Activation and Functional Analysis of Janus Kinase 2 in BA/F3 Cells Using the Coumermycin/Gyrase B System

Janus kinase 2 (Jak2) protein tyrosine kinase plays an important role in interleukin-3– or granulocyte–macrophage colony-stimulating factor–mediated signal transduction pathways leading to cell proliferation, activation of early response genes, and inhibition of apoptosis. However, it is unclear whether Jak2 can activate these signaling pathways directly without the involvement of cytokine receptor phosphorylation. To investigate the specific role of Jak2 in the regulation of signal transduction pathways, we generated gyrase B (GyrB)–Jak2 fusion proteins, dimerized through the addition of coumermycin. Coumermycin induced autophosphorylation of GyrB–Jak2 fusion proteins, thus bypassing receptor activation. Using different types of chimeric Jak2 molecules, we observed that although the kinase domain of Jak2 is sufficient for autophosphorylation, the N-terminal regions are essential for the phosphorylation of Stat5 and for the induction of short-term cell proliferation. Moreover, coumermycin-induced activation of Jak2 can also lead to increased levels of c-myc and CIS mRNAs in BA/F3 cells stably expressing the Jak2 fusion protein with the intact N-terminal region. Conversely, activation of the chimeric Jak2 induced neither phosphorylation of Shc or SHP-2 nor activation of the c-fos promoter. Here, we showed that the GyrB–Jak2 system can serve as an excellent model to dissect signals of receptor-dependent and -independent events. We also obtained evidence indicating a role for the N-terminal region of Jak2 in downstream signaling events.

Kinetic and structural analysis of mutant CD4 receptors that are defective in HIV gp120 binding

The T-cell antigen coreceptor CD4 also serves as the receptor for the envelope glycoprotein gp120 of HIV. Extensive mutational analysis of CD4 has implicated residues from a portion of the extracellular amino-terminal domain (D1) in gp120 binding. However, none of these proteins has been fully characterized biophysically, and thus the precise effects on molecular structure and binding interactions are unknown. In the present study, we produced soluble versions of three mutant CD4 molecules (F43V, G47S, and A55F) and characterized their structural properties, thermostability, and ability to bind gp120. Crystallographic and thermodynamic analysis showed minimal structural alterations in the F43V and G47S mutant proteins, which have solvent-exposed mutant side chains. In contrast, some degree of disorder appears to exist in the folded state of A55F, as a result of mutating a buried side chain. Real time kinetic measurements of the interaction of the mutant proteins with gp120 showed affinity decreases of 5-fold for G47S, 50-fold for A55F, and 200-fold for F43V. Although both rate constants for the binding reaction were affected by these mutations, the loss in affinity was mainly due to a decrease in on rates, with less drastic changes occurring in the off rates. These observations suggest the involvement of conformational adaptation in the CD4–gp120 interaction. Together, the structural and kinetic data confirm that F43V is a critical residue in gp120 recognition site, which may also include main chain interactions at residue Gly-47.

Three-dimensional modeling of and ligand docking to vitamin D receptor ligand binding domain

The ligand binding domain of the human vitamin D receptor (VDR) was modeled based on the crystal structure of the retinoic acid receptor. The ligand binding pocket of our VDR model is spacious at the helix 11 site and confined at the β-turn site. The ligand 1α,25-dihydroxyvitamin D3 was assumed to be anchored in the ligand binding pocket with its side chain heading to helix 11 (site 2) and the A-ring toward the β-turn (site 1). Three residues forming hydrogen bonds with the functionally important 1α- and 25-hydroxyl groups of 1α,25-dihydroxyvitamin D3 were identified and confirmed by mutational analysis: the 1α-hydroxyl group is forming pincer-type hydrogen bonds with S237 and R274 and the 25-hydroxyl group is interacting with H397. Docking potential for various ligands to the VDR model was examined, and the results are in good agreement with our previous three-dimensional structure-function theory.

