αC Helix Research Articles

Docking Interactions Induce Exposure of Activation Loop in the MAP Kinase ERK2

MAP kinases bind activating kinases, phosphatases, and substrates through docking interactions. Here, we report a 1.9 A crystallographic analysis of inactive ERK2 bound to a "D motif" docking peptide (pepHePTP) derived from hematopoietic tyrosine phosphatase, a negative regulator of ERK2. In this complex, the complete D motif interaction defined by mutagenic analysis is observed, including extensive electrostatic interactions with the "CD" site of the kinase. Large conformational changes occur in the activation loop where the dual phosphorylation sites, which are buried in the inactive form of ERK2, become exposed to solvent in the complex. Similar conformational changes occur in a complex between ERK2 and a MEK2 (MAP/ERK kinase-2)-derived D motif peptide (pepMEK2). D motif peptides are known to bind homologous loci in the MAP kinases p38alpha and JNK1, also inducing conformational changes in these enzymes. However, the binding interactions and conformational changes are unique to each, thus contributing to specificity among MAP kinases.

Biochemical and NMR analyses of an SF3b155–p14–U2AF-RNA interaction network involved in branch point definition during pre-mRNA splicing

The p14 subunit of the essential splicing factor 3b (SF3b) can be cross-linked to the branch-point adenosine of pre-mRNA introns within the spliceosome. p14 stably interacts with the SF3b subunit SF3b155, which also binds the 65-kDa subunit of U2 auxiliary splicing factor (U2AF65). We combined biochemical and NMR techniques to study the conformation of p14 either alone or complexed with SF3b155 fragments, as well as an interaction network involving p14, SF3b155, U2AF65, and U2 snRNA/pre-mRNA. p14 comprises a canonical RNA recognition motif (RRM) with an additional C-terminal helix (alphaC) and a beta hairpin insertion. SF3b155 binds to the beta-sheet surface of p14, thereby occupying the canonical RNA-binding site of the p14 RRM. The minimal region of SF3b155 interacting with p14 (i.e., residues 381-424) consists of four alpha-helices, which are partially preformed in isolation. Helices alpha2 and alpha3 (residues 401-415) constitute the core p14-binding epitope. Regions of SF3b155 binding to p14 and U2AF65 are nonoverlapping. This allows for a simultaneous interaction of SF3b155 with both proteins, which may support the stable association of U2 snRNP with the pre-mRNA. p14-RNA interactions are modulated by SF3b155 and the RNA-binding site of the p14-SF3b155 complex involves the noncanonical beta hairpin insertion of the p14 RRM, consistent with the beta-sheet surface being occupied by the helical SF3b155 peptide and p14 helix alphaC. Our data suggest that p14 lacks inherent specificity for recognizing the branch point, but that some specificity may be achieved by scaffolding interactions involving other components of SF3b.

Structural Basis of Lytic Cycle Activation by the Epstein-Barr Virus ZEBRA Protein

Epstein-Barr virus (EBV) causes infectious mononucleosis and is linked to several human malignancies. EBV has a biphasic infection cycle consisting of a latent and a lytic, replicative phase. The switch from latent to lytic infection is triggered by the EBV immediate-early transcription factor ZEBRA (BZLF1, Zta, Z, EB1). We present the crystal structure of ZEBRA's DNA binding domain bound to an EBV lytic gene promoter element. ZEBRA exhibits a variant of the basic-region leucine zipper (bZIP) fold in which a C-terminal moiety stabilizes the coiled coil involved in dimer formation. The structure provides insights into ZEBRA's broad target site specificity, preferential activation of specific EBV promoters in their methylated state, ability to dimerize despite lacking a leucine zipper motif, and failure to heterodimerize with cellular bZIP proteins. The structure will allow for the design of new therapeutic agents that block activation of the EBV lytic cycle.

