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U Lindberg

Publications and source records attributed to U Lindberg.

At least 37 records · Page 2Linked to original sources
PubMed1997

The crystal structure of a major allergen from plants.

BACKGROUND: Profilins are small eukaryotic proteins involved in modulating the assembly of actin microfilaments in the cytoplasm. They are able to bind both phosphatidylinositol-4,5-bisphosphate and poly-L-proline (PLP) and thus play a critical role in signaling pathways. Plant profilins are of interest because immunological cross-reactivity between pollen and human profilin may be the cause of hay fever and broad allergies to pollens. RESULTS: The determination of the Arabidopsis thaliana profilin isoform I structure, using multiwavelength anomalous diffraction (MAD) to obtain structure-factor phases, is reported here. The structure of Arabidopsis profilin is similar to that of previously determined profilin structures. Conserved amino acid residues in profilins from plants, mammals, and lower eukaryotes are critically important in dictating the geometry of the PLP-binding site and the overall polypeptide fold. The main feature distinguishing plant profilins from other profilins is a solvent-filled pocket located in the most variable region of the fold. CONCLUSIONS: Comparison of the structures of SH3 domains with those of profilins from three distinct sources suggests that the mode of PLP binding may be similar. A comparison of three profilin structures from different families reveals only partial conservation of the actin-binding surface. The proximity of the semi-conserved actin-binding site and the binding pocket characteristic of plant profilins suggests that epitopes encompassing both features are responsible for the cross-reactivity of antibodies between human and plant profilins thought to be responsible for type I allergies.

Actins↗
PubMed1996

The structure of an open state of beta-actin at 2.65 A resolution.

The structure of an "open state" of crystalline profilin:beta-actin has been solved to 2.65 A by X-ray crystallography. The open-state crystals, in 1.8 M potassium phosphate, have an expanded unit cell dimension in the c direction of 185.7 A compared with 171.9 A in the previously solved ammonium sulphate-stabilized "tight-state" structure. The unit cell change between the open and the tight states is accompanied by large subdomain movements in actin. Furthermore, the nucleotide in the open state is significantly more exposed to solvent, and local conformational changes in the hydrophobic pocket surrounding cysteine 374 occur during the transition to the tight state. Significant changes were observed at the N terminus and in the DNase-I binding loop. Neither the structure of profilin nor its contact with beta-actin are affected by the changes in the unit cell. Applying osmotic pressure to profilin:beta-actin crystals brings about a collapse of the unit cell comparable with that seen in the open to tight-state transition, enabling an estimate of the work required to cause this transformation of beta-actin in the crystals. The slight difference in energy between the open and collapsed states explains the extreme sensitivity of profilin:beta-actin crystals to changes in chemical and thermal environment.

Actins↗
PubMed1996

Effects of the type of divalent cation, Ca2+ or Mg2+, bound at the high-affinity site and of the ionic composition of the solution on the structure of F-actin.

F-actins containing either Ca2+ or Mg2+ at the single high-affinity site for a divalent cation differ in their dynamic properties [Carlier (1991) J. Biol. Chem. 266, 1-4]. In an attempt to obtain information on the structural basis of this difference, we probed the conformation of specific sites in the subunits of Mg- and Ca-F-actin with limited proteolysis by subtilisin and trypsin. The influence of the kind of polymerizing salt was also investigated. At high proteinase concentrations required for digestion of actin in the polymer form, subtilisin gives a complex fragmentation pattern. In addition to the earlier known cleavage between Met47 and Gly48 in the DNAse-I-binding loop, cleavage of F-actin between Ser234 and Ser235 in subdomain 4 has recently been reported [Vahdat, Miller, Phillips, Muhlrad and Reisler (1995) FEBS Lett. 365, 149-151]. Here we show that actually a larger segment, comprising residues 227-235, is removed and the bond between Leu67 and Lys68 in subdomain 2 is split in both G- and F-actin, and that the differences in the fragmentation patterns of the G- and F-forms are accounted for by the protection of the bond 47-48 in F-actin. The subtilisin and trypsin cleavage sites in segment 61-69, subtilisin sites in segment 227-235 and trypsin sites between Lys373 and Cys374 were less accessible in Mg-F-actin than in Ca-F-actin. These are intramolecular effects, as similar changes were observed on Ca2+/Mg2+ replacement in G-actin. The cation-dependent effects, in particular those on segment 61-69, were however less pronounced in F-actin than in G-actin. The results suggest that substitution of Mg2+ for Ca2+, and KCl-induced polymerization of CaATP-G-actin, bring about a similar change in the conformation of subdomain 2 of the monomer. The presence of Mg2+ at the high-affinity site also resulted in an increased protection of the bond 47-48. This latter appears to be an intermolecular effect because it is specific for F-actin. The susceptibility to subtilisin and trypsin was also strongly influenced by the kind and concentration of polymerizing salt. The digestion patterns suggest that the exposure and/or flexibility of the regions containing the cleavage sites diminish with enhancement of the ionic strength of the solution. The results are discussed in terms of the current models of F-actin.

