Weak Sequence Research Articles

Getting the most out of publicly available T‐DNA insertion lines

In the course of several different projects, we came to realize that there is a significant amount of untapped potential in the publicly available T-DNA insertion lines. In addition to the GABI-Kat lines, which were designed specifically for activation tagging, lines from the SAIL and FLAGdb collections are also useful for this purpose. As well as the 35S promoter chosen for activation tagging in GABI-Kat lines, we found that the 1'2' bidirectional promoter is capable of activating expression of flanking genomic sequences in both GABI-Kat and SAIL lines. Thus these lines have added potential for activation tagging. We also show that these lines are capable of generating antisense transcripts and so have the potential to be used for suppression (loss/reduction of function) studies. By virtue of weak terminator sequences in some T-DNA constructs, transcript read-through from selectable markers is also possible, which again has the potential to be exploited in activation/suppression studies. Finally, we show that, by selecting and characterizing lines in which the T-DNA insertions are present specifically within introns of a target gene, an allelic series of mutants with varying levels of reduced expression can be generated, due to differences in efficiency of intron splicing. Taken together, our analyses demonstrate that there is a wealth of untapped potential within existing insertion lines for studies on gene function, and the effective exploitation of these resources is discussed.

PP4R4/KIAA1622 Forms a Novel Stable Cytosolic Complex with Phosphoprotein Phosphatase 4

Protein serine/threonine phosphatase 4 (PP4c) is an essential polypeptide involved in critical cellular processes such as microtubule growth and organization, DNA damage checkpoint recovery, apoptosis, and tumor necrosis factor alpha signaling. Like other phosphatases of the PP2A family, PP4c interacts with regulatory proteins, which specify substrate targeting and intracellular localization. The identification of these regulatory proteins is, therefore, key to fully understanding the function of this enzyme class. Here, using a sensitive affinity purification/mass spectrometry approach, we identify a novel, stable cytosolic PP4c interacting partner, KIAA1622, which we have renamed PP4R4. PP4R4 displays weak sequence homology with the A (scaffolding) subunit of the PP2A holoenzyme and specifically associates with PP4c (and not with the related PP2Ac or PP6c phosphatases). The PP4c.PP4R4 interaction is disrupted by mutations analogous to those abrogating the association of PP2Ac with PP2A A subunit. However, unlike the PP2A A subunit, which plays a scaffolding role, PP4R4 does not bridge PP4c with previously characterized PP4 regulatory subunits. PP4c.PP4R4 complexes exhibit phosphatase activity toward a fluorogenic substrate and gammaH2AX, but this activity is lower than that associated with the PP4c.PP4R2.PP4R3 complex, which itself is less active than the free PP4c catalytic subunit. Our data demonstrate that PP4R4 forms a novel cytosolic complex with PP4c, independent from the complexes containing PP4R1, PP4R2.PP4R3, and alpha4, and that the regulatory subunits of PP4c have evolved different modes of interaction with the catalytic subunit.

Evolution of Peptidase Diversity

A wide variety of peptidases associate with vital biological pathways, but the origin and evolution of their tremendous diversity are poorly defined. Application of the MEROPS classification to a comprehensive set of genomes yields a simple pattern of peptidase distribution and provides insight into the organization of proteolysis in all forms of life. Unexpectedly, a near ubiquitous core set of peptidases is shown to contain more types than those unique to higher multicellular organisms. From this core group, an array of eukaryote-specific peptidases evolved to yield well known intracellular and extracellular processes. The paucity of peptidase families unique to higher metazoa suggests gains in proteolytic network complexity required a limited number of biochemical inventions. These findings provide a framework for deeper investigation into the evolutionary forces that shaped each peptidase family and a roadmap to develop a timeline for their expansion as an interconnected system.

Evolutionary Conserved Role for TARPs in the Gating of Glutamate Receptors and Tuning of Synaptic Function

Neurotransmission in the brain is critically dependent on excitatory synaptic signaling mediated by AMPA-class ionotropic glutamate receptors (AMPARs). AMPARs are known to be associated with Transmembrane AMPA receptor Regulatory Proteins (TARPs). In vertebrates, at least four TARPs appear to have redundant roles as obligate chaperones for AMPARs, thus greatly complicating analysis of TARP participation in synaptic function. We have overcome this limitation by identifying and mutating the essential set of TARPs in C. elegans (STG-1 and STG-2). In TARP mutants, AMPAR-mediated currents and worm behaviors are selectively disrupted despite apparently normal surface expression and clustering of the receptors. Reconstitution experiments indicate that both STG-1 and STG-2 can functionally substitute for vertebrate TARPs to modify receptor function. Thus, we show that TARPs are obligate auxiliary subunits for AMPARs with a primary, evolutionarily conserved functional role in the modification of current kinetics.

