Secondary literature sources for the PDZ domain

For each of the hand selected primary papers associated with the PDZ domain, 100 neighbouring papers are retrieved from PubMed. If one of these neighbouring papers is referenced by more than two primary papers, it is shown in the table below.

The biology of vision of Drosophila.

Zuker CS
Proc Natl Acad Sci U S A. 1996; 93: 571-6

Phototransduction systems in vertebrates and invertebrates share a great deal of similarity in overall strategy but differ significantly in the underlying molecular machinery. Both are rhodopsin-based G protein-coupled signaling cascades displaying exquisite sensitivity and broad dynamic range. However, light activation of vertebrate photoreceptors leads to activation of a cGMP-phosphodiesterase effector and the generation of a hyperpolarizing response. In contrast, activation of invertebrate photoreceptors, like Drosophila, leads to stimulation of phospholipase C and the generation of a depolarizing receptor potential. The comparative study of these two systems of phototransduction offers the opportunity to understand how similar biological problems may be solved by different molecular mechanisms of signal transduction. The study of this process in Drosophila, a system ideally suited to genetic and molecular manipulation, allows us to dissect the function and regulation of such a complex signaling cascade in its normal cellular environment. In this manuscript I review some of our recent findings and the strategies used to dissect this process.

Domain repertoires as a tool to derive protein recognition rules.

Zucconi A, Panni S, Paoluzi S, Castagnoli L, Dente L, Cesareni G
FEBS Lett. 2000; 480: 49-54

Several approaches, some of which are described in this issue, have been proposed to assemble a complete protein interaction map. These are often based on high throughput methods that explore the ability of each gene product to bind any other element of the proteome of the organism. Here we propose that a large number of interactions can be inferred by revealing the rules underlying recognition specificity of a small number (a few hundreds) of families of protein recognition modules. This can be achieved through the construction and characterization of domain repertoires. A domain repertoire is assembled in a combinatorial fashion by allowing each amino acid position in the binding site of a given protein recognition domain to vary to include all the residues allowed at that position in the domain family. The repertoire is then searched by phage display techniques with any target of interest and from the primary structure of the binding site of the selected domains one derives rules that are used to infer the formation of complexes between natural proteins in the cell.

Sequence-specific recognition of the internalization motif of the Alzheimer's amyloid precursor protein by the X11 PTB domain.

Zhang Z, Lee CH, Mandiyan V, Borg JP, Margolis B, Schlessinger J, Kuriyan J
EMBO J. 1997; 16: 6141-50

The crystal structure of the phosphotyrosine-binding domain (PTB) of the X11 protein has been determined, in complex with unphosphorylated peptides corresponding to a region of beta-amyloid precursor protein (betaAPP) that is required for receptor internalization. The mode of binding to X11 of the unphosphorylated peptides, which contain an NPxY motif, resembles that of phosphorylated peptides bound to the Shc and IRS-1 PTB domains. Eight peptide residues make specific contacts with the X11 PTB domain, and they collectively achieve high affinity (KD = 0.32 microM) and specificity. These results suggest that, in contrast to the SH2 domains, the PTB domains are primarily peptide-binding domains that have, in some cases, acquired specificity for phosphorylated tyrosines.

Semaphorin signaling: a little less per-plexin.

Yu HH, Kolodkin AL
Neuron. 1999; 22: 11-4

A role for surface hydrophobicity in protein-protein recognition.

Young L, Jernigan RL, Covell DG
Protein Sci. 1994; 3: 717-29

The role of hydrophobicity as a determinant of protein-protein interactions is examined. Surfaces of apo-protein targets comprising 9 classes of enzymes, 7 antibody fragments, hirudin, growth hormone, and retinol-binding protein, and their associated ligands with available X-ray structures for their complexed forms, are scanned to determine clusters of surface-accessible amino acids. Clusters of surface residues are ranked on the basis of the hydrophobicity of their constituent amino acids. The results indicate that the location of the co-crystallized ligand is commonly found to correspond with one of the strongest hydrophobic clusters on the surface of the target molecule. In 25 of 38 cases, the correspondence is exact, with the position of the most hydrophobic cluster coinciding with more than one-third of the surface buried by the bound ligand. The remaining 13 cases demonstrate this correspondence within the top 6 hydrophobic clusters. These results suggest that surface hydrophobicity can be used to identify regions of a protein's surface most likely to interact with a binding ligand. This fast and simple procedure may be useful for identifying small sets of well-defined loci for possible ligand attachment.

Identification of a novel PSD-95/Dlg/ZO-1 (PDZ)-like protein interacting with the C terminus of presenilin-1.

Xu X, Shi Yc, Wu X, Gambetti P, Sui D, Cui MZ
J Biol Chem. 1999; 274: 32543-6

Presenilin-1 (PS-1) is the most causative Alzheimer gene product, and its function is not well understood. In an attempt to elucidate the function of PS-1, we screened a human brain cDNA library for PS-1-interacting proteins using the yeast two-hybrid system and isolated a novel protein containing a PSD-95/Dlg/ZO-1 (PDZ)-like domain. This novel PS-1-associated protein (PSAP) shares a significant similarity with a Caenorhabditis elegans protein of unknown function. Northern blot analysis revealed that PSAP is predominantly expressed in the brain. Deletion of the first four C-terminal amino acid residues of PS-1, which contain the PDZ domain-binding motif (Gln-Phe-Tyr-Ile), reduced the binding activity of PS-1 toward PSAP 4-fold. These data suggest that PS-1 may associate with a PDZ-like domain-containing protein in vivo and thus may participate in receptor or channel clustering and intracellular signaling events in the brain.

Identification of a human LNX protein containing multiple PDZ domains.

Xie Y, Zhao W, Wang W, Zhao S, Tang R, Ying K, Zhou Z, Mao Y
Biochem Genet. 2001; 39: 117-26

Recently PDZ domains have been recognized as crucial organizers of protein complexes at the plasma membrane. Here we report the cloning of a novel 3.7-kb cDNA LNX from a human fetal brain cDNA library. The deduced amino acid sequence shares about 88% identity with the p70 form of Lnx, which is the mouse ligand of Numb protein X. Motif analysis reveals five protein-interaction modules conservative to those in the Lnx protein, including four PDZ domains and a NPXY motif for binding of the Numb PTB domain. Northern blot analysis shows that the length of the transcript in brain is distinct from those in other tissues. By radiation hybrid mapping, we localize the LNX gene to human chromosome 4q12 between marker D4S1577 and marker D4S1594. We predict that LNX may be important for the organization of signaling complexes in Numb-involved pathways or pathways regulated by other PTB-containing proteins.

The ins and outs of the transmembrane 4 superfamily.

Wright MD, Tomlinson MG
Immunol Today. 1994; 15: 588-94

The recently discovered transmembrane 4 superfamily comprises a group of cell-surface proteins that are characterized by the presence of four hydrophobic domains, which are presumed to be membrane spanning. At least seven of these molecules are expressed on leukocytes, and it seems likely that they mediate signal transduction events that play a role in the regulation of cell development, activation, growth and motility.

Formation of a native-like beta-hairpin finger structure of a peptide from the extended PDZ domain of neuronal nitric oxide synthase in aqueous solution.

Wang P, Zhang Q, Tochio H, Fan JS, Zhang M
Eur J Biochem. 2000; 267: 3116-22

Neuronal nitric oxide synthase (nNOS) is targeted to the cell membrane via interactions of its extended PDZ domain with PDZ domains of membrane-associated proteins including PSD-95 and alpha1-syntrophin. The formation of heterodimers between the nNOS PDZ domain and the PDZ domains of nNOS-binding proteins requires a stretch of continuous amino-acid residues C-terminal to the canonical nNOS PDZ domain. In this work, we show that a 27-residue peptide comprising the C-terminal extension of the extended nNOS PDZ domain is capable of binding to PSD-95. The structure of the 27-residue peptide in aqueous solution was determined using multidimensional NMR-spectroscopic techniques. The free peptide adopts a native-like beta-hairpin finger structure in aqueous solution. The results indicate that the C-terminal extension peptide of the nNOS PDZ domain may represent a relatively independent structural unit in the mediation of the interaction between nNOS and PDZ domain-containing proteins including PSD-95 and alpha1-syntrophin.

Cloning and characterization of MUPP1, a novel PDZ domain protein.

Ullmer C, Schmuck K, Figge A, Lubbert H
FEBS Lett. 1998; 424: 63-8

Using the yeast two-hybrid system we isolated a cDNA clone encoding a novel protein interacting with the C-terminal domain of the 5-HT2C receptor. The protein, named MUPP1 (multi-PDZ-domain protein), contains thirteen PDZ domains and no obvious catalytic domain; it is related to hINADL and a putative C. elegans polypeptide referred to as C52A11.4 containing six or ten PDZ domains, respectively. Domains highly similar to those of MUPP1 are arrayed in the same order in all three proteins. The MUPP1 gene is localized on human chromosome 9p24-p22. Transcripts encoding MUPP1 are abundant in the brain as well as in several peripheral organs.

Crete, channels, cells, circuits and computers.

Ullman S, Roth A, Thomson A, Linne ML
Trends Neurosci. 1997; 20: 53-4

Intracellular and extracellular control of activin function by novel regulatory molecules.

Tsuchida K, Matsuzaki T, Yamakawa N, Liu Z, Sugino H
Mol Cell Endocrinol. 2001; 180: 25-31

Activin signal transduction is regulated through multiple mechanisms. We have identified novel regulatory proteins that control activin functions either intracellularly or extracellularly. As intracellular molecules, PSD-95/Dlg/ZO-1 (PDZ) proteins that specifically associate with activin type II receptors (ActRIIs) were identified. We have named the molecules as activin receptor-interacting proteins (ARIPs). ARIP1 has two WW domains and five PDZ domains, associates not only with ActRIIs but also with Smads, and controls activin functions intracellularly in neuronal cells. Another ARIP we have found has only one PDZ domain, and is likely to be involved in intracellular trafficking and sorting of activin receptor complexes in the cell. As an extracellular regulatory protein, we have identified a novel follistatin-like protein, named follistatin-related gene (FLRG). Like follistatins, FLRG binds activins and bone morphogenetic proteins (BMPs) and controls their functions extracellularly. The mode of association of follistatin and FLRG with activins and their expression patterns are different, suggesting the distinct functions of follistatin and FLRG in vivo.