Actins↗
PubMed1996

Two GTPases, Cdc42 and Rac, bind directly to a protein implicated in the immunodeficiency disorder Wiskott-Aldrich syndrome.

BACKGROUND: Members of the Rho family of small GTPases play an essential role in controlling the motile behaviour of animal cells. Specifically, Cdc42 and Rac have been shown to induce the formation of filopodia and lamellipodia, respectively, at the cell periphery of Swiss 3T3 fibroblasts. In addition, both GTPases are required for progression through G1 phase of the cell cycle, possibly by regulating the activity of the Jun N-terminal kinase (JNK) signalling pathway. In order to examine more closely the mechanisms underlying the diverse functions of Rho GTPases in mammalian cells, we searched for downstream targets of these proteins. RESULTS: A yeast two-hybrid screen for proteins interacting with the human Cdc42 GTPase identified WASP, a protein implicated in the immunodeficiency disorder Wiskott-Aldrich syndrome (WAS). Recombinant WASP, expressed in Escherichia coli, also bound to Cdc42 and weakly to Rac, but not at all to Rho. The Cdc42/Rac-binding domain was identified in a region between amino acids 201-321 of WASP, and binding was dependent on Cdc42 and Rac being in the GTP-bound conformation. Furthermore, WASP did not catalyze GTPase activation or nucleotide exchange activity on Cdc42. CONCLUSIONS: Positional cloning has implicated WASP in causing WAS, and the protein is defective in patients suffering from the disease. WASP is expressed exclusively in cells of hematopoietic lineage, and lymphocytes from WAS patients have a distorted cell-surface and exhibit reduced proliferative capacity. WASP has recently been found to bind to the Src-homology 3 (SH3) domain of the adapter protein Nck. This observation, and the results presented here, suggest that WAS is the result of defects in signal transduction pathways regulated by Cdc42/Rac and Nck.

Amino Acid Sequence↗
PubMed1995

The effect on actin ATPase of phalloidin and tetramethylrhodamine phalloidin.

Actin polymerization has been studied in the absence of excess nucleotide. Using G-actin ATP monomers, it was shown that mechanical shearing stimulates ATP hydrolysis. The procedures used enabled the detection of differential effects of phalloidin and tetramethylrhodamine-phalloidin, on the P(i)-release step of the actin ATPase. It is concluded that tetramethylrhodamine, in contrast to phalloidin, accelerates P(i)-release from actin filaments.

Actins↗
PubMed1995

Structural studies on the ribbon-to-helix transition in profilin: actin crystals.

Knowledge of the structure of actin in its various conformational states is important for understanding the diverse motile activities carried out by eukaryotic cells. Profilin:actin crystals provide a unique system for studying conformational states of actin, because they exhibit a high degree of polymorphism in response to environmental conditions while maintaining crystalline order. A preliminary comparison of two states of profilin:beta-actin crystals shows that crystal polymorphism involves movements of actin subdomains at hinge points homologous to those found in hexokinase, a protein whose polypeptide fold is related to actin. The homology of the hinge points in actin to those in hexokinase suggests that actin subdomain movements in profilin:beta-actin crystals have functional significance. We discuss how these movements could be related to structural transitions between states of filamentous actin in muscle contraction.

Actins↗
PubMed1994

Crystallization and structure determination of bovine profilin at 2.0 A resolution.