A Family Portrait of the RIO Kinases

The RIO family of atypical serine protein kinases has been first characterized only recently. It consists of enzymes that contain a unique domain with a characteristic kinase sequence motif and usually some additional domains. At least two RIO proteins, Rio1 and Rio2, are present in organisms varying from Archaea to humans, with a third Rio3 subfamily present only in multicellular eukaryotes. Yeast Rio1 and Rio2 proteins have been implicated in the processing of 20 S pre-rRNA and are necessary for survival of the cells. Crystal structures of Archaeoglobus fulgidus Rio1 and Rio2 have shown that whereas the overall fold of these enzymes resembles typical protein kinases, some of the structural domains, particularly those involved in peptide substrate binding, are not present. The mode of binding of nucleotides also differs from that found in typical protein kinases. Although it has been shown that both Rio1 and Rio2 have the enzymatic activity of kinases and are capable of autophosphorylation, the biological substrates of RIO proteins and their full biological role still remain to be discovered.

Crystal Structures of the Mnk2 Kinase Domain Reveal an Inhibitory Conformation and a Zinc Binding Site

Human mitogen-activated protein kinases (MAPK)-interacting kinases 1 and 2 (Mnk1 and Mnk2) target the translational machinery by phosphorylation of the eukaryotic initiation factor 4E (eIF4E). Here, we present the 2.1 A crystal structure of a nonphosphorylated Mnk2 fragment that encompasses the kinase domain. The results show Mnk-specific features such as a zinc binding motif and an atypical open conformation of the activation segment. In addition, the ATP binding pocket contains an Asp-Phe-Asp (DFD) in place of the canonical magnesium binding Asp-Phe-Gly (DFG) motif. The phenylalanine of this motif sticks into the ATP binding pocket and blocks ATP binding as observed with inhibitor bound and, thus, inactive p38 kinase. Replacement of the DFD by the canonical DFG motif affects the conformation of Mnk2, but not ATP binding and kinase activity. The results suggest that the ATP binding pocket and the activation segment of Mnk2 require conformational switches to provide kinase activity.

Crystal Structure of Human Taspase1, a Crucial Protease Regulating the Function of MLL

Taspase1 catalyzes the proteolytic processing of the mixed lineage leukemia (MLL) nuclear protein, which is required for maintaining Hox gene expression patterns. Chromosomal translocations of the MLL gene are associated with leukemia in infants. Taspase1, a threonine aspartase, is a member of the type 2 asparaginase family, but is the only protease in this family. We report here the crystal structures of human activated Taspase1 and its proenzyme, as well as the characterization of the effects of mutations in the active site region using a newly developed fluorogenic assay. The structure of Taspase1 has significant differences from other asparaginases, especially near the active site. Mutation of the catalytic nucleophile, Thr234, abolishes autocatalytic processing in cis but does not completely block proteolysis in trans. The structure unexpectedly showed the binding of a chloride ion in the active site, and our kinetic studies confirm that chlorides ions are inhibitors of this enzyme at physiologically relevant concentrations.

Hsp90 restrains ErbB‐2/HER2 signalling by limiting heterodimer formation

ErbB-2/HER2 is an oncogenic tyrosine kinase that regulates a signalling network by forming ligand-induced heterodimers with several growth factor receptors of the ErbB family. Hsp90 and co-chaperones regulate degradation of ErbB-2 but not other ErbB members. Here, we report that the role of Hsp90 in modulating the ErbB network extends beyond regulation of protein stability. The capacity of ErbB-2 to recruit ligand-bound receptors into active heterodimers is limited by Hsp90, which is dissociated from ErbB-2 following ligand-induced heterodimerization. We show that Hsp90 binds a specific loop within the kinase domain of ErbB-2, thereby restraining heterodimer formation and catalytic function. These results define a role for Hsp90 as a molecular switch regulating the ErbB signalling network by limiting formation of ErbB-2-centred receptor complexes.

The structure of MSK1 reveals a novel autoinhibitory conformation for a dual kinase protein.