Sorting out receptor trafficking.

Trimmer JS
Neuron. 1999; 22: 411-2

Formation of nNOS/PSD-95 PDZ dimer requires a preformed beta-finger structure from the nNOS PDZ domain.

Tochio H, Mok YK, Zhang Q, Kan HM, Bredt DS, Zhang M
J Mol Biol. 2000; 303: 359-70

PDZ domains are modular protein units that play important roles in organizing signal transduction complexes. PDZ domains mediate interactions with both C-terminal peptide ligands and other PDZ domains. Here, we used PDZ domains from neuronal nitric oxide synthase (nNOS) and postsynaptic density protein-95 (PSD-95) to explore the mechanism for PDZ-dimer formation. The nNOS PDZ domain terminates with a approximately 30 residue amino acid beta-finger peptide that is shown to be required for nNOS/PSD-95 PDZ dimer formation. In addition, formation of the PDZ dimer requires this beta-finger peptide to be physically anchored to the main body of the canonical nNOS PDZ domain. A buried salt bridge between the beta-finger and the PDZ domain induces and stabilizes the beta-hairpin structure of the nNOS PDZ domain. In apo-nNOS, the beta-finger peptide is partially flexible and adopts a transient beta-strand like structure that is stabilized upon PDZ dimer formation. The flexibility of the NOS PDZ beta-finger is likely to play a critical role in supporting the formation of nNOS/PSD-95 complex. The experimental data also suggest that nNOS PDZ and the second PDZ domain of PSD-95 form a "head-to-tail" dimer similar to the nNOS/syntrophin complex characterized by X-ray crystallography.

Solution structure and backbone dynamics of the second PDZ domain of postsynaptic density-95.

Tochio H, Hung F, Li M, Bredt DS, Zhang M
J Mol Biol. 2000; 295: 225-37

The second PDZ domain of postsynaptic density-95 (PSD-95 PDZ2) plays a critical role in coupling N-methyl-D-aspartate receptors to neuronal nitric oxide synthase (nNOS). In this work, the solution structure of PSD-95 PDZ2 was determined to high resolution by NMR spectroscopy. The structure of PSD-95 PDZ2 was compared in detail with that of alpha1-syntrophin PDZ domain, as the PDZ domains share similar target interaction properties. The interaction of the PSD-95 PDZ2 with a carboxyl-terminal peptide derived from a cytoplasmic protein CAPON was studied by NMR titration experiments. Complex formation between PSD-95 PDZ2 and the nNOS PDZ was modelled on the basis of the crystal structure of the alpha1-syntrophin PDZ/nNOS PDZ dimer. We found that the prolonged loop connecting the betaB and betaC strands of PSD-95 PDZ2 is likely to play a role in both the binding of the carboxyl-terminal peptide and the nNOS beta-finger. Finally, the backbone dynamics of the PSD-95 PDZ2 in the absence of bound peptide were studied using a model-free approach. The "GLGF"-loop and the loop connecting alphaB and betaF of the protein display some degree of flexibility in solution. The rest of the protein is rigid and lacks detectable slow time-scale (microseconds to milliseconds) motions. In particular, the loop connecting betaB and betaC loop adopts a well-defined, rigid structure in solution. It appears that the loop adopts a pre-aligned conformation for the PDZ domain to interact with its targets.

Receptors: structure and function.

Stone DK
Am J Med. 1998; 105: 244-50

Regulation of ion transport by protein-protein interaction domains.

Staub O, Rotin D
Curr Opin Nephrol Hypertens. 1997; 6: 447-54

Protein-protein interaction via specific modular domains has been implicated in the regulation of many signalling pathways. Recently, such modules, in particular WW, Src homology-3 and PDZ domains, have been shown to regulate localization, clustering and function of several ion channels and transporters, including the epithelial Na+ channel, K+ channel and ionotropic neurotransmitter receptors.

Protein-peptide interactions.

Stanfield RL, Wilson IA
Curr Opin Struct Biol. 1995; 5: 103-13

Proteins can interact with short peptide sequences in a variety of ways that can be sequence dependent or independent. The bound peptides are frequently in an extended conformation but may also adopt beta-turns or alpha-helices as motifs for recognition. The peptides can be completely buried in cavities, bound in grooves or pockets, or form beta-strand type interactions at the protein surface. These various recognition motifs are illustrated by peptide interactions with antibodies, calmodulin, OppA periplasmic binding protein, PapD chaperone, MHC class I and class II molecules, and Src homology (SH) domains 2 and 3.

Identification, sequence, and mapping of the mouse multiple PDZ domain protein gene, Mpdz.

Simpson EH, Suffolk R, Jackson IJ
Genomics. 1999; 59: 102-4

The PDZ domain gained its name from the three proteins that were first seen to have homology by virtue of these domains, the mammalian postsynaptic density protein, PSD-95, the Drosophila discs-large septate junction protein, DLG, and the mammalian epithelial tight-junction protein zona occludens, ZO-1. Over 50 PDZ domain-containing genes have been recognized so far from almost any organism subjected to sequencing, including mammals, nematodes, yeast, plants, and bacteria. The domain consists of an approximately 90-amino-acid-residue unit, which is often repeated in the protein. The majority of residues form a conserved spatial structure while a few amino acids in critical positions confer protein binding specificity. A subgroup of PDZ domains have been shown to recognize a short carboxy-terminal amino acid motif, T/SXV (Ser/Thr-X-Val-COO-), where X is any amino acid. We have identified and completely sequenced a gene, Mpdz, that encodes a mouse protein containing 13 such domains. We have also mapped the gene to a series of overlapping deletions on mouse chromosome 4 and can therefore determine that its function is not essential for embryonic development or neonatal survival.

N-terminal PDZ domain is required for NHERF dimerization.

Shenolikar S, Minkoff CM, Steplock DA, Evangelista C, Liu M, Weinman EJ
FEBS Lett. 2001; 489: 233-6

NHERF, a 55 kDa PDZ-containing protein, binds receptors and ion transporters to mediate signal transduction at the plasma membrane. Recombinant NHERF demonstrated an apparent size of 150 kDa on gel filtration, which could be reduced to approximately 55 kDa by protein denaturing agents, consistent with the formation of NHERF dimers. Biosensor studies established the time- and concentration-dependent dimerization of NHERF. Overlays of recombinant NHERF fragments suggested that NHERF dimerization was principally mediated by the N-terminal PDZ-I domain. In PS120 cells, reversible protein phosphorylation modulated NHERF dimerization and suggested a role for NHERF dimers in hormonal signaling.

Ion channel targeting in neurons.

Sheng M, Wyszynski M
Bioessays. 1997; 19: 847-53

Electrical signaling by neurons depends on the precisely ordered distribution of a wide variety of ion channels on the neuronal surface. The mechanisms underlying the targeting of particular classes of ion channels to specific subcellular sites are poorly understood. Recent studies have identified a new class of protein-protein interaction mediated by PDZ domains, protein binding modules that recognize specific sequences at the C terminus of membrane proteins. The PDZ domains of a family of synaptic cytoskeleton-associated proteins, typified by PSD-95, bind to the intracellular C-terminal tails of NMDA receptors and Shaker-type K+ channels. This interaction appears to be important in the clustering and localization of these ion channels at synaptic sites. Recognition of specific C-terminal peptide sequences by different PDZ domain-containing proteins may be a general mechanism for differential targeting of proteins to a variety of subcellular locations.

PDZ domains and the organization of supramolecular complexes.

Sheng M, Sala C
Annu Rev Neurosci. 2001; 24: 1-29

PDZ domains are modular protein interaction domains that bind in a sequence-specific fashion to short C-terminal peptides or internal peptides that fold in a beta-finger. The diversity of PDZ binding specificities can be explained by variable amino acids lining the peptide-binding groove of the PDZ domain. Abundantly represented in Caenorhabditis elegans, Drosophila melanogaster, and mammalian genomes, PDZ domains are frequently found in multiple copies or are associated with other protein-binding motifs in multidomain scaffold proteins. PDZ-containing proteins are typically involved in the assembly of supramolecular complexes that perform localized signaling functions at particular subcellular locations. Organization around a PDZ-based scaffold allows the stable localization of interacting proteins and enhances the rate and fidelity of signal transduction within the complex. Some PDZ-containing proteins are more dynamically regulated in distribution and may also be involved in the trafficking of interacting proteins within the cell.

Specific interactions between the syntrophin PDZ domain and voltage-gated sodium channels.

Schultz J, Hoffmuller U, Krause G, Ashurst J, Macias MJ, Schmieder P, Schneider-Mergener J, Oschkinat H
Nat Struct Biol. 1998; 5: 19-24

Syntrophins are modular proteins belonging to the dystrophin associated glycoprotein complex and are thought to be involved in the regulation of the muscular system. Screening of peptide libraries revealed selectivity of the synotrophin PDZ domain toward the motif R/K/Q-E-S/T-X-V-COO- found to be highly conserved in the alpha-subunit C-terminus of vertebrate voltage gated sodium channels (VGSCs). The solution structure of the domain in complex with the peptide G-V-K-E-S-L-V shows specific interactions between the conserved residues in the peptide and syntrophin-characteristic residues in the domain. We propose that syntrophins localize VGSCs to the dystrophin network through its PDZ domain.

Mutagenesis and selection of PDZ domains that bind new protein targets.

Schneider S, Buchert M, Georgiev O, Catimel B, Halford M, Stacker SA, Baechi T, Moelling K, Hovens CM
Nat Biotechnol. 1999; 17: 170-5

PDZ domains are a recently characterized protein-recognition module. In most cases, PDZ domains bind to the C-terminal end of target proteins and are thought thereby to link these target proteins into functional signaling networks. We report the isolation of artificial PDZ domains selected via a mutagenesis screen in vivo, each recognizing a different C-terminal peptide. We demonstrate that the PDZ domains isolated can bind selectively to their target peptides in vitro and in vivo. Two of the target peptides chosen are the C-terminal ends of two cellular transmembrane proteins with which no known PDZ domains have been reported to interact. By targeting these artificial PDZ domains to the nucleus, interacting target peptides were efficiently transported to the same subcellular localization. One of the isolated PDZ domains was tested and shown to be efficiently directed to the plasma membrane when cotransfected with the full-length transmembrane protein in mammalian cells. Thus, artificial PDZ domains can be engineered and used to target intracellular proteins to different subcellular compartments.

