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Andrey V Kajava

Publications and source records attributed to Andrey V Kajava.

At least 19 recordsLinked to original sources
PubMed2006

The turn of the screw: variations of the abundant beta-solenoid motif in passenger domains of Type V secretory proteins.

Many virulence factors of gram-negative bacteria are secreted by the Type V secretion system via the autotransporter (AT) and two-partner secretion (TPS) pathways. AT proteins effect their own secretion. They comprise three domains: the amino-terminal leader sequence; the secreted passenger domain; and the translocator domain that forms the secretory channel. In the TPS pathway, the passenger and translocator domains are translated as separate proteins. In a previous publication, we proposed a beta-helical structure for the TPS passenger domain of the filamentous hemagglutinin (FHA) of Bordetella pertussis which contains two tracts, R1 and R2, of 19-residue sequence repeats and built molecular models for the R1 and R2 beta-helices. Here, we compare the structure predicted for R1 with the recently determined crystal structure of a fragment containing three R1 repeats and find close agreement, with an RMSD of 1.1A. In the interim, the number of known AT and TPS protein sequences has increased to >1000. To investigate the incidence of beta-helical structures among them, we carried out a sequence-based analysis and conclude that, despite wide diversity in the sizes and sequences of passenger domains, most of them contain beta-solenoids that we classify into thirteen types based on distinctive properties of their beta-coils (repeat length, numbers and lengths of beta-strands and turns, cross-sectional shape, presence of specific residues in certain positions) summarized in a 2D coil template. Some coil types are typical for conventional AT proteins, others, for TPS or trimeric AT proteins. Some beta-solenoids consist of stacked subdomains with coils of different types. To illustrate model-building from a coil template, we modeled a type-T4 beta-solenoid for TibA of Escherichia coli which is predicted to have two conserved polar residues, Thr and Gln, in interior positions.

Adhesins, Bacterial↗
PubMed2006

Standard conformations of beta-arches in beta-solenoid proteins.

Strand-turn-strand motifs found in beta-helical (more generally, beta-solenoid) proteins differ fundamentally from those found in globular proteins. The latter are primarily beta-hairpins in which the two strands form an antiparallel beta-sheet. In the former, the two strands are relatively rotated by approximately 90 degrees around the strand axes so that they interact via the side-chains, not via the polypeptide backbones. We call the latter structures, beta-arches, and their turns, beta-arcs. In beta-solenoid proteins, beta-arches stack in-register to form beta-arcades in which parallel beta-sheets are assembled from corresponding strands in successive layers. The number of beta-solenoids whose three-dimensional structures have been determined is now large enough to support a detailed analysis and classification of beta-arc conformations. Here, we present a systematic account of beta-arcs distinguished by the number of residues, their conformations, and their propensity to stack into arcades with other like or unlike arches. The trends to emerge from this analysis have implications for sequence-based detection and structural prediction of other beta-solenoid proteins as well as for identification of amyloidogenic sequences and elucidation of amyloid fibril structures.

Amino Acid Sequence↗
PubMed2006

Synthetic RGD-containing alpha-helical coiled coil peptides promote integrin-dependent cell adhesion.

Integrin receptors are the main mediators of cell adhesion to the extracellular matrix. They bind to their ligands by interacting with short amino acid sequences, such as the RGD sequence. Soluble, small RGD-based peptides have been used to block integrin-binding to ligands, thereby interfering with cell adhesion, migration and survival, while substrate-immobilized RGD sequences have been used to enhance cell binding to artificial surfaces. This approach has several important medical applications, e.g. in suppression of tumor angiogenesis or stimulation of bone formation around implants. However, the relatively weak affinity of short RGD-containing peptides often results in incomplete integrin inhibition or ineffective ligation. In this work, we designed and synthesized several new multivalent RGD-containing molecules and tested their ability to inhibit or to promote integrin-dependent cell adhesion when used in solution or immobilized on substrates, respectively. These molecules consist of an oligomeric structure formed by alpha-helical coiled coil peptides fused at their amino-terminal ends with an RGD-containing fragment. When immobilized on a substrate, these peptides specifically promoted integrin alphaVbeta3-dependent cell adhesion, but when used in solution, they blocked alphaVbeta3-dependent cell adhesion to the natural substrates fibronectin and vitronectin. One of the peptides was nearly 10-fold more efficient than fibronectin or vitronectin in promoting cell adhesion, and almost 100-fold more efficient than a linear RGD tripeptide in blocking adhesion. These results indicate that alpha-helical coiled coil peptides carrying an amino-terminal RGD motif can be used as soluble antagonists or surface-immobilized agonists to efficiently inhibit or promote integrin alphaVbeta3-mediated cell adhesion, respectively.

