between p110alpha and p85alpha or between the kinase domain of p110alpha and other domains within the catalytic subunit. Disruptions of these interactions are likely to affect the regulation of kinase activity by p85 or the catalytic activity of the enzyme, respectively. In addition to providing new insights about the structure of PI3Kalpha, these results suggest specific mechanisms for the effect of oncogenic mutations in p110alpha and p85alpha.     

Protein kinase C contains a pseudosubstrate prototope in its regulatory domain

The regulatory domain of protein kinase C contains an amino acid sequence between residues 19 and 36 that resembles a substrate phosphorylation site in its distribution of basic residue recognition determinants. The corresponding synthetic peptide (Arg19-Phe-Ala-Arg-Lys-Gly-Ala25-Leu-Arg-Gln-Lys-Asn-Val-His -Glu-Val-Lys-Asn36) acts as a potent substrate antagonist with an inhibitory constant of 147 +/- 9 nM. It is a specific inhibitor of protein kinase C and inhibits both autophosphorylation and protein substrate phosphorylation. Substitution of Ala25 with serine transforms the pseudosubstrate into a potent substrate. These results demonstrate that the conserved region of the regulatory domain (residues 19 to 36) of protein kinase C has the secondary structural features of a pseudosubstrate and may be responsible for maintaining the enzyme in the inactive form in the absence of allosteric activators such as phospholipids.     

Primary structure of the beta subunit of the DHP-sensitive calcium channel from skeletal muscle

Complementary DNAs for the beta subunit of the dihydropyridine-sensitive calcium channel of rabbit skeletal muscle were isolated on the basis of peptide sequences derived from the purified protein. The deduced primary structure is without homology to other known protein sequences and is consistent with the beta subunit being a peripheral membrane protein associated with the cytoplasmic aspect of the sarcolemma. The protein contains sites that might be expected to be preferentially phosphorylated by protein kinase C and guanosine 3',5'-monophosphate-dependent protein kinase. A messenger RNA for this protein appears to be expressed in brain.     

Defect in phosphorylation of insulin receptors in cells from an insulin-resistant patient with normal insulin binding

Mononuclear blood cells were obtained from a patient with type A insulin resistance. The cells showed a normal ability to bind iodine 125-labeled insulin. Analysis of solubilized insulin receptors from the patient's cells revealed a defect in insulin-stimulated tyrosine kinase activity, which is closely associated with the receptor itself. The enzyme failed to phosphorylate the insulin receptor and showed a markedly reduced ability to phosphorylate exogenously added substrates. It appears that receptors from this insulin-resistant patient have a defect distal to the insulin-binding site (the alpha subunit of the receptor). The defect could be located in the beta subunit, which has an adenosine triphosphate-binding site, or in another receptor component that transfers a signal of insulin binding into kinase activity. This dissociation between the normal binding and the defective protein kinase component of the insulin receptor represents the first biochemical defect of the receptor distal to ligand binding.     

Structural mechanism for STI-571 inhibition of abelson tyrosine kinase

The inadvertent activation of the Abelson tyrosine kinase (Abl) causes chronic myelogenous leukemia (CML). A small-molecule inhibitor of Abl (STI-571) is effective in the treatment of CML. We report the crystal structure of the catalytic domain of Abl, complexed to a variant of STI-571. Critical to the binding of STI-571 is the adoption by the kinase of an inactive conformation, in which a centrally located "activation loop" is not phosphorylated. The conformation of this loop is distinct from that in active protein kinases, as well as in the inactive form of the closely related Src kinases. These results suggest that compounds that exploit the distinctive inactivation mechanisms of individual protein kinases can achieve both high affinity and high specificity.     

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.     

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.     

Crystal structure of the eukaryotic 60S ribosomal subunit in complex with initiation factor 6

Protein synthesis in all organisms is catalyzed by ribosomes. In comparison to their prokaryotic counterparts, eukaryotic ribosomes are considerably larger and are subject to more complex regulation. The large ribosomal subunit (60S) catalyzes peptide bond formation and contains the nascent polypeptide exit tunnel. We present the structure of the 60S ribosomal subunit from Tetrahymena thermophila in complex with eukaryotic initiation factor 6 (eIF6), cocrystallized with the antibiotic cycloheximide (a eukaryotic-specific inhibitor of protein synthesis), at a resolution of 3.5 angstroms. The structure illustrates the complex functional architecture of the eukaryotic 60S subunit, which comprises an intricate network of interactions between eukaryotic-specific ribosomal protein features and RNA expansion segments. It reveals the roles of eukaryotic ribosomal protein elements in the stabilization of the active site and the extent of eukaryotic-specific differences in other functional regions of the subunit. Furthermore, it elucidates the molecular basis of the interaction with eIF6 and provides a structural framework for further studies of ribosome-associated diseases and the role of the 60S subunit in the initiation of protein synthesis.     

