Zinc Finger-like Motif Conserved in a Family of RNA Binding Proteins

NP220s compose a family of RNA binding proteins together with matrin 3, one of major proteins of the nuclear matrix. They have repeats of RNA recognition motif (RRM; MH2) homologous to RRM in heterogeneous nuclear RNPs I/L in addition to MH1 and MH3 with unknown function. In search of additional homologous sequences, we found the reported sequence of rat matrin 3 is partially incorrect. Correction of this sequence showed that the NP220 family has a fourth homologous motif with the characteristics of a Cys₂-His₂ zinc finger-like motif. The sequence of this motif is perfectly conserved in human and mouse NP220s despite their 75% overall sequence homology.     

Functional Significance of the E Loop,a Novel Motif Conserved in the Lantibiotic Immunity ATP-Binding Cassette Transport Systems

Lantibiotics are peptide-derived antibacterial substances produced by some Gram-positive bacteria and characterized by the presence of unusual amino acids, like lanthionines and dehydrated amino acids. Because lantibiotic producers may be attacked by self-produced lantibiotics, they express immunity proteins on the cytoplasmic membrane. An ATP-binding cassette (ABC) transport system mediated by the LanFEG protein complex is a major system in lantibiotic immunity. Multiple-sequence alignment analysis revealed that LanF proteins contain the E loop, a variant of the Q loop, which is a well-conserved motif in the nucleotide-binding domains (NBDs) of general ABC transporters. To elucidate E loop function, we introduced a mutation in the NukF protein, which is involved in the nukacin-ISK-1 immunity system. Amino acid replacement of glutamic acid in the E loop with glutamine (E85Q) resulted in slight decreases in the immunity level and transport activity. Additionally, the E85A mutation severely impaired the immunity level and transport activity. On the other hand, ATPase activities of purified E85Q and E85A mutants were almost similar to that of the wild type. These results suggested that the E loop found in ABC transporters involved in lantibiotic immunity plays a significant role in the function of these transporters, especially in the structural change of transmembrane domains.Lantibiotics are antibacterial peptides produced by some Gram-positive bacteria and are characterized by the presence of unusual amino acids, such as lanthionine and dehydrated amino acid residues (4, 9, 20). The unusual amino acids are introduced after translation by a modification enzyme(s), and their subsequent processing and secretion are carried out by a leader peptidase and transporter, respectively. Since the secreted mature lantibiotics have the potential to attack producer cells, lantibiotic-producer cells express self-protection systems against their own lantibiotics. These self-protection systems have 2 major mechanisms: a lantibiotic transport mechanism mediated by an ATP-binding cassette (ABC) transporter (LanFEG) and a lantibiotic-binding mechanism mediated by a lipoprotein (LanI) or a membrane protein (LanH) (2, 8, 26, 33, 34).Transport by LanFEG is a common and major mechanism in the lantibiotic immunity systems. LanFEG and LanI are needed for full immunity against nisin and subtilin, which are type A(I) lantibiotics (33, 34). However, the immunity level conferred by LanFEG is much higher than that conferred by LanI. LanFEG and LanH are expressed against nukacin ISK-1, which is a type A(II) lantibiotic produced by Staphylococcus warneri ISK-1 (2). As in the case of nisin and subtilin, LanFEG plays a major role in the level of immunity against nukacin ISK-1. Moreover, against lacticin 481, which is also a type A(II) lantibiotic, only LanFEG is expressed and it confers full immunity (9).ABC transporters function as molecular pumps and transport various substrates, such as nutrients, lipids, and antibiotics coupled to ATP hydrolysis (10, 31). Bacterial ABC transporters consist of 2 transmembrane domains (TMDs) and 2 nucleotide-binding domains (NBDs). They utilize ATP hydrolysis as a source of energy for the transport. The NBD of an ABC transporter has several conserved motifs, such as Walker A, Walker B, Q loop, Signature, and H loop, in its amino acid sequence, and these motifs are involved in the functions of ABC transporters (31). Although the detailed substrate-binding mechanism is still unknown, the dimerization of NBDs concomitant with ATP binding leads to the conformational change of 2 TMDs, resulting in transport of the substrate (31). Sequence similarities and hydrophobicity profiles suggest that LanFEG consists of 2 heterodimeric subunits containing TMDs (LanEG) and 2 homodimeric subunits containing NBDs (LanF) (4, 27).In general, ABC transporters that had been identified together with their substrates mediate the transport of the substrate across the membrane. An exception reported previously is the Lol system, which releases lipoproteins from the inner membrane to the outer membrane in Gram-negative bacteria (40). However, LanFEG proteins are believed to scavenge lantibiotics present on the membrane. This hypothesis is strongly supported by the mode of action of lantibiotics: many lantibiotics, especially type A(I) lantibiotics, show pore-forming activity against model membranes (4). Taken together, the transport mechanism of LanFEG seems to be different from that of general ABC transporters.The immunity mechanism against nukacin ISK-1 mediated by NukFEG and NukH has been investigated before (2, 21-23, 39). On the basis of our analysis, we suggested that NukFEG transports both nukacin ISK-1 on the membrane and nukacin ISK-1 captured by NukH (2, 22). Since the transport reaction depended on the metabolic energy of the cells, we presumed that ATP hydrolysis by NukF is a driving force for the transport (22).Using multiple sequence alignment analysis, we have found that all the LanF proteins have the E loop as a variant of the Q loop in general ABC transporters. Therefore, in this study, we investigated the function of the E loop existing in NukF by using site-directed mutagenesis. A bioassay using nukacin ISK-1 and recombinant Lactococcus lactis expressing nukF and its mutants showed that the E loop is important for immunity. Additionally, a transport assay with fluorescein isothiocyanate (FITC)-labeled nukacin ISK-1 indicated that the E loop is involved in transport activity. Since purified NukF and E loop mutants showed similar ATPase activity, we proposed that the E loop has an important role in the function of LanFEG, especially in coupled movement with the transmembrane subunit NukEG.     

