Birgit Eisenhaber

Sequence properties of GPI-anchored proteins near the omega-site: constraints for the polypeptide binding site of the putative transamidase.

Glycosylphosphatidylinositol (GPI) anchoring is a common post-translational modification of extracellular eukaryotic proteins. Attachment of the GPI moiety to the carboxyl terminus (omega-site) of the polypeptide occurs after proteolytic cleavage of a C-terminal propeptide. In this work, the sequence pattern for GPI-modification was analyzed in terms of physical amino acid properties based on a database analysis of annotated proprotein sequences. In addition to a refinement of previously described sequence signals, we report conserved sequence properties in the regions omega - 11...omega - 1 and omega + 4...omega + 5. We present statistical evidence for volume-compensating residue exchanges with respect to the positions omega - 1...omega + 2. Differences between protozoan and metazoan GPI-modification motifs consist mainly in variations of preferences to amino acid types at the positions near the omega-site and in the overall motif length. The variations of polypeptide substrates are exploited to suggest a model of the polypeptide binding site of the putative transamidase, the enzyme catalyzing the GPI-modification. The volume of the active site cleft accommodating the four residues omega - 1...omega + 2 appears to be approximately 540 A3.

Prediction of peroxisomal targeting signal 1 containing proteins from amino acid sequence.

Peroxisomal matrix proteins have to be imported into their target organelle post-translationally. The major translocation pathway depends on a C-terminal targeting signal, termed PTS1. Our previous analysis of sequence variability in the PTS1 motif revealed that, in addition to the known C-terminal tripeptide, at least nine residues directly upstream are important for signal recognition in the PTS1-Pex5 receptor complex. The refined PTS1 motif description was implemented in a prediction tool composed of taxon-specific functions (metazoa, fungi, remaining taxa), capable of recognising potential PTS1s in query sequences. The composite score function consists of classical profile terms and additional terms penalising deviations from the derived physical property pattern over sequence segments. The prediction algorithm has been validated with a self-consistency and three different cross-validation tests. Additionally, we tested the tool on a large set of non-peroxisomal negatives, on mutation data, and compared the prediction rate to the PTS1 component of the PSORT2 program. The sensitivity of our predictor in recognising documented PTS1 signal containing proteins is close to 90% for reliable prediction. The predictor distinguishes even SKL-appended non-peroxisomally targeted proteins such as a mouse dihydrofolate reductase-SKL construct. The corresponding rate of false positives is not worse than 0.8%; thus, the tool can be applied for large-scale unsupervised sequence database annotation. A scan of public protein databases uncovered a number of yet uncharacterised proteins for which the PTS1 signal might be critical for biological function. The predicted presence of a PTS1 signal implies peroxisomal localisation in the absence of N-terminal targeting sequences such as the mitochondrial import signal.

Glycosylphosphatidylinositol Lipid Anchoring of Plant Proteins. Sensitive Prediction from Sequence- and Genome-Wide Studies for Arabidopsis and Rice

Posttranslational glycosylphosphatidylinositol (GPI) lipid anchoring is common not only for animal and fungal but also for plant proteins. The attachment of the GPI moiety to the carboxyl-terminus after proteolytic cleavage of a C-terminal propeptide is performed by the transamidase complex. Its four known subunits also have obvious full-length orthologs in the Arabidopsis and rice (Oryza sativa) genomes; thus, the mechanism of substrate protein processing appears similar for all eukaryotes. A learning set of plant proteins (substrates for the transamidase complex) has been collected both from the literature and plant sequence databases. We find that the plant GPI lipid anchor motif differs in minor aspects from the animal signal (e.g. the plant hydrophobic tail region can contain a higher fraction of aromatic residues). We have developed the “big-Π plant” program for prediction of compatibility of query protein C-termini with the plant GPI lipid anchor motif requirements. Validation tests show that the sensitivity for transamidase targets is approximately 94%, and the rate of false positive prediction is about 0.1%. Thus, the big-Π predictor can be applied as unsupervised genome annotation and target selection tool. The program is also suited for the design of modified protein constructs to test their GPI lipid anchoring capacity. The big-Π plant predictor Web server and lists of potential plant precursor proteins in Swiss-Prot, SPTrEMBL, Arabidopsis, and rice proteomes are available at http://mendel.imp.univie.ac.at/gpi/plants/gpi_plants.html. Arabidopsis and rice protein hits have been functionally classified. Several GPI lipid-anchored arabinogalactan-related proteins have been identified in rice.