Backwards and forwards binding.

Saraste M, Musacchio A
Nat Struct Biol. 1994; 1: 835-7

PDZ domains bind carboxy-terminal sequences of target proteins.

Saras J, Heldin CH
Trends Biochem Sci. 1996; 21: 455-8

[Chemical synthesis of proteins]

Sakakibara S
Tanpakushitsu Kakusan Koso. 1995; 40: 304-16

Lano, a novel LAP protein directly connected to MAGUK proteins in epithelial cells.

Saito H, Santoni MJ, Arsanto JP, Jaulin-Bastard F, Le Bivic A, Marchetto S, Audebert S, Isnardon D, Adelaide J, Birnbaum D, Borg JP
J Biol Chem. 2001; 276: 32051-5

Protein networks asymetrically distributed to basolateral and apical epithelial membranes maintain cell polarity and homeostasis of epithelial tissues. Genetic studies in non-vertebrates assigned two families of basolateral proteins, MAGUK (membrane-associated and guanylate kinase) and LAP (leucine-rich repeats and PDZ) proteins, to a common pathway crucial for the epithelial architecture and acting as a gatekeeper to malignancy. In mammals, three LAP proteins have been described, Densin-180, Erbin, and hScribble. Here, we identify a protein called Lano (LAP and no PDZ) only present in vertebrates and presenting strong identities with LAP proteins. Despite the lack of PDZ domain, Lano is located at the basolateral side of epithelial cells in a similar manner to Erbin and hScribble. Using in vitro and in vivo experiments, we demonstrate that Lano directly interacts with the PDZ domains of MAGUK proteins, including hDLG (human disc large), in epithelial cells. A second pool of Lano is complexed to Erbin. These LAP-MAGUK protein complexes coexist at the basolateral side of epithelial cells. We provide evidence for a direct interaction between LAP and MAGUK proteins, and we propose that various LAP-MAGUK networks targeted to the basolateral side of epithelial cells participate to homeostasis of epithelial tissues and tumor growth.

Latrophilin-like receptor from the parasitic nematode Haemonchus contortus as target for the anthelmintic depsipeptide PF1022A.

Saeger B, Schmitt-Wrede HP, Dehnhardt M, Benten WP, Krucken J, Harder A, Von Samson-Himmelstjerna G, Wiegand H, Wunderlich F
FASEB J. 2001; 15: 1332-4

Classification of membrane transporters.

Sadee W, Graul RC, Lee AY
Pharm Biotechnol. 1999; 12: 29-58

Disparate cell types use a shared complex of PDZ proteins for polarized protein localization.

Rongo C
Cytokine Growth Factor Rev. 2001; 12: 349-59

Based on their morphology and function, epithelial cells and neurons appear to have very little in common; however, growing evidence indicates that these two disparate cell types share an underlying polarization pathway responsible for sorting proteins to specific subcellular sites. An evolutionarily conserved complex of PDZ domain-containing proteins thought to be responsible for polarized protein localization has been identified from both brain and epithelial tissue, both from mammals and from the nematode C. elegans. Some of the most recent data on PDZ proteins and the proteins with which they interact are summarized. In particular, some of the more recently proposed models for their function in cells, and the in vivo and in vitro data that support these models are focussed upon.

PDZ-mediated interactions retain the epithelial GABA transporter on the basolateral surface of polarized epithelial cells.

Perego C, Vanoni C, Villa A, Longhi R, Kaech SM, Frohli E, Hajnal A, Kim SK, Pietrini G
EMBO J. 1999; 18: 2384-93

The PDZ target motifs located in the C-terminal end of many receptors and ion channels mediate protein-protein interactions by binding to specific PDZ-containing proteins. These interactions are involved in the localization of surface proteins on specialized membrane domains of neuronal and epithelial cells. However, the molecular mechanism responsible for this PDZ protein-dependent polarized localization is still unclear. This study first demonstrated that the epithelial gamma-aminobutyric acid (GABA) transporter (BGT-1) contains a PDZ target motif that mediates the interaction with the PDZ protein LIN-7 in Madin-Darby canine kidney (MDCK) cells, and then investigated the role of this interaction in the basolateral localization of the transporter. It was found that although the transporters from which the PDZ target motif was deleted were still targeted to the basolateral surface, they were not retained but internalized in an endosomal recycling compartment. Furthermore, an interfering BGT peptide determined the intracellular relocation of the native transporter. These data indicate that interactions with PDZ proteins determine the polarized surface localization of target proteins by means of retention and not targeting mechanisms. PDZ proteins may, therefore, act as a sort of membrane protein sorting machinery which, by recognizing retention signals (the PDZ target sequences), prevents protein internalization.

Mapping protein family interactions: intramolecular and intermolecular protein family interaction repertoires in the PDB and yeast.

Park J, Lappe M, Teichmann SA
J Mol Biol. 2001; 307: 929-38

In the postgenomic era, one of the most interesting and important challenges is to understand protein interactions on a large scale. The physical interactions between protein domains are fundamental to the workings of a cell: in multi-domain polypeptide chains, in multi-subunit proteins and in transient complexes between proteins that also exist independently. To study the large-scale patterns and evolution of interactions between protein domains, we view interactions between protein domains in terms of the interactions between structural families of evolutionarily related domains. This allows us to classify 8151 interactions between individual domains in the Protein Data Bank and the yeast Saccharomyces cerevisiae in terms of 664 types of interactions, between protein families. At least 51 interactions do not occur in the Protein Data Bank and can only be derived from the yeast data. The map of interactions between protein families has the form of a scale-free network, meaning that most protein families only interact with one or two other families, while a few families are extremely versatile in their interactions and are connected to many families. We observe that almost half of all known families engage in interactions with domains from their own family. We also see that the repertoires of interactions of domains within and between polypeptide chains overlap mostly for two specific types of protein families: enzymes and same-family interactions. This suggests that different types of protein interaction repertoires exist for structural, functional and regulatory reasons. Copyright 12001 Academic Press.

Voltage-dependent activation of ion channels.

Papazian DM, Bezanilla F
Adv Neurol. 1999; 79: 481-91

PDZ domains in bacterial proteins.

Pallen MJ, Ponting CP
Mol Microbiol. 1997; 26: 411-3

The C-terminus of human glutaminase L mediates association with PDZ domain-containing proteins.

Olalla L, Aledo JC, Bannenberg G, Marquez J
FEBS Lett. 2001; 488: 116-22

The enzyme glutaminase in brain is responsible for the synthesis of neurotransmitter glutamate. We used the two-hybrid genetic selection system in yeast to look for interactors of glutaminase in human brain. We have identified two proteins containing PDZ domains, alpha1-syntrophin and a glutaminase-interacting protein, named GIP, that showed association with human glutaminase L, as deduced from specificity test of the two-hybrid system. The complete GIP cDNA clone has 1315 nucleotides with a 372-base open reading frame encoding a 124-amino acids protein. Glutaminase associates with both PDZ proteins through its C-terminal end; mutagenesis of single amino acids revealed the sequence -ESXV as essential for the interaction. These data suggest the possibility that PDZ domain-containing proteins are involved in the regulation of glutaminase in brain.

Emerging roles of Dlg-like PDZ proteins in the organization of the NMDA-type glutamatergic synapse.

Nagano T, Jourdi H, Nawa H
J Biochem (Tokyo). 1998; 124: 869-75

A group of proteins found at cell-cell junctions have a common structural domain, called PDZ-a stretch of 80-90 amino acid residues initially identified in the three proteins PSD-95, Dlg, and ZO-1. This domain is found in various proteins from bacteria to mammals and is involved in protein-protein interaction. Recently, many proteins containing this domain were identified in the nervous system by molecular cloning and shown to interact with other synaptic proteins, including various transmitter receptors, ion channels, and signal transducers. These PDZ-containing proteins are mostly located near the synaptic membrane and are, therefore, speculated to transport associated proteins to the synapse and/or anchor them at the synaptic sites. Alternatively, as a single molecule often contains multiple PDZ domains that can interact with each other, it may cluster all these synaptic molecules and facilitate their signaling at synaptic sites. This review focuses on the best characterized PDZ-containing proteins that interact with N-methyl-D-aspartate (NMDA)-type glutamate receptors and discusses their functions in synaptic organization.

Structure of interleukin 16 resembles a PDZ domain with an occluded peptide binding site.

Muhlhahn P, Zweckstetter M, Georgescu J, Ciosto C, Renner C, Lanzendorfer M, Lang K, Ambrosius D, Baier M, Kurth R, Holak TA
Nat Struct Biol. 1998; 5: 682-6

The structure of a folded core of IL-16 is similar to that of intracellular protein modules called PDZ domains. IL-16 is thus the first extracellular protein found to have a PDZ-like fold. However, it does not exhibit normal peptide binding properties of PDZ domains. This is due to alterations of the structure at the 'PDZ-like binding site' of IL-16 (the GLGF cleft): the GLGF cleft of IL-16 is much smaller than those of PDZ-domains and is additionally blocked with a tryptophan side chain at its center. Our experiments indicate also that IL-16 nonspecifically aggregates in solution; but formation of a homo-tetrameric protein is not required, in contrast to previous suggestions, for its chemo-attractant activity.

Ptch2, a second mouse Patched gene is co-expressed with Sonic hedgehog.

Motoyama J, Takabatake T, Takeshima K, Hui C
Nat Genet. 1998; 18: 104-6

Structure of the VHS domain of human Tom1 (target of myb 1): insights into interactions with proteins and membranes.