Specific Prenylation of Tomato Rab Proteins by Geranylgeranyl Type-II Transferase Requires a Conserved Cysteine-Cysteine Motif

Posttranslational isoprenylation of some small GTP-binding proteins is required for their biological activity. Rab geranylgeranyl transferase (Rab GGTase) uses geranylgeranyl pyrophosphate to modify Rab proteins, its only known substrates. Geranylgeranylation of Rabs is believed to promote their association with target membranes and interaction with other proteins. Plants, like other eukaryotes, contain Rab-like proteins that are associated with intracellular membranes. However, to our knowledge, the geranylgeranylation of Rab proteins has not yet been characterized from any plant source. This report presents an activity assay that allows the characterization of prenylation of Rab-like proteins in vitro, by protein extracts prepared from plants. Tomato Rab1 proteins and mammalian Rab1a were modified by geranylgeranyl pyrophosphate but not by farnesyl pyrophosphate. This modification required a conserved cysteine-cysteine motif. A mutant form lacking the cysteine-cysteine motif could not be modified, but inhibited the geranylgeranylation of its wild-type homolog. The tomato Rab proteins were modified in vitro by protein extract prepared from yeast, but failed to become modified when the protein extract was prepared from a yeast strain containing a mutant allele for the [alpha] subunit of yeast Rab GGTase (bet4 ts). These results demonstrate that plant cells, like other eukaryotes, contain Rab GGTase-like activity.     

The "CPC Clip Motif": A Conserved Structural Signature for Heparin-Binding Proteins

Glycosaminoglycans (GAGs) are essential molecules that regulate diverse biological processes including cell adhesion, differentiation, signaling and growth, by interaction with a wide variety of proteins. However, despite the efforts committed to understand the molecular nature of the interactions in protein-GAG complexes, the answer to this question remains elusive.In the present study the interphases of 20 heparin-binding proteins have been analyzed searching for a conserved structural pattern. We have found that a structural motif encompassing one polar and two cationic residues (which has been named the CPC clip motif) is conserved among all the proteins deposited in the PDB. The distances between the α carbons and the side chain center of gravity of the residues composing this motif are also conserved. Furthermore, this pattern can be found in other proteins suggested to bind heparin for which no structural information is available. Hence we propose that the CPC clip motif, working like a staple, is a primary contributor to the attachment of heparin and other sulfated GAGs to heparin-binding proteins.     

Functional Annotation of Conserved Hypothetical Proteins from Haemophilus influenzae Rd KW20

Haemophilus influenzae is a Gram negative bacterium that belongs to the family Pasteurellaceae, causes bacteremia, pneumonia and acute bacterial meningitis in infants. The emergence of multi-drug resistance H. influenzae strain in clinical isolates demands the development of better/new drugs against this pathogen. Our study combines a number of bioinformatics tools for function predictions of previously not assigned proteins in the genome of H. influenzae. This genome was extensively analyzed and found 1,657 functional proteins in which function of 429 proteins are unknown, termed as hypothetical proteins (HPs). Amino acid sequences of all 429 HPs were extensively annotated and we successfully assigned the function to 296 HPs with high confidence. We also characterized the function of 124 HPs precisely, but with less confidence. We believed that sequence of a protein can be used as a framework to explain known functional properties. Here we have combined the latest versions of protein family databases, protein motifs, intrinsic features from the amino acid sequence, pathway and genome context methods to assign a precise function to hypothetical proteins for which no experimental information is available. We found these HPs belong to various classes of proteins such as enzymes, transporters, carriers, receptors, signal transducers, binding proteins, virulence and other proteins. The outcome of this work will be helpful for a better understanding of the mechanism of pathogenesis and in finding novel therapeutic targets for H. influenzae.     

