TPRpred: a tool for prediction of TPR-, PPR- and SEL1-like repeats from protein sequences

BACKGROUND: Solenoid repeat proteins of the Tetratrico Peptide Repeat (TPR) family are involved as scaffolds in a broad range of protein-protein interactions. Several resources are available for the prediction of TPRs, however, they often fail to detect divergent repeat units. RESULTS: We have developed TPRpred, a profile-based method which uses a P-value-dependent score offset to include divergent repeat units and which exploits the tendency of repeats to occur in tandem. TPRpred detects not only TPR-like repeats, but also the related Pentatrico Peptide Repeats (PPRs) and SEL1-like repeats. The corresponding profiles were generated through iterative searches, by varying the threshold parameters for inclusion of repeat units into the profiles, and the best profiles were selected based on their performance on proteins of known structure. We benchmarked the performance of TPRpred in detecting TPR-containing proteins and in delineating the individual repeats therein, against currently available resources. CONCLUSION: TPRpred performs significantly better in detecting divergent repeats in TPR-containing proteins, and finds more individual repeats than the existing methods. The web server is available at , and the C++ and Perl sources of TPRpred along with the profiles can be downloaded from

Exploring protein domain evolution by designing new TPR-like domains

Proteine stellen die am häufigsten vorkommende Gruppe der Biomoleküle, die aufgrund ihrer Diversität an der großen Mehrzahl der biochemischen Prozesse beteiligt ist. Die Faltungseinheit der Proteine ist die Domäne. Neuartige Proteine entstehen oft aus der Rekombination, dem Zufügen oder Entfernen vorhandener Domänen; sie sind daher stabile Bausteine der Evolution. Wie Domänen, die schon eine beträchtliche Komplexität haben, selbst entstanden sind, ist allerdings weitgehend unbekannt. Die scheinbar endlose Vielfalt der Proteine reduziert sich auf eine begrenzte Zahl struktureller Formen, sogenannte Folds. Folds setzen sich aus Supersekundärstrukturen zusammen, die in einigen Fällen auch aus repetitiven Einheiten bestehen. Dies weist darauf hin, dass sie durch Fusion und Rekombination dieser Einheiten entstanden sein könnten. Solenoidproteine, die aus sich wiederholenden Einheiten von Tetratricopeptiden (TPR) bestehen, stellen ein attraktives Modell dar um diese Frage zu untersuchen. TPR Domänen sind aus repetitiven alpha-hairpins geformt, die als einzelne Elemente häufig in anderem Kontext in Proteinen vorkommen. Die Wiederholung und Verknüpfung von Proteinfragmenten, die ihren Ursprung in anderen Polypeptiden haben, könnte somit, nicht nur für TPR Domänen, ein wichtiges Prinzip der Evolution von Folds und Domänen darstellen. Zur Beantwortung dieser Frage benötigen wir die Kenntnis, welche aa-hairpins TPR-ähnlich sind. Da die verfügbaren Resourcen oft divergierende Repeats nicht erkennen, haben wir “TPRpred” entwickelt, eine Methode auf der Basis von Profilen, die hierzu in der Lage ist. TPRpred war nicht nur besser im Erkennen divergierender Repeats in TRP Proteinen, sondern erkannte auch eine höhere Zahl einzelner Repeat-Einheiten. Wir identifizierten in nicht-TPR Proteinen mehrere alpha-hairpins, die einer TPR Einheit ähnelten, und wählten für weitere Untersuchungen die besten fünf aus: Mitochondriale Außenmembrantranslokase Tom20, ribosomales Protein S20 (RPS20), Phospholipase C (PLC), Heat shock protein 20 (HSC) und bakterielle Gucoamylase (BGA). Mit diesen Hairpins konstruierten wir jeweils drei künstliche Gene mit einer, zwei bzw. drei verknüpften Einheiten. Die resultierenden Proteine wurden nach Expression in Escherichia coli gereinigt und biophysikalisch charakterisiert. Die Eigenschaften dieser TPR-ähnlichen Domänen korrelieren mit der statistischen Signifikanz, mit der sie der TPR-Einheit ähneln. Proteine, die aus Tom20 und RPS20 hervorgingen, haben vermutlich nativen Charakter, entsprechend einem gefalteten Protein. RPS20 ist auch deswegen bedeutsam, da ribosomale Proteine mit die ältesten bekannten Proteine sind, deren Fragmente daher die Bausteine in der frühen Evolution von Domänen gebildet haben könnten.Proteins are the most abundant and diverse class of biomolecules that mediate the vast majority of biochemical processes. The functional units within a protein are the "domains" which fold autonomously from the rest of the linear amino acid sequence in the protein. Novelty in protein function often arises as a result of gain, loss or re-shuffling of existing domains. Thus, protein domains can arguably be seen as stable units of evolution. However, the evolutionary origin of domains themselves is more challenging and is largely unexplored area of research. Domains often adopt to a limited number of structural forms called folds, despite the seemingly endless diversity of the proteins. These folds are largely formed by a limited "vocabulary" of recurring supersecondary structural elements, often by repetition of the same element and, increasingly, elements similar in both structure and sequence are discovered. This suggests that modern protein domains evolved by fusion and recombination from a more ancient peptide world and that many of the core folds observed today may contain homologous building blocks. Solenoid repeat proteins of Tetratrico Peptide Repeat (TPR) domain represent an attractive model to explore this issue. TPR domains are formed by repetition of an alpha-hairpin, a supersecondary structural element. Since alpha-hairpins are frequent in proteins, therefore TPR-like domains might have arisen by the repetition of protein fragments that were originally used in a different structural context. In order to explore this question, we require a better ability to judge, which alpha-hairpins are TPR-like. Currently, several resources are available for the prediction of TPRs, however, they often fail to detect divergent repeat units. We therefore developed "TPRpred", a profile-based method which uses a P-value-dependent score offset to include divergent repeat units, and also exploits the tendency of the repeats to occur in tandem. We benchmarked the performance of TPRpred in detecting TPR-containing proteins and in delineating the individual repeats within a protein, against currently available resources. TPRpred not only performed significantly better in detecting divergent repeats in TPR-containing proteins, but also detected more number of individual repeat units. We identified several promising alpha-hairpins in non-TPR proteins which resemble the repeating unit of TPR, by using TPRpred in conjunction with structure-structure comparisons, and we further selected the best five hairpins namely, the mitochondrial outer membrane translocase Tom20, the ribosomal protein S20 (RPS20), the phospholipase C (PLC), the heat shock protein 20 (HSC) and the bacterial glucoamylase (BGA), to experimentally construct new TPR-like domains by repetition. Using each of these hairpins, we constructed three different artificial genes coding for one, two and three copies. The resulting artificial proteins were expressed, purified and then characterised using circular dichroism, thermal denaturation and fluorescence spectroscopy experiments. The biophysical properties of these TPR-like domains can also be correlated to the statistical significance of the parental hairpin likely to be a repeating unit of TPR. Although high-resolution structures have not yet been determined, proteins made from the hairpins of Tom20 and RPS20 appear to have native-like properties. The hairpin of RPS20 is significant in our study, because ribosomal proteins are among the most ancient proteins known, and since many of the modern non-ribosomal proteins contain fragments from the ribosomes, they might have been the building blocks in early protein domain evolution

