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Broadly, computational approaches for ortholog assignment is a three steps process: (i) identify all putative homologs between the genomes, (ii) identify gene anchors and (iii) link anchors to identify best gene matches given their order and context. In this article, we engineer two methods to improve two important aspects of this pipeline [specifically steps (ii) and (iii)]. First, computing sequence similarity data [step (i)] is a computationally intensive task for large sequence sets, creating a bottleneck in the ortholog assignment pipeline. We have designed a fast and highly scalable sort-join method (afree) based on k-mer counts to rapidly compare all pairs of sequences in a large protein sequence set to identify putative homologs. Second, availability of complex genomes containing large gene families with prevalence of complex evolutionary events, such as duplications, has made the task of assigning orthologs and co-orthologs difficult. Here, we have developed an iterative graph matching strategy where at each iteration the best gene assignments are identified resulting in a set of orthologs and co-orthologs. We find that the afree algorithm is faster than existing methods and maintains high accuracy in identifying similar genes. The iterative graph matching strategy also showed high accuracy in identifying complex gene relationships. Standalone afree available from http://vbc.med.monash.edu.au/∼kmahmood/afree. EGM2, complete ortholog assignment pipeline (including afree and the iterative graph matching method) available from http://vbc.med.monash.edu.au/∼kmahmood/EGM2.

The ability to catalytically cleave protein substrates after synthesis is fundamental for all forms of life. Accordingly, site-specific proteolysis is one of the most important post-translational modifications. The key to understanding the physiological role of a protease is to identify its natural substrate(s). Knowledge of the substrate specificity of a protease can dramatically improve our ability to predict its target protein substrates, but this information must be utilized in an effective manner in order to efficiently identify protein substrates by in silico approaches. To address this problem, we present PROSPER, an integrated feature-based server for in silico identification of protease substrates and their cleavage sites for twenty-four different proteases. PROSPER utilizes established specificity information for these proteases (derived from the MEROPS database) with a machine learning approach to predict protease cleavage sites by using different, but complementary sequence and structure characteristics. Features used by PROSPER include local amino acid sequence profile, predicted secondary structure, solvent accessibility and predicted native disorder. Thus, for proteases with known amino acid specificity, PROSPER provides a convenient, pre-prepared tool for use in identifying protein substrates for the enzymes. Systematic prediction analysis for the twenty-four proteases thus far included in the database revealed that the features we have included in the tool strongly improve performance in terms of cleavage site prediction, as evidenced by their contribution to performance improvement in terms of identifying known cleavage sites in substrates for these enzymes. In comparison with two state-of-the-art prediction tools, PoPS and SitePrediction, PROSPER achieves greater accuracy and coverage. To our knowledge, PROSPER is the first comprehensive server capable of predicting cleavage sites of multiple proteases within a single substrate sequence using machine learning techniques. It is freely available at http://lightning.med.monash.edu.au/PROSPER/.

Summary: Protein topology diagrams are 2D representations of protein structure that are particularly useful in understanding and analysing complex protein folds. Generating such diagrams presents a major problem in graph drawing, with automatic approaches often resulting in errors or uninterpretable results. Here we apply a breakthrough in diagram layout to protein topology cartoons, providing clear, accurate, interactive and editable diagrams, which are also an interface to a structural search method., Availability: Pro-origami is available via a web server at http://munk.csse.unimelb.edu.au/pro-origami, Contact: a.stivala@pgrad.unimelb.edu.au; pjs@csse.unimelb.edu.au.

Over 3% of human proteins contain single amino acid repeats (repeat-containing proteins, RCPs). Many repeats (homopeptides) localize to important proteins involved in transcription, and the expansion of certain repeats, in particular poly-Q and poly-A tracts, can also lead to the development of neurological diseases. Previous studies have suggested that the homopeptide makeup is a result of the presence of G+C-rich tracts in the encoding genes and that expansion occurs via replication slippage. Here, we have performed a large-scale genomic analysis of the variation of the genes encoding RCPs in 13 species and present these data in an online database (http://repeats.med.monash.edu.au/genetic_analysis/). This resource allows rapid comparison and analysis of RCPs, homopeptides, and their underlying genetic tracts across the eukaryotic species considered. We report three major findings. First, there is a bias for a small subset of codons being reiterated within homopeptides, and there is no G+C or A+T bias relative to the organism's transcriptome. Second, single base pair transversions from the homocodon are unusually common and may represent a mechanism of reducing the rate of homopeptide mutations. Third, homopeptides that are conserved across different species lie within regions that are under stronger purifying selection in contrast to nonconserved homopeptides.

