Scar, a WASp-related protein, activates nucleation of actin filaments by the Arp2/3 complex

The Arp2/3 complex, a stable assembly of two actin-related proteins (Arp2 and Arp3) with five other subunits, caps the pointed end of actin filaments and nucleates actin polymerization with low efficiency. WASp and Scar are two similar proteins that bind the p21 subunit of the Arp2/3 complex, but their effect on the nucleation activity of the complex was not known. We report that full-length, recombinant human Scar protein, as well as N-terminally truncated Scar proteins, enhance nucleation by the Arp2/3 complex. By themselves, these proteins either have no effect or inhibit actin polymerization. The actin monomer-binding W domain and the p21-binding A domain from the C terminus of Scar are both required to activate Arp2/3 complex. A proline-rich domain in the middle of Scar enhances the activity of the W and A domains. Preincubating Scar and Arp2/3 complex with actin filaments overcomes the initial lag in polymerization, suggesting that efficient nucleation by the Arp2/3 complex requires assembly on the side of a preexisting filament—a dendritic nucleation mechanism. The Arp2/3 complex with full-length Scar, Scar containing P, W, and A domains, or Scar containing W and A domains overcomes inhibition of nucleation by the actin monomer-binding protein profilin, giving active nucleation over a low background of spontaneous nucleation. These results show that Scar and, likely, related proteins, such as the Cdc42 targets WASp and N-WASp, are endogenous activators of actin polymerization by the Arp2/3 complex.

Intron “sliding” and the diversity of intron positions

Alignments of homologous genes typically reveal a great diversity of intron locations, far more than could fit comfortably in a single gene. Thus, a minority of these intron positions could be inherited from a single ancestral gene, but the larger share must be attributed to subsequent events of intron gain or intron “sliding” (movement from one position to another within a gene). Intron sliding has been argued from cases of discordant introns and from putative spatial clustering of intron positions. A list of 32 cases of discordant introns is presented here. Most of these cases are found to be artefactual. The spatial and phylogenetic distributions of intron positions from five published compilations of gene data, comprising 205 intron positions, have been examined systematically for evidence of intron sliding. The results suggest that sliding, if it occurs at all, has contributed little to the diversity of intron positions.

Intron positions correlate with module boundaries in ancient proteins

We analyze the three-dimensional structure of proteins by a computer program that finds regions of sequence that contain module boundaries, defining a module as a segment of polypeptide chain bounded in space by a specific given distance. The program defines a set of “linker regions” that have the property that if an intron were to be placed into each linker region, the protein would be dissected into a set of modules all less than the specified diameter. We test a set of 32 proteins, all of ancient origin, and a corresponding set of 570 intron positions, to ask if there is a statistically significant excess of intron positions within the linker regions. For 28-Å modules, a standard size used historically, we find such an excess, with P < 0.003. This correlation is neither due to a compositional or sequence bias in the linker regions nor to a surface bias in intron positions. Furthermore, a subset of 20 introns, which can be putatively identified as old, lies even more explicitly within the linker regions, with P < 0.0003. Thus, there is a strong correlation between intron positions and three-dimensional structural elements of ancient proteins as expected by the introns-early approach. We then study a range of module diameters and show that, as the diameter varies, significant peaks of correlation appear for module diameters centered at 21.7, 27.6, and 32.9 Å. These preferred module diameters roughly correspond to predicted exon sizes of 15, 22, and 30 residues. Thus, there are significant correlations between introns, modules, and a quantized pattern of the lengths of polypeptide chains, which is the prediction of the “Exon Theory of Genes.”