Identification of a 1.6 kb genome locus of guinea pig cytomegalovirus required for efficient viral growth in animals but not in cell culture

Guinea pig cytomegalovirus (GPCMV) provides a useful model for studies of congenital CMV infection. During characterization of the GPCMV genome sequence, we identified two types of strains in a virus stock purchased from ATCC. One of them, GPCMV/del, lacks a 1.6 kb locus that positionally corresponds to murine CMV (MCMV) M129–M133. Growth of GPCMV/del in cell culture was marginally better than that of the other strain, GPCMV/full, which harbors the 1.6 kb locus. However, in animals infected intraperitoneally with virus stocks containing both strains, GPCMV/full disseminated more efficiently than GPCMV/del, including 200-fold greater viral load in salivary glands. Viral DNA, transcripts of the immediate-early 2 gene homolog, and viral antigens were more abundant in animals infected with GPCMV/full than in those infected with GPCMV/del. Although the observed phenomena have some similarity with the growth properties of MCMV strains defective in mck-1/mck-2(M129/131) and those defective in sgg(M132), no M129–M132 homologs were found in the 1.6 kb locus. Since one of the ORFs in the locus has a weak sequence similarity with HCMV UL130, which relates to cell tropism, further studies will be required to learn the mechanism for efficient GPCMV growth in animal.

Maps: An integrated system for protein sequence annotation using support vector machine

An Integrated environment for biological data is valuable to the function annotation of protein sequences. In this paper, we present a protein sequence annotation system, named MAPS (Multiple Annotation for Protein Sequences), which provides a mechanism to extract multiple annotations from various types of biological data including SwissProt keywords, InterPro signatures and GO terms. Furthermore, MAPS can automatically eliminate the annotation errors generated by a pre‐trained SVM classifier. It assigns an annotation to the protein sequence at question by considering not only a single similar protein but also all similar proteins with the annotation. In other words, we take account of the evolutionary information of the protein of interest to reduce the error annotations inferred from weak sequence similarities and from sequence identities in non‐functional segments. The experimental results show that the error annotations can be eliminated effectively while keeping high accuracy for different types of annotations.

Thrilling Moment of an Inhibitory Channel

Long thought to be potassium channels with calming influence quietly working away in the background, in this issue of Neuron, Thomas et al. demonstrate that K(2P)2.1 (TREK-1) channels have two endogenous isoforms due to a mechanism called alternative translation initiation (ATI). Hence, they can actually be excitable.

Design of Protein–Ligand Binding Based on the Molecular-Mechanics Energy Model

While the molecular-mechanics field has standardized on a few potential energy functions, computational protein design efforts are based on potentials that are unique to individual laboratories. Here we show that a standard molecular-mechanics potential energy function without any modifications can be used to engineer protein–ligand binding. A molecular-mechanics potential is used to reconstruct the coordinates of various binding sites with an average root-mean-square error of 0.61 Å and to reproduce known ligand-induced side-chain conformational shifts. Within a series of 34 mutants, the calculation can always distinguish between weak ( K d > 1 mM) and tight ( K d < 10 μM) binding sequences. Starting from partial coordinates of the ribose-binding protein lacking the ligand and the 10 primary contact residues, the molecular-mechanics potential is used to redesign a ribose-binding site. Out of a search space of 2 × 10 12 sequences, the calculation selects a point mutant of the native protein as the top solution (experimental K d = 17 μM) and the native protein as the second best solution (experimental K d = 210 nM). The quality of the predictions depends on the accuracy of the generalized Born electrostatics model, treatment of protonation equilibria, high-resolution rotamer sampling, a final local energy minimization step, and explicit modeling of the bound, unbound, and unfolded states. The application of unmodified molecular-mechanics potentials to protein design links two fields in a mutually beneficial way. Design provides a new avenue for testing molecular-mechanics energy functions, and future improvements in these energy functions will presumably lead to more accurate design results.