Glycoprotein (GP) Ib-IX-V binds von Willebrand factor (VWF), initiating thrombosis at high shear stress. The VWF-A1 domain binds the N-terminal domain of GPIbalpha (His1-Glu282); this region contains seven leucine-rich repeats (LRR) plus N- and C-terminal flanking sequences and an anionic sequence containing three sulfated tyrosines. Our previous analysis of canine/human and human/canine chimeras of GPIbalpha expressed on Chinese hamster ovary (CHO) cells demonstrated that LRR2-4 (Leu60-Glu128) were crucial for GPIbalpha-dependent adhesion to VWF. Paradoxically, co-crystal structures of the GPIbalpha N-terminal domain and GPIbalpha-binding VWF-A1 under static conditions revealed that the LRR2-4 sequence made minimal contact with VWF-A1. To resolve the specific functional role of LRR2-4, we compared wild-type human GPIbalpha with human GPIbalpha containing a homology domain swap of canine for human sequence within Leu60-Glu128 and a reverse swap (canine GPIbalpha with human Leu60-Glu128) for the ability to support adhesion to VWF under flow. Binding of conformation-specific anti-GPIbalpha antibodies and VWF binding in the presence of botrocetin (which does not discriminate between species) confirmed equivalent expression of wild-type and mutant receptors in a functional form competent to bind ligand. Compared with CHO cells expressing wild-type GPIbalpha, cells expressing GPIbalpha, where human Leu60-Glu128 sequence was replaced by canine sequence, supported adhesion to VWF at low shear rates but became increasingly ineffective as shear increased from 50 to 2000 s(-1). Together, these data demonstrate that LRR2-4, encompassing a pronounced negative charge patch on human GPIbalpha, is essential for GPIbalpha.VWF-dependent adhesion as hydrodynamic shear increases.

The crystallization of macromolecules remains a major bottleneck in structural biology. The routine screening of more than one thousand crystallization conditions and subsequent optimization by fine screening presents a challenge to conventional laboratory notebook keeping. In addition, the development of high-throughput robotic crystallization and imaging systems presents a pressing need for low-cost laboratory information management system (LIMS). Here we describe CLIMS2, a crystallization LIMS that features a simple, user-friendly graphical interface, allowing the storage, management, retrieval and mining of crystallization data. The CLIMS2 executable and documentation is freely available at http://clims.med.monash.edu.au., (2005 Wiley-Liss, Inc.)

The sequence of a genome contains the plans of the possible life of an organism, but implementation of genetic information depends on the functions of the proteins and nucleic acids that it encodes. Many individual proteins of known sequence and structure present challenges to the understanding of their function. In particular, a number of genes responsible for diseases have been identified but their specific functions are unknown. Whole-genome sequencing projects are a major source of proteins of unknown function. Annotation of a genome involves assignment of functions to gene products, in most cases on the basis of amino-acid sequence alone. 3D structure can aid the assignment of function, motivating the challenge of structural genomics projects to make structural information available for novel uncharacterized proteins. Structure-based identification of homologues often succeeds where sequence-alone-based methods fail, because in many cases evolution retains the folding pattern long after sequence similarity becomes undetectable. Nevertheless, prediction of protein function from sequence and structure is a difficult problem, because homologous proteins often have different functions. Many methods of function prediction rely on identifying similarity in sequence and/or structure between a protein of unknown function and one or more well-understood proteins. Alternative methods include inferring conservation patterns in members of a functionally uncharacterized family for which many sequences and structures are known. However, these inferences are tenuous. Such methods provide reasonable guesses at function, but are far from foolproof. It is therefore fortunate that the development of whole-organism approaches and comparative genomics permits other approaches to function prediction when the data are available. These include the use of protein-protein interaction patterns, and correlations between occurrences of related proteins in different organisms, as indicators of functional properties. Even if it is possible to ascribe a particular function to a gene product, the protein may have multiple functions. A fundamental problem is that function is in many cases an ill-defined concept. In this article we review the state of the art in function prediction and describe some of the underlying difficulties and successes.