The interaction of Arp2/3 complex with actin: Nucleation, high affinity pointed end capping, and formation of branching networks of filaments

The Arp2/3 complex is a stable assembly of seven protein subunits including two actin-related proteins (Arp2 and Arp3) and five novel proteins. Previous work showed that this complex binds to the sides of actin filaments and is concentrated at the leading edges of motile cells. Here, we show that Arp2/3 complex purified from Acanthamoeba caps the pointed ends of actin filaments with high affinity. Arp2/3 complex inhibits both monomer addition and dissociation at the pointed ends of actin filaments with apparent nanomolar affinity and increases the critical concentration for polymerization at the pointed end from 0.6 to 1.0 μM. The high affinity of Arp2/3 complex for pointed ends and its abundance in amoebae suggest that in vivo all actin filament pointed ends are capped by Arp2/3 complex. Arp2/3 complex also nucleates formation of actin filaments that elongate only from their barbed ends. From kinetic analysis, the nucleation mechanism appears to involve stabilization of polymerization intermediates (probably actin dimers). In electron micrographs of quick-frozen, deep-etched samples, we see Arp2/3 bound to sides and pointed ends of actin filaments and examples of Arp2/3 complex attaching pointed ends of filaments to sides of other filaments. In these cases, the angle of attachment is a remarkably constant 70 ± 7°. From these in vitro biochemical properties, we propose a model for how Arp2/3 complex controls the assembly of a branching network of actin filaments at the leading edge of motile cells.

CKAAPs DB: a conserved key amino acid positions database

The Conserved Key Amino Acid Positions DataBase (CKAAPs DB) provides access to an analysis of structurally similar proteins with dissimilar sequences where key residues within a common fold are identified. The derivation and significance of CKAAPs starting from pairwise structure alignments is described fully in Reddy et al. [Reddy,B.V.B., Li,W.W., Shindyalov,I.N. and Bourne,P.E. (2000) Proteins, in press]. The CKAAPs identified from this theoretical analysis are provided to experimentalists and theoreticians for potential use in protein engineering and modeling. It has been suggested that CKAAPs may be crucial features for protein folding, structural stability and function. Over 170 substructures, as defined by the Combinatorial Extension (CE) database, which are found in approximately 3000 representative polypeptide chains have been analyzed and are available in the CKAAPs DB. CKAAPs DB also provides CKAAPs of the representative set of proteins derived from the CE and FSSP databases. Thus the database contains over 5000 representative poly­peptide chains, covering all known structures in the PDB. A web interface to a relational database permits fast retrieval of structure-sequence alignments, CKAAPs and associated statistics. Users may query by PDB ID, protein name, function and Enzyme Classification number. Users may also submit protein alignments of their own to obtain CKAAPs. An interface to display CKAAPs on each structure from a web browser is also being implemented. CKAAPs DB is maintained by the San Diego Supercomputer Center and accessible at the URL http://ckaaps.sdsc.edu.

Nonuniformity of the constitutive law parameters for shear rupture and quasistatic nucleation to dynamic rupture: a physical model of earthquake generation processes.

Based on the recent high-resolution laboratory experiments on propagating shear rupture, the constitutive law that governs shear rupture processes is discussed in view of the physical principles and constraints, and a specific constitutive law is proposed for shear rupture. It is demonstrated that nonuniform distributions of the constitutive law parameters on the fault are necessary for creating the nucleation process, which consists of two phases: (i) a stable, quasistatic phase, and (ii) the subsequent accelerating phase. Physical models of the breakdown zone and the nucleation zone are presented for shear rupture in the brittle regime. The constitutive law for shear rupture explicitly includes a scaling parameter Dc that enables one to give a common interpretation to both small scale rupture in the laboratory and large scale rupture as earthquake source in the Earth. Both the breakdown zone size Xc and the nucleation zone size L are prescribed and scaled by Dc, which in turn is prescribed by a characteristic length lambda c representing geometrical irregularities of the fault. The models presented here make it possible to understand the earthquake generation process from nucleation to unstable, dynamic rupture propagation in terms of physics. Since the nucleation process itself is an immediate earthquake precursor, deep understanding of the nucleation process in terms of physics is crucial for the short-term (or immediate) earthquake prediction.

The nucleation of receptor-mediated endocytosis.