Motif Refinement of the Peroxisomal Targeting Signal 1 and Evaluation of Taxon-specific Differences

Eukaryote peroxisomes, plant glyoxysomes and trypanosomal glycosomes belong to the microbody family of organelles that compartmentalise a variety of biochemical processes. The interaction between the PTS1 signal and its cognate receptor Pex5 initiates the major import mechanism for proteins into the matrix of these organelles. Relying on the analysis of amino acid sequence variability of known PTS1-targeted proteins and PTS1-containing peptides that interact with Pex5 in the yeast two-hybrid assay, on binding site studies of the Pex5-ligand complex crystal structure, 3D models and sequences of Pex5 proteins from various taxa, we derived the requirements for a C-terminal amino acid sequence to interact productively with Pex5. We found evidence that, at least the 12 C-terminal residues of a given substrate protein are implicated in PTS1 signal recognition. This motif can be structurally and functionally divided into three regions: (i) the C-terminal tripeptide, (ii) a region interacting with the surface of Pex5 (about four residues further upstream), and (iii) a polar, solvent-accessible and unstructured region with linker function (the remaining five residues). Specificity differences are confined to taxonomic subgroups (metazoa and fungi) and are connected with amino acid type preferences in region 1 and deviating hydrophobicity patterns in region 2.

The ring between ring fingers (RBR) protein family.

SummaryProteins of the ring between ring fingers (RBR)-domain family are characterized by three groups of specifically clustered (typically eight) cysteine and histidine residues. Whereas the amino-terminal ring domain (N-RING) binds two zinc ions and folds into a classical cross-brace ring finger, the carboxy-terminal ring domain (C-RING) involves only one zinc ion. The three-dimensional structure of the central ring domain, the IBR domain, is still unsolved. About 400 genes coding for RBR proteins have been identified in the genomes of uni- and multicellular eukaryotes and some of their viruses, but the family has not been found in archaea or bacteria. The RBR proteins are classified into 15 major subfamilies (besides some orphan cases) by the phylogenetic relationships of the RBR segments and the conservation of their sequence architecture. The RBR domain mediates protein-protein interactions and a subset of RBR proteins has been shown to function as E3 ubiquitin ligases. RBR proteins have attracted interest because of their involvement in diseases such as parkinsonism, dementia with Lewy bodies, and Alzheimers disease, and in susceptibility to some intracellular bacterial pathogens. Here, we present an overview of the RBR-domain containing proteins and their subcellular localization, additional domains, function, specificity, and regulation.

Prediction of Posttranslational Modification of Proteins from Their Amino Acid Sequence

If posttranslational modifications (PTMs) are chemical alterations of the protein primary structure during the proteins life cycle as a result of an enzymatic reaction, then the motif in the substrate protein sequence that is recognized by the enzyme can serve as basis for predictor construction that recognizes PTM sites in database sequences. The recognition motif consists generally of two regions: first, a small, central segment that enters the catalytic cleft of the enzyme and that is specific for this type of PTM and, second, a sequence environment of about 10 or more residues with linker characteristics (a trend for small and polar residues with flexible backbone) on either side of the central part that are needed to provide accessibility of the central segment to the enzymes catalytic site. In this review, we consider predictors for cleavage of targeting signals, lipid PTMs, phosphorylation, and glycosylation.

Posttranslational modifications and subcellular localization signals: indicators of sequence regions without inherent 3D structure?