On the upper bounds of WCS coefficients in Orlicz function spaces

Let Φ(u) be an N-function and let $L^{(\Phi)}[0, 1]$ and $L^{\Phi}[0, 1]$ be the Orlicz function spaces equipped with the gauge norm and the Orlicz norm respectively. The author refines some results on the upper bounds of the weak convergent sequence coefficients $WCS(L^{(\Phi)}[0, 1])$ and $WCS(L^{\Phi}[0, 1])$ given in [8] and [7, Ch.3.2]. Theorems 1 and 2 are given in Section 2. Several examples, given in Section 3, are used to make comments upon the papers of Yan [11], [12] and [13]. Keywords Orlicz space, WCS coefficient and normal structure coefficient Mathematics Subject Classification (2000) 46B30

Molecular Insights into the Biosynthesis of the F420 Coenzyme

Coenzyme F(420), a hydride carrier, is found in Archaea and some bacteria and has crucial roles in methanogenesis, antibiotic biosynthesis, DNA repair, and activation of antitubercular compounds. CofD, 2-phospho-l-lactate transferase, catalyzes the last step in the biosynthesis of F(420)-0 (F(420) without polyglutamate), by transferring the lactyl phosphate moiety of lactyl(2)diphospho-(5')guanosine to 7,8-didemethyl-8-hydroxy-5-deazariboflavin ribitol (Fo). CofD is highly conserved among F(420)-producing organisms, and weak sequence homologs are also found in non-F(420)-producing organisms. This superfamily does not share any recognizable sequence conservation with other proteins. Here we report the first crystal structures of CofD, the free enzyme and two ternary complexes, with Fo and P(i) or with Fo and GDP, from Methanosarcina mazei. The active site is located at the C-terminal end of a Rossmann fold core, and three large insertions make significant contributions to the active site and dimer formation. The observed binding modes of Fo and GDP can explain known biochemical properties of CofD and are also supported by our binding assays. The structures provide significant molecular insights into the biosynthesis of the F(420) coenzyme. Large structural differences in the active site region of the non-F(420)-producing CofD homologs suggest that they catalyze a different biochemical reaction.

In Vitro Assembly Studies of FtsZ/Tubulin-like Proteins (TubZ) from Bacillus Plasmids: EVIDENCE FOR A CAPPING MECHANISM

Proteins with a weak sequence similarity to tubulin and FtsZ are expressed from large plasmids of Bacillus anthracis and Bacillus thuringiensis and are probably involved in plasmid segregation. Previously designated RepX and TubZ, we designate them here as TubZ-Ba and TubZ-Bt. We have expressed and purified the proteins for in vitro studies. TubZ-Ba and TubZ-Bt share only 21% amino acid identity, but they have remarkably similar biochemical properties. They both assemble into two-stranded filaments and larger bundles above a critical concentration, and they hydrolyze GTP at a very high rate, approximately 20 GTP min(-1) TubZ(-1). Assembly is also supported by GTPgammaS. A tiny amount of GTPgammaS stabilizes polymers assembled in GTP and inhibits the GTPase by a mechanism involving cooperativity. The nucleotide in the polymers is almost 100% GDP, which is similar to microtubules but very different from the 20-30% GDP in FtsZ polymers. This suggests that the TubZ polymers have a capping mechanism that may be related to the GTP cap that produces dynamic instability of microtubules.

Structures of the Shigella flexneri Type 3 Secretion System Protein MxiC Reveal Conformational Variability Amongst Homologues

Many Gram-negative pathogenic bacteria use a complex macromolecular machine, known as the type 3 secretion system (T3SS), to transfer virulence proteins into host cells. The T3SS is composed of a cytoplasmic bulb, a basal body spanning the inner and outer bacterial membranes, and an extracellular needle. Secretion is regulated by both cytoplasmic and inner membrane proteins that must respond to specific signals in order to ensure that virulence proteins are not secreted before contact with a eukaryotic cell. This negative regulation is mediated, in part, by a family of proteins that are thought to physically block the entrance to the secretion apparatus until an appropriate signal is received following host cell contact. Despite weak sequence homology between proteins of this family, the crystal structures of Shigella flexneri MxiC we present here confirm the conservation of domain topology with the homologue from Yersinia sp. Interestingly, comparison of the Shigella and Yersinia structures reveals a significant structural change that results in substantial domain re-arrangement and opening of one face of the molecule. The conservation of a negatively charged patch on this face suggests it may have a role in binding other components of the T3SS.