A theory of the mechanical origins of receptor-mediated endocytosis shows that a spontaneous membrane complex formation can provide the stimulus for a local membrane motion toward the cytosol. This motion is identified with a nucleation stage of receptor-mediated endocytosis. When membrane complexes cluster, membrane deformation is predicted to be most rapid. The rate of growth of membrane depressions depends upon the relative rates of approach of aqueous cytosolic and extracellular fluids toward the cell membrane. With cytosolic and extracellular media characterized by apparent viscosities, the rate of growth of membrane depressions is predicted to increase as the extracellular viscosity nears the apparent viscosity of the cytosol and then to decrease when the extracellular viscosity exceeds that of the cytosol. To determine whether these trends would be apparent in the overall endocytosis rate constant, an experimental study of transferrin-mediated endocytosis in two different cell lines was conducted. The experimental results reveal the same dependence of internalization rate on extracellular viscosity as predicted by the theory. These and other comparisons with experimental data suggest that the nucleation stage of receptor-mediated endocytosis is important in the overall endocytosis process.

On the nucleation and growth of amyloid beta-protein fibrils: detection of nuclei and quantitation of rate constants.

We have studied the fibrillogenesis of synthetic amyloid beta-protein-(1-40) fragment (A beta) in 0.1 M HCl. At low pH, A beta formed fibrils at a rate amenable to detailed monitoring by quasi-elastic light-scattering spectroscopy. Examination of the fibrils with circular dichroism spectroscopy and electron microscopy showed them to be highly similar to those found in amyloid plaques. We determined the hydrodynamic radii of A beta aggregates during the entire process of fibril nucleation and growth. Above an A beta concentration of approximately 0.1 mM, the initial rate of elongation and the final size of fibrils were independent of A beta concentration. Below an A beta concentration of 0.1 mM, the initial elongation rate was proportional to the peptide concentration, and the resulting fibrils were significantly longer than those formed at higher concentration. We also found that the surfactant n-dodecylhexaoxyethylene glycol monoether (C12E6) slowed nucleation and elongation of fibrils in a concentration-dependent manner. Our observations are consistent with a model of A beta fibrillogenesis that includes the following key steps: (i) peptide micelles form above a certain critical A beta concentration, (ii) fibrils nucleate within these micelles or on heterogeneous nuclei (seeds), and (iii) fibrils grow by irreversible binding of monomers to fibril ends. Interpretation of our data enabled us to determine the sizes of fibril nuclei and A beta micelles and the rates of fibril nucleation (from micelles) and fibril elongation. Our approach provides a powerful means for the quantitative assay of A beta fibrillogenesis.

Optimization of rates of protein folding: the nucleation-condensation mechanism and its implications.

Small, single-module proteins that fold in a single cooperative step may be paradigms for understanding early events in protein-folding pathways generally. Recent experimental studies of the 64-residue chymotrypsin inhibitor 2 (CI2) support a nucleation mechanism for folding, as do some computer stimulations. CI2 has a nucleation site that develops only in the transition state for folding. The nucleus is composed of a set of adjacent residues (an alpha-helix), stabilized by long-range interactions that are formed as the rest of the protein collapses around it. A simple analysis of the optimization of the rate of protein folding predicts that rates are highest when the denatured state has little residual structure under physiological conditions and no intermediates accumulate. This implies that any potential nucleation site that is composed mainly of adjacent residues should be just weakly populated in the denatured state and become structured only in a high-energy intermediate or transition state when it is stabilized by interactions elsewhere in the protein. Hierarchical mechanisms of folding in which stable elements of structure accrete are unfavorable. The nucleation-condensation mechanism of CI2 fulfills the criteria for fast folding. On the other hand, stable intermediates do form in the folding of more complex proteins, and this may be an unavoidable consequence of increasing size and nucleation at more than one site.

Changing nucleosome positions through modification of the DNA rotational information.

The effects of the rotational information of DNA in determining the in vitro localization of nucleosomal core particles (ncps) have been studied in the Saccharomyces cerevisiae 5S rRNA repeat gene. We have altered the distribution of the phased series of flexibility signals present on this DNA by inserting a 25-bp tract, and we have analyzed the effects of this mutation on the distribution and on the frequencies of ncps, as compared with the wild type and a reference 21-bp insertion mutant. The variation of the standard free energy of nucleosome reconstitution was determined. The results show that the DNA rotational information is a major determinant of ncps positioning, define how many rotationally phased signals are required for the formation of a stable particle, and teach how to modify their distribution through the alteration of the rotational signals.

