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Low Sequence Identity (low + sequence_identity)
Selected AbstractsActin-binding domain of mouse plectinFEBS JOURNAL, Issue 10 2004Crystal structure, binding to vimentin Plectin, a large and widely expressed cytolinker protein, is composed of several subdomains that harbor binding sites for a variety of different interaction partners. A canonical actin-binding domain (ABD) comprising two calponin homology domains (CH1 and CH2) is located in proximity to its amino terminus. However, the ABD of plectin is unique among actin-binding proteins as it is expressed in the form of distinct, plectin isoform-specific versions. We have determined the three-dimensional structure of two distinct crystalline forms of one of its ABD versions (pleABD/2,) from mouse, to a resolution of 1.95 and 2.0 Å. Comparison of pleABD/2, with the ABDs of fimbrin and utrophin revealed structural similarity between plectin and fimbrin, although the proteins share only low sequence identity. In fact, pleABD/2, has been found to have the same compact fold as the human plectin ABD and the fimbrin ABD, differing from the open conformation described for the ABDs of utrophin and dystrophin. Plectin harbors a specific binding site for intermediate filaments of various types within its carboxy-terminal R5 repeat domain. Our experiments revealed an additional vimentin-binding site of plectin, residing within the CH1 subdomain of its ABD. We show that vimentin binds to this site via the amino-terminal part of its rod domain. This additional amino-terminal intermediate filament protein binding site of plectin may have a function in intermediate filament dynamics and assembly, rather than in linking and stabilizing intermediate filament networks. [source] Efficient recognition of protein fold at low sequence identity by conservative application of Psi-BLAST: validation,JOURNAL OF MOLECULAR RECOGNITION, Issue 2 2005F. J. Stevens Abstract A substantial fraction of protein sequences derived from genomic analyses is currently classified as representing ,hypothetical proteins of unknown function'. In part, this reflects the limitations of methods for comparison of sequences with very low identity. We evaluated the effectiveness of a Psi-BLAST search strategy to identify proteins of similar fold at low sequence identity. Psi-BLAST searches for structurally characterized low-sequence-identity matches were carried out on a set of over 300 proteins of known structure. Searches were conducted in NCBI's non-redundant database and were limited to three rounds. Some 614 potential homologs with 25% or lower sequence identity to 166 members of the search set were obtained. Disregarding the expect value, level of sequence identity and span of alignment, correspondence of fold between the target and potential homolog was found in more than 95% of the Psi-BLAST matches. Restrictions on expect value or span of alignment improved the false positive rate at the expense of eliminating many true homologs. Approximately three-quarters of the putative homologs obtained by three rounds of Psi-BLAST revealed no significant sequence similarity to the target protein upon direct sequence comparison by BLAST, and therefore could not be found by a conventional search. Although three rounds of Psi-BLAST identified many more homologs than a standard BLAST search, most homologs were undetected. It appears that more than 80% of all homologs to a target protein may be characterized by a lack of significant sequence similarity. We suggest that conservative use of Psi-BLAST has the potential to propose experimentally testable functions for the majority of proteins currently annotated as ,hypothetical proteins of unknown function';. Copyright © 2004 John Wiley & Sons, Ltd. [source] Efficient recognition of protein fold at low sequence identity by conservative application of Psi-BLAST: application,JOURNAL OF MOLECULAR RECOGNITION, Issue 2 2005F. J. Stevens Abstract Based on a study involving structural comparisons of proteins sharing 25% or less sequence identity, three rounds of Psi-BLAST appear capable of identifying remote evolutionary homologs with greater than 95% confidence provided that more than 50% of the query sequence can be aligned with the target sequence. Since it seems that more than 80% of all homologous protein pairs may be characterized by a lack of significant sequence similarity, the experimental biologist is often confronted with a lack of guidance from conventional homology searches involving pair-wise sequence comparisons. The ability to disregard levels of sequence identity and expect value in Psi-BLAST if at least 50% of the query sequence has been aligned allows for generation of new hypotheses by consideration of matches that are conventionally disregarded. In one example, we suggest a possible evolutionary linkage between the cupredoxin and immunoglobulin fold families. A thermostable hypothetical protein of unknown function may be a circularly permuted homolog to phosphotriesterase, an enzyme capable of detoxifying organophosphate nerve agents. In a third example, the amino acid sequence of another hypothetical protein of unknown function reveals the