Misra S, Beach BM, Hurley JH
Biochemistry. 2000; 39: 11282-90

VHS domains are found at the N-termini of select proteins involved in intracellular membrane trafficking. We have determined the crystal structure of the VHS domain of the human Tom1 (target of myb 1) protein to 1.5 A resolution. The domain consists of eight helices arranged in a superhelix. The surface of the domain has two main features: (1) a basic patch on one side due to several conserved positively charged residues on helix 3 and (2) a negatively charged ridge on the opposite side, formed by residues on helix 2. We compare our structure to the recently obtained structure of tandem VHS-FYVE domains from Hrs [Mao, Y., Nickitenko, A., Duan, X., Lloyd, T. E., Wu, M. N., Bellen, H., and Quiocho, F. A. (2000) Cell 100, 447-456]. Key features of the interaction surface between the FYVE and VHS domains of Hrs, involving helices 2 and 4 of the VHS domain, are conserved in the VHS domain of Tom1, even though Tom1 does not have a FYVE domain. We also compare the structures of the VHS domains of Tom1 and Hrs to the recently obtained structure of the ENTH domain of epsin-1 [Hyman, J., Chen, H., Di Fiore, P. P., De Camilli, P., and Brunger, A. T. (2000) J. Cell Biol. 149, 537-546]. Comparison of the two VHS domains and the ENTH domain reveals a conserved surface, composed of helices 2 and 4, that is utilized for protein-protein interactions. In addition, VHS domain-containing proteins are often localized to membranes. We suggest that the conserved positively charged surface of helix 3 in VHS and ENTH domains plays a role in membrane binding.

Yersiniabactin synthetase: probing the recognition of carrier protein domains by the catalytic heterocyclization domains, Cy1 and Cy2, in the chain-initiating HWMP2 subunit.

Miller DA, Walsh CT
Biochemistry. 2001; 40: 5313-21

The HMWP2 subunit of yersiniabactin (Ybt) synthetase, a 230 kDa nonribosomal peptide synthetase (NRPS) making the N-terminus of the Ybt siderophore of Yersinia pestis, has one cysteine-specific adenylation (A) domain, three carrier protein domains (ArCP, PCP1, PCP2), and two heterocyclization domains (Cy1, Cy2). The A domain loads the two PCP domains with cysteines that get heterocyclized by the Cy domains to yield a tricyclic hydroxyphenylthiazolinylthiazolinyl (HPTT) chain lodged in thioester linkage to the PCP2 domain. The interdomain recognition by the Cy1 and Cy2 domains for the three carrier proteins was tested using inactivating mutations at the conserved serine that is phosphopantetheinylated in each carrier domain (S52A, S1439A, and S1977A). These mutant forms of HMWP2 were tested for in trans complementation by carrier protein fragments: holo-ArCPs (S52A), holo-PCP1 and analogues (S1439A), and holo-PCP2 and analogues (S1977A). The S52A mutant tests the recognition of the Cy1 domain for donor acyl-ArCP substrates, while the S1439A mutant tests the specificity of the same Cy1 domain for downstream substrates presented by distinct PCPs. The S1439A likewise tests the recognition of Cy2 for its upstream PCP-tethered acyl donor. The S1977A mutant analogously tests the Cy2 domain for downstream Cys-PCP recognition. In all cases in trans complementation was successful with the carrier protein fragments, allowing kinetic probes of catalytic efficiency for PCP scaffolds and for uncoupling of the condensation and heterocyclization functions of Cy1 and Cy2. Overall, the Cy domains tested showed a definite selectivity for the upstream protein scaffold but were more relaxed toward the downstream acceptor protein. This work points to the importance of protein-protein interactions in mediating directional chain growth in NRPS and presents the first systematic exploration of how the protein scaffolds affect catalytic efficiency.

The function of PTB domain proteins.

Margolis B, Borg JP, Straight S, Meyer D
Kidney Int. 1999; 56: 1230-7

Phosphotyrosine binding (PTB) domains have been identified in a large number of proteins. In proteins like Shc and IRS-1, the PTB domain binds in a phosphotyrosine-dependent fashion to peptides that form a b turn. In these proteins, PTB domains play an important role in signal transduction by growth factor receptors. However, in several other proteins, the PTB domains have been found to participate in phosphotyrosine-independent interactions. The X11 family of proteins contains a PTB domain that binds peptides in a phosphotyrosine-independent fashion. The homologue of X11 in C. elegans is the lin-10 gene, a gene crucial for receptor targeting to the basolateral surface of body wall epithelia. The X11/Lin-10 proteins are found in a complex with two other proteins, Lin-2 and Lin-7, which have also been implicated in basolateral targeting in worm epithelia. This protein complex is also likely to be important in the targeting of cell surface proteins in mammalian neurons and epithelia. The ability of the PTB domain to bind peptides in a phosphotyrosine-dependent and -independent fashion allows this domain to be involved in diverse cellular functions.

Modular organization of the PDZ domains in the human discs-large protein suggests a mechanism for coupling PDZ domain-binding proteins to ATP and the membrane cytoskeleton.

Marfatia SM, Morais Cabral JH, Lin L, Hough C, Bryant PJ, Stolz L, Chishti AH
J Cell Biol. 1996; 135: 753-66

The human homologue (hDIg) of the Drosophila discs-large tumor suppressor (DIg) is a multidomain protein consisting of a carboxyl-terminal guanylate kinase-like domain, an SH3 domain, and three slightly divergent copies of the PDZ (DHR/GLGF) domain. Here have examined the structural organization of the three PDZ domains of hDIg using a combination of protease digestion and in vitro binding measurements. Our results show that the PDZ domains are organized into two conformationally stable modules one (PDZ, consisting of PDZ domains 1 and 2, and the other (PDZ) corresponding to the third PDZ domain. Using amino acid sequencing and mass spectrometry, we determined the boundaries of the PDZ domains after digestion with endoproteinase Asp-N, trypsin, and alpha-chymotrypsin. The purified PDZ1+2, but not the PDZ3 domain, contains a high affinity binding site for the cytoplasmic domain of Shaker-type K+ channels. Similarly, we demonstrate that the PDZ1+2 domain can also specifically bind to ATP. Furthermore, we provide evidence for an in vivo interaction between hDIg and protein 4.1 and show that the hDIg protein contains a single high affinity protein 4.1-binding site that is not located within the PDZ domains. The results suggest a mechanism by which PDZ domain-binding proteins may be coupled to ATP and the membrane cytoskeleton via hDlg.

The PDZ domain of human erythrocyte p55 mediates its binding to the cytoplasmic carboxyl terminus of glycophorin C. Analysis of the binding interface by in vitro mutagenesis.

Marfatia SM, Morais-Cabral JH, Kim AC, Byron O, Chishti AH
J Biol Chem. 1997; 272: 24191-7

The PDZ domain, also known as the GLGF repeat/DHR domain, is an approximately 90-amino acid motif discovered in a recently identified family of proteins termed MAGUKs (membrane-associated guanylate kinase homologues). Sequence comparison analysis has since identified PDZ domains in over 50 proteins. Like SH2 and SH3 domains, the PDZ domains mediate specific protein-protein interactions, whose specificities appear to be dictated by the primary structure of the PDZ domain as well as its binding target. Using recombinant fusion proteins and a blot overlay assay, we show that a single copy of the PDZ domain in human erythrocyte p55 binds to the carboxyl terminus of the cytoplasmic domain of human erythroid glycophorin C. Deletion mutagenesis of 21 amino acids at the amino terminus of the p55 PDZ domain completely abrogates its binding activity for glycophorin C. Using an alanine scan and surface plasmon resonance technique, we identify residues in the cytoplasmic domain of glycophorin C that are critical for its interaction with the PDZ domain. The recognition specificity of the p55 PDZ domain appears to be unique, since the three PDZ domains of hDlg (human lymphocyte homologue of the Drosophila discs large tumor suppressor) do not bind the cytoplasmic domain of glycophorin C. Taken together with our previous studies, these results complete the identification of interacting domains in the ternary complex between p55, glycophorin C, and protein 4.1. Implications of these findings are discussed in terms of binding specificity and the regulation of cytoskeleton-membrane interactions.

Identification of the protein 4.1 binding interface on glycophorin C and p55, a homologue of the Drosophila discs-large tumor suppressor protein.

Marfatia SM, Leu RA, Branton D, Chishti AH
J Biol Chem. 1995; 270: 715-9

Protein 4.1 is the prototype of a family of proteins that include ezrin, talin, brain tumor suppressor merlin, and tyrosine phosphatases. All members of the protein 4.1 superfamily share a highly conserved N-terminal 30-kDa domain whose biological function is poorly understood. It is believed that the attachment of the cytoskeleton to the membrane may be mediated via this 30-kDa domain, a function that requires formation of multiprotein complexes at the plasma membrane. In this investigation, synthetically tagged peptides and bacterially expressed proteins were used to map the protein 4.1 binding site on human erythroid glycophorin C, a transmembrane glycoprotein, and on human erythroid p55, a palmitoylated peripheral membrane phosphoprotein. The results show that the 30-kDa domain of protein 4.1 binds to a 12-amino acid segment within the cytoplasmic domain of glycophorin C and to a positively charged, 39-amino acid motif in p55. Sequences similar to this charged motif are conserved in other members of the p55 superfamily, including the Drosophila discs-large tumor suppressor protein. Our data provide new insights into how protein 4.1, glycophorin C, p55, and their non-erythroid homologues, interact with the cytoskeleton to exert their physiological effects.

PDZ Domain Protein GIPC Interacts with the Cytoplasmic Tail of Melanosomal Membrane Protein gp75 (Tyrosinase-related Protein-1).

Liu TF, Kandala G, Setaluri V
J Biol Chem. 2001; 276: 35768-77

Tyrosinase and tyrosinase-related proteins (TRPs) are a family of melanosomal membrane proteins involved in mammalian pigmentation. Whereas the melanogenic functions of TRPs are localized in their amino-terminal domains that reside within the lumen of melanosomes, the sorting and targeting of these proteins to melanosomes is mediated by signals in their cytoplasmic domains. To identify proteins that interact with the cytoplasmic tail of gp75 (TRP-1), the most abundant melanosomal membrane protein, we performed yeast two-hybrid screening of a melanocyte cDNA library. Here, we show that the cytoplasmic domain of gp75 interacts with a PDZ domain-containing protein. The gp75-interacting protein is identical to GIPC, an RGS (regulator of G protein signaling)/GAIP-interacting protein, and to SEMCAP-1, a transmembrane semaphorin-binding protein. Carboxyl-terminal amino acid residues, Ser-Val-Val, of gp75 are necessary and sufficient for interaction of gp75 with the single PDZ domain in GIPC. Although endogenous and transfected GIPCs bind efficiently to transiently expressed gp75, only a small amount of GIPC is found associated with gp75 at steady state. Using a strategy to selectively synchronize the biosynthesis of endogenous gp75, we demonstrate that only newly synthesized gp75 associates with GIPC, primarily in the juxtanuclear Golgi region. Our data suggest that GIPC/SEMCAP-1 plays a role in biosynthetic sorting of proteins, specifically gp75, to melanosomes.

