The ankyrin repeat: a diversity of interactions on a common structural framework.

The ankyrin repeat is one of the most common protein sequence motifs. Recent X-ray and NMR structures of ankyrin-repeat proteins and their complexes have provided invaluable insights into the molecular basis of the extraordinary variety of biological activities of these molecules. In particular, they have begun to reveal how a large family of structurally related proteins can interact specifically with such a diverse array of macromolecular targets.     

A leucine-rich repeat peptide derived from the Drosophila Toll receptor forms extended filaments with a beta-sheet structure.

Leucine-rich repeats (LRRs) are 22-28 amino acid-long sequence motifs found in a family of cytoplasmic, membrane and extracellular proteins. There is evidence that LRRs function in signal transduction, cellular adhesion and protein-protein interactions. Here we report unusual properties of a synthetic LRR peptide derived from the sequence of the Drosophila membrane receptor Toll. In neutral solution the peptide forms a gel revealed by electron microscopy to consist of extended filaments approximately 8 nm in thickness. As the gel forms, the circular dichroism spectrum of the peptide solution changes from one characteristic of random coil to one associated with beta-sheet structures. Molecular modelling suggests that the peptide form an amphipathic structure with a predominantly apolar and charged surface. Based on these results, models for the gross structure of the peptides filaments and a possible molecular mechanism for cellular adhesion are proposed.     

Sequence-structure analysis of FAD-containing proteins

We have analyzed structure-sequence relationships in 32 families of flavin adenine dinucleotide (FAD)-binding proteins, to prepare for genomic-scale analyses of this family. Four different FAD-family folds were identified, each containing at least two or more protein families. Three of these families, exemplified by glutathione reductase (GR), ferredoxin reductase (FR), and p-cresol methylhydroxylase (PCMH) were previously defined, and a family represented by pyruvate oxidase (PO) is newly defined. For each of the families, several conserved sequence motifs have been characterized. Several newly recognized sequence motifs are reported here for the PO, GR, and PCMH families. Each FAD fold can be uniquely identified by the presence of distinctive conserved sequence motifs. We also analyzed cofactor properties, some of which are conserved within a family fold while others display variability. Among the conserved properties is cofactor directionality: in some FAD-structural families, the adenine ring of the FAD points toward the FAD-binding domain, whereas in others the isoalloxazine ring points toward this domain. In contrast, the FAD conformation and orientation are conserved in some families while in others it displays some variability. Nevertheless, there are clear correlations among the FAD-family fold, the shape of the pocket, and the FAD conformation. Our general findings are as follows: (a) no single protein 'pharmacophore' exists for binding FAD; (b) in every FAD-binding family, the pyrophosphate moiety binds to the most strongly conserved sequence motif, suggesting that pyrophosphate binding is a significant component of molecular recognition; and (c) sequence motifs can identify proteins that bind phosphate-containing ligands.     

Neuron-specific antigen HPC-1 from bovine brain reveals strong homology to epimorphin, an essential factor involved in epithelial morphogenesis: identification of a novel protein family.

We have already cloned the cDNA for the HPC-1 antigen, a neuron-specific protein antigen from the rat brain. Here we report the molecular cloning of the bovine HPC-1 antigen homologue, and much strong sequence conservation between rat and bovine. By searching the recent protein data base, it was found that the HPC-1 antigen revealed unusual similarity to epimorphin which was mesenchymal factor related to the morphogenesis of primitive epidermal tissues in embryonic stages. We also found that the HPC-1 antigen was identical to p35A (syntaxin) which bound both to a synaptic vesicle protein and to N-type calcium channel. Although the relationship of the physiological functions, structures and topologies along cellular membrane between the HPC-1 antigen and epimorphin have not been consistent yet, these two proteins belong to a novel protein family.     

Common occurrence of internal repeat symmetry in membrane proteins

Symmetry plays significant roles in protein structure and function. Particularly, symmetric interfaces are known to act as switches for two-state conformational change. Membrane proteins often undergo two-state conformational change during the transport process of ion channels or the active/inactive transitions in receptors. Here, we provide the first comprehensive analyses of internal repeat symmetry in membrane proteins. We examined the known membrane protein structures and found that, remarkably, nearly half of them have internal repeat symmetry. Moreover, we found that the conserved cores of these internal repeats are positioned at the interface of symmetric units when they are mapped on structures. Because of the large sequence divergence that occurs between internal repeats, the inherent symmetry present in protein sequences often has only been detected after structure determination. We therefore developed a sensitive procedure to predict the internal repeat symmetry from sequence information and identified 4653 proteins that are likely to have internal repeat symmetry.     