ATP binding-site, metal binding site, and catalytic sidechain consistent with kinase activity of unknown specificity. This approach significantly expands the utility of existing sequence data to define the primary structure degeneracy of binding sites for substrates, cofactors and other proteins. Copyright © 2004 John Wiley & Sons, Ltd. [source] Molecular Characterization of a Strain of Squash Leaf Curl China Virus from the PhilippinesJOURNAL OF PHYTOPATHOLOGY, Issue 10 2003T. Kon Abstract The complete nucleotide sequence of infectious cloned DNA components (A and B) of the causal agent of squash leaf curl disease in the Philippines was determined. DNA-A and DNA-B comprise 2739 and 2705 nucleotides, respectively; the common region is 174 bases in length. Five ORFs were found in DNA-A and two in DNA-B. Partial dimeric clones containing DNA-A and DNA-B, constructed in a binary vector and transformed into Agrobacterium tumefaciens, induced systemic infection in agro-inoculated pumpkin plants (Cucurbita moschata). The total DNA-A sequence was most closely related to that of Squash leaf curl China virus (SLCCNV) (88% identity), although the existence of B component of SLCCNV has not been reported. The deduced coat protein was like that of SLCCNV (98% amino acid sequence identity) and the Philippines virus has low sequence identity to Squash leaf curl virus (SLCV) and Squash mild leaf curl virus (SMLCV) (63 and 64% total nucleotide sequence identities, respectively). From these results, we propose that the Philippines virus be designated Squash leaf curl China virus -[Philippines] (SLCCNV-[PH]). [source] Dynameomics: Large-scale assessment of native protein flexibilityPROTEIN SCIENCE, Issue 12 2008Noah C. Benson Abstract Structure is only the first step in understanding the interactions and functions of proteins. In this paper, we explore the flexibility of proteins across a broad database of over 250 solvated protein molecular dynamics simulations in water for an aggregate simulation time of ,6 ,s. These simulations are from our Dynameomics project, and these proteins represent approximately 75% of all known protein structures. We employ principal component analysis of the atomic coordinates over time to determine the primary axis and magnitude of the flexibility of each atom in a simulation. This technique gives us both a database of flexibility for many protein fold families and a compact visual representation of a particular protein's native-state conformational space, neither of which are available using experimental methods alone. These tools allow us to better understand the nature of protein motion and to describe its relationship to other structural and dynamical characteristics. In addition to reporting general properties of protein flexibility and detailing many dynamic motifs, we characterize the relationship between protein native-state flexibility and early events in thermal unfolding and show that flexibility predicts how a protein will begin to unfold. We provide evidence that fold families have conserved flexibility patterns, and family members who deviate from the conserved patterns have very low sequence identity. Finally, we examine novel aspects of highly inflexible loops that are as important to structural integrity as conventional secondary structure. These loops, which are difficult if not impossible to locate without dynamic data, may constitute new structural motifs. [source] The epitope space of the human proteomePROTEIN SCIENCE, Issue 4 2008Lisa Berglund Abstract In the post-genome era, there is a great need for protein-specific affinity reagents to explore the human proteome. Antibodies are suitable as reagents, but generation of antibodies with low cross-reactivity to other human proteins requires careful selection of antigens. Here we show the results from a proteome-wide effort to map linear epitopes based on uniqueness relative to the entire human proteome. The analysis was based on a sliding window sequence similarity search using short windows (8, 10, and 12 amino acid residues). A comparison of exact string matching (Hamming distance) and a heuristic method (BLAST) was performed, showing that the heuristic method combined with a grid strategy allows for whole proteome analysis with high accuracy and feasible run times. The analysis shows that it is possible to find unique antigens for a majority of the human proteins, with relatively strict rules involving low sequence identity of the possible linear epitopes. The implications for human antibody-based proteomics efforts are discussed. [source] Diagnostic cross-linking of paired cysteine pairs demonstrates homologous structures for two chemoreceptor domains with low sequence identityPROTEIN SCIENCE, Issue 1 2006Wing-Cheung Lai Abstract Hundreds of bacterial chemoreceptors from many species have periplasmic, ligand-recognition domains of approximately the same size, but little or no sequence identity. The only structure determined is for the periplasmic domain of chemoreceptor Tar from Salmonella and Escherichia coli. Do sequence-divergent but similarly sized chemoreceptor periplasmic domains have related structures? We addressed this issue for the periplasmic domain