The carboxyl terminus of B class ephrins constitutes a PDZ domain binding motif.

Lin D, Gish GD, Songyang Z, Pawson T
J Biol Chem. 1999; 274: 3726-33

Ephrin B proteins function as ligands for B class Eph receptor tyrosine kinases and are postulated to possess an intrinsic signaling function. The sequence at the carboxyl terminus of B-type ephrins contains a putative PDZ binding site, providing a possible mechanism through which transmembrane ephrins might interact with cytoplasmic proteins. To test this notion, a day 10.5 mouse embryonic expression library was screened with a biotinylated peptide corresponding to the carboxyl terminus of ephrin B3. Three of the positive cDNAs encoded polypeptides with multiple PDZ domains, representing fragments of the molecule GRIP, the protein syntenin, and PHIP, a novel PDZ domain-containing protein related to Caenorhabditis elegans PAR-3. In addition, the binding specificities of PDZ domains previously predicted by an oriented library approach (Songyang, Z., Fanning, A. S., Fu, C., Xu, J., Marfatia, S. M., Chishti, A. H., Crompton, A., Chan, A. C., Anderson, J. M., and Cantley, L. C. (1997) Science 275, 73-77) identified the tyrosine phosphatase FAP-1 as a potential binding partner for B ephrins. In vitro studies demonstrated that the fifth PDZ domain of FAP-1 and full-length syntenin bound ephrin B1 via the carboxyl-terminal motif. Lastly, syntenin and ephrin B1 could be co-immunoprecipitated from transfected COS-1 cells, suggesting that PDZ domain binding of B ephrins can occur in cells. These results indicate that the carboxyl-terminal motif of B ephrins provides a binding site for specific PDZ domain-containing proteins, which might localize the transmembrane ligands for interactions with Eph receptors or participate in signaling within ephrin B-expressing cells.

The second PDZ domain of INAD is a type I domain involved in binding to eye protein kinase C. Mutational analysis and naturally occurring variants.

Kumar R, Shieh BH
J Biol Chem. 2001; 276: 24971-7

INAD is a scaffolding protein containing five PSD95/dlg/zonular occludens-1 (PDZ) domains that tether NORPA (phospholipase Cbeta(4)), the TRP calcium channel, and eye-PKC in Drosophila photoreceptors. We previously showed that eye-PKC interacted with the second PDZ domain (PDZ2) of INAD. Sequence comparison with a prototypical type I PDZ domain predicts that PDZ2 is the best candidate among the five PDZ domains to recognize eye-PKC that contains a type I PDZ ligand, Ile-Thr-Ile-Ile, at its carboxyl terminus. Replacement of Ile(-3) in eye-PKC with charged residues resulted in a drastic reduction of the PDZ2 interaction. Substitution of a conserved His with Arg at the second alpha-helix of PDZ2 led to a reduced binding; however, a Leu replacement resulted in an enhanced eye-PKC association. We isolated and sequenced the InaD gene. The coding sequence of InaD contains nine exons spanning 3 kilobases. Translation of coding sequences from three wild-type alleles revealed three SNPs affecting residues, 282, 319, and 333 of INAD. These polymorphisms are localized in PDZ2. Interestingly, we found two of three PDZ2 variants displayed a greater affinity for eye-PKC. In summary, we evaluated the molecular basis of the eye-PKC and PDZ2 association by mutational analysis and concluded that PDZ2 of INAD is a type I domain important for the eye-PKC interaction.

Solution structure of the PDZ2 domain from human phosphatase hPTP1E and its interactions with C-terminal peptides from the Fas receptor.

Kozlov G, Gehring K, Ekiel I
Biochemistry. 2000; 39: 2572-80

The solution structure of the second PDZ domain (PDZ2) from human phosphatase hPTP1E has been determined using 2D and 3D heteronuclear NMR experiments. The binding of peptides derived from the C-terminus of the Fas receptor to PDZ2 was studied via changes in backbone peptide and protein resonances. The structure is based on a total of 1387 nonredundant experimental NMR restraints including 1261 interproton distance restraints, 45 backbone hydrogen bonds, and 81 torsion angle restraints. Analysis of 30 lowest-energy structures resulted in rmsd values of 0.41 +/- 0.09 A for backbone atoms (N, Calpha, C') and 1.08 +/- 0.10 A for all heavy atoms, excluding the disordered N- and C-termini. The hPTP1E PDZ2 structure is similar to known PDZ domain structures but contains two unique structural features. In the peptide binding domain, the first glycine of the GLGF motif is replaced by a serine. This serine appears to replace a bound water observed in PDZ crystal structures that hydrogen bonds to the bound peptide's C-terminus. The hPTP1E PDZ2 structure also contains an unusually large loop following strand beta2 and proximal to the peptide binding site. This well-ordered loop folds back against the PDZ domain and contains several residues that undergo large amide chemical shift changes upon peptide binding. Direct observation of peptide resonances demonstrates that as many as six Fas peptide residues interact with the PDZ2 domain.

The aquaporin family of molecular water channels.

Knepper MA
Proc Natl Acad Sci U S A. 1994; 91: 6255-8

Synaptic targeting of ionotropic neurotransmitter receptors.

Kirsch J, Meyer G, Betz H
Mol Cell Neurosci. 1996; 8: 93-8

Functional relevance of the disulfide-linked complex of the N-terminal PDZ domain of InaD with NorpA.

Kimple ME, Siderovski DP, Sondek J
EMBO J. 2001; 20: 4414-22

In Drosophila, phototransduction is mediated by G(q)-activation of phospholipase C and is a well studied model system for understanding the kinetics of signal initiation, propagation and termination controlled by G proteins. The proper intracellular targeting and spatial arrangement of most proteins involved in fly phototransduction require the multi-domain scaffolding protein InaD, composed almost entirely of five PDZ domains, which independently bind various proteins including NorpA, the relevant phospho lipase C-beta isozyme. We have determined the crystal structure of the N-terminal PDZ domain of InaD bound to a peptide corresponding to the C-terminus of NorpA to 1.8 A resolution. The structure highlights an intermolecular disulfide bond necessary for high affinity interaction as determined by both in vitro and in vivo studies. Since other proteins also possess similar, cysteine-containing consensus sequences for binding PDZ domains, this disulfide-mediated 'dock-and-lock' interaction of PDZ domains with their ligands may be a relatively ubiquitous mode of coordinating signaling pathways.

Organization and regulation of proteins at synapses.

Kim JH, Huganir RL
Curr Opin Cell Biol. 1999; 11: 248-54

The organization and regulation of synaptic connections in the mammalian nervous system entail complicated and co-ordinated molecular and cellular processes. The unveiling of various protein-protein interactions and their functional consequences at synapses have led to a greater understanding of the process of synapse formation and the modulation of synaptic transmission. Recent studies indicate that the major excitatory neurotransmitter receptors in the brain, the glutamate receptors, are associated with many different molecules that are involved in the formation of elaborate synaptic cytoskeletal networks and signal transduction cascades. These complex protein networks may play critical roles in the regulation of neurotransmitter receptor function and the efficacy of synaptic transmission.

Characterizing Class I WW domains defines key specificity determinants and generates mutant domains with novel specificities.

Kasanov J, Pirozzi G, Uveges AJ, Kay BK
Chem Biol. 2001; 8: 231-41

Introduction: WW domains are small protein interaction modules found in a wide range of eukaryotic signaling and structural proteins. Five classes of WW domains have been annotated to date, where each class is largely defined by the type of peptide ligand selected, rather than by similarities within WW domains. Class I WW domains bind Pro-Pro-Xxx-Tyr containing ligands, and it would be of interest to determine residues within the domains that determine this specificity.Results: Fourteen WW domains selected Leu/Pro-Pro-Xxx-Tyr containing peptides ligands via phage display and were thus designated as Class 1 WW domains. These domains include those present in human YAP (hYAP) and WWP3, as well as those found in ubiquitin protein ligases of the Nedd4 family, including mouse Nedd4 (mNedd4), WWP1, WWP2 and Rsp5. Comparing the primary structures of these WW domains highlighted a set of highly conserved residues, in addition to those originally noted to occur within WW domains. Substitutions at two of these conserved positions completely inhibited ligand binding, whereas substitution at a non-conserved position did not. Moreover, mutant WW domains containing substitutions at conserved positions bound novel peptide ligands.Conclusions: Class I WW domains contain a highly conserved set of residues that are important in selecting Pro-Xxx-Tyr containing peptide ligands. The presence of these residues within an uncharacterized WW domain can be used to predict its ability to bind Pro-Xxx-Tyr containing peptide ligands.

Exocytosis: SNAREs drum up!

Johannes L, Galli T, Ludger J
Eur J Neurosci. 1998; 10: 415-22

The tetranucleotide UCAY directs the specific recognition of RNA by the Nova K-homology 3 domain.

Jensen KB, Musunuru K, Lewis HA, Burley SK, Darnell RB
Proc Natl Acad Sci U S A. 2000; 97: 5740-5

The Nova family of proteins are target antigens in the autoimmune disorder paraneoplastic opsoclonus-myoclonus ataxia and contain K-homology (KH)-type RNA binding domains. The Nova-1 protein has recently been shown to regulate alternative splicing of the alpha2 glycine receptor subunit pre-mRNA by binding to an intronic element containing repeats of the tetranucleotide UCAU. Here, we have used selection-amplification to demonstrate that the KH3 domain of Nova recognizes a single UCAY element in the context of a 20-base hairpin RNA; the UCAY tetranucleotide is optimally presented as a loop element of the hairpin scaffold and requires protein residues C-terminal to the previously defined KH domain. These results suggest that KH domains in general recognize tetranucleotide motifs and that biological RNA targets of KH domains may use either RNA secondary structure or repeated sequence elements to achieve high affinity and specificity of protein binding.