Mitogen and stress-activated kinase-1 (MSK1) is a serine/threonine protein kinase that is activated by either p38 or p42ERK MAPKs in response to stress or mitogenic extracellular stimuli. MSK1 belongs to a family of protein kinases that contain two distinct kinase domains in one polypeptide chain. We report the 1.8 A crystal structure of the N-terminal kinase domain of MSK1. The crystal structure reveals a unique inactive conformation with the ATP binding site blocked by the nucleotide binding loop. This inactive conformation is stabilized by the formation of a new three-stranded beta sheet on the N lobe of the kinase domain. The three beta strands come from residues at the N terminus of the kinase domain, what would be the alphaB helix in the active conformation, and the activation loop. The new three-stranded beta sheet occupies a position equivalent to the N terminus of the alphaC helix in active protein kinases.

Functional interaction of calmodulin with a plant cyclic nucleotide gated cation channel

A family of plant ligand gated nonselective cation channels (cngcs) can be activated by direct, and reversible binding of cyclic nucleotide. These proteins have a cytoplasm-localized cyclic nucleotide binding domain (CNBD) at the carboxy-terminus of the polypeptide. A portion of the cngc CNBD also acts as a calmodulin (CaM) binding domain (CaMBD). The objective of this work is to further characterize interaction of cyclic nucleotide and CaM in gating plant cngc currents. The three-dimensional structure of an Arabidopsis thaliana cngc (Atcngc2) CNBD was modeled, indicating cAMP binding to the Atcngc2 CNBD in a pocket formed by a β barrel structure appressing a shortened (relative to animal cngc CNBDs) αC helix. The Atcngc2 CaMBD was expressed as a fusion peptide linking blue and green fluorescent proteins, and used to quantify CaM ( A. thaliana CaM isoform 4) binding. CaM bound the fusion protein in a Ca 2+–dependent manner with a K d of 7.6 nM and a Ca 2+ binding K d of 200 nM. Functional characterization (voltage clamp analysis) of Atcngc2 was undertaken by expression in human embryonic kidney cells. CaM reversed cAMP activation of Atcngc2 currents. This functional interaction was dependent on free cytosolic Ca 2+. Increasing cytosolic Ca 2+ was found to inhibit cAMP activation of the channel in the absence of added CaM. We conclude that the physical interaction of Ca 2+/CaM with plant cngcs blocks cyclic nucleotide activation of these channels. Thus, the cytosolic secondary messengers CaM, cAMP, and Ca 2+ can act in an integrated fashion to gate currents through these plant ion channels.

Crystal structure of the tyrosine kinase domain of the hepatocyte growth factor receptor c-Met and its complex with the microbial alkaloid K-252a.

The protooncogene c-met codes for the hepatocyte growth factor receptor tyrosine kinase. Binding of its ligand, hepatocyte growth factor/scatter factor, stimulates receptor autophosphorylation, which leads to pleiotropic downstream signaling events in epithelial cells, including cell growth, motility, and invasion. These events are mediated by interaction of cytoplasmic effectors, generally through Src homology 2 (SH2) domains, with two phosphotyrosine-containing sequence motifs in the unique C-terminal tail of c-Met (supersite). There is a strong link between aberrant c-Met activity and oncogenesis, which makes this kinase an important cancer drug target. The furanosylated indolocarbazole K-252a belongs to a family of microbial alkaloids that also includes staurosporine. It was recently shown to be a potent inhibitor of c-Met. Here we report the crystal structures of an unphosphorylated c-Met kinase domain harboring a human cancer mutation and its complex with K-252a at 1.8-A resolution. The structure follows the well established architecture of protein kinases. It adopts a unique, inhibitory conformation of the activation loop, a catalytically noncompetent orientation of helix alphaC, and reveals the complete C-terminal docking site. The first SH2-binding motif (1349YVHV) adopts an extended conformation, whereas the second motif (1356YVNV), a binding site for Grb2-SH2, folds as a type II Beta-turn. The intermediate portion of the supersite (1353NATY) assumes a type I Beta-turn conformation as in an Shc-phosphotyrosine binding domain peptide complex. K-252a is bound in the adenosine pocket with an analogous binding mode to those observed in previously reported structures of protein kinases in complex with staurosporine.