Members of the membrane attack complex/perforin-like (MACPF) protein superfamily have long captured interest because of their unique ability to assemble into large oligomeric pores on the surfaces of cells. The best characterised of these act in vertebrate immunity where they function to deliver pro-apoptotic factors or induce the cytolysis and death of targeted cells. Less appreciated, however, is that rather than causing cell death, MACPF proteins have also evolved to control cellular signalling pathways and influence developmental programmes such as pattern formation and neurogenesis. Torso-like (Tsl) from the fruit fly Drosophila, for example, functions to localise the activity of a growth factor for patterning its embryonic termini. It remains unclear whether these developmental proteins employ an attenuated form of the classical MACPF lytic pore, or if they have evolved to function via alternative mechanisms of action. In this minireview, we examine the evidence that links pore-forming MACPF proteins to the control of growth factor and cytokine signalling. We will then attempt to reconcile how the MACPF domain may have been repurposed during evolution for developmental events rather than cell killing. [ABSTRACT FROM AUTHOR]

Dichelobacter nodosus is the principal causative agent of ovine footrot and its extracellular proteases are major virulence factors. Virulent isolates of D. nodosus secrete three subtilisin-like serine proteases: AprV2, AprV5 and BprV. These enzymes are each synthesized as precursor molecules that include a signal (pre-) peptide, a pro-peptide and a C-terminal extension, which are processed to produce the mature active forms. The function of the C-terminal regions of these proteases and the mechanism of protease processing and secretion are unknown. AprV5 contributes to most of the protease activity secreted by D. nodosus. To understand the role of the C-terminal extension of AprV5, we constructed a series of C-terminal-deletion mutants in D. nodosus by allelic exchange. The proteases present in the resultant mutants and their complemented derivatives were examined by protease zymogram analysis, western blotting and mass spectrometry. The results showed that the C-terminal region of AprV5 is required for the normal expression of protease activity, deletion of this region led to a delay in the processing of these enzymes. D. nodosus is an unusual bacterium in that it produces three closely related extracellular serine proteases. We have now shown that one of these enzymes, AprV5, is responsible for its own maturation, and for the optimal cleavage of AprV2 and BprV, to their mature active forms. These studies have increased our understanding of how this important pathogen processes these virulence-associated extracellular proteases and secretes them into its external environment. [ABSTRACT FROM AUTHOR]

As an organism that has evolved to live in environments ranging from soil to the cytosol of mammalian cells, Listeria monocytogenes must regulate the secretion and activity of protein products that promote survival within these habitats. The post-translocation chaperone PrsA2 has been adapted to assist in the folding and activity of L. monocytogenes secreted proteins required for bacterial replication within host cells. Here we present the first structure/function investigation of the contributions of PrsA2 to protein secretion and activity as well as to bacterial virulence. Domain swap experiments with the closely related L. monocytogenes PrsA1 protein combined with targeted mutagenesis indicate distinct functional roles for the PrsA2 peptidyl-prolyl isomerase (PPIase) and the N- and C-terminal domains in pathogenesis. In contrast to other PrsA-like proteins described thus far in the literature, an absolute in vivo requirement for PrsA2 PPIase activity is evident in mouse infection models. This work illustrates the diversity of function associated with L. monocytogenes PrsA2 that serves to promote bacterial life within the infected host. [ABSTRACT FROM AUTHOR]

Many bacterial pathogens utilize a type III secretion system to deliver multiple effector proteins into host cells. Here we found that the type III effectors, NleE from enteropathogenic E. coli (EPEC) and OspZ from Shigella, blocked translocation of the p65 subunit of the transcription factor, NF-κB, to the host cell nucleus. NF-κB inhibition by NleE was associated with decreased IL-8 expression in EPEC-infected intestinal epithelial cells. Ectopically expressed NleE also blocked nuclear translocation of p65 and c-Rel, but not p50 or STAT1/2. NleE homologues from other attaching and effacing pathogens as well OspZ from Shigella flexneri 6 and Shigella boydii, also inhibited NF-κB activation and p65 nuclear import; however, a truncated form of OspZ from S. flexneri 2a that carries a 36 amino acid deletion at the C-terminus had no inhibitory activity. We determined that the C-termini of NleE and full length OspZ were functionally interchangeable and identified a six amino acid motif, IDSY(M/I)K, that was important for both NleE- and OspZ-mediated inhibition of NF-κB activity. We also established that NleB, encoded directly upstream from NleE, suppressed NF-κB activation. Whereas NleE inhibited both TNFa and IL-1β stimulated p65 nuclear translocation and IkB degradation, NleB inhibited the TNFa pathway only. Neither NleE nor NleB inhibited AP-1 activation, suggesting that the modulatory activity of the effectors was specific for NF-κB signaling. Overall our data show that EPEC and Shigella have evolved similar T3SS-dependent means to manipulate host inflammatory pathways by interfering with the activation of selected host transcriptional regulators. [ABSTRACT FROM AUTHOR]