Cell Adhesion↗
PubMed2006

Beta-structures in fibrous proteins.

The beta-form of protein folding, one of the earliest protein structures to be defined, was originally observed in studies of silks. It was then seen in early studies of synthetic polypeptides and, of course, is now known to be present in a variety of guises as an essential component of globular protein structures. However, in the last decade or so it has become clear that the beta-conformation of chains is present not only in many of the amyloid structures associated with, for example, Alzheimer's Disease, but also in the prion structures associated with the spongiform encephalopathies. Furthermore, X-ray crystallography studies have revealed the high incidence of the beta-fibrous proteins among virulence factors of pathogenic bacteria and viruses. Here we describe the basic forms of the beta-fold, summarize the many different new forms of beta-structural fibrous arrangements that have been discovered, and review advances in structural studies of amyloid and prion fibrils. These and other issues are described in detail in later chapters.

Amyloid↗
PubMed2006

Beta-rolls, beta-helices, and other beta-solenoid proteins.

Beta-rolls and beta-helices belong to a larger group of topologically similar proteins with solenoid folds: because their regular secondary structure elements are exclusively beta-strands, they are referred to as beta-solenoids. The number of beta-solenoids whose structures are known is now large enough to support a systematic analysis. Here we survey the distinguishing structural features of beta-solenoids, also documenting their notable diversity. Appraisal of these structures suggests a classification based on handedness, twist, oligomerization state, and coil shape. In addition, beta-solenoids are distinguished by the number of chains that wind around a common axis: the majority are single-stranded but there is a recently discovered subset of triple-stranded beta-solenoids. This survey has revealed some relationships of the amino acid sequences of beta-solenoids with their structures and functions-in particular, the repetitive character of the coil sequences and conformations that recur in tracts of tandem repeats. We have proposed the term beta-arc for the distinctive turns found in beta-solenoids and beta-arch for the corresponding strand-turn-strand motifs. The evolutionary mechanisms underlying these proteins are also discussed. This analysis has direct implications for sequence-based detection, structural prediction, and de novo design of other beta-solenoid proteins. The abundance of virulence factors, toxins and allergens among beta-solenoids, as well as commonalities of beta-solenoids with amyloid fibrils, imply that this class of folds may have a broader role in human diseases than was previously recognized. Thus, identification of genes with putative beta-solenoid domains promises to be a fertile direction in the search for viable targets in the development of new antibiotics and vaccines.

Models, Molecular↗
PubMed2006

Structure, function, and amyloidogenesis of fungal prions: filament polymorphism and prion variants.

Infectious proteins (prions) became an important medical issue when they were identified as agents of the transmissible spongiform encephalopathies. More recently, prions have been found in fungi and their investigation has been facilitated by greater experimental tractability. In each case, the normal form of the prion protein may be converted into the infectious form (the prion itself) in an autocatalytic process; conversion may either occur spontaneously or by transmission from an already infected cell. Four fungal prion proteins have been studied in some depth-Ure2p, Sup35p, and Rnq1p of Saccharomyces cerevisiae and HET-s of Podospora anserina. Each has a "prion domain" that governs infectivity and a "functional domain" that contributes the protein's activity in a wild-type cell, if it has one. This activity is repressed in prion-infected cells for loss-of-activity prions, [URE3] (the prion of Ure2p) and [PSI] (the prion of Sup35p). For gain-of-activity prions, [PIN] (the prion of Rnq1p) and [Het-s] (the prion of HET-s), the prion domain is also involved in generating a new activity in infected cells. In prion conversion, prion domains polymerize into an amyloid filament, switching from a "natively unfolded" conformation into an amyloid conformation (stable, protease-resistant, rich in cross-beta structure). For Ure2p and probably also Sup35p, the functional domain retains its globular fold but is inactivated by a steric mechanism. We review the evidence on which this scenario is based with emphasis on filament structure, summarizing current experimental constraints and appraising proposed models. We conclude that the parallel superpleated beta-structure and a specific beta-helical formulation are valid candidates while other proposals are excluded. In both the Ure2p and Sup35p systems, prion domain amyloid filaments exhibit polymorphic variation. However, once a certain structure is nucleated, it is maintained throughout that filament. Electron microscopy of several Ure2p-related constructs indicates that the basis for polymorphism lies mainly if not entirely in the prion domain. Filament polymorphism appears to underlie the phenomenon of prion "variants" which differ in the severity of their phenotype, that is, for Ure2p and Sup35p, the stringency with which their activity is switched off. We discuss a possible structural basis for this phenomenon.