Crystal Structure of a C-terminal Deletion Mutant of Human Protein Kinase CK2 Catalytic Subunit

Protein kinase CK2 (formerly called: casein kinase 2) is a heterotetrameric enzyme composed of two separate catalytic chains (CK2α) and a stable dimer of two non-catalytic subunits (CK2β). CK2α is a highly conserved member of the superfamily of eukaryotic protein kinases. The crystal structure of a C-terminal deletion mutant of human CK2α was solved and refined to 2.5 Å resolution. In the crystal the CK2α mutant exists as a monomer in agreement with the organization of the subunits in the CK2 holoenzyme. The refined structure shows the helix αC and the activation segment, two main regions of conformational plasticity and regulatory importance in eukaryotic protein kinases, in active conformations stabilized by extensive contacts to the N-terminal segment. This arrangement is in accordance with the constitutive activity of the enzyme. By structural superimposition of human CK2α in isolated form and embedded in the human CK2 holoenzyme the loop connecting the strands β4 and β5 and the ATP-binding loop were identified as elements of structural variability. This structural comparison suggests that the ATP-binding loop may be the key region by which the non-catalytic CK2β dimer modulates the activity of CK2α. The β4/β5 loop was found in a closed conformation in contrast to the open conformation observed for the CK2α subunits of the CK2 holoenzyme. CK2α monomers with this closed β4/β5 loop conformation are unable to bind CK2β dimers in the common way for sterical reasons, suggesting a mechanism to protect CK2α from integration into CK2 holoenzyme complexes. This observation is consistent with the growing evidence that CK2α monomers and CK2β dimers can exist in vivo independently from the CK2 holoenzyme and may possess physiological roles of their own.

Molecular Mechanism for the Regulation of Protein Kinase B/Akt by Hydrophobic Motif Phosphorylation

Protein kinase B/Akt plays crucial roles in promoting cell survival and mediating insulin responses. The enzyme is stimulated by phosphorylation at two regulatory sites: Thr 309 of the activation segment and Ser 474 of the hydrophobic motif, a conserved feature of many AGC kinases. Analysis of the crystal structures of the unphosphorylated and Thr 309 phosphorylated states of the PKB kinase domain provides a molecular explanation for regulation by Ser 474 phosphorylation. Activation by Ser 474 phosphorylation occurs via a disorder to order transition of the αC helix with concomitant restructuring of the activation segment and reconfiguration of the kinase bilobal structure. These conformational changes are mediated by a phosphorylation-promoted interaction of the hydrophobic motif with a channel on the N-terminal lobe induced by the ordered αC helix and are mimicked by peptides corresponding to the hydrophobic motif of PKB and potently by the hydrophobic motif of PRK2.

A Novel Missense Mutation in Human Lactate Dehydrogenase B-Subunit Gene

Reduced activity of serum lactate dehydrogenase (LDH; EC 1.1.1.27) was found in a male medical student during practical examinations of his own blood. Serum LDH isoenzyme pattern showed reductions in activities of the isoenzymes with lower subunit A/B ratios such as LDH1 and LDH2. These findings were indicative of a partial LDH-B subunit deficiency, which was confirmed in erythrocyte hemolysates by Western blotting. Polymerase chain reaction (PCR)-based DNA sequence analysis of the LDH-B subunit gene revealed a heterozygous nucleotide change: a guanine to adenine substitution in codon 69 (GGG → GAG) at the third exon of the LDH-B subunit gene that resulted in a glycine to glutamic acid substitution (G69E). The mutation was confirmed by PCR–restriction fragment length polymorphism (RFLP) analysis using a mismatched primer to introduce a new NcoI restriction site. The same heterozygous mutation was found in his mother but not in other family members. This mutation involves a residue belonging to αC helix in LDH-B subunit protein molecule that functions as an interface for other subunits.