Seven newly discovered intron positions in the triose-phosphate isomerase gene: evidence for the introns-late theory.

The gene encoding the glycolytic enzyme triose-phosphate isomerase (TPI; EC 5.3.1.1) has been central to the long-standing controversy on the origin and evolutionary significance of spliceosomal introns by virtue of its pivotal support for the introns-early view, or exon theory of genes. Putative correlations between intron positions and TPI protein structure have led to the conjecture that the gene was assembled by exon shuffling, and five TPI intron positions are old by the criterion of being conserved between animals and plants. We have sequenced TPI genes from three diverse eukaryotes--the basidiomycete Coprinus cinereus, the nematode Caenorhabditis elegans, and the insect Heliothis virescens--and have found introns at seven novel positions that disrupt previously recognized gene/protein structure correlations. The set of 21 TPI introns now known is consistent with a random model of intron insertion. Twelve of the 21 TPI introns appear to be of recent origin since each is present in but a single examined species. These results, together with their implication that as more TPI genes are sequenced more intron positions will be found, render TPI untenable as a paradigm for the introns-early theory and, instead, support the introns-late view that spliceosomal introns have been inserted into preexisting genes during eukaryotic evolution.

Types of Listeria monocytogenes predicted by the positions of EcoRI cleavage sites relative to ribosomal RNA sequences.

By using taxonomic characters derived from EcoRI restriction endonuclease digestion of genomic DNA and hybridization with a labeled rRNA operon from Escherichia coli, a polymorphic structure of Listeria monocytogenes, characterized by fragments with different frequencies of occurrence, was observed. This structure was expanded by creating predicted patterns through a recursive process of observation, expectation, prediction, and assessment of completeness. This process was applied, in turn, to normalized strain patterns, fragment bands, and positions of EcoRI recognition sites relative to rRNA regions. Analysis of 1346 strains provided observed patterns, fragment sizes, and their frequencies of occurrence in the patterns. Fragment size statistics led to the creation of unobserved combinations of bands, predicted pattern types. The observed fragment bands revealed positions of EcoRI sites relative to rRNA sequences. Each EcoRI site had a frequency of occurrence, and unobserved fragment sizes were postulated on the basis of knowing the restriction site locations. The result of the recursion process applied to the components of the strain data was an extended classification with observed and predicted members.

Crystal structure of an H-2Kb-ovalbumin peptide complex reveals the interplay of primary and secondary anchor positions in the major histocompatibility complex binding groove.

Sequence analysis of peptides naturally presented by major histocompatibility complex (MHC) class I molecules has revealed allele-specific motifs in which the peptide length and the residues observed at certain positions are restricted. Nevertheless, peptides containing the standard motif often fail to bind with high affinity or form physiologically stable complexes. Here we present the crystal structure of a well-characterized antigenic peptide from ovalbumin [OVA-8, ovalbumin-(257-264), SIINFEKL] in complex with the murine MHC class I H-2Kb molecule at 2.5-A resolution. Hydrophobic peptide residues Ile-P2 and Phe-P5 are packed closely together into binding pockets B and C, suggesting that the interplay of peptide anchor (P5) and secondary anchor (P2) residues can couple the preferred sequences at these positions. Comparison with the crystal structures of H-2Kb in complex with peptides VSV-8 (RGYVYQGL) and SEV-9 (FAPGNYPAL), where a Tyr residue is used as the C pocket anchor, reveals that the conserved water molecule that binds into the B pocket and mediates hydrogen bonding from the buried anchor hydroxyl group could not be likewise positioned if the P2 side chain were of significant size. Based on this structural evidence, H-2Kb has at least two submotifs: one with Tyr at P5 (or P6 for nonamer peptides) and a small residue at P2 (i.e., Ala or Gly) and another with Phe at P5 and a medium-sized hydrophobic residue at P2 (i.e., Ile). Deciphering of these secondary submotifs from both crystallographic and immunological studies of MHC peptide binding should increase the accuracy of T-cell epitope prediction.