Crystal structure of SEL1L: Insight into the roles of SLR motifs in ERAD pathway

Terminally misfolded proteins are selectively recognized and cleared by the endoplasmic reticulum-associated degradation (ERAD) pathway. SEL1L, a component of the ERAD machinery, plays an important role in selecting and transporting ERAD substrates for degradation. We have determined the crystal structure of the mouse SEL1L central domain comprising five Sel1-Like Repeats (SLR motifs 5 to 9; hereafter called SEL1Lcent). Strikingly, SEL1Lcent forms a homodimer with two-fold symmetry in a head-to-tail manner. Particularly, the SLR motif 9 plays an important role in dimer formation by adopting a domain-swapped structure and providing an extensive dimeric interface. We identified that the full-length SEL1L forms a self-oligomer through the SEL1Lcent domain in mammalian cells. Furthermore, we discovered that the SLR-C, comprising SLR motifs 10 and 11, of SEL1L directly interacts with the N-terminus luminal loops of HRD1. Therefore, we propose that certain SLR motifs of SEL1L play a unique role in membrane bound ERAD machinery.ope

Ras GTPase-like protein MglA, a controller of bacterial social-motility in Myxobacteria, has evolved to control bacterial predation by Bdellovibrio

Bdellovibrio bacteriovorus invade Gram-negative bacteria in a predatory process requiring Type IV pili (T4P) at a single invasive pole, and also glide on surfaces to locate prey. Ras-like G-protein MglA, working with MglB and RomR in the deltaproteobacterium Myxococcus xanthus, regulates adventurous gliding and T4P-mediated social motility at both M. xanthus cell poles. Our bioinformatic analyses suggested that the GTPase activating protein (GAP)-encoding gene mglB was lost in Bdellovibrio, but critical residues for MglABd GTP-binding are conserved. Deletion of mglABd abolished prey-invasion, but not gliding, and reduced T4P formation. MglABd interacted with a previously uncharacterised tetratricopeptide repeat (TPR) domain protein Bd2492, which we show localises at the single invasive pole and is required for predation. Bd2492 and RomR also interacted with cyclic-di-GMP-binding receptor CdgA, required for rapid prey-invasion. Bd2492, RomRBd and CdgA localize to the invasive pole and may facilitate MglA-docking. Bd2492 was encoded from an operon encoding a TamAB-like secretion system. The TamA protein and RomR were found, by gene deletion tests, to be essential for viability in both predatory and non-predatory modes. Control proteins, which regulate bipolar T4P-mediated social motility in swarming groups of deltaproteobacteria, have adapted in evolution to regulate the anti-social process of unipolar prey-invasion in the “lone-hunter” Bdellovibrio. Thus GTP-binding proteins and cyclic-di-GMP inputs combine at a regulatory hub, turning on prey-invasion and allowing invasion and killing of bacterial pathogens and consequent predatory growth of Bdellovibrio

Origin of a folded repeat protein from an intrinsically disordered ancestor.