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.     

Autophosphorylation of protein kinase C at three separated regions of its primary sequence

The major autophosphorylation sites of the rat beta II isozyme of protein kinase C were identified. The modified threonine and serine residues were found in the amino-terminal peptide, the carboxyl-terminal tail, and the hinge region between the regulatory lipid-binding domain and the catalytic kinase domain. Because this autophosphorylation follows an intrapeptide mechanism, extraordinary flexibility of the protein is necessary to phosphorylate the three regions. Comparison of the sequences surrounding the modified residues showed no obvious recognition motif nor any similarity to substrate phosphorylation sites, suggesting that proximity to the active site may be the primary criterion for their phosphorylation.     

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.     

A calcium-dependent protein kinase with a regulatory domain similar to calmodulin.

Calcium can function as a second messenger through stimulation of calcium-dependent protein kinases. A protein kinase that requires calcium but not calmodulin or phospholipids for activity has been purified from soybean. The kinase itself binds calcium with high affinity. A complementary DNA clone for this kinase has been identified; it encodes a protein with a predicted molecular mass of 57,175 daltons. This protein contains a catalytic domain similar to that of calmodulin-dependent kinases and a calmodulin-like region with four calcium binding domains (EF hands). The predicted structure of this kinase explains its direct regulation via calcium binding and establishes it as a prototype for a new family of calcium-regulated protein kinases.     

Unexpected modes of PDZ domain scaffolding revealed by structure of nNOS-syntrophin complex

The PDZ protein interaction domain of neuronal nitric oxide synthase (nNOS) can heterodimerize with the PDZ domains of postsynaptic density protein 95 and syntrophin through interactions that are not mediated by recognition of a typical carboxyl-terminal motif. The nNOS-syntrophin PDZ complex structure revealed that the domains interact in an unusual linear head-to-tail arrangement. The nNOS PDZ domain has two opposite interaction surfaces-one face has the canonical peptide binding groove, whereas the other has a beta-hairpin "finger." This nNOS beta finger docks in the syntrophin peptide binding groove, mimicking a peptide ligand, except that a sharp beta turn replaces the normally required carboxyl terminus. This structure explains how PDZ domains can participate in diverse interaction modes to assemble protein networks.     

刺五加ATP合酶β亚基基因的克隆与序列分析

[目的]克隆刺五加叶绿体的ATP合酶β亚基cDNA并对其进行生物信息学分析。[方法]根据已知物种叶绿体ATP合酶β亚基基因的序列,设计1对同源引物,通过RT-PCR扩增得到刺五加ATP合酶β亚基cDNA,并对其进行序列比对及结构预测分析。[结果]RT-PCR扩增获得了长1 099 bp的刺五加ATP合酶β亚基cDNA,该基因编码366个氨基酸。序列比对及结构预测分析表明,刺五加ATP合酶β亚基基因编码的氨基酸与水稻的同源性最高,达96.41%。其二级结构中含有171个α螺旋(alpha helix),占46.72%;53个延伸链(extended strand),占14.48%;27个β折叠(beta turn),占7.38%;115个无规则蜷曲(random coil),占31.42%。第262～271位氨基酸为ATP合酶β亚基的标志性位点。整个多肽链无明显的疏水区域,初步认定为亲水性蛋白。[结论]该试验克隆得到的ATP合酶β亚基基因为叶绿体ATP合酶β亚基基因,为研究刺五加能量代谢对植物次生代谢的影响及了解植物ATP合酶的结构与功能提供了必要的信息。     

Essential cytoplasmic translocation of a cytokine receptor-assembled signaling complex

Cytokine signaling is thought to require assembly of multicomponent signaling complexes at cytoplasmic segments of membrane-embedded receptors, in which receptor-proximal protein kinases are activated. Indeed, CD40, a tumor necrosis factor receptor (TNFR) family member, forms a complex containing adaptor molecules TRAF2 and TRAF3, ubiquitin-conjugating enzyme Ubc13, cellular inhibitor of apoptosis proteins 1 and 2 (c-IAP1/2), IkappaB kinase regulatory subunit IKKgamma (also called NEMO), and mitogen-activated protein kinase (MAPK) kinase kinase MEKK1 upon ligation. TRAF2, Ubc13, and IKKgamma were required for complex assembly and activation of MEKK1 and MAPK cascades. However, these kinases were not activated unless the multicomponent signaling complex translocated from CD40 to the cytosol upon c-IAP1/2-induced degradation of TRAF3. This two-stage signaling mechanism may apply to other innate immune receptors, accounting for spatial and temporal separation of MAPK and IKK signaling.