PIRLs: a novel class of plant intracellular leucine-rich repeat proteins

Leucine-rich repeat (LRR) proteins feature tandem leucine-rich motifs that form a protein-protein interaction domain. Plants contain diverse classes of LRR proteins, many of which take part in signal transduction. We have identified a novel family of nine Arabidopsis LRR proteins that, based on predicted intracellular location and LRR motif consensus sequence, are related to Ras-binding LRR proteins found in signaling complexes in animals and yeast. This new class has been named plant intracellular Ras group-related LRR proteins (PIRLs). We have characterized PIRL cDNAs, rigorously defined gene and protein annotations, investigated gene family evolution and surveyed mRNA expression. While LRR regions suggested a relationship to Ras group LRR proteins, outside of their LRR domains PIRLs differed from Ras group proteins, exhibiting N- and C-terminal regions containing low complexity stretches and clusters of charged amino acids. PIRL genes grouped into three subfamilies based on sequence relationships and gene structures. Related gene pairs and dispersed chromosomal locations suggested family expansion by ancestral genomic or segmental duplications. Expression surveys revealed that all PIRL mRNAs are actively transcribed, with three expressed differentially in leaves, roots or flowers. These results define PIRLs as a distinct, plant-specific class of intracellular LRR proteins that probably mediate protein interactions, possibly in the context of signal transduction. T-DNA knock-out mutants have been isolated as a starting point for systematic functional analysis of this intriguing family.     

Novel protein domains and motifs in the marine planctomycete Rhodopirellula baltica

The planctomycetes are a phylum of bacteria that have a unique cell compartmentalisation and yeast-like budding cell division and peptidoglycan-less proteinaceous cell walls. We wished to further our understanding of these unique organisms at the molecular level by searching for conserved amino acid sequence motifs and domains in the proteins encoded by Rhodopirellula baltica. Using BLAST and single-linkage clustering, we have discovered several new protein domains and sequence motifs in this planctomycete. R. baltica has multiple members of the newly discovered GEFGR protein family and the ASPIC C-terminal domain family, whilst most other organisms for which whole genome sequence is available have no more than one. Many of the domains and motifs appear to be restricted to the planctomycetes. It is possible that these protein domains and motifs may have been lost or replaced in other phyla, or they may have undergone multiple duplication events in the planctomycete lineage. One of the novel motifs probably represents a novel N-terminal export signal peptide. With their unique cell biology, it may be that the planctomycete cell compartmentalisation plan in particular needs special membrane transport mechanisms. The discovery of these new domains and motifs, many of which are associated with secretion and cell-surface functions, will help to stimulate experimental work and thus enhance further understanding of this fascinating group of organisms.     

Major epiplasmic proteins of ciliates are articulins: cloning, recombinant expression, and structural characterization

The cytoskeleton of certain protists comprises an extensive membrane skeleton, the epiplasm, which contributes to the cell shape and patterning of the species-specific cortical architecture. The isolated epiplasm of the ciliated protist Pseudomicrothorax dubius consists of two major groups of proteins with molecular masses of 78-80 kD and 11- 13 kD, respectively. To characterize the structure of these proteins, peptide sequences of two major polypeptides (78-80 kD) as well as a cDNA representing the entire coding sequence of a minor and hitherto unidentified component (60 kD; p60) of the epiplasm have been determined. All three polypeptides share sequence similarities. They contain repeated valine- and proline-rich motifs of 12 residues with the consensus VPVP--V-V-V-. In p60 the central core domain consists of 24 tandemly repeated VPV motifs. Within the repeat motifs positively and negatively charged residues, when present, show an alternating pattern in register with the V and P positions. Recombinant p60 was purified in 8 M urea and dialyzed against buffer. Infrared spectroscopic measurements indicate 30% beta-sheet. Electron microscopy reveals short filamentous polymers with a rather homogenous diameter (approximately 15-20 nm), but variable lengths. The small polymers form thicker filaments, ribbons, and larger sheets or tubes. A core domain similar to that of P. dubius p60 is also found in the recently described epiplasmic proteins of the flagellate Euglena, the so-called articulins. Our results show that the members of this protein family are not restricted to flagellates, but are also present in the distantly related ciliates where they are major constituents of the epiplasm. Comparison of flagellate and ciliate articulins highlights common features of this novel family of cytoskeletal proteins.     