M-TASSER: An Algorithm for Protein Quaternary Structure Prediction

In a cell, it has been estimated that each protein on average interacts with roughly 10 others, resulting in tens of thousands of proteins known or suspected to have interaction partners; of these, only a tiny fraction have solved protein structures. To partially address this problem, we have developed M-TASSER, a hierarchical method to predict protein quaternary structure from sequence that involves template identification by multimeric threading, followed by multimer model assembly and refinement. The final models are selected by structure clustering. M-TASSER has been tested on a benchmark set comprising 241 dimers having templates with weak sequence similarity and 246 without multimeric templates in the dimer library. Of the total of 207 targets predicted to interact as dimers, 165 (80%) were correctly assigned as interacting with a true positive rate of 68% and a false positive rate of 17%. The initial best template structures have an average root mean-square deviation to native of 5.3, 6.7, and 7.4Å for the monomer, interface, and dimer structures. The final model shows on average a root mean-square deviation improvement of 1.3, 1.3, and 1.5Å over the initial template structure for the monomer, interface, and dimer structures, with refinement evident for 87% of the cases. Thus, we have developed a promising approach to predict full-length quaternary structure for proteins that have weak sequence similarity to proteins of solved quaternary structure.

Coelenterazine-binding protein of Renilla muelleri: cDNA cloning, overexpression, and characterization as a substrate of luciferase

The Renilla bioluminescent system in vivo is comprised of three proteins--the luciferase, green-fluorescent protein, and coelenterazine-binding protein (CBP), previously called luciferin-binding protein (LBP). This work reports the cloning of the full-size cDNA encoding CBP from soft coral Renilla muelleri, its overexpression and properties of the recombinant protein. The apo-CBP was quantitatively converted to CBP by simple incubation with coelenterazine. The physicochemical properties of this recombinant CBP are determined to be practically the same as those reported for the CBP (LBP) of R. reniformis. CBP is a member of the four-EF-hand Ca(2+)-binding superfamily of proteins with only three of the EF-hand loops having the Ca(2+)-binding consensus sequences. There is weak sequence homology with the Ca(2+)-regulated photoproteins but only as a result of the necessary Ca(2+)-binding loop structure. In combination with Renilla luciferase, addition of only one Ca(2+) is sufficient to release the coelenterazine as a substrate for the luciferase for bioluminescence. This combination of the two proteins generates bioluminescence with higher reaction efficiency than using free coelenterazine alone as the substrate for luciferase. This increased quantum yield, a difference of bioluminescence spectra, and markedly different kinetics, implicate that a CBP-luciferase complex might be involved.

Three-dimensional sequence analysis of a subsurface carbonate ramp, Mississippian Appalachian foreland basin, West Virginia, USA

Well-cuttings, wireline logs and limited core and outcrop data were used to generate a regional, three-dimensional sequence framework for Upper Mississippian (Chesterian), Greenbrier Group carbonates in the Appalachian foreland basin, West Virginia, USA. The resulting maps were used to document the stratigraphic response of the basin to tectonics and to glacio-eustasy during the transition into ice-house conditions. The ramp facies include inner ramp red beds and aeolianites, lagoonal muddy carbonates, mid-ramp ooid and skeletal grainstone shoal complexes, and outer ramp wackestone–mudstone, that grades downslope into laminated silty lime mudstone. The facies make up fourth-order sequences, a few metres to over 90 m (300 ft) thick. The sequences are bounded along the ramp margin by lowstand sandstones and calcareous siltstones. On the ramp, sequence boundaries are overlain by thin transgressive siliciclastics and aeolianites, and only a few are calichified. Maximum flooding surfaces on the outer ramp lie beneath deeper water facies that overlie lowstand to transgressive siliciclastic or carbonate units. On the shallow ramp, maximum flooding surfaces overlie siliciclastic-prone transgressive systems tracts, that are overlain by highstand carbonates with significant grainstone units interlayered with lagoonal lime mudstones. The fourth-order sequences are the major mappable subsurface units; they are bundled into weak composite sequences which are bounded by red beds. In spite of differential subsidence rates across the foreland basin (1 to 3 cm/k.y. up to 25 cm/k.y.), eustatic sea-level changes controlled regional sequence development. Thrust-load induced differential subsidence of fault-blocks, coupled with in-plane stress, controlled the rapid basinward thickening of the depositional wedge, whose thickness and facies were influenced by subtle structures such as arches trending at high angles as well as parallel to the margin.