Studies on the role of the hydrophobic domain of Ost4p in interactions with other subunits of yeast oligosaccharyl transferase

In the yeast, Saccharomyces cerevisiae, oligosaccharyl transferase (OT), which catalyzes the transfer of dolichol-linked oligosaccharide chains to nascent polypeptides in the endoplasmic reticulum, consists of nine nonidentical membrane protein subunits. Genetic and biochemical evidence indicated these nine proteins exist in three subcomplexes. Three of the OT subunits (Ost4p, Ost3p, and Stt3p) have been proposed to exist in one subcomplex. To investigate the interaction of these three membrane proteins, initially we carried out a mutational analysis of Ost4p, which is an extraordinarily small membrane protein containing only 36 amino acid residues. This analysis indicated that when single amino acid residues in a region close to the luminal face of the putative transmembrane domain of Ost4p were changed into an ionizable amino acid such as Lys or Asp, growth at 37°C and OT activity measured in vitro were impaired. In addition, using immunoprecipitation techniques and Western blot analysis, we found that with these mutations the interaction between Ost4p, Ost3p, and Stt3p was disrupted. Introduction of Lys or Asp residues at other positions in the putative transmembrane domain or at the N or C terminus of Ost4p had no effect on disrupting subunit interactions or impairing the activity of OT. These findings suggest that a localized region of the putative transmembrane domain of Ost4p mediates in stabilization of the interaction with the two other OT subunits (Ost3p and Stt3p) in a subcomplex in the endoplasmic reticulum membrane.

Analysis of receptor signaling pathways by mass spectrometry: Identification of Vav-2 as a substrate of the epidermal and platelet-derived growth factor receptors

Oligomerization of receptor protein tyrosine kinases such as the epidermal growth factor receptor (EGFR) by their cognate ligands leads to activation of the receptor. Transphosphorylation of the receptor subunits is followed by the recruitment of signaling molecules containing src homology 2 (SH2) or phosphotyrosine interaction domains (PID). Additionally, several cytoplasmic proteins that may or may not associate with the receptor undergo tyrosine phosphorylation. To identify several components of the EGFR signaling pathway in a single step, we have immunoprecipitated molecules that are tyrosine phosphorylated in response to EGF and analyzed them by one-dimensional gel electrophoresis followed by mass spectrometry. Combining matrix-assisted laser desorption/ionization (MALDI) and nanoelectrospray tandem mass spectrometry (MS/MS) led to the identification of nine signaling molecules, seven of which had previously been implicated in EGFR signaling. Several of these molecules were identified from low femtomole levels of protein loaded onto the gel. We identified Vav-2, a recently discovered guanosine nucleotide exchange factor that is expressed ubiquitously, as a substrate of the EGFR. We demonstrate that Vav-2 is phosphorylated on tyrosine residues in response to EGF and associates with the EGFR in vivo. Binding of Vav-2 to the EGFR is mediated by the SH2 domain of Vav-2. In keeping with its ubiquitous expression, Vav-2 seems to be a general signaling molecule, since it also associates with the platelet-derived growth factor (PDGF) receptor and undergoes tyrosine phosphorylation in fibroblasts upon PDGF stimulation. The strategy suggested here can be used for routine identification of downstream components of cell surface receptors in mammalian cells.

Phytochrome B binds with greater apparent affinity than phytochrome A to the basic helix–loop–helix factor PIF3 in a reaction requiring the PAS domain of PIF3

The signaling pathways by which the phytochrome (phy) family of photoreceptors transmits sensory information to light-regulated genes remain to be fully defined. Evidence for a relatively direct pathway has been provided by the binding of one member of the family, phyB, to a promoter-element-bound, basic helix–loop–helix protein, PIF3, specifically upon light-induced conversion of the photoreceptor molecule to its biologically active conformer (Pfr). Here, we show that phyA also binds selectively and reversibly to PIF3 upon photoconversion to Pfr, but that the apparent affinity of PIF3 for phyA is 10-fold lower than for phyB. This result is consistent with previous in vivo data from PIF3-deficient Arabidopsis, indicating that PIF3 has a major role in phyB signaling, but a more minor role in phyA signaling. We also show that phyB binds stoichiometrically to PIF3 at an equimolar ratio, suggesting that the resultant complex is the unit active in transcriptional regulation at target promoters. Deletion mapping suggests that a 37-aa segment present at the N terminus of phyB, but absent from phyA, contributes strongly to the high binding affinity of phyB for PIF3. Conversely, deletion mapping and point mutation analysis of PIF3 for determinants involved in recognition of phyB indicates that the PAS domain of PIF3 is a major contributor to this interaction, but that a second determinant in the C-terminal domain is also necessary.