of chemoreceptor TrgE from E. coli, which has a low level of sequence similarity to Tar, by combining homology modeling and diagnostic cross-linking between pairs of introduced cysteines. A homology model of the TrgE domain was created using the homodimeric, four-helix bundle structure of the TarS domain from Salmonella. In this model, we chose four pairs of positions at which introduced cysteines would be sufficiently close to form disulfides across each of four different helical interfaces. For each pair we chose a second pair, in which one cysteine of the original pair was shifted by one position around the helix and thus would be less favorably placed for disulfide formation. We created genes coding for proteins containing four such pairs of cysteine pairs and investigated disulfide formation in vivo as well as functional consequences of the substitutions and disulfides between neighboring helices. Results of the experimental tests provided strong support for the accuracy of the model, indicating that the TrgE periplasmic domain is very similar to the TarS domain. Diagnostic cross-linking of paired pairs of introduced cysteines could be applied generally as a stringent test of homology models. [source] In silico protein design by combinatorial assembly of protein building blocksPROTEIN SCIENCE, Issue 10 2004Hui-Hsu (Gavin) Tsai Abstract Utilizing concepts of protein building blocks, we propose a de novo computational algorithm that is similar to combinatorial shuffling experiments. Our goal is to engineer new naturally occurring folds with low homology to existing proteins. A selected protein is first partitioned into its building blocks based on their compactness, degree of isolation from the rest of the structure, and hydrophobicity. Next, the protein building blocks are substituted by fragments taken from other proteins with overall low sequence identity, but with a similar hydrophobic/hydrophilic pattern and a high structural similarity. These criteria ensure that the designed protein has a similar fold, low sequence identity, and a good hydrophobic core compared with its native counterpart. Here, we have selected two proteins for engineering, protein G B1 domain and ubiquitin. The two engineered proteins share ,20% and ,25% amino acid sequence identities with their native counterparts, respectively. The stabilities of the engineered proteins are tested by explicit water molecular dynamics simulations. The algorithm implements a strategy of designing a protein using relatively stable fragments, with a high population time. Here, we have selected the fragments by searching for local minima along the polypeptide chain using the protein building block model. Such an approach provides a new method for engineering new proteins with similar folds and low homology. [source] Structural conservation in the major facilitator superfamily as revealed by comparative modelingPROTEIN SCIENCE, Issue 7 2004Eyal Vardy Abstract The structures of membrane transporters are still mostly unsolved. Only recently, the first two high-resolution structures of transporters of the major facilitator superfamily (MFS) were published. Despite the low sequence similarity of the two proteins involved, lactose permease and glycerol-3-phosphate transporter, the reported structures are highly similar. This leads to the hypothesis that all members of the MFS share a similar structure, regardless of their low sequence identity. To test this hypothesis, we generated models of two other members of the MFS, the Tn10-encoded metal-tetracycline/H+ antiporter (TetAB) and the rat vesicular monoamine transporter (rVMAT2). The models are based on the two MFS structures and on experimental data. The models for both proteins are in good agreement with the data available and support the notion of a shared fold for all MFS proteins. [source] Structure of the diaminopimelate epimerase DapF from Mycobacterium tuberculosisACTA CRYSTALLOGRAPHICA SECTION D, Issue 4 2009Veeraraghavan Usha The meso (or d,l) isomer of diaminopimelic acid (DAP), a precursor of l -lysine, is a key component of the pentapeptide linker in bacterial peptidoglycan. While the peptidoglycan incorporated in the highly complex cell wall of the pathogen Mycobacterium tuberculosis structurally resembles that of Escherichia coli, it is unique in that it can contain penicillin-resistant meso -DAP,meso -DAP linkages. The interconversion of l,l -DAP and meso -DAP is catalysed by the DAP epimerase DapF, a gene product that is essential in M. tuberculosis. Here, the crystal structure of the ligand-free form of M. tuberculosis DapF (MtDapF) refined to a resolution of 2.6,Å is reported. MtDapF shows small if distinct deviations in secondary structure from the two-domain ,/,-fold of the known structures of Haemophilus influenzae DapF and Bacillus anthracis DapF, which are in line with its low sequence identity (,27%) to the former. Modelling the present structure onto that of l,l -aziridino-DAP-bound H. influenzae DapF illustrates that a rigid-body movement of domain II and a rearrangement of the B4,A2 loop (residues 80,90) of domain I are likely to accompany the transition from the present inactive form to a catalytically competent