Schwannomin isoform-1 interacts with syntenin via PDZ domains.

Jannatipour M, Dion P, Khan S, Jindal H, Fan X, Laganiere J, Chishti AH, Rouleau GA
J Biol Chem. 2001; 276: 33093-100

The neurofibromatosis type 2 gene (NF2) is involved in the pathogenesis of benign tumors of the human nervous system. The NF2 protein, called schwannomin or merlin, is inactivated in virtually all schwannomas and meningiomas. The molecular mechanisms by which schwannomin functions as a tumor suppressor is unknown but believed to involve plasma membrane-cytoskeletal interactions. Two major alternatively spliced isoforms of schwannomin differing in their C termini have been reported. Using the yeast two-hybrid system, we have identified syntenin as a binding partner for schwannomin isoform-1 (sch-1). Syntenin is an adapter protein that couples transmembrane proteoglycans to cytoskeletal components and is involved in intracellular vesicle transport. The C terminus 25 amino acids of sch-1 and the two PDZ domains of syntenin mediate their binding, and mutations introduced within the VAFFEEL region of sch-1 defined a sequence crucial for syntenin recognition. We have showed that the two proteins interacted in vitro and in vivo and localized underneath the plasma membrane. Fibroblast cells expressing heterologous antisense syntenin display alterations in the subcellular distribution of sch-1. Together, these results provide the first functional clue to the existence of schwannomin isoforms and could unravel novel pathways for the transport and subcellular localization of schwannomin in vivo.

Binding of neuroligins to PSD-95.

Irie M, Hata Y, Takeuchi M, Ichtchenko K, Toyoda A, Hirao K, Takai Y, Rosahl TW, Sudhof TC
Science. 1997; 277: 1511-5

PSD-95 is a component of postsynaptic densities in central synapses. It contains three PDZ domains that localize N-methyl-D-aspartate receptor subunit 2 (NMDA2 receptor) and K+ channels to synapses. In mouse forebrain, PSD-95 bound to the cytoplasmic COOH-termini of neuroligins, which are neuronal cell adhesion molecules that interact with beta-neurexins and form intercellular junctions. Neuroligins bind to the third PDZ domain of PSD-95, whereas NMDA2 receptors and K+ channels interact with the first and second PDZ domains. Thus different PDZ domains of PSD-95 are specialized for distinct functions. PSD-95 may recruit ion channels and neurotransmitter receptors to intercellular junctions formed between neurons by neuroligins and beta-neurexins.

The PDZ domain of the SpoIVB serine peptidase facilitates multiple functions.

Hoa NT, Brannigan JA, Cutting SM
J Bacteriol. 2001; 183: 4364-73

During spore formation in Bacillus subtilis, the SpoIVB protein is a critical component of the sigma(K) regulatory checkpoint. SpoIVB has been shown to be a serine peptidase that is synthesized in the spore chamber and which self-cleaves, releasing active forms. These forms can signal proteolytic processing of the transcription factor sigma(K) in the outer mother cell chamber of the sporulating cell. This forms the basis of the sigma(K) checkpoint and ensures accurate sigma(K)-controlled gene expression. SpoIVB has also been shown to activate a second distinct process, termed the second function, which is essential for the formation of heat-resistant spores. In addition to the serine peptidase domain, SpoIVB contains a PDZ domain. We have altered a number of conserved residues in the PDZ domain by site-directed mutagenesis and assayed the sporulation phenotype and signaling properties of mutant SpoIVB proteins. Our work has revealed that the SpoIVB PDZ domain could be used for up to four distinct processes, (i) targeting of itself for trans proteolysis, (ii) binding to the protease inhibitor BofC, (iii) signaling of pro-sigma(K) processing, and (iv) signaling of the second function of SpoIVB.

Unexpected modes of PDZ domain scaffolding revealed by structure of nNOS-syntrophin complex.

Hillier BJ, Christopherson KS, Prehoda KE, Bredt DS, Lim WA
Science. 1999; 284: 812-5

The PDZ protein interaction domain of neuronal nitric oxide synthase (nNOS) can heterodimerize with the PDZ domains of postsynaptic density protein 95 and syntrophin through interactions that are not mediated by recognition of a typical carboxyl-terminal motif. The nNOS-syntrophin PDZ complex structure revealed that the domains interact in an unusual linear head-to-tail arrangement. The nNOS PDZ domain has two opposite interaction surfaces-one face has the canonical peptide binding groove, whereas the other has a beta-hairpin "finger." This nNOS beta finger docks in the syntrophin peptide binding groove, mimicking a peptide ligand, except that a sharp beta turn replaces the normally required carboxyl terminus. This structure explains how PDZ domains can participate in diverse interaction modes to assemble protein networks.

Characterisation of porcine aminopeptidase A: a type II integral membrane protein.

Hesp JR, Hooper NM
Biochem Soc Trans. 1995; 23: 550-550

Characterization of the allosteric modulatory protein associated with non-NMDA receptors.

Henley JM
Biochem Soc Trans. 1993; 21: 89-93

Synaptic PDZ domain-containing proteins.

Hata Y, Nakanishi H, Takai Y
Neurosci Res. 1998; 32: 1-7

Synaptic junctions accumulate various molecules involved in neurotransmissions and synaptic plasticity. The proper organizations of these molecules at synaptic junctions sustain the interneuronal signal transmissions and the subsequent signal cascades underling learning and memory. Recent studies have revealed that a protein-interacting module named a PDZ domain plays important roles in the interactions among the components of synaptic junctions. In this article, we summarize synaptic PDZ domain-containing proteins and discussed their functions.

The Drosophila cation channel trpl expressed in insect Sf9 cells is stimulated by agonists of G-protein-coupled receptors.

Harteneck C, Obukhov AG, Zobel A, Kalkbrenner F, Schultz G
FEBS Lett. 1995; 358: 297-300

Structures and regulations of vertebrate channels responsible for sustained calcium elevations after hormone stimulation are largely unknown. Therefore, the Drosophila photoreceptor channels, trp and trpl, which are assumed to be involved in calcium influx, serve as model system, trpl expressed in Sf9 cells showed spontaneous activity. Hormonal stimulations of calcium influx (detected by fura-2) and of an outwardly rectifying current were observed in Sf9 cells coinfected with baculoviruses encoding trpl and various heptahelical receptors for histamine, thrombin, and thromboxane A2, all known to cause phospholipase C-beta activation in mammalian cells. Although the identity of the G-proteins and of possible second messengers involved need to be clarified, it is clear that trpl represents a receptor/G-protein regulated cation channel.

Energetic determinants of internal motif recognition by PDZ domains.

Harris BZ, Hillier BJ, Lim WA
Biochemistry. 2001; 40: 5921-30

PDZ domains are protein-protein interaction modules that organize intracellular signaling complexes. Most PDZ domains recognize specific peptide motifs followed by a required COOH-terminus. However, several PDZ domains have been found which recognize specific internal peptide motifs. The best characterized example is the syntrophin PDZ domain which, in addition to binding peptide ligands with the consensus sequence -E-S/T-X-V-COOH, also binds the neuronal nitric oxide synthase (nNOS) PDZ domain in a manner that does not depend on its precise COOH-terminal sequence. In the structure of the syntrophin-nNOS PDZ heterodimer complex, the two PDZ domains interact in a head-to-tail fashion, with an internal sequence from the nNOS PDZ domain binding precisely at the peptide binding groove of the syntrophin PDZ domain. To understand the energetic basis of this alternative mode of PDZ recognition, we have undertaken an extensive mutagenic and biophysical analysis of the nNOS PDZ domain and its interaction with the syntrophin PDZ domain. Our data indicate that the presentation of the nNOS internal motif within the context of a rigid beta-hairpin conformation is absolutely essential to binding; amino acids crucial to the structural integrity of the hairpin are as important or more important than residues that make direct contacts. The results reveal the general rules of PDZ recognition of diverse ligand types.

Developing three-dimensional models of ion channel proteins.

Guy HR, Durell SR
Ion Channels. 1996; 4: 1-40

Cyclic peptides as non-carboxyl-terminal ligands of syntrophin PDZ domains.

Gee SH, Sekely SA, Lombardo C, Kurakin A, Froehner SC, Kay BK
J Biol Chem. 1998; 273: 21980-7

Syntrophins, a family of intracellular peripheral membrane proteins of the dystrophin-associated protein complex (DAPC), each contain a single PDZ domain. Syntrophin PDZ domains bind C-terminal peptide sequences with the consensus R/K-E-S/T-X-V-COOH, an interaction that mediates association of skeletal muscle sodium channels with the DAPC. Here, we have isolated cyclic peptide ligands for syntrophin PDZ domains from a library of combinatorial peptides displayed at the N terminus of protein III of bacteriophage M13. Affinity selection from a library of X10C peptides yielded ligands with the consensus X-(R/K)-E-T-C-L/M-A-G-X-Psi-C, where Psi represents any hydrophobic amino acid. These peptides contain residues (underlined) similar to the C-terminal consensus sequence for binding to syntrophin PDZ domains and bind to the same site on syntrophin PDZ domains as C-terminal peptides, but do not bind to other closely related PDZ domains. PDZ binding is dependent on the formation of an intramolecular disulfide bond in the peptides, since treatment with dithiothreitol, or substitution of either of the two cysteines with alanines, eliminated this activity. Furthermore, amino acid replacements revealed that most residues in the phage-selected peptides are required for binding. Our results define a new mode of binding to PDZ domains and suggest that proteins containing similar conformationally constrained sequences may be ligands for PDZ domains.

Single-amino acid substitutions alter the specificity and affinity of PDZ domains for their ligands.