Amino Acid Sequence↗
PubMed2005

Tubulin polyglutamylase enzymes are members of the TTL domain protein family.

Polyglutamylation of tubulin has been implicated in several functions of microtubules, but the identification of the responsible enzyme(s) has been challenging. We found that the neuronal tubulin polyglutamylase is a protein complex containing a tubulin tyrosine ligase-like (TTLL) protein, TTLL1. TTLL1 is a member of a large family of proteins with a TTL homology domain, whose members could catalyze ligations of diverse amino acids to tubulins or other substrates. In the model protist Tetrahymena thermophila, two conserved types of polyglutamylases were characterized that differ in substrate preference and subcellular localization.

Amino Acid Motifs↗
PubMed2005

The parallel superpleated beta-structure as a model for amyloid fibrils of human amylin.

Human amylin is a 37 amino acid residue peptide hormone whose fibrillogenesis has been correlated with type 2 diabetes. These fibrils are rope-like bundles of several 5nm diameter protofilaments. Here, we propose, as a model for the protofilament, a variant of the parallel superpleated beta-structure previously derived for amyloid filaments of the yeast prion Ure2p. In the amylin model, individual polypeptides from residues 9 to 37 have a planar S-shaped fold with three beta-strands. These serpentines are stacked in register, with a 0.47 nm axial rise and a small rotational twist per step, generating an array of three parallel beta-sheets in cross-beta conformation. The interior, the two "bays" sandwiched between adjacent sheets, are occupied by non-polar and by polar/uncharged residues that are predicted to form H-bonded ladders, similar to those found in beta-helical proteins. The N-terminal peptide containing a disulfide bond occupies an extraneous peripheral position in the protofilament. The left-handed twist of the beta-sheets is shown to underlie left-handed coiling of amylin protofilaments in fibrils. The model is consistent with current biophysical, biochemical and genetic data and, in particular, affords a plausible explanation for why rodent amylin does not form fibrils.

Amino Acid Sequence↗
PubMed2005

The axial channel of the 20S proteasome opens upon binding of the PA200 activator.

Proteasomes consist of a proteolytic core called the 20 S particle and ancillary factors that regulate its activity in various ways. PA200 has been identified as a large (200 kDa) nuclear protein that stimulates proteasomal hydrolysis of peptides. To characterize its interaction with the 20 S core, we have visualized PA200-20 S complexes by electron microscopy. Monomers of PA200 bind to one or both ends of the 20 S core. Reconstructed in three dimensions to 23 A resolution from cryo-electron micrographs of the singly bound complex, PA200 has an asymmetric dome-like structure with major and minor lobes. Taking into account previous bioinformatic analysis, it is likely to represent an irregular folding of an alpha-helical solenoid composed of HEAT-like repeats. PA200 makes contact with all alpha-subunits except alpha7, and this interaction induces an opening of the axial channel through the alpha-ring. Thus, the activation mechanism of PA200 is expressed via its allosteric effects on the 20 S core particle, perhaps facilitating release of digestion products or the entrance of substrates.

Allosteric Site↗
PubMed2004

In vivo homodimerisation of HTLV-1 Gag and MA gives clues to the retroviral capsid and TM envelope protein arrangement.

During retroviral particle formation, the capsid precursors (Gag) associate with the cell membrane via their matrix (MA) domain to form viral assembling particles. After budding, Gag and its proteolytically matured MA, form a shell in the released immature and mature particles, respectively. Although the arrangement of Gag domains in vitro and their radial organisation in retroviral particles have been extensively studied, little is known concerning Gag inter-subunit interactions in authentic retroviruses. We report that human T-cell leukemia virus type 1 Gag homodimerises in the cell via a disulphide bonding at cysteine 61 in the MA domain. Most Gags are homodimeric after budding and MAs are also dimeric in mature authentic virions. Molecular modelling of the MA domain indicates that non-covalent interactions at the MA dimer interface may also be important for Gag (and MA) dimerisation. In addition, all amino acids previously reported to be involved in MA-transmembrane (TM) interactions are located on the MA face opposite to the dimer interface. The model reveals that homodimerisation is compatible with a hexameric network of Gag and MA dimers that look like the hexameric networks observed for other retroviruses. These data, together with previous studies, lead us to propose a supra-molecular arrangement model in which the transmembrane glycoproteins of the virion envelope are anchored in a hexameric cage hole formed by the MA.