Given the huge number of sequences of otherwise uncharacterized protein sequences, computer-aided prediction of posttranslational modifications (PTMs) and translocation signals from amino acid sequence becomes a necessity. We have contributed to this multi-faceted, worldwide effort with the development of predictors for GPI lipid anchor sites, for N-terminal N-myristoylation sites, for farnesyl and geranylgeranyl anchor attachment as well as for the PTS1 peroxisomal signal. Although the substrate protein sequence signals for various PTMs or translocation systems vary dramatically, we found that their principal architecture is similar for all the cases studied. Typically, a small stretch of the amino acid residues is buried in the catalytic cleft of the protein-modifying enzyme (or the binding site of the transporter). This piece most intensely interacts with the enzyme and its sequence variability is most restricted. This stretch is surrounded by linker segments that connect the part bound by the enzyme with the rest of the substrate protein. These residues are, as a trend, small with a flexible backbone and polar. Due to the mechanistic requirements of binding to the enzyme, we suggest that most PTM sites are necessarily embedded into intrinsically disordered regions (except for cases of autocatalytic PTMs, PTMs executed in the unfolded state or non-enzymatic PTMs) and this issue requires consideration in structural studies of proteins with complex architecture. Surprisingly, some proteins carry sequence signals for posttranslational modification or translocation that remain hidden in the normal biological context but can become fully functional in certain conditions.

Proteins with two SUMO-like domains in chromatin-associated complexes: The RENi (Rad60-Esc2-NIP45) family

BackgroundPost-translational modification by Small Ubiquitin-like Modifiers (SUMO) has been implicated in protein targeting, in the maintenance of genomic integrity and in transcriptional control. But the specific molecular effects of SUMO modification on many target proteins remain to be elucidated. Recent findings point at the importance of SUMO-mediated histone NAD-dependent deacetylase (HDAC) recruitment in transcriptional regulation.ResultsWe describe the RENi family of SUMO-like domain proteins (SDP) with the unique feature of typically containing two carboxy-terminal SUMO-like domains. Using sequence analytic evidence, we collect family members from animals, fungi and plants, most prominent being yeast R ad60, E sc2 and mouse NI P45 http://mendel.imp.univie.ac.at/SEQUENCES/reni/. Different proteins of the novel family are known to interact directly with histone NAD-dependent deacetylases (HDACs), structural maintenance of chromosomes (SMC) proteins, and transcription factors. In particular, the highly non-trivial designation of the first of the two successive SUMO-domains in non-plant RENi provides a rationale for previously published functionally impaired mutant variants.ConclusionsTill now, SUMO-like proteins have been studied exclusively in the context of their covalent conjugation to target proteins. Here, we present the exciting possibility that SUMO domain proteins, similarly to ubiquitin modifiers, have also evolved in a second line – namely as multi-domain proteins that are non-covalently attached to their target proteins. We suggest that the SUMO stable fusion proteins of the RENi family, which we introduce in this work, might mimic SUMO and share its interaction motifs (in analogy to the way that ubiquitin-like domains mimic ubiquitin). This presumption is supported by parallels in the spectrum of modified or bound proteins e.g. transcription factors and chromatin-associated proteins and in the recruitment of HDAC-activity.

SPACER: server for predicting allosteric communication and effects of regulation

The SPACER server provides an interactive framework for exploring allosteric communication in proteins with different sizes, degrees of oligomerization and function. SPACER uses recently developed theoretical concepts based on the thermodynamic view of allostery. It proposes easily tractable and meaningful measures that allow users to analyze the effect of ligand binding on the intrinsic protein dynamics. The server shows potential allosteric sites and allows users to explore communication between the regulatory and functional sites. It is possible to explore, for instance, potential effector binding sites in a given structure as targets for allosteric drugs. As input, the server only requires a single structure. The server is freely available at http://allostery.bii.a-star.edu.sg/.

ANNIE: integrated de novo protein sequence annotation

Function prediction of proteins with computational sequence analysis requires the use of dozens of prediction tools with a bewildering range of input and output formats. Each of these tools focuses on a narrow aspect and researchers are having difficulty obtaining an integrated picture. ANNIE is the result of years of close interaction between computational biologists and computer scientists and automates an essential part of this sequence analytic process. It brings together over 20 function prediction algorithms that have proven sufficiently reliable and indispensable in daily sequence analytic work and are meant to give scientists a quick overview of possible functional assignments of sequence segments in the query proteins. The results are displayed in an integrated manner using an innovative AJAX-based sequence viewer. ANNIE is available online at: http://annie.bii.a-star.edu.sg. This website is free and open to all users and there is no login requirement.