Gee SH, Quenneville S, Lombardo CR, Chabot J
Biochemistry. 2000; 39: 14638-46

PDZ domains are modular protein-protein interaction domains that bind to specific C-terminal sequences of membrane proteins and/or to other PDZ domains. Certain PDZ domains in PSD-95 and syntrophins interact with C-terminal peptide ligands and heterodimerize with the extended nNOS PDZ domain. The capacity to interact with nNOS correlates with the presence of a Lys residue in the carboxylate- binding loop of these PDZ domains. Here, we report that substitution of an Arg for Lys-165 in PSD-95 PDZ2 disrupted its interaction with nNOS, but not with the C terminus of the Shaker-type K(+) channel Kv1.4. The same mutation affected nNOS binding to alpha1- and beta1-syntrophin PDZ domains to a lesser extent, due in part to the stabilizing effect of tertiary interactions with the canonical nNOS PDZ domain. PDZ domains with an Arg in the carboxylate-binding loop do not bind nNOS; however, substitution with Lys or Ala was able to confer nNOS binding. Our results indicate that the carboxylate-binding loop Lys or Arg is a critical determinant of nNOS binding and that the identity of this residue can profoundly alter one mode of PDZ recognition without affecting another. We also analyzed the effects of mutating Asp-143, a residue in the alphaB helix of alpha1-syntrophin that forms a tertiary contact with the nNOS PDZ domain. This residue is important for both nNOS and C-terminal peptide binding and confers a preference for peptides with a positively charged residue at position -4. On this basis, we have identified the C terminus of the Kir2.1 channel as a possible binding partner for syntrophin PDZ domains. Together, our results demonstrate that single-amino acid substitutions alter the specificity and affinity of PDZ domains for their ligands.

PDZ domains in synapse assembly and signalling.

Garner CC, Nash J, Huganir RL
Trends Cell Biol. 2000; 10: 274-80

Synaptic junctions are highly specialized structures designed to promote the rapid and efficient transmission of signals from the presynaptic terminal to the postsynaptic membrane within the central nervous system. Proteins containing PDZ domains play a fundamental organizational role at both the pre- and postsynaptic plasma membranes. This review focuses on recent advances in our understanding of the mechanisms underlying the assembly of synapses in the central nervous system.

Analysis of PDZ domain-ligand interactions using carboxyl-terminal phage display.

Fuh G, Pisabarro MT, Li Y, Quan C, Lasky LA, Sidhu SS
J Biol Chem. 2000; 275: 21486-91

PDZ domains mediate protein-protein interactions at specialized subcellular sites, such as epithelial cell tight junctions and neuronal post-synaptic densities. Because most PDZ domains bind extreme carboxyl-terminal sequences, the phage display method has not been amenable to the study of PDZ domain binding specificities. For the first time, we demonstrate the functional display of a peptide library fused to the carboxyl terminus of the M13 major coat protein. We used this library to analyze carboxyl-terminal peptide recognition by two PDZ domains. For each PDZ domain, the library provided specific ligands with sub-micromolar binding affinities. Synthetic peptides and homology modeling were used to dissect and rationalize the binding interactions. Our results establish carboxyl-terminal phage display as a powerful new method for mapping PDZ domain binding specificity.

[Biologically active peptide fragments of functional proteins produced by proteolysis in vitro]

Filippova MM, Karelin AA, Ivanov VT
Bioorg Khim. 1997; 23: 388-409

Over 100 various peptides were identified as a result of numerous studies of in vitro proteolytic digestion of some proteins and tissue preparations. Many of them exhibit wide spectra of biological effects, which are similar to those described for some groups of endogenous peptide bioregulators. The possibility of in vitro modeling of endogenous processes of proteolytic digestion of proteins that provides biologically active peptides is discussed.

Identification of functional PDZ domain binding sites in several human proteins.

Fabre S, Reynaud C, Jalinot P
Mol Biol Rep. 2000; 27: 217-24

TIP-15 was previously identified as a cellular protein that can bind to the C-terminal end of the HTLV-1 Tax protein via its two PDZ domains. The sequence of the N-terminal part of TIP-15 is identical to that of the synaptic protein PSD-95. Both proteins are likely to be produced from the same gene by alternative splicing. Whereas expression of the PSD-95 mRNA was detected only with brain RNAs, that of TIP-15 was detected with RNAs from thymus, brain, skeletal muscle and Jurkat cells. The TIP-15 protein exhibits an apparent molecular weight of 40 kD and is weakly expressed in T cell lines. A two-hybrid screen performed with TIP-15 as bait revealed the presence of a PDZ binding site (PDZ-BS) in the following proteins: Lysyl tRNA synthetase, 6-phosphogluconolactonase (6-GPL), Stress-activated protein kinase 3 (SAPK3), NET-1, Diacylglycerol kinase zeta, MTMR1, MCM7, and hSec8. The sequence at the C-terminal ends of these proteins matches the X-S/T-X-V-COOH consensus previously defined for PDZ-BSs, with the exception of 6-GPL and SAPK3 which include a leucine as the C-terminal residue. For Lysyl tRNA synthetase, NET1, MTMR1 and hSec8, binding to TIP-15 was confirmed by co-immunoprecipitation experiments performed with the extracts of transfected COS7 cells. These results show the existence of functional PDZ-BSs in these proteins, but future studies will be necessary to establish whether or not TIP-15 represents a physiological partner. The significance of the presence of a PDZ-BS in these various proteins is discussed with respect to their function.

Prediction and comparison of the haem-binding sites in membrane haemoproteins.

Esposti MD
Biochim Biophys Acta. 1989; 977: 249-65

This article contains a comparative review of the structural properties of membrane haemoproteins, with particular emphasis on the possible similarities of the haem-binding peptides. A procedure is suggested for identifying the peptides which may bind membrane-buried haems on the basis of the primary sequences of the proteins. The integration of this procedure with the information deduced by refined hydropathy analysis indicates that the basic structural model for the haemoproteins which interact with quinones may be a transmembrane helical bundle containing the haem(s) at its centre. Structural similarities exist in the sequence of hydrophobic segments that are predicted to bind the membrane-buried haems of b-cytochromes which interact with quinones. The predicted haem-binding sites show similarities also with the peptides that bind the non-haem iron in the bacterial reaction centres, and this may be correlated to the common function of interacting with quinones and their intermediates. The analysis of the amino-acid composition of the proposed ligand peptides in the membrane haemoproteins examined has provided a molecular rationale for explaining the highly anisotropic low-spin EPR signal which is characteristic of many membrane-bound b-cytochromes.

Identification of 4-coumarate:coenzyme A ligase (4CL) substrate recognition domains.

Ehlting J, Shin JJ, Douglas CJ
Plant J. 2001; 27: 455-65

4-coumarate:CoA ligase (4CL), the last enzyme of the general phenylpropanoid pathway, provides precursors for the biosynthesis of a large variety of plant natural products. 4 CL catalyzes the formation of CoA thiol esters of 4-coumarate and other hydroxycinnamates in a two step reaction involving the formation of an adenylate intermediate. 4 CL shares conserved peptide motifs with diverse adenylate-forming enzymes such as firefly luciferases, non-ribosomal peptide synthetases, and acyl:CoA synthetases. Amino acid residues involved in 4 CL catalytic activities have been identified, but domains involved in determining substrate specificity remain unknown. To address this question, we took advantage of the difference in substrate usage between the Arabidopsis thaliana 4 CL isoforms At4CL1 and At4CL2. While both enzymes convert 4-coumarate, only At4CL1 is also capable of converting ferulate. Employing a domain swapping approach, we identified two adjacent domains involved in substrate recognition. Both substrate binding domain I (sbd I) and sbd II of At4CL1 alone were sufficient to confer ferulate utilization ability upon chimeric proteins otherwise consisting of At4CL2 sequences. In contrast, sbd I and sbd II of At4CL2 together were required to abolish ferulate utilization in the context of At4CL1. Sbd I corresponds to a region previously identified as the substrate binding domain of the adenylation subunit of bacterial peptide synthetases, while sbd II centers on a conserved domain of so far unknown function in adenylate-forming enzymes (GEI/LxIxG). At4CL1 and At4CL2 differ in nine amino acids within sbd I and four within sbd II, suggesting that these play roles in substrate recognition.

L27, a novel heterodimerization domain in receptor targeting proteins Lin-2 and Lin-7.

Doerks T, Bork P, Kamberov E, Makarova O, Muecke S, Margolis B
Trends Biochem Sci. 2000; 25: 317-8

Signaling pathways are focused at specialized regions of the plasma membrane by scaffolding proteins of the MAGUK family.

Dimitratos SD, Woods DF, Stathakis DG, Bryant PJ
Bioessays. 1999; 21: 912-21

The MAGUKs (membrane-associated guanylate kinase homologs) are a family of proteins that act as molecular scaffolds for signaling pathway components at the plasma membrane of animal cells. They are localized in and required for the formation of several types of cell junctions, including epithelial tight and septate junctions as well as synaptic and neuromuscular junctions. They are also localized at the plasma membrane of other cell types, including erythrocytes, where they contribute to cell shape maintenance. MAGUKs function mainly by binding directly to the cytoplasmic termini of transmembrane proteins as well as to other signal transduction proteins. They appear to hold together elements of individual signaling pathways, thereby contributing to the efficiency and specificity of signaling interactions while simultaneously maintaining the structural specializations of the plasma membrane. BioEssays 1999;21:912-921.

Nitric oxide signalling in insects.

Davies S
Insect Biochem Mol Biol. 2000; 30: 1123-38

Adaptors and integrators.

Cowburn D
Structure. 1996; 4: 1005-8

The PTB domain expands both the PH-domain set and peptide-protein recognition motifs; the PDZ domain shows an intriguing resemblance.

Protein-binding domains of the tight junction protein, ZO-2, are highly conserved between avian and mammalian species.

Collins JR, Rizzolo LJ
Biochem Biophys Res Commun. 1998; 252: 617-22

The tight junction is composed of many proteins and includes three members of the MAGUK (membrane-associated, guanylate kinase-like) protein family: ZO-1, ZO-2, and ZO-3. ZO-2 was cloned and sequenced from embryonic chicken retina. Antibodies against a short ZO-2 peptide immunolabeled the outer limiting membrane (an adherens junction of the neural retina) and the apical junctional complexes of the retinal pigment epithelium. Each ZO family member contains a homologous series of protein-binding domains: three distinct PDZ domains and src homology 3 (SH3), guanylate kinase-like (GuK), and acidic domains. Compared with human and canine ZO-2s, the PDZ and SH3 domains are the most conserved (90-95% amino acid sequence identity). These domains are only 50-71% identical with the homologous domains of ZO-1 and ZO-3. Although the sequence is less conserved for regions that link the protein-binding domains, the length of those regions is conserved in ZO-2s. The postacidic (C-terminal) region is the least conserved. The evolutionary pressure to maintain the sequence of the protein-binding domains suggests that homologous domains have different functions in ZO-1, ZO-2, and ZO-3.

