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.     

A Family of Novel DNA-Binding Nuclear Proteins Having Polypyrimidine Tract-Binding Motif and Arginine/Serine-Rich Motif

NP220 is a DNA-binding nuclear protein originally cloned from human cell lines. Human NP220 (hNP220) has an arginine/serine-rich motif found in non-small nuclear RNP splicing factors (SR proteins) and shares three domains (MH1, MH2 and MH3) with an acidic nuclear matrix protein, matrin 3. The MH2 domain repeats three times and has homology to the polypyrimidine tract-binding motif of heterogeneous nuclear RNP I/L. NP220 also has a DNA-binding domain and nine repeats of the sequence LVTVDEVIEEEDL (acidic repeat). We have now isolated mouse equivalents of NP220 (mNP220s) and found that NP220s form a family of proteins with four members produced by alternative splicing of a common pre-mRNA. Two longer forms (NP220α and NP220β) have all functional domains mentioned above while two shorter forms (NP220γ and NP220δ) lack the DNA-binding domain and the acidic repeat. The structural aspects of NP220s are distinct from that of the SR proteins but rather resemble U2AF and Tra2 which activate a specific 3′-splicing site of specific genes in response to differentiation-dependent signals.     

An Evolutionary Analysis of Flightin Reveals a Conserved Motif Unique and Widespread in Pancrustacea

Flightin is a thick filament protein that in Drosophila melanogaster is uniquely expressed in the asynchronous, indirect flight muscles (IFM). Flightin is required for the structure and function of the IFM and is indispensable for flight in Drosophila. Given the importance of flight acquisition in the evolutionary history of insects, here we study the phylogeny and distribution of flightin. Flightin was identified in 69 species of hexapods in classes Collembola (springtails), Protura, Diplura, and insect orders Thysanura (silverfish), Dictyoptera (roaches), Orthoptera (grasshoppers), Pthiraptera (lice), Hemiptera (true bugs), Coleoptera (beetles), Neuroptera (green lacewing), Hymenoptera (bees, ants, and wasps), Lepidoptera (moths), and Diptera (flies and mosquitoes). Flightin was also found in 14 species of crustaceans in orders Anostraca (water flea), Cladocera (brine shrimp), Isopoda (pill bugs), Amphipoda (scuds, sideswimmers), and Decapoda (lobsters, crabs, and shrimps). Flightin was not identified in representatives of chelicerates, myriapods, or any species outside Pancrustacea (Tetraconata, sensu Dohle). Alignment of amino acid sequences revealed a conserved region of 52 amino acids, referred herein as WYR, that is bound by strictly conserved tryptophan (W) and arginine (R) and an intervening sequence with a high content of tyrosines (Y). This motif has no homologs in GenBank or PROSITE and is unique to flightin and paraflightin, a putative flightin paralog identified in decapods. A third motif of unclear affinities to pancrustacean WYR was observed in chelicerates. Phylogenetic analysis of amino acid sequences of the conserved motif suggests that paraflightin originated before the divergence of amphipods, isopods, and decapods. We conclude that flightin originated de novo in the ancestor of Pancrustacea > 500 MYA, well before the divergence of insects (~400 MYA) and the origin of flight (~325 MYA), and that its IFM-specific function in Drosophila is a more recent adaptation. Furthermore, we propose that WYR represents a novel myosin coiled-coil binding motif.     

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.     

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.     

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.     

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.