The domain structure of protoporphyrinogen oxidase, the molecular target of diphenyl ether-type herbicides

Protoporphyrinogen oxidase (EC 1–3-3–4), the 60-kDa membrane-bound flavoenzyme that catalyzes the final reaction of the common branch of the heme and chlorophyll biosynthesis pathways in plants, is the molecular target of diphenyl ether-type herbicides. It is highly resistant to proteases (trypsin, endoproteinase Glu-C, or carboxypeptidases A, B, and Y), because the protein is folded into an extremely compact form. Trypsin maps of the native purified and membrane-bound yeast protoporphyrinogen oxidase show that this basic enzyme (pI > 8.5) was cleaved at a single site under nondenaturing conditions, generating two peptides with relative molecular masses of 30,000 and 35,000. The endoproteinase Glu-C also cleaved the protein into two peptides with similar masses, and there was no additional cleavage site under mild denaturing conditions. N-terminal peptide sequence analysis of the proteolytic (trypsin and endoproteinase Glu-C) peptides showed that both cleavage sites were located in putative connecting loop between the N-terminal domain (25 kDa) with the βαβ ADP-binding fold and the C-terminal domain (35 kDa), which possibly is involved in the binding of the isoalloxazine moiety of the FAD cofactor. The peptides remained strongly associated and fully active with the Km for protoporphyrinogen and the Ki for various inhibitors, diphenyl-ethers, or diphenyleneiodonium derivatives, identical to those measured for the native enzyme. However, the enzyme activity of the peptides was much more susceptible to thermal denaturation than that of the native protein. Only the C-terminal domain of protoporphyrinogen oxidase was labeled specifically in active site-directed photoaffinity-labeling experiments. Trypsin may have caused intramolecular transfer of the labeled group to reactive components of the N-terminal domain, resulting in nonspecific labeling. We suggest that the active site of protoporphyrinogen oxidase is in the C-terminal domain of the protein, at the interface between the C- and N-terminal domains.

Catalytic mechanism of the adenylyl and guanylyl cyclases: Modeling and mutational analysis

The adenylyl and guanylyl cyclases catalyze the formation of 3′,5′-cyclic adenosine or guanosine monophosphate from the corresponding nucleoside 5′-triphosphate. The guanylyl cyclases, the mammalian adenylyl cyclases, and their microbial homologues function as pairs of homologous catalytic domains. The crystal structure of the rat type II adenylyl cyclase C2 catalytic domain was used to model by homology a mammalian adenylyl cyclase C1-C2 domain pair, a homodimeric adenylyl cyclase of Dictyostelium discoideum, a heterodimeric soluble guanylyl cyclase, and a homodimeric membrane guanylyl cyclase. Mg2+ATP or Mg2+GTP were docked into the active sites based on known stereochemical constraints on their conformation. The models are consistent with the activities of seven active-site mutants. Asp-310 and Glu-432 of type I adenylyl cyclase coordinate a Mg2+ ion. The D310S and D310A mutants have 10-fold reduced Vmax and altered [Mg2+] dependence. The NTP purine moieties bind in mostly hydrophobic pockets. Specificity is conferred by a Lys and an Asp in adenylyl cyclase, and a Glu, an Arg, and a Cys in guanylyl cyclase. The models predict that an Asp from one domain is a general base in the reaction, and that the transition state is stabilized by a conserved Asn-Arg pair on the other domain.