Comparison of the efficiency of evolutionary change-based and side chain orientation-based fold recognition potentials

The present article describes residue level knowledge based potential SORDIS. SORDIS incorporates the information on side-chain orientation in relation to hydrophobic core centres, distance of residue from the globule centre and secondary structure. SORDIS has been tested and compared with widespread evolutionary change-based substitution matrices (BLOSUM, PAM, GONNET, Johnson-Overington, BLAJ, HSDM, and STROMA) in fold recognition experiments within the zone of weak sequence similarity (<16%). The obtained results show that the lower is the amino acid similarity between homologous pairs the higher is the performance of SORDIS in comparison with the potentials, based on the information about the evolutionary changes. Therefore, we propose that the employment of SORDIS in fold recognition can be useful.

RhaU of Rhizobium leguminosarum Is a Rhamnose Mutarotase

Of the nine genes comprising the L-rhamnose operon of Rhizobium leguminosarum, rhaU has not been assigned a function. The construction of a Delta rhaU strain revealed a growth phenotype that was slower than that of the wild-type strain, although the ultimate cell yields were equivalent. The transport of L-rhamnose into the cell and the rate of its phosphorylation were unaffected by the mutation. RhaU exhibits weak sequence similarity to the formerly hypothetical protein YiiL of Escherichia coli that has recently been characterized as an L-rhamnose mutarotase. To characterize RhaU further, a His-tagged variant of the protein was prepared and subjected to mass spectrometry analysis, confirming the subunit size and demonstrating its dimeric structure. After crystallization, the structure was refined to a 1.6-A resolution to reveal a dimer in the asymmetric unit with a very similar structure to that of YiiL. Soaking a RhaU crystal with L-rhamnose resulted in the appearance of beta-L-rhamnose in the active site.

RNA Polymerase I: A Multifunctional Molecular Machine

In this issue, Kuhn et al. (2007) report the complete structure of the 14-subunit yeast RNA polymerase (Pol) I enzyme at 12 A resolution using cryo-electron microscopy (cryo-EM). Their study reveals that three subunits of Pol I perform functions in transcription elongation that are outsourced to the transcription factors TFIIF and TFIIS in the analogous Pol II transcription system.

Crystal Structure of Human XLF: A Twist in Nonhomologous DNA End-Joining

DNA double-strand breaks represent one of the most severe forms of DNA damage in mammalian cells. One pathway for repairing these breaks occurs via nonhomologous end-joining (NHEJ) and depends on XRCC4, LigaseIV, and Cernunnos, also called XLF. Although XLF stimulates XRCC4/LigaseIV to ligate mismatched and noncohesive DNA ends, the mechanistic basis for this function remains unclear. Here we report the structure of a partially functional 224 residue N-terminal fragment of human XLF. Despite only weak sequence similarity, XLF(1-170) shares structural homology with XRCC4(1-159). However, unlike the highly extended 130 A helical domain observed in XRCC4, XLF adopts a more compact, folded helical C-terminal region involving two turns and a twist, wrapping back to the structurally conserved N terminus. Mutational analysis of XLF and XRCC4 reveals a potential interaction interface, suggesting a mechanism for how XLF stimulates the ligation of mismatched ends.

Human mitochondrial transcription factor A possesses multiple subcellular targeting signals

The mitochondrial transcription factor A (TFAM) is a member of a high-mobility group (HMG) family represented mostly by nuclear proteins. Although nuclear localization of TFAM has been demonstrated in some tumors and after treatment of tumor cells with anticancer drugs, the significance of these observations has not been fully elucidated. Here we report that both TFAM overexpression and impairment of its mitochondrial targeting can result in nuclear accumulation of the protein. Both M1 and M7 methionines of human TFAM (hTFAM) can be used for translation initiation with almost equal efficiency resulting in two polypeptides. The shorter polypeptide, however, is not located in the nucleus, despite truncation in the mitochondrial targeting sequence, and both isoforms are targeted to mitochondria with similar efficiency. We further demonstrate that nuclear TFAM confers significant cytoprotection against the chemotherapeutic drugs etoposide, camptothecin, and cisplatin. Three regions of hTFAM [HMG-like domain 1 (HMG1) and HMG-like domain 2 (HMG2), as well as the tail region] can effect nuclear accumulation of enhanced green fluorescent protein (EGFP) fusions. The HMG1 domain contains a bipartite nuclear localization sequence whose identity is supported by site-directed mutagenesis. However, this bipartite nuclear localization sequence is weak, and both N-terminal and C-terminal flanking sequences enhance the nuclear targeting of EGFP. Finally, several mutations in the HMG1 domain increased the mitochondrial targeting of the EGFP fusions, suggesting that the mitochondrial targeting sequence of hTFAM may extend beyond the cleavable presequence.