The native state of serpins represents a long-lived intermediate or metastable structure on the serpin folding pathway. Upon interaction with a protease, the serpin trap is sprung and the molecule continues to fold into a more stable conformation. However, thermodynamic stability can also be achieved through alternative, unproductive folding pathways that result in the formation of inactive conformations. Our increasing understanding of the mechanism of protease inhibition and the dynamics of native serpin structures has begun to reveal how evolution has harnessed the actual process of protein folding (rather than the final folded outcome) to elegantly achieve function. The cost of using metastability for function, however, is an increased propensity for misfolding. [Copyright &y& Elsevier]

Macromolecular crystallography requires simple yet effective means of organizing and managing the large amounts of data generated by crystallization experiments. There are several freely available web- based Laboratory Information Management Systems (LIMS) that assist in these tasks. These, however, rely on the limited user interfaces allowed in HTML-based web pages. To address this limitation, a new LIMS for protein crystallization, which features a novel rich graphical user interface (GUI) to a relational database, has been developed. This application, which is called CLIMS (Crystallography LIMS), assists in all aspects of protein-crystallization projects: protein expression, handling, crystallization optimization visualization of results and preliminary diffraction data. Extensive use of templates, particularly for commercial screens and common optimization grid screens, exploits the redundancy in experimental setups. The crystallization tray is the central focus of the graphical interface, thus facilitating rapid visualization and annotation of results. CLIMS was developed specifically to cater for the needs of individual laboratories requiring an intuitive and robust system for managing crystallization experiments and is freely available. [ABSTRACT FROM AUTHOR]

Macrophage-expressed gene 1 (MPEG1/Perforin-2) is a perforin-like protein that functions within the phagolysosome to damage engulfed microbes. MPEG1 is thought to form pores in target membranes, however, its mode of action remains unknown. We use cryo-Electron Microscopy (cryo-EM) to determine the 2.4 Å structure of a hexadecameric assembly of MPEG1 that displays the expected features of a soluble prepore complex. We further discover that MPEG1 prepore-like assemblies can be induced to perforate membranes through acidification, such as would occur within maturing phagolysosomes. We next solve the 3.6 Å cryo-EM structure of MPEG1 in complex with liposomes. These data reveal that a multi-vesicular body of 12 kDa (MVB12)-associated β-prism (MABP) domain binds membranes such that the pore-forming machinery of MPEG1 is oriented away from the bound membrane. This unexpected mechanism of membrane interaction suggests that MPEG1 remains bound to the phagolysosome membrane while simultaneously forming pores in engulfed bacterial targets. Macrophage-expressed gene 1 (MPEG1) functions within the phagolysosome to damage engulfed microbes, presumably via forming pores in target membranes. In order to provide insights into the mechanism of MPEG1 function and membrane binding, the authors present structures of hexadecameric MPEG1 prepores both in solution and in complex with liposomes. [ABSTRACT FROM AUTHOR]

Presents a letter to the editor commenting on the post-genomic era that has lead to an explosion in protein expression and structural initiatives from both a biotechnology and an academic perspective.

Ataxin-3 belongs to the family of polyglutamine proteins, which are associated with nine different neurodegenerative disorders. Relatively little is known about the structural and functional properties of ataxin-3, and only recently have these aspects of the protein begun to be explored. We have performed a preliminary investigation into the conserved N-terminal domain of ataxin-3, termed Josephin. We show that Josephin is a monomeric domain which folds into a globular conformation and possesses ubiquitin protease activity. In addition, we demonstrate that the presence of the polyglutamine region of the protein does not alter the structure of the protein. However, its presence destabilizes the Josephin domain. The implications of these data in the pathogenesis of polyglutamine repeat proteins are discussed. [Copyright &y& Elsevier]