Pentatricopeptide repeat proteins and their emerging roles in plants.

Several protein families with tandem repeat motifs play a very important role in plant development and defense. The pentatricopeptide repeat (PPR) protein family, one of the largest families, is the most perplexing one in plants. PPR proteins have been implicated in many crucial functions broadly involving organelle biogenesis and plant development. PPR motifs are degenerate motifs, each with 35-amino-acid sequences and are present in tandem arrays of 2-27 repeats per protein. Although PPR proteins are found in other eukaryotes, their large number is probably required in plants to meet the specific needs of organellar gene expression. The repeats of PPR proteins form a superhelical structure to bind a specific ligand, probably a single-stranded RNA molecule, and modulate its expression. Functional studies on different PPR proteins have revealed their role in organellar RNA processing, fertility restoration in CMS plants, embryogenesis, and plant development. Functional genomic techniques can help identify the diverse roles of the PPR family of proteins in nucleus-organelle interaction and in plant development.     

Conserved pore-forming regions in polypeptide-transporting proteins

Transport of solutes and polypeptides across membranes is an essential process for every cell. In the past, much focus has been placed on helical transporters. Recently, the beta-barrel-shaped transporters have also attracted some attention. The members of this family are found in the outer bacterial membrane and the outer membrane of endosymbiotically derived organelles. Here we analyze the features and the evolutionary development of a specified translocator family, namely the beta-barrel-shaped polypeptide-transporters. We identified sequence motifs, which characterize all transporters of this family, as well as motifs specific for a certain subgroup of proteins of this class. The general motifs are related to the structural composition of the pores. Further analysis revealed a defined distance of two motifs to the C-terminal portion of the proteins. Furthermore, the evolutionary relationship of the proteins and the motifs are discussed.     

Sequence and phylogenetic analysis of squid myosin-V: a vesicle motor in nerve cells

We have shown that vesicles in the axoplasm of the squid giant axon move on actin filaments and that movement is inhibited by myosin V-specific antibodies [Tabb et al., 1998]. In the study reported in this article, experiments were performed to clone and sequence the cDNA for squid brain myosin V. Five proteolytic fragments of purified squid brain myosin V were analyzed by direct protein sequencing [Tabb et al., 1998]. Based on this sequence information, degenerate primers were constructed and used to isolate cDNA clones by PCR. Five clones, representing overlapping segments of the gene, were sequenced. The sequence data and the previous biochemical characterization of the molecule support the classification of this vesicle-associated myosin as a member of the class V myosins. Motif analysis of the head, neck, and tail domains revealed that squid MyoV has consensus sequences for all the motifs found in vertebrate members of the myosin V family of motor proteins. A phylogenetic tree was constructed from a sequence alignment by the neighbor-joining method, using Megalign (DNAStar, Madison, WI); the resulting phylogenetic tree showed that squid MyoV is more closely related to vertebrate MyoV (mouse dilute, chicken dilute, rat myr6, and human myo5a) than Drosophila and yeast (myo2, and myo4) myosins V. These new data on the phylogenetic relationships of squid myosin V to vertebrate myosin V strengthens the argument that myosin V functions as a vesicle motor in vertebrate neurons.     

Cys2/His2 zinc-finger protein family of petunia: evolution and general mechanism of target-sequence recognition.

The EPF family is a group of Cys2/His2zinc-finger proteins in petunia. In these proteins, characteristically long spacer regions have been found to separate the zinc fingers. Our previous DNA-binding studies demonstrated that two-fingered proteins (ZPT2-1 and ZPT2-2), which have spacers of different lengths, bind to two separate AGT core motifs in a spacing specific manner. To investigate the possibility that these proteins might distinguish between the target sequences on the basis of spacing between the core motifs, we screened petunia cDNA library for other proteins belonging to this family. Initial screening by PCR and subsequent cloning of full-length cDNAs allowed us to identify the genes for 10 new proteins that had two, three or four zinc fingers. Among the two-fingered proteins the spacing between zinc fingers varied from 19 to 65 amino acids. The variation in the length of spacers was even more extensive in three- and four-fingered proteins. The presence of such proteins is consistent with our hypothesis that the spacing between the core motifs might be important for target sequence recognition. Furthermore, comparison of diverse protein structures suggests that three- and two-fingered proteins might have resulted due to successive loss of fingers from a four-fingered protein during molecular evolution. We also demonstrate that a highly conserved motif (QALGGH) among the members of EPF family and other Cys2/His2 zinc-finger proteins in plants is critical for the DNA-binding activity.     