enzyme. Despite a highly conserved active-site architecture, the model indicates that stabilization of the DAP backbone occurs in MtDapF through a tyrosine residue that is specific to mycobacterial DAP epimerases. [source] The structure of l -rhamnulose-1-phosphate aldolase (class II) solved by low-resolution SIR phasing and 20-fold NCS averagingACTA CRYSTALLOGRAPHICA SECTION D, Issue 5 2002Markus Kroemer The enzyme l -rhamnulose-1-phosphate aldolase catalyzes the reversible cleavage of l -rhamnulose-1-phosphate to dihydroxyacetone phosphate and l -lactaldehyde. It is a homotetramer with an Mr of 30,000 per subunit and crystallized in space group P3221. The enzyme shows a low sequence identity of 18% with the structurally known l -fuculose-1-phosphate aldolase that splits a stereoisomer in a similar reaction. Structure analysis was initiated with a single heavy-atom derivative measured to 6,Å resolution. The resulting poor electron density, a self-rotation function and the working hypothesis that both enzymes are C4 symmetric with envelopes that resemble one another allowed the location of the 20 protomers of the asymmetric unit. The crystal-packing unit was a D4 -symmetric propeller consisting of five D4 -symmetric octamers around an internal crystallographic twofold axis. Presumably, the propellers associate laterally in layers, which in turn pile up along the 32 axis to form the crystal. The non-crystallographic symmetry was used to extend the phases to the 2.7,Å resolution limit and to establish a refined atomic model of the enzyme. The structure showed that the two enzymes are indeed homologous and that they possess chemically similar active centres. [source] Structure of Stenotrophomonas maltophilia FeoA complexed with zinc: a unique prokaryotic SH3-domain protein that possibly acts as a bacterial ferrous iron-transport activating factorACTA CRYSTALLOGRAPHICA SECTION F (ELECTRONIC), Issue 6 2010Yi-Che Su Iron is vital to the majority of prokaryotes, with ferrous iron believed to be the preferred form for iron uptake owing to its much better solubility. The major route for bacterial ferrous iron uptake is found to be via an Feo (ferrous iron-transport) system comprising the three proteins FeoA, FeoB and FeoC. Although the structure and function of FeoB have received much attention recently, the roles played by FeoA and FeoC have been little investigated to date. Here, the tertiary structure of FeoA from Stenotrophomonas maltophilia (Sm), a vital opportunistic pathogen in immunodepressed hosts, is reported. The crystal structure of SmFeoA has been determined to a resolution of 1.7,Å using an Se single-wavelength anomalous dispersion (Se-SAD) approach. Although SmFeoA bears low sequence identity to eukaryotic proteins, its structure is found to adopt a eukaryotic SH3-domain-like fold. It also bears weak similarity to the C-terminal SH3 domain of bacterial DtxR (diphtheria toxin regulator), with some unique characteristics. Intriguingly, SmFeoA is found to adopt a unique dimer cross-linked by two zinc ions and six anions (chloride ions). Since FeoB has been found to contain a G-protein-like domain with low GTPase activity, FeoA may interact with FeoB through the SH3,G-protein domain interaction to act as a ferrous iron-transport activating factor. [source] Purification, crystallization and preliminary X-ray studies of the putative lysozyme SP0987 from Streptococcus pneumoniaeACTA CRYSTALLOGRAPHICA SECTION F (ELECTRONIC), Issue 3 2010Siqiang Niu Streptococcus pneumoniae SP0987, which was identified as a hypothetical protein, has a very low sequence identity to other well characterized lysozyme structures. Since determination of three-dimensional structure is a powerful means of functional characterization, X-ray crystallography has been used to accomplish this task. Here, the expression, purification, crystallization and preliminary crystallographic analysis of SP0987 from Streptococcus pneumoniae TIGR4 are reported. The crystal belonged to space group P212121 (with unit-cell parameters a = 36.46, b = 40.89, c = 147.44,Å) and diffracted to a resolution of 1.85,Å. The crystals are most likely to contain one molecule in the asymmetric unit, with a VM value of 2.02,Å3,Da,1. [source] Crystallization and preliminary X-ray analysis of a cohesin-like module from AF2375 of the archaeon Archaeoglobus fulgidusACTA CRYSTALLOGRAPHICA SECTION F (ELECTRONIC), Issue 3 2009Milana Voronov-Goldman A cohesin-like module of 160 amino-acid residues from the hypothetical protein AF2375 of the noncellulolytic, hyperthermophilic, sulfate-reducing archaeon Archaeoglobus fulgidus was cloned, expressed, purified, crystallized and subjected to X-ray structural study in order to compare its structure with those of cellulolytic cohesins. The crystals had cubic symmetry, with unit-cell parameters a = b = c = 101.75,Å in space group P4332, and diffracted to 1.82,Å resolution. The asymmetric unit contained a single cohesin molecule. A model assembled from six cohesin structures (PDB entries 1anu, 1aoh, 1g1k, 1qzn, 1zv9 and 1tyj) of very low sequence identity to the cohesin-like module was used in molecular-replacement attempts, producing a marginal solution. [source] | ||||||||||