MENT is a developmentally regulated heterochromatin-associated protein that condenses chromatin in terminally differentiated avian blood cells. Its homology to the serpin protein family suggests that the conserved serpin reactive center loop (RCL) and the unique M-loop are important for its function. To examine the role of these domains, we studied the interaction of wild-type and mutant MENT with naked DNA and biochemically defined nucleosome arrays reconstituted from 12-mer repeats containing nucleosome positioning sequences. Wild-type MENT folded the naked DNA duplexes into closely juxtaposed parallel structures ("tramlines"). Deletion of the M-loop, but not inactivation of the RCL, prevented tramline formation and the cooperative interaction of MENT with DNA. Reconstitution of wildtype MENT with nucleosome arrays caused their tight folding and self. association. M-loop deletion inhibited nucleosome array folding, whereas the inactive RCL mutant was competent to fold the nucleosome arrays, but had a significantly impaired ability to cause their self-association. Bifunctional chemical cross-linking of MENT revealed oligomerization of wild-type MENT in the presence of chromatin and DNA. This oligomerization was severely reduced in the RCL mutant. We propose that the mechanism of MENT-induced heterochromatin formation involves two independent events: bringing together nucleosome linkers within a chromatin fiber and formation of protein bridges between chromatin fibers. Ordered binding of MENT to linker DNA via its unique M-loop domain promotes the folding of chromatin, whereas bridging of chromatin fibers is facilitated by MENT oligomerization mediated by the RCL. [ABSTRACT FROM AUTHOR]

The waxy cell wall is crucial to the survival of mycobacteria within the infected host. The cell wall is a complex structure rich in unusual molecules that includes two related lipoglycans, the phosphatidylinositol mannosides (PIMs) and lipoarabinomannans (LAMs). Many proteins implicated in the PIM/LAM biosynthetic pathway, while attractive therapeutic targets, are poorly defined. The 2.4Å resolution crystal structure of an essential lipoprotein, LpqW, implicated in LAM biosynthesis is reported here. LpqW adopts a scaffold reminiscent of the distantly related, promiscuous substrate-binding proteins of the ATP-binding cassette import system. Nevertheless, the unique closed conformation of LpqW suggests that mycobacteria and other closely related pathogens have hijacked this scaffold for use in key processes of cell wall biosynthesis. In silico docking provided a plausible model in which the candidate PIM ligand binds within a marked electronegative region located on the surface of LpqW. We suggest that LpqW represents an archetypal lipoprotein that channels intermediates from a pathway for mature PIM production into a pathway for LAM biosynthesis, thus controlling the relative abundance of these two important components of the cell wall. [Copyright &y& Elsevier]

Abstract: The expression and harvesting of proteins from insoluble inclusion bodies by solubilization and refolding is a technique commonly used in the production of recombinant proteins. To bring clarity to the large and widespread quantity of published protein refolding data, we have recently established the REFOLD database (http://refold.med.monash.edu.au), which is a freely available, open repository for protocols describing the refolding and purification of recombinant proteins. Refolding methods are currently published in many different formats and resources—REFOLD provides a standardized system for the structured reporting and presentation of these data. Furthermore, data in REFOLD are readily accessible using a simple search function, and the database also enables analyses which identify and highlight particular trends between suitable refolding and purification conditions and specific protein properties. This information may in turn serve to facilitate the rational design and development of new refolding protocols for novel proteins. There are approximately 200 proteins currently listed in REFOLD, and it is anticipated that with the continued contribution of data by researchers this number will grow significantly, thus strengthening the emerging trends and patterns and making this database a valuable tool for the scientific community. [Copyright &y& Elsevier]

Expansion of ‘low complex’ repeats of amino acids such as glutamine (Poly-Q) is associated with protein misfolding and the development of degenerative diseases such as Huntington's disease. The mechanism by which such regions promote misfolding remains controversial, the function of many repeat-containing proteins (RCPs) remains obscure, and the role (if any) of repeat regions remains to be determined. Here, a Web-accessible database of RCPs is presented. The distribution and evolution of RCPs that contain homopeptide repeats tracts are considered, and the existence of functional patterns investigated. Generally, it is found that while polyamino acid repeats are extremely rare in prokaryotes, several eukaryote putative homologs of prokaryote RCP—involved in important housekeeping processes—retain the repetitive region, suggesting an ancient origin for certain repeats. Within eukarya, the most common uninterrupted amino acid repeats are glutamine, asperagines, and alanine. Interestingly, while poly-Q repeats are found [n vertebrates and nonvertebrates, poly-N repeats are only common in more primitive nonvertebrate organisms, such as insects and nematodes. We have assigned function to eukaryote RCPs using Online Mendelian Inheritance in Man (OMIM), the Human Reference Protein Database (HRPD), FlyBase, and Wormpep. Prokaryote RCPs were annotated using BLASTp searches and Gene Ontology. These data reveal that the majority of RCPs are involved in processes that require the assembly of large, multiprotein complexes, such as transcription and signaling. [ABSTRACT FROM AUTHOR]