Radical SAM, a novel protein superfamily linking unresolved steps in familiar biosynthetic pathways with radical mechanisms: functional characterization using new analysis and information visualization methods

A novel protein superfamily with over 600 members was discovered by iterative profile searches and analyzed with powerful bioinformatics and information visualization methods. Evidence exists that these proteins generate a radical species by reductive cleavage of S-adenosylmethionine (SAM) through an unusual Fe-S center. The superfamily (named here Radical SAM) provides evidence that radical-based catalysis is important in a number of previously well- studied but unresolved biochemical pathways and reflects an ancient conserved mechanistic approach to difficult chemistries. Radical SAM proteins catalyze diverse reactions, including unusual methylations, isomerization, sulfur insertion, ring formation, anaerobic oxidation and protein radical formation. They function in DNA precursor, vitamin, cofactor, antibiotic and herbicide biosynthesis and in biodegradation pathways. One eukaryotic member is interferon-inducible and is considered a candidate drug target for osteoporosis; another is observed to bind the neuronal Cdk5 activator protein. Five defining members not previously recognized as homologs are lysine 2,3-aminomutase, biotin synthase, lipoic acid synthase and the activating enzymes for pyruvate formate-lyase and anaerobic ribonucleotide reductase. Two functional predictions for unknown proteins are made based on integrating other data types such as motif, domain, operon and biochemical pathway into an organized view of similarity relationships.

A rapid classification protocol for the CATH Domain Database to support structural genomics

In order to support the structural genomic initiatives, both by rapidly classifying newly determined structures and by suggesting suitable targets for structure determination, we have recently developed several new protocols for classifying structures in the CATH domain database (http://www.biochem.ucl.ac.uk/bsm/cath). These aim to increase the speed of classification of new structures using fast algorithms for structure comparison (GRATH) and to improve the sensitivity in recognising distant structural relatives by incorporating sequence information from relatives in the genomes (DomainFinder). In order to ensure the integrity of the database given the expected increase in data, the CATH Protein Family Database (CATH-PFDB), which currently includes 25 320 structural domains and a further 160 000 sequence relatives has now been installed in a relational ORACLE database. This was essential for developing more rigorous validation procedures and for allowing efficient querying of the database, particularly for genome analysis. The associated Dictionary of Homologous Superfamilies [Bray,J.E., Todd,A.E., Pearl,F.M.G., Thornton,J.M. and Orengo,C.A. (2000) Protein Eng., 13, 153–165], which provides multiple structural alignments and functional information to assist in assigning new relatives, has also been expanded recently and now includes information for 903 homo­logous superfamilies. In order to improve coverage of known structures, preliminary classification levels are now provided for new structures at interim stages in the classification protocol. Since a large proportion of new structures can be rapidly classified using profile-based sequence analysis [e.g. PSI-BLAST: Altschul,S.F., Madden,T.L., Schaffer,A.A., Zhang,J., Zhang,Z., Miller,W. and Lipman,D.J. (1997) Nucleic Acids Res., 25, 3389–3402], this provides preliminary classification for easily recognisable homologues, which in the latest release of CATH (version 1.7) represented nearly three-quarters of the non-identical structures.

KH domain: one motif, two folds

The K homology (KH) module is a widespread RNA-binding motif that has been detected by sequence similarity searches in such proteins as heterogeneous nuclear ribonucleoprotein K (hnRNP K) and ribosomal protein S3. Analysis of spatial structures of KH domains in hnRNP K and S3 reveals that they are topologically dissimilar and thus belong to different protein folds. Thus KH motif proteins provide a rare example of protein domains that share significant sequence similarity in the motif regions but possess globally distinct structures. The two distinct topologies might have arisen from an ancestral KH motif protein by N- and C-terminal extensions, or one of the existing topologies may have evolved from the other by extension, displacement and deletion. C-terminal extension (deletion) requires β-sheet rearrangement through the insertion (removal) of a β-strand in a manner similar to that observed in serine protease inhibitors serpins. Current analysis offers a new look on how proteins can change fold in the course of evolution.

The Sm domain is an ancient RNA-binding motif with oligo(U) specificity

Sm and Sm-like proteins are members of a family of small proteins that is widespread throughout eukaryotic kingdoms. These proteins form heteromers with one another and bind, as heteromeric complexes, to various RNAs, recognizing primarily short U-rich stretches. Interestingly, completion of several genome projects revealed that archaea also contain genes that may encode Sm-like proteins. Herein, we studied the properties of one Sm-like protein derived from the archaebacterium Archaeoglobus fulgidus and overexpressed in Escherichia coli. This single small protein closely reflects the properties of an Sm or Sm-like protein heteromer. It binds to RNA with a high specificity for oligo(U), and assembles onto the RNA to form a complex that exhibits, as judged by electron microscopy, a ring-like structure similar to the ones observed with the Sm core ribonucleoprotein and the like Sm (LSm) protein heteromer. Importantly, multivariate statistical analysis of negative-stain electron-microscopic images revealed a sevenfold symmetry for the observed ring structure, indicating that the proteins form a homoheptamer. These results support the structural model of the Sm proteins derived from crystallographic studies on Sm heterodimers and demonstrate that the Sm protein family evolved from a single ancestor that was present before the eukaryotic and archaeal kingdoms separated.