Alignment and structure prediction of divergent protein families: periplasmic and outer membrane proteins of bacterial efflux pumps

Broad-specificity efflux pumps have been implicated in multidrug-resistant strains of Pseudomonas aeruginosa and other Gram-negative bacteria. Most Gram-negative pumps of clinical relevance have three components, an inner membrane transporter, an outer membrane channel protein, and a periplasmic protein, which together coordinate efflux from the cytoplasmic membrane across the outer membrane through an unknown mechanism. The periplasmic efflux proteins (PEPs) and outer membrane efflux proteins (OEPs) are not obviously related to proteins of known structure, and understanding the structure and function of these proteins has been hindered by the difficulty of obtaining reasonable multiple alignments. We present a general strategy for the alignment and structure prediction of protein families with low mutual sequence similarity using the PEP and OEP families as detailed examples. Gibbs sampling, hidden Markov models, and other analysis techniques were used to locate motifs, generate multiple alignments, and assign PEP or OEP function to hypothetical proteins in several species. We also developed an automated procedure which combines multiple alignments with structure prediction algorithms in order to identify conserved structural features in protein families. This process was used to identify a probable alpha-helical hairpin in the PEP family and was applied to the detection of transmembrane beta-strands in OEPs. We also show that all OEPs contain a large tandem duplication, and demonstrate that the OEP family is unlikely to adopt a porin fold, in contrast to previous predictions.     

The potential for manipulating RNA with pentatricopeptide repeat proteins

The pentatricopeptide repeat (PPR) protein family, which is particularly prevalent in plants, includes many sequence‐specific RNA‐binding proteins involved in all aspects of organelle RNA metabolism, including RNA stability, processing, editing and translation. PPR proteins consist of a tandem array of 2‐30 PPR motifs, each of which aligns to one nucleotide in the RNA target. The amino acid side chains at two or three specific positions in each motif confer nucleotide specificity in a predictable and programmable manner. Thus, PPR proteins appear to provide an extremely promising opportunity to create custom RNA‐binding proteins with tailored specificity. We summarize recent progress in understanding RNA recognition by PPR proteins, with a particular focus on potential applications of PPR‐based tools for manipulating RNA, and on the challenges that remain to be overcome before these tools may be routinely used by the scientific community.     

Structure and sequence relationships in the lipocalins and related proteins.

The lipocalins and fatty acid-binding proteins (FABPs) are two recently identified protein families that both function by binding small hydrophobic molecules. We have sought to clarify relationships within and between these two groups through an analysis of both structure and sequence. Within a similar overall folding pattern, we find large parts of the lipocalin and FABP structures to be quantitatively equivalent. The three largest structurally conserved regions within the lipocalin common core correspond to characteristic sequence motifs that we have used to determine the constitution of this family using an iterative sequence analysis procedure. This afforded a new interpretation of the family, which highlighted the difficulties of determining a comprehensive and coherent classification of the lipocalins. The first of the three conserved sequence motifs is also common to the FABPs and corresponds to a conserved structural element characteristic of both families. Similarities of structure and sequence within the two families suggests that they form part of a larger "structural superfamily"; we have christened this overall group the calycins to reflect the cup-shaped structure of its members.     

Signals involved in targeting membrane proteins to synaptic vesicles

1. Synaptic vesicles (SVs) mediate fast regulated secretion of classical neurotransmitters. In order to perform their task SVs rely on a restrict set of membrane proteins. The mechanisms responsible for targeting these proteins to the SV membrane are still poorly understood.2. Likewise, little is known about the intracellular routes taken by these proteins in their way to SV membrane. Recently, several domains and motifs necessary for correct localization of SV proteins have been identified.3. In this review we summarize the sequence motifs that have been identified in the cytoplasmic domains of SV proteins that are involved in endocytosis and targeting of SVs. We suggest that the vesicular acetylcholine transporter, a protein found predominantly in synaptic vesicles, is perhaps a model protein to understand the pathways and interactions that are used for synaptic vesicle targeting.