Requirement for the PDZ domain protein, INAD, for localization of the TRP store-operated channel to a signaling complex.

Chevesich J, Kreuz AJ, Montell C
Neuron. 1997; 18: 95-105

In Drosophila, the store-operated Ca2+ channel, TRP, is required in photoreceptor cells for a sustained response to light. Here, we show that TRP forms a complex with phospholipase C-beta (NORPA), rhodopsin (RH1), calmodulin, and the PDZ domain containing protein INAD. Proteins with PDZ domains have previously been shown to cluster ion channels in vitro. We show that in InaD mutant flies, TRP is no longer spatially restricted to its normal subcellular compartment, the rhabdomere. These results provide evidence that a PDZ domain protein is required, in vivo, for anchoring of an ion channel to a signaling complex. Furthermore, disruption of this interaction results in retinal degeneration. We propose that the TRP channel is linked to NORPA and RH1 to facilitate feedback regulation of these upstream signaling molecules.

Craniofacial dysmorphogenesis including cleft palate in mice with an insertional mutation in the discs large gene.

Caruana G, Bernstein A
Mol Cell Biol. 2001; 21: 1475-83

The discs large (Dlg) protein, or synapse-associated protein 97 (SAP97), is a member of the membrane-associated guanylate kinase family of multidomain scaffolding proteins which recruits transmembrane and signaling molecules to localized plasma membrane sites. Murine dlg is the homologue of the Drosophila dlg tumor suppressor gene. The loss of dlg function in Drosophila disrupts cellular growth control, apicobasal polarity, and cell adhesion of imaginal disc epithelial cells, resulting in embryonic lethality. In this study, we isolated a mutational insertion in the murine dlg locus by gene trapping in totipotent embryonic stem cells. This insertion results in a truncated protein product that contains the N-terminal three PSD-95/DLG/ZO-1 domains of Dlg fused to the LacZ reporter and subsequently lacks the src homology 3 (SH3), protein 4.1 binding, and guanylate kinase (GUK)-like domains. The Dlg-LacZ fusion protein is expressed in epithelial, mesenchymal, neuronal, endothelial, and hematopoietic cells during embryogenesis. Mice homozygous for the dlg mutation exhibit growth retardation in utero, have hypoplasia of the premaxilla and mandible, have a cleft secondary palate, and die perinatally. Consistent with this phenotype, Dlg-LacZ is expressed in mesenchymal and epithelial cells throughout palatal development. Our genetic and phenotypic analysis of dlg mutant mice suggests that protein-protein interactions involving the SH3, protein 4.1 binding, and/or GUK-like domains are essential to the normal function of murine Dlg within craniofacial and palatal morphogenesis.

Membrane polarity in epithelial cells: protein sorting and establishment of polarized domains.

Caplan MJ
Am J Physiol. 1997; 272: 4259-4259

Epithelial cells go to great trouble to organize the subdomains of their plasma membranes. The apical and basolateral surfaces of polarized epithelia are equipped with markedly distinct populations of channels, carriers, and pumps. This anisotropy is an absolute prerequisite for vectorial solute and fluid transport. The physiological properties of an individual epithelial cell type are determined not only by its census of transport proteins but also by the manner in which these proteins are segregated between the apical and basolateral portions of the plasmalemma (Curr. Top. Membr. 39: 37-86, 1991). To achieve this asymmetry, an epithelial cell must be able to establish distinct surface domains, to target newly synthesized transport proteins to their appropriate sites of functional residence, and to retain them there following their delivery. Studies of the cellular pathways involved in generating and maintaining the polarized state have begun to illuminate an elegant network of cell biological specializations that may be involved not only in establishing the distributions of transport proteins but in dynamically regulating their function as well.

Fungal Zuotin proteins evolved from MIDA1-like factors by lineage-specific loss of MYB domains.

Braun EL, Grotewold E
Mol Biol Evol. 2001; 18: 1401-12

Proteins are often characterized by the presence of multiple domains, which make specific contributions to their cellular function. While the gain of domains in proteins by duplication and shuffling is well established, domain loss is poorly documented. Here, we provide evidence that domain loss has played an important role in the evolution of protein architecture and function by demonstrating that fungal Zuotin proteins evolved from MIDA1-like proteins, present in animals and plants, by complete loss of the carboxyl-terminal MYB domains. Phylogenetic analyses of the DnaJ motif (the J domain) present in both Zuotin and MIDA1 proteins were complicated by the limited length and profound differences in evolutionary rates exhibited by this domain. To rigorously examine J domain phylogeny, we combined the nonparametric bootstrap with Monte Carlo simulation. This method, which we have designated the resampled parametric bootstrap, allowed us to assess type I and type II error associated with these analyses. These results revealed significant support for domain loss rather than domain gain or gene loss involving paralogs. The absence of sequences related to the MIDA1 MYB domains in Saccharomyces cerevisiae further indicates that the domains have been completely lost, consistent with known functional differences between Zuotin and MIDA1 proteins. These analyses suggest that the description of additional examples of complete domain loss may provide a method to identify orthologous proteins exhibiting functional differences using genomic sequence data.

Identification of G protein-gated and G protein-modulated ionic channels. Molecular basis for G protein action.

Birnbaumer L, VanDongen AM, Codina J, Yatani A, Mattera R, Graf R, Brown AM
Soc Gen Physiol Ser. 1989; 44: 17-54

PDZ proteins and polarity: functions from the fly.

Bilder D
Trends Genet. 2001; 17: 511-9

Proteins that contain PDZ domains have been implicated in the localization of interacting partners to specific regions of the cell membrane. Although PDZ proteins that bind to a number of important mammalian proteins have been isolated, the significance of these interactions is unclear. In recent years, the fruit fly has emerged as a rich system for genetic analysis of PDZ protein function. Here, I discuss what the Drosophila data tell us about the roles and mechanisms of PDZ protein activity, and how this informs our understanding of PDZ function in other organisms.

[An objective method for isolating globular protein domains]

Berezovskii IN, Tumanian VG
Biofizika. 1995; 40: 1171-7

Regions of more stability and nearest environs were localized on the basis of the calculation of intramolecular interactions in the frame of pair-wise approach. A criterium for definition of domains boundary in globular proteins is establishing if residues from k to l are included into one domain and residues from l+1 to l+1+m are included into another domain. According to this criterium, the boundary between two domains is set between two residues in the case when the interaction energy is minimal when first residue is included into the first domain and the second one into another. Structural domains are founded and probability of existing of domains boundary is studied for the binase, barnase, and complex of barnase with dioxy-dinucleotide inhibitor. It is determinated that the biggest structural interaction between domains coinsiders with location of domains at 1-43, 44-110 residues correspondently.

Effect of proteolysis on the activity of the sodium channel in isolated lobster nerve membrane.

Barnola FV, Torres ME
J Neurosci Res. 1983; 10: 73-81

The effect of enzymatic proteolysis on structural and functional properties of the isolated lobster nerve membrane was investigated. The membranes were treated with different amounts of either trypsin or unspecific protease. Sodium channel activity was determined by measuring the veratridine-tetrodotoxin-sensitive sodium influx in proteoliposomes prepared with nerve membrane and soybean lipids. The changes in the membrane proteins were followed by electrophoresis in polyacrylamide gradient gels. From the densitometric scan of the gels the relative area for each protein was obtained, and the ratio of enzyme-treated to control areas was evaluated. Under a similar degree of proteolysis catalyzed either by trypsin or by unspecific protease, the sensitive sodium influx is not affected by trypsin, whereas it is about 60% diminished by the unspecific protease. In this condition the zones corresponding to molecular weights of 240,000 and 166,000 daltons appear modified in the electrophoretic gels by both enzymes. The 117,000-dalton range is modified only by the unspecific protease. Increasing trypsin concentration diminishes sodium influx about 60%; and the 240,000-, 166,000-, and 117,000-dalton zones appear modified. A further increase of the protease concentration totally abolishes the sensitive sodium influx and modifies practically all of the membrane proteins. The present results indicate the rather high sensitivity of the membrane sodium channel activity to proteolytic action, and show that the membrane sites that respond to veratridine appear to be highly affected by proteolysis. In contrast, the tetrodotoxin receptor retains its binding capacity even after treatment of the membrane with protease concentrations 1,000 times higher than those affecting the sensitive sodium influx [Benzer and Raftery, 1972; Villegas et al, 1973].

Regulatory potential, phyletic distribution and evolution of ancient, intracellular small-molecule-binding domains.

Anantharaman V, Koonin EV, Aravind L
J Mol Biol. 2001; 307: 1271-92

Central cellular functions such as metabolism, solute transport and signal transduction are regulated, in part, via binding of small molecules by specialized domains. Using sensitive methods for sequence profile analysis and protein structure comparison, we exhaustively surveyed the protein sets from completely sequenced genomes for all occurrences of 21 intracellular small-molecule-binding domains (SMBDs) that are represented in at least two of the three major divisions of life (bacteria, archaea and eukaryotes). These included previously characterized domains such as PAS, GAF, ACT and ferredoxins, as well as three newly predicted SMBDs, namely the 4-vinyl reductase (4VR) domain, the NIFX domain and the 3-histidines (3H) domain. Although there are only a limited number of different superfamilies of these ancient SMBDs, they are present in numerous distinct proteins combined with various enzymatic, transport and signal-transducing domains. Most of the SMBDs show considerable evolutionary mobility and are involved in the generation of many lineage-specific domain architectures. Frequent re-invention of analogous architectures involving functionally related, but not homologous, domains was detected, such as, fusion of different SMBDs to several types of DNA-binding domains to form diverse transcription regulators in prokaryotes and eukaryotes. This is suggestive of similar selective forces affecting the diverse SMBDs and resulting in the formation of multidomain proteins that fit a limited number of functional stereotypes. Using the "guilt by association approach", the identification of SMBDs allowed prediction of functions and mode of regulation for a variety of previously uncharacterized proteins. Copyright 2001 Academic Press.