Structure and function of the C-terminal PABC domain of human poly(A)-binding protein

We have determined the solution structure of the C-terminal quarter of human poly(A)-binding protein (hPABP). The protein fragment contains a protein domain, PABC [for poly(A)-binding protein C-terminal domain], which is also found associated with the HECT family of ubiquitin ligases. By using peptides derived from PABP interacting protein (Paip) 1, Paip2, and eRF3, we show that PABC functions as a peptide binding domain. We use chemical shift perturbation analysis to identify the peptide binding site in PABC and the major elements involved in peptide recognition. From comparative sequence analysis of PABC-binding peptides, we formulate a preliminary PABC consensus sequence and identify human ataxin-2, the protein responsible for type 2 spinocerebellar ataxia (SCA2), as a potential PABC ligand.

X-ray structure of the human hyperplastic discs protein: An ortholog of the C-terminal domain of poly(A)-binding protein

The poly(A)-binding protein (PABP) recognizes the 3′ mRNA poly(A) tail and plays an essential role in eukaryotic translation initiation and mRNA stabilization/degradation. PABP is a modular protein, with four N-terminal RNA-binding domains and an extensive C terminus. The C-terminal region of PABP is essential for normal growth in yeast and has been implicated in mediating PABP homo-oligomerization and protein–protein interactions. A small, proteolytically stable, highly conserved domain has been identified within this C-terminal segment. Remarkably, this domain is also present in the hyperplastic discs protein (HYD) family of ubiquitin ligases. To better understand the function of this conserved region, an x-ray structure of the PABP-like segment of the human HYD protein has been determined at 1.04-Å resolution. The conserved domain adopts a novel fold resembling a right-handed supercoil of four α-helices. Sequence profile searches and comparative protein structure modeling identified a small ORF from the Arabidopsis thaliana genome that encodes a structurally similar but distantly related PABP/HYD domain. Phylogenetic analysis of the experimentally determined (HYD) and homology modeled (PABP) protein surfaces revealed a conserved feature that may be responsible for binding to a PABP interacting protein, Paip1, and other shared interaction partners.

Solution 19F nuclear Overhauser effects in structural studies of the cytoplasmic domain of mammalian rhodopsin

19F nuclear Overhauser effects (NOEs) between fluorine labels on the cytoplasmic domain of rhodopsin solubilized in detergent micelles are reported. Previously, high-resolution solution 19F NMR spectra of fluorine-labeled rhodopsin in detergent micelles were described, demonstrating the applicability of this technique to studies of tertiary structure in the cytoplasmic domain. To quantitate tertiary contacts we have applied a transient one-dimensional difference NOE solution 19F NMR experiment to this system, permitting assessment of proximities between fluorine labels specifically incorporated into different regions of the cytoplasmic face. Three dicysteine substitution mutants (Cys-140–Cys-316, Cys-65–Cys-316, and Cys-139–Cys-251) were labeled by attachment of the trifluoroethylthio group through a disulfide linkage. Each mutant rhodopsin was prepared (8–10 mg) in dodecylmaltoside and analyzed at 20°C by solution 19F NMR. Distinct chemical shifts were observed for all of the rhodopsin 19F labels in the dark. An up-field shift of the Cys-316 resonance in the Cys-65–Cys-316 mutant suggests a close proximity between the two residues. When analyzed for 19F-19F NOEs, a moderate negative enhancement was observed for the Cys-65–Cys-316 pair and a strong negative enhancement was observed for the Cys-139–Cys-251 pair, indicating proximity between these sites. No NOE enhancement was observed for the Cys-140–Cys-316 pair. These NOE effects demonstrate a solution 19F NMR method for analysis of tertiary contacts in high molecular weight proteins, including membrane proteins.