Amino Acid Sequence↗
PubMed2004

A model for Ure2p prion filaments and other amyloids: the parallel superpleated beta-structure.

In its prion form, Ure2p, a regulator of nitrogen catabolism in Saccharomyces cerevisiae, polymerizes into filaments whereby its C-terminal regulatory domain is inactivated but retains its native fold. The filament has an amyloid fibril backbone formed by the Asn-rich, N-terminal, "prion" domain. The prion domain is also capable of forming fibrils when alone or when fused to other proteins. We have developed a model for the fibril that we call a parallel superpleated beta-structure. In this model, the prion domain is divided into nine seven-residue segments, each with a four-residue strand and a three-residue turn, that zig-zag in a planar serpentine arrangement. Serpentines are stacked axially, in register, generating an array of parallel beta-sheets, with a small and potentially variable left-hand twist. The interior of the filament is mostly stabilized not by packing of apolar side chains but by H-bond networks generated by the stacking of Asn side chains: charged residues are excluded. The model is consistent with current biophysical, biochemical, and structural data (notably, mass-per-unit-length measurements by scanning transmission electron microscopy that gave one subunit rise per 0.47 nm) and is readily adaptable to other amyloids, for instance the core of Sup35p filaments and glutamine expansions in huntingtin.

Amino Acid Sequence↗
PubMed2004

NMR solution structure of Mob1, a mitotic exit network protein and its interaction with an NDR kinase peptide.

Proteins of the Mob1/phocein family are found in all eukaryotic cells. In yeast, they are activating subunits of Dbf2-related protein kinases involved in cell cycle control. Despite the wide occurrence of these proteins, their biological functions remain poorly understood. Here we report the solution structure of the Mob1 protein from Xenopus laevis solved by heteronuclear multidimensional NMR. The structure reveals a fold constituted by a central left-handed four-helix bundle, one connecting helix, two flanking helices and a long flexible loop. The clustering of two Cys and two His residues, and zinc measurement by atomic absorption spectroscopy support the existence of a zinc ion binding site. Our NMR structure is in good agreement with the recently described X-ray structure of human Mob1-A. Chemical shift perturbations observed upon addition of a peptide encompassing the basic region of the N-terminal regulatory domain of NDR kinase were used to identify and map a specific interaction between Mob1 and this kinase. The chemical shift changes indicate that the main interaction occurs on the acidic and conserved surface of Mob1. This surface was previously hypothesized to be the interaction surface according to the X-ray structure and was identified as functionally important in yeast. Our data suggest that the NDR kinase is a functional Dbf2 homologue in animal cells and contributes to the understanding of the molecular function of Mob1 proteins.

Amino Acid Sequence↗
PubMed2004

Organization of designed nanofibrils assembled from alpha-helical peptides as determined by electron microscopy.

Self-assembling peptides present attractive platforms for engineering materials with controlled nanostructures. Recently, an alpha-helical fibril forming peptide (alphaFFP) was designed that self-assembles into nanofibrils at acid pH. Circular dichroism spectroscopy, electron-microscopy and x-ray fibre diffraction data showed that the most likely structure of alphaFFP fibrils is a five-stranded coiled coil rope. In the present study, scanning transmission electron microscopy (STEM) was used to improve our understanding of the alphaFFP fibril structure. The measurements of fibril mass per length suggest that there are ten alpha-helices in transverse sections of the fibrils. Based on the known data, it is proposed that a predominant fibrillar structure of alphaFFP is a dimer of alpha-helical five stranded protofilaments wrapped around a common axis. It is shown that these structures have an axial dimension of 58 +/- 16 nm and a width of 4 +/- 1 nm. A small number of thin fibrils is also observed in the negative stained preparation and STEM images. The thin fibrils may correspond to the single protofilament.

Microscopy, Electron, Scanning↗
PubMed2004

New HEAT-like repeat motifs in proteins regulating proteasome structure and function.