Profilin regulates the behavior of the eukaryotic microfilament system through its interaction with non-filamentous actin. It also binds several ligands, including poly(L-proline) and the membrane phospholipid phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2). Bovine profilin crystals (space group C2; a = 69.15 A, b = 34.59 A, c = 52.49 A; alpha = gamma = 90 degrees, beta = 92.56 degrees) were grown from a mixture of poly(ethylene glycol) 400 and ammonium sulfate. X-ray diffraction data were collected on an imaging plate scanner at the DORIS storage ring (DESY, Hamburg), and were phased by molecular replacement, using a search model derived from the 2.55 A structure of profilin complexed to beta-actin. The refined model of bovine profilin has a crystallographic R-factor of 16.5% in the resolution range 6.0 to 2.0 A and includes 128 water molecules, several of which form hydrogen bonds to stabilize unconventional turns. The structure of free bovine profilin is similar to that of bovine profilin complexed to beta-actin, and C alpha atoms from the two structures superimpose with an r.m.s. deviation of 1.25 A. This value is reduced to 0.51 A by omitting Ala1 and the N-terminal acetyl group, which lie at a profilin-actin interface in crystals of the complex. These residues display a strained conformation in crystalline profilin-actin but may allow the formation of a hydrogen bond between the N-acetyl carbonyl group of profilin and the phenol hydroxyl group of Tyr188 in actin. Several other actin-binding residues of profilin show different side-chain rotomer conformations in the two structures. The polypeptide fold of bovine profilin is generally similar to those observed by NMR for profilin from other sources, although the N terminus of Acanthamoeba profilin isoform I lies in a distorted helix and the C-terminal helix is less tilted with respect to the strands in the central beta-pleated sheet than is observed in bovine profilin. The majority of the aromatic residues in profilin are exposed to solvent and lie in either of two hydrophobic patches, neither of which takes part in an interface with actin. One of these patches is required for binding poly(L-proline) and contains an aromatic cluster comprising the highly conserved residues Trp3, Tyr6, Trp31 and Tyr139. In forming this cluster, Trp31 adopts a sterically strained rotamer conformation.(ABSTRACT TRUNCATED AT 400 WORDS)

Actins↗
PubMed1994

Structural aspects of actin-binding proteins.

The three-dimensional structures of myosin subfragment 1 (S1), gelsolin segment 1 complexed with alpha-actin, villin fragment 14T, Acanthamoeba profilin-I, and bovine profilin complexed with beta-actin were completed last year. Together, they expand our understanding of the structural organization of actin-binding proteins. In addition, the segment 1 and bovine profilin complexes provide atomic-level descriptions of their interfaces with actin.

Actins↗
PubMed1993

The structure of crystalline profilin-beta-actin.

The three-dimensional structure of bovine profilin-beta-actin has been solved to 2.55 A resolution by X-ray crystallography. There are several significant local changes in the structure of beta-actin compared with alpha-actin as well as an overall 5 degrees rotation between its two major domains. Actin molecules in the crystal are organized into ribbons through intermolecular contacts like those found in oligomeric protein assemblies. Profilin forms two extensive contacts with the actin ribbon, one of which appears to correspond to the solution contact in vitro.

Actins↗
PubMed1993

Mutagenesis of human profilin locates its poly(L-proline)-binding site to a hydrophobic patch of aromatic amino acids.

The actin-binding protein, profilin, contains a src-homology (SH) 3-like fold (Schutt, C.E. et al., submitted), and its tight interaction with poly(L-proline) is reminiscent of the binding activity exhibited by SH3-domains. Here we demonstrate that replacements of aromatic amino acids in a hydrophobic patch on the surface of the profilin molecule abolish its poly(L-proline)-binding capacity. However, the location of this hydrophobic patch is found in another region of the molecule than that displaying structural similarities with SH3 domains.

Amino Acids↗
PubMed1993

Mutations in beta-actin: influence on polymer formation and on interactions with myosin and profilin.

Two beta-actin mutants, one with proline 38 replaced with alanine (P38A) and the other with cysteine-374 replaced with serine (C374S), as well as the wild-type beta-actin, were expressed in the yeast, S. cerevisiae, purified to homogeneity, and analyzed in vitro for polymerizability and interaction with DNase I, myosin, and profilin. Both mutations interfered with the polymerization of the actin, and with its interaction with myosin. The C374S mutation had the most pronounced effect; it reduced the polymerizability of the actin, abolished its binding to profilin, and filaments containing this mutation moved at reduced rates in the in vitro 'motility assay'. The ATPase activity measured in solutions containing myosin subfragment 1 was similar for both the mutant and wild-type actins.

