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.

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)

Overexpression of tubulin in yeast: differences in subunit association.

Microtubules are cytoskeletal organelles composed principally of polymerized alpha beta-tubulin heterodimers. The specific roles and the detailed structures of the individual alpha- and beta-tubulin subunits have not been established, since the conditions necessary for separating the heterodimer result in loss of the subunits' ability to repolymerize. We have overcome this obstacle by constructing plasmids which allow regulated overexpression of individual tubulin subunits in the yeast Saccharomyces cerevisiae under control of the galactose promoter. Overproduction was monitored with alpha- and beta-tubulin-specific antibodies using immunoblotting of cell extracts, and the state of association of the individual subunits in vivo was determined by immunofluorescence microscopy. Cells overproducing only beta-tubulin accumulated fibrous structures associated with the cell membrane, whereas cells overproducing only alpha-tubulin displayed a diffuse signal throughout the cytoplasm. Cells simultaneously overexpressing alpha- and beta-tubulin subunits accumulated membrane-associated, filamentous arrays in which both subunits were incorporated. When cells with the fibrous tubulin-containing structures are treated with zymolyase, a yeast cell wall disrupting enzyme, the fibers appear to splay apart, suggesting that the immunofluorescent rings represent bundles of fibers. Cells overproducing beta-tubulin alone or both alpha- and beta-tubulin were examined at various times after galactose induction, and significant differences were found in the tubulin association state prior to the formation of fibers. For alpha beta-tubulin, fibers form directly from a nuclear structure, whereas beta-tubulin alone first accumulates in the cytoplasm. The differences in patterns of tubulin accumulation and assembly presumably reflect a difference in the intrinsic association properties of the alpha- and beta-subunits.

Conservation of tubulin alpha-beta differences in zinc-induced sheets with variations in pH and microtubule-associated protein content.

Zinc-induced tubulin sheets were grown at pH values of 5.7 and 6.4 from porcine brain tubulin purified by phosphocellulose chromatography as well as from microtubule protein containing tubulin plus 20% (w/w) unfractionated microtubule-associated proteins (MAPs). Electron micrographs of negatively stained sheets were analyzed by a combination of real space cross-correlation and Fourier space methods, providing two-dimensional reconstructions to approximately 16 A resolution. The reconstructed images revealed that the protofilaments comprising zinc-induced sheets are composed of two clearly distinguishable alternating subunits, presumably corresponding to the alpha- and beta-tubulin monomers, whose morphologies are not significantly influenced by pH or the presence of MAPs during sheet assembly. Sheets assembled at pH 5.7, either with or without MAPs, were divided into two domains by a protofilament discontinuity which was not present in sheets assembled at pH 6.4, and displayed a 2.4 A reduction of the interprotofilament distance in projection relative to sheets assembled at pH 6.4. We conclude that morphological differences between tubulin subunits represent intrinsic structural features not contributed by MAPs, and that pH is more important than MAP content in influencing the lattice parameters of zinc-induced sheets.

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.

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.