Identification of a Novel Protein-Protein Interaction Motif Mediating Interaction of GPCR-Associated Sorting Proteins with G Protein-Coupled Receptors

GPCR desensitization and down-regulation are considered key molecular events underlying the development of tolerance in vivo. Among the many regulatory proteins that are involved in these complex processes, GASP-1 have been shown to participate to the sorting of several receptors toward the degradation pathway. This protein belongs to the recently identified GPCR-associated sorting proteins (GASPs) family that comprises ten members for which structural and functional details are poorly documented. We present here a detailed structure–function relationship analysis of the molecular interaction between GASPs and a panel of GPCRs. In a first step, GST-pull down experiments revealed that all the tested GASPs display significant interactions with a wide range of GPCRs. Importantly, the different GASP members exhibiting the strongest interaction properties were also characterized by the presence of a small, highly conserved and repeated “GASP motif” of 15 amino acids. We further showed using GST-pull down, surface plasmon resonance and co-immunoprecipitation experiments that the central domain of GASP-1, which contains 22 GASP motifs, is essential for the interaction with GPCRs. We then used site directed mutagenesis and competition experiments with synthetic peptides to demonstrate that the GASP motif, and particularly its highly conserved core sequence SWFW, is critically involved in the interaction with GPCRs. Overall, our data show that several members of the GASP family interact with GPCRs and highlight the presence within GASPs of a novel protein-protein interaction motif that might represent a new target to investigate the involvement of GASPs in the modulation of the activity of GPCRs.     

The Adeno-Associated Virus Type 5 Small Rep Proteins Expressed via Internal Translation Initiation Are Functional

Although precluded from using splicing to produce multiple small Rep proteins, adeno-associated virus type 5 (AAV5) generates a Rep40-like protein by alternative translation initiation at an internal AUG. A defined region upstream of the internal AUG was both required and sufficient to program internal initiation within AAV5 and may act similarly in heterologous contexts. The internally initiated AAV5 Rep40-like protein was functional and had helicase activity similar to that of AAV2 Rep40. Surprisingly, both the AAV5 Rep40-like protein and Rep52 were able to be translated from the AAV5 upstream P7-generated RNAs; however, the relative level of small to large Rep proteins was reduced compared to that of the wild type. A P19 mutant AAV5 infectious clone generated near-wild-type levels of the double-stranded monomer replicative form (mRF) replicative intermediate but reduced levels of virus, consistent with the previously defined role of Rep40-like proteins in genome encapsidation. Levels of mutant virus were dramatically reduced upon amplification.     

An Acidic 39-kDa Protein Secreted from Stigmas of Tobacco Has an Amino-Terminal Motif That is Conserved among Thaumatin-Like Proteins

An acidic 39-kDa polypeptide (SE39b) secreted from stigmas oftobacco was identified by two-dimensional poly-acrylamide gelelectrophoresis (2D PAGE), and its sugar chain was detectedwith concanavalin A (Con A) and peroxidase. The amino-terminalamino acid sequence of SE39b resembled those of thaumatin-likeproteins. (Received June 10, 1996; Accepted November 7, 1996)     

Functional Significance May Underlie the Taxonomic Utility of Single Amino Acid Substitutions in Conserved Proteins

We hypothesized that some amino acid substitutions in conserved proteins that are strongly fixed by critical functional roles would show lineage-specific distributions. As an example of an archetypal conserved eukaryotic protein we considered the active site of β-tubulin. Our analysis identified one amino acid substitution—β-tubulin F224—which was highly lineage specific. Investigation of β-tubulin for other phylogenetically restricted amino acids identified several with apparent specificity for well-defined phylogenetic groups. Intriguingly, none showed specificity for “supergroups” other than the unikonts. To understand why, we analysed the β-tubulin Neighbor-Net and demonstrated a fundamental division between core β-tubulins (plant-like) and divergent β-tubulins (animal and fungal). F224 was almost completely restricted to the core β-tubulins, while divergent β-tubulins possessed Y224. Thus, our specific example offers insight into the restrictions associated with the co-evolution of β-tubulin during the radiation of eukaryotes, underlining a fundamental dichotomy between F-type, core β-tubulins and Y-type, divergent β-tubulins. More broadly our study provides proof of principle for the taxonomic utility of critical amino acids in the active sites of conserved proteins.     