NF-Y binding to twin CCAAT boxes: role of Q-rich domains and histone fold helices

NF-Y (CBF) is a CCAAT-binding trimer that activates 25% of eukaryotic promoters. It contains putative histone fold motifs (HFMs) and distorts DNA. By using electrophoretic mobility shift assays with the twin CCAAT boxes of the human γ-globin promoter and several combinations of subunit mutants, we dissected some of the structural features of CCAAT-box binding. NF-YA and NF-YC Q-rich domains significantly influence bending angles quantitatively, but not qualitatively, since they do not modify DNA orientation. They are both required for co-operative interactions among NF-Y molecules: for this, a precise alignement of two CCAAT boxes, 32 bp, three turns of the helix, is essential. Unlike the wild-type (wt) protein, steric hindrance does not impede simultaneous binding of the mutant composed of the short homology domains to CCAAT boxes closer than 22 bp: the addition of 11 amino acid residues to NF-YB and 13 to NF-YC flanking the HFM, restores wt behaviour. These stretches are predicted to form H2B-like αC and H2A-like αN fourth helices. A further support to this hypothesis comes from off-rates analysis of mutant combinations: the half-life of NF-Y, which is dependent on the type of NF-YB used, is extremely shortened, when the putative αC is present, nearly as much as in the wt NF-YB. These data (i) provide further evidence that NF-YB-NF-YC belong to the H2B-H2A subclasses, (ii) uncover new features of Q-rich domains, and (iii) define rules for NF-Y synergy that are potentially important for the regulation of many eukaryotic promoters.

Mapping of the binding of platelet-derived growth factor to distinct domains of the basement membrane proteins BM-40 and perlecan and distinction from the BM-40 collagen-binding epitope.

A surface plasmon resonance assay was used to analyze the binding of platelet-derived growth factor (PDGF)-AA and PDGF-BB to various proteins of the extracellular matrix. This identified several collagen types; laminin-1, nidogen, perlecan and BM-40 as potential ligands for PDGF with Kd values in the range 2-3200 nM. Perlecan and BM-40 were used to examine the domain specificity and other parameters of the interactions. Recombinant human and mouse BM-40 were shown to bind both PDGFs in a similar manner with a Kd of about 5-10 nM. Studies with deletion mutants of human BM-40 demonstrated binding to its C-terminal extracellular calcium-binding (EC) module, yet the interaction did not require calcium. This distinguishes this from the binding of the EC module to various collagen types, which is strictly calcium dependent. Furthermore, deletion of helix alphaC or two point mutations in helix alphaA of the EC module either enhanced or abolished binding to collagen IV. Since these mutations had no effects on binding to PDGF, it demonstrated the presence of two different binding epitopes. Binding of PDGF-BB to the perlecan core protein could be mapped to its domain III-2 (Kd = 8 nM) with lower affinities shown for domains I, IV-1 and V (Kd = 34-64 nM). Other perlecan domains (II, III-1, III-3, IV-2) were inactive. PDGF-AA was also shown to bind domain III-2 but not III-1. Neither nidogen, BM-40 or perlecan domain III-2 interfered with the binding of PDGF to its alpha and beta receptors, however, suggesting that these interactions may be mainly used for storage of PDGF in the extracellular matrix.

N.m.r. studies of the helix-coil transition of polypeptides in non-protonating solvent mixtures

High resolution nuclear magnetic resonance (n.m.r.) spectra have been obtained for poly(β-benzyl aspartate), poly(γ-benzyl glutamate) and several of their copolymers in the solvent system dimethylsulphoxide/chloroform, in which no amide protonation can occur. ‘Double-peak’ αC H and peptide N H resonances are observed in the helix-coil transition region of the same form as seen in trifluoroacetic acid/chloroform solutions. It is concluded that protonation of peptide groups is not essential, either for inducing the helix to coil transition itself or for observation of different and characteristic helix and coil αC H and N H shifts.