Actins↗
PubMed1993

A new perspective on muscle contraction.

Recent experimental findings suggest that the myosin cross-bridge theory may no longer be adequate to account for certain basic facts concerning muscle contraction. A newly-proposed mechanism based on length changes in actin filaments might be the basis for a simpler explanation for how the free energy of ATP hydrolysis can be transduced into work by muscle fibers.

Actins↗
PubMed1993

Location of profilin at presynaptic sites in the cerebellar cortex; implication for the regulation of the actin-polymerization state during axonal elongation and synaptogenesis.

Profilin is a 15 kDa protein that binds actin monomers and inhibits their polymerization in vitro. The actin-profilin complex can be rapidly dissociated in vitro by phosphatidylinositol-4,5-bis-phosphate, providing a mechanism for regulating actin assembly-disassembly cycles during cell motile events. We have used a polyclonal antibody to calf spleen profilin to analyse the developmental expression and cellular distribution of profilin in the rat cerebellum and cultured cortical neurons. Immature neurons contain large amount of profilin both in vivo and in vitro. Immunofluorescence showed it to be present in developing neurites and growth cones but not in the filopodia of cortical neurons in culture. Profilin immunoreactivity was intense in the parallel fibres, the granule cell axons of the cerebellar cortex, at the time when they are elongating. Purkinje cell dendrites were not labelled. Profilin immunostaining was present in presynaptic varicosities, but not in dendritic spines within the molecular layer of juvenile and adult rats. The profilin concentration was higher in synaptosomes than in the total cerebellum during the second and third postnatal weeks, a period of intense synaptogenesis. Thus, profilin may help regulate actin polymerization and depolymerization during axonal elongation and synaptogenesis. Its restriction to the presynaptic site in the adult suggests that it may also be involved in the regulation of the release of synaptic vesicles.

Actins↗
PubMed1992

The use of alternative substrates in the characterization of actin-methylating and carnosine-methylating enzymes.

Actin isolated from nearly every eukaryotic species contains approximately 1 mol 3-methylhistidine/mol protein. His73 in actin has been shown, by protein sequencing, to be the site of methylation. The methylation occurs enzymically and post-translationally. A rabbit skeletal muscle myofibrillary fraction has previously been shown to contain a histidine methyltransferase activity that is actin specific. Detailed study of this enzyme has been hampered by lack of a suitable substrate assay. Naturally occurring actins are poor substrates for the enzyme, presumably due to prexistent methylation at His73. In this study, two potential alternative substrates have been investigated. These are a chicken beta-actin expressed in Escherichia coli as a fusion protein with 80 amino acids of an influenza protein, NS1, and a synthetic peptide, Tyr-Pro-Ile-Glu-His-Gly-Ile-Ile-Thr, corresponding to residues 69-77 of actin. Both substrates were covalently methylated at histidine residues in the presence of S-adenosylmethionine and partially purified enzyme fractions from rabbit muscle. In methylation experiments employing the fusion actin in the form of inclusion bodies, 3-methylhistidine is the major product, as is the case when soluble muscle or non-muscle actin is used. However, for the synthetic peptide, the methylated product primarily contained 1-methylhistidine and only a small amount of the isomeric 3-methylhistidine. Further investigations revealed that the peptide was recognized by carnosine N-methyltransferase, another histidine methyltransferase found in muscle tissue. Carnosine N-methyltransferase appears to copurify with the actin-methylating enzyme in preliminary fractionation experiments. Separation of the two methyltransferase activities is described.

Actins↗
PubMed1992

Characterization of yeast-expressed beta-actins, site-specifically mutated at the tumor-related residue Gly245.

The tumorigenic cell line HUT14 expresses a beta-actin carrying a mutation at position 245. In this study, two mutant beta-actins with amino acid changes at position 245 replacing the wild-type glycine by an aspartic acid and a lysine residue, respectively, were produced in the yeast Saccharomyces cerevisiae, purified to homogeneity and characterized with respect to polymerization behaviour and interaction with myosin. The major functional effect of these mutations appears to be an impaired polymerization, while the interaction with myosin seems less influenced. In addition, the results also suggest the presence of a Ca(2+)-binding site in the region of residue 245 in actin.

Actins↗