Functional domains of yeast plasmid-encoded Rep proteins

Both of the Saccharomyces cerevisiae 2 microm circle-encoded Rep1 and Rep2 proteins are required for efficient distribution of the plasmid to daughter cells during cellular division. In this study two-hybrid and in vitro protein interaction assays demonstrate that the first 129 amino acids of Rep1 are sufficient for self-association and for interaction with Rep2. Deletion of the first 76 amino acids of Rep1 abolished the Rep1-Rep2 interaction but still allowed some self-association, suggesting that different but overlapping domains specify these interactions. Amino- or carboxy-terminally truncated Rep1 fusion proteins were unable to complement defective segregation of a 2 microm-based stability vector with rep1 deleted, supporting the idea of the requirement of Rep protein interaction for plasmid segregation but indicating a separate required function for the carboxy-terminal portion of Rep1. The results of in vitro baiting assays suggest that Rep2 contains two nonoverlapping domains, both of which are capable of mediating Rep2 self-association. The amino-terminal domain interacts with Rep1, while the carboxy-terminal domain was shown by Southwestern analysis to have DNA-binding activity. The overlapping Rep1 and Rep2 interaction domains in Rep1, and the ability of Rep2 to interact with Rep1, Rep2, and DNA, suggest a model in which the Rep proteins polymerize along the 2 microm circle plasmid stability locus, forming a structure that mediates plasmid segregation. In this model, competition between Rep1 and Rep2 for association with Rep1 determines the formation or disassembly of the segregation complex.     

A Novel Conserved Phosphotyrosine Motif in the Drosophila Fibroblast Growth Factor Signaling Adaptor Dof with a Redundant Role in Signal Transmission