We have identified repeat motifs in the large proteasome-binding proteins PA200 and Ecm29 by applying a sensitive sequence profile method. These repeat motifs, especially those of PA200, resemble HEAT/ARM repeats in length and other properties but differ from them in the occupancy of certain positions. The HEAT motif consists of two alpha-helices and two turns: molecular modeling suggests that in the PA200 and Ecm29 repeats, the alpha-helices may be slightly turned relative to their orientations in typical HEAT repeats. Both PA200 and Ecm29 are composed almost entirely of such repeats, and therefore are likely to have alpha-helical solenoid structures. These observations lead us to speculate on how PA200 and Ecm29 may associate with proteasomes.

Amino Acid Motifs↗
PubMed2004

Interactions of the human calcitonin fragment 9-32 with phospholipids: a monolayer study.

Human calcitonin and its C-terminal fragment 9-32 (hCT(9-32)) administered in a spray translocate into respiratory nasal epithelium with an effect similar to intravenous injection. hCT(9-32) is an efficient carrier to transfer the green fluorescent protein into excised bovine nasal mucosa. To understand the translocation of hCT(9-32) across plasma membranes, we investigated its interactions with phospholipids and its interfacial structure using model lipid monolayers. A combination of physicochemical methods was applied including surface tension measurements on adsorbed and spread monolayers at the air-water interface, Fourier transform infrared, circular dichroism, and atomic force microscopy on Langmuir-Blodgett monolayers. The results disclose that hCT(9-32) preferentially interacts with negatively charged phospholipids and does not insert spontaneously into lipid monolayers. This supports a nonreceptor-mediated endocytic internalization pathway as previously suggested. Structural studies revealed a random coil conformation of hCT(9-32) in solution, transforming to alpha-helices when the peptide is localized at lipid-free or lipid-containing air-water interfaces. Atomic force microscopy studies of monolayers of the peptide alone or mixed with dioleoylphosphatidylcholine revealed that hCT(9-32) forms filaments rolled into spirals. In contrast, when interacting with dioleoylphosphatidylglycerol, hCT(9-32) does not adopt filamentous structures. A molecular model and packing is proposed for the spiral-forming hCT(9-32).

Calcitonin↗
PubMed2003

The carboxy terminus of Prospero regulates its subcellular localization.

Subcellular localization of the transcription factor Prospero is dynamic. For example, the protein is cytoplasmic in neuroblasts, nuclear in sheath cells, and degraded in newly formed neurons. The carboxy terminus of Prospero, including the homeodomain and Prospero domain, plays roles in regulating these changes. The homeodomain has two distinct subdomains, which exclude proteins from the nucleus, while the intact homeo/Prospero domain masks this effect. One subdomain is an Exportin-dependent nuclear export signal requiring three conserved hydrophobic residues, which models onto helix 1. Another, including helices 2 and 3, requires proteasome activity to degrade nuclear protein. Finally, the Prospero domain is missing in pros(I13) embryos, thus unmasking nuclear exclusion, resulting in constitutively cytoplasmic protein. Multiple processes direct Prospero regulation of cell fate in embryonic nervous system development.

Active Transport, Cell Nucleus↗
PubMed2002

Processing of Escherichia coli alkaline phosphatase. Sequence requirements and possible conformations of the -6 to -4 region of the signal peptide.

Analysis of the precursors of bacterial exported proteins revealed that those having bulky hydrophobic residues at position -5 have a high incidence of Pro residues at positions -6 and -4, Val at position -3, and Ser at positions -4 and -2. This led to a hypothesis that the previously observed inhibition of processing by bulky residues at position -5 can be suppressed by introduction of Pro, Ser, or Val in the corresponding nearby positions. Subsequent mutational analysis of Escherichia coli alkaline phosphatase showed that, as it was predicted, Pro on either side of bulky hydrophobic -5 Leu, Ile, or Tyr completely restores efficiency of the maturation. Introduction of Val at position -3 also partially suppresses the inhibition imposed by -5 Leu, while a Ser residue at position -4 or -2 does not restore processing. In addition, effective maturation of a mutant with Pro residues at positions from -6 throughout -4 proved that polyproline conformation of this region is permissive for processing. To understand the effects of the mutations, we modeled a peptide substrate into the active site of the signal peptidase using the known position of the beta-lactam inhibitor. The inhibitory effect of the -5 residue and its suppression by either Pro -6 or Pro -4 can be explained if we assume that Pro-containing -6 to -4 regions adopt a polyproline conformation whereas the region without Pro residues has a beta-conformation. These results permit us to specify sequence requirements at -6, -5, and -4 positions for efficient processing and to improve the prediction of yet unknown cleavage sites.

Alkaline Phosphatase↗