Repetitive proteins are thought to have arisen through the amplification of subdomain-sized peptides. Many of these originated in a non-repetitive context as cofactors of RNA-based replication and catalysis, and required the RNA to assume their active conformation. In search of the origins of one of the most widespread repeat protein families, the tetratricopeptide repeat (TPR), we identified several potential homologs of its repeated helical hairpin in non-repetitive proteins, including the putatively ancient ribosomal protein S20 (RPS20), which only becomes structured in the context of the ribosome. We evaluated the ability of the RPS20 hairpin to form a TPR fold by amplification and obtained structures identical to natural TPRs for variants with 2-5 point mutations per repeat. The mutations were neutral in the parent organism, suggesting that they could have been sampled in the course of evolution. TPRs could thus have plausibly arisen by amplification from an ancestral helical hairpin

Modulation of the surface proteome through multiple ubiquitylation pathways in African Trypanosomes

Recently we identified multiple suramin-sensitivity genes with a genome wide screen in Trypanosoma brucei that includes the invariant surface glycoprotein ISG75, the adaptin-1 (AP-1) complex and two deubiquitylating enzymes (DUBs) orthologous to ScUbp15/HsHAUSP1 and pVHL-interacting DUB1 (type I), designated TbUsp7 and TbVdu1, respectively. Here we have examined the roles of these genes in trafficking of ISG75, which appears key to suramin uptake. We found that, while AP-1 does not influence ISG75 abundance, knockdown of TbUsp7 or TbVdu1 leads to reduced ISG75 abundance. Silencing TbVdu1 also reduced ISG65 abundance. TbVdu1 is a component of an evolutionarily conserved ubiquitylation switch and responsible for rapid receptor modulation, suggesting similar regulation of ISGs in T. brucei. Unexpectedly, TbUsp7 knockdown also blocked endocytosis. To integrate these observations we analysed the impact of TbUsp7 and TbVdu1 knockdown on the global proteome using SILAC. For TbVdu1, ISG65 and ISG75 are the only significantly modulated proteins, but for TbUsp7 a cohort of integral membrane proteins, including the acid phosphatase MBAP1, that is required for endocytosis, and additional ISG-related proteins are down-regulated. Furthermore, we find increased expression of the ESAG6/7 transferrin receptor and ESAG5, likely resulting from decreased endocytic activity. Therefore, multiple ubiquitylation pathways, with a complex interplay with trafficking pathways, control surface proteome expression in trypanosomes

Multi-genome identification and characterization of chlamydiae-specific type III secretion substrates: the Inc proteins

<p>Abstract</p> <p>Background</p> <p><it>Chlamydiae </it>are obligate intracellular bacteria that multiply in a vacuolar compartment, the inclusion. Several chlamydial proteins containing a bilobal hydrophobic domain are translocated by a type III secretion (TTS) mechanism into the inclusion membrane. They form the family of Inc proteins, which is specific to this phylum. Based on their localization, Inc proteins likely play important roles in the interactions between the microbe and the host. In this paper we sought to identify and analyze, using bioinformatics tools, all putative Inc proteins in published chlamydial genomes, including an environmental species.</p> <p>Results</p> <p>Inc proteins contain at least one bilobal hydrophobic domain made of two transmembrane helices separated by a loop of less than 30 amino acids. Using bioinformatics tools we identified 537 putative Inc proteins across seven chlamydial proteomes. The amino-terminal segment of the putative Inc proteins was recognized as a functional TTS signal in 90% of the <it>C. trachomatis </it>and <it>C. pneumoniae </it>sequences tested, validating the data obtained <it>in silico</it>. We identified a <it>macro </it>domain in several putative Inc proteins, and observed that Inc proteins are enriched in segments predicted to form coiled coils. A surprisingly large proportion of the putative Inc proteins are not constitutively translocated to the inclusion membrane in culture conditions.</p> <p>Conclusions</p> <p>The Inc proteins represent 7 to 10% of each proteome and show a great degree of sequence diversity between species. The abundance of segments with a high probability for coiled coil conformation in Inc proteins support the hypothesis that they interact with host proteins. While the large majority of Inc proteins possess a functional TTS signal, less than half may be constitutively translocated to the inclusion surface in some species. This suggests the novel finding that translocation of Inc proteins may be regulated by as-yet undetermined mechanisms.</p