The fibroblast growth factor receptor (FGFR) signals through adaptors constitutively associated with the receptor. In Drosophila melanogaster, the FGFR-specific adaptor protein Downstream-of-FGFR (Dof) becomes phosphorylated upon receptor activation at several tyrosine residues, one of which recruits Corkscrew (Csw), the Drosophila homolog of SHP2, which provides a molecular link to mitogen-activated protein kinase (MAPK) activation. However, the Csw pathway is not the only link from Dof to MAPK. In this study, we identify a novel phosphotyrosine motif present in four copies in Dof and also found in other insect and vertebrate signaling molecules. We show that these motifs are phosphorylated and contribute to FGF signal transduction. They constitute one of three sets of phosphotyrosines that act redundantly in signal transmission: (i) a Csw binding site, (ii) four consensus Grb2 recognition sites, and (iii) four novel tyrosine motifs. We show that Src64B binds to Dof and that Src kinases contribute to FGFR-dependent MAPK activation. Phosphorylation of the novel tyrosine motifs is required for the interaction of Dof with Src64B. Thus, Src64B recruitment to Dof through the novel phosphosites can provide a new link to MAPK activation and other cellular responses. This may give a molecular explanation for the involvement of Src kinases in FGF-dependent developmental events.Fibroblast growth factor (FGF) receptors (FGFRs) are highly conserved molecules that belong to the family of receptor tyrosine kinases (RTKs). They form dimers which are activated by autophosphorylation upon ligand binding. Activated RTKs can directly recruit signaling molecules via their phosphorylated tyrosine residues. They can also phosphorylate other signaling molecules, and the phosphosites of those molecules can serve as additional interaction surfaces for downstream signal transducers (8).RTKs activate conserved intracellular signaling cascades, one of the most frequently activated being the Ras-mitogen-activated protein kinase (MAPK) pathway. Ras activation at the plasma membrane occurs via the recruitment of the phosphotyrosine binding adaptor molecule Grb2 in a complex with the Ras GTP exchange factor (RasGEF) Sos (26). The FGF signal transduction pathway also uses the Ras-MAPK cascade, but while most other RTKs access this cascade by using phosphotyrosines in their intracellular domains to recruit either Grb2 itself or signaling molecules such as Shc or SHP2, which can then recruit Grb2 upon phosphorylation, this is not the case for FGF receptors (8).Instead, FGF receptors use constitutively bound adaptor proteins, which are tyrosine phosphorylated upon receptor activation at several sites and provide binding surfaces for many signaling molecules, including the known activators of the Ras-MAPK pathway (8). Surprisingly, although the FGF receptor and its downstream signal transducers are conserved between vertebrates and insects, and in both cases an adaptor is required to connect them, the adaptor function is carried out by unrelated molecules in vertebrates and insects. In vertebrates, the adaptor protein FRS2 constitutively binds to the juxtamembrane region of the FGF receptor and becomes tyrosine phosphorylated by the activated receptor (18, 29, 30). The homolog of FRS2 in Drosophila melanogaster is not involved in FGF signaling (A. Michelson, personal communication). Instead, the adaptor molecule Dof/Sms (Downstream-of-FGFR, or Stumps) is the obligate partner of fly FGF receptors (27, 40, 41). Dof shows no sequence similarity to its vertebrate functional correlate FRS2 but is related to two other vertebrate adaptors, BCAP and BANK, which are involved in B-cell receptor signaling (2).Dof is essential in all FGF signal-mediated processes in the fly, including mesoderm formation and development of the tracheal network during embryogenesis, which we use here as an in vivo system to study signaling through Dof (40). Dof binds to both Drosophila FGF receptors, Heartless and Breathless, via its conserved DBB (Dof-BCAP-BANK) domain and becomes phosphorylated upon receptor activation at several tyrosine residues (32, 41). Corkscrew (Csw), the Drosophila homolog of SHP2, has been shown to bind to one of these phosphosites, providing a molecular link to MAPK activation (32). However, other functional structural studies show that the Csw pathway is not the only one employed by Dof to activate MAPK and indicate that different parts of the molecule might act redundantly (41).Src family kinases are also known activators of the MAPK pathway in both Drosophila and vertebrates (4, 22, 42). Furthermore, vertebrate FGF receptor signaling has been described to activate Src family kinases in many different systems (4, 5) and genetic studies of Drosophila point to an involvement of Src kinases in FGF-dependent processes (9, 36). However, there is no evidence on whether and how MAPK activation by Src can be driven directly by Drosophila FGF receptors. This prompted us to investigate whether the adaptor protein Dof might contribute to the recruitment of Src to activated FGF receptors as an alternative way of activating MAPK.Cells are able to fine-tune the strength and duration of signaling, providing a means to generate signal- or cell type-specific readouts in response to different signals, even though the signaling cascades use common components (reviewed in reference 6). For example, in PC12 cells, EGF signaling generates a transient pulse of MAPK activation, inducing cell proliferation, whereas FGF stimulation generates a sustained MAPK activation, which leads to differentiation (25, 44). One way of achieving this modulation is to be able to use several parallel links to a downstream effector. Two different types of parallel signaling connections have been observed. Adaptors can have multiple binding sites for a given signaling molecule, which allows an increase in signaling strength via the recruitment of larger numbers of the same molecule. For example, FRS2α contains four Grb2 binding sites (12), and BCAP has four PI3K interaction sites (17). Adaptor molecules can also display diverse binding sites for multiple different signaling molecules, allowing activation of the same signaling cascade along alternative routes: FRS2α contains both Grb2 and SHP2 binding sites for MAPK activation (12). In Dof we find both cases. It contains a Csw binding site, which has been shown to contribute to its function, and four Grb2 consensus binding sites, for which the relevance for MAPK activation has not been demonstrated so far (32, 41).The most frequently used binding sites in RTK signaling that allow signal-dependent transient protein-protein interactions are phosphorylatable tyrosine motifs, which provide binding surfaces for signal transducers bearing phosphotyrosine binding domains. Although all proteins containing phosphotyrosine binding domains can bind phosphotyrosines, these domains differ in their specificity for binding sites. The target recognition sites of the two most common phosphotyrosine binding domains, the PTB and SH2 domains, are determined by short stretches of amino acids upstream or downstream of the phosphorylated tyrosine (35). The motif recognized by PTB domains usually contains conserved amino acids N-terminal to a phosphotyrosine (39), whereas conserved residues C-terminal to a phosphotyrosine contribute to the binding specificity of SH2 domains (37, 38). All of the recognizable motifs surrounding phosphotyrosines in Dof suggest interactions with SH2 domains. Although the binding specificity of many SH2 domains is characterized by phosphopeptide library screens and crystallography studies (31), the interaction partners for several known phosphorylated tyrosines in signaling molecules have not yet been identified.We present here a systematic mapping of FGFR-dependent tyrosine phosphorylation of Dof by mutational analysis and show that a novel phosphotyrosine motif contributes in a redundant manner to FGF signaling by allowing Dof to utilize alternative signal transduction routes to activate the MAPK pathway.