X-ray crystal structure of the hypothetical phosphotyrosine phosphatase MDP-1 of the haloacid dehalogenase superfamily

The haloacid dehalogenase (HAD) superfamily is comprised of structurally homologous enzymes that share several conserved sequence motifs (loops I-IV) in their active site. The majority of HAD members are phosphohydrolases and may be divided into three subclasses depending on domain organization. In classes I and II, a mobile "cap" domain reorients upon substrate binding, closing the active site to bulk solvent. Members of the third class lack this additional domain. Herein, we report the 1.9 A X-ray crystal structures of a member of the third subclass, magnesium-dependent phosphatase-1 (MDP-1) both in its unliganded form and with the product analogue, tungstate, bound to the active site. The secondary structure of MDP-1 is similar to that of the "core" domain of other type I and type II HAD members with the addition of a small, 28-amino acid insert that does not close down to exclude bulk solvent in the presence of ligand. In addition, the monomeric oligomeric state of MDP-1 does not allow the participation of a second subunit in the formation and solvent protection of the active site. The binding sites for the phosphate portion of the substrate and Mg(II) cofactor are also similar to those of other HAD members, with all previously observed contacts conserved. Unlike other subclass III HAD members, MDP-1 appears to be equally able to dephosphorylate phosphotyrosine and closed-ring phosphosugars. Modeling of possible substrates in the active site of MDP-1 reveals very few potential interactions with the substrate leaving group. The mapping of conserved residues in sequences of MDP-1 from different eukaryotic organisms reveals that they colocalize to a large region on the surface of the protein outside the active site. This observation combined with the modeling studies suggests that the target of MDP-1 is most likely a phosphotyrosine in an unknown protein rather than a small sugar-based substrate.     

The crystal structure of the yeast Hsp40 Ydj1 complexed with its peptide substrate

The mechanisms by which Hsp40 functions as a molecular chaperone to recognize and bind non-native polypeptides is not understood. We have identified a peptide substrate for Ydj1, a member of the type I Hsp40 from yeast. The structure of the Ydj1 peptide binding fragment and its peptide substrate complex was determined to 2.7 A resolution. The complex structure reveals that Ydj1 peptide binding fragment forms an L-shaped molecule constituted by three domains. The domain I exhibits a similar protein folds as domain III while the domain II contains two Zinc finger motifs. The peptide substrate binds Ydj1 by forming an extra beta strand with domain I of Ydj1. The Leucine residue in the middle of the peptide substrate GWLYEIS inserts its side chain into a hydrophobic pocket formed on the molecular surface of Ydj1 domain I. The Zinc finger motifs located in the Ydj1 domain II are not in the vicinity of peptide substrate binding site.     

SH3P7 Is a Cytoskeleton Adapter Protein and Is Coupled to Signal Transduction from Lymphocyte Antigen Receptors

Lymphocytes respond to antigen receptor engagement with tyrosine phosphorylation of many cellular proteins, some of which have been identified and functionally characterized. Here we describe SH3P7, a novel substrate protein for Src and Syk family kinases. SH3P7 migrates in sodium dodecyl sulfate-polyacrylamide gel electrophoresis as a 55-kDa protein that is preferentially expressed in brain, thymus, and spleen. It contains multiple amino acid sequence motifs, including two consensus tyrosine phosphorylation sites of the YXXP type and one SH3 domain. A region of sequence similarity, which we named SCAD, was found in SH3P7 and three actin-binding proteins. The SCAD region may represent a new type of protein-protein interaction domain that mediates binding to actin. Consistent with this possibility, SH3P7 colocalizes with actin filaments of the cytoskeleton. Altogether, our data implicate SH3P7 as an adapter protein which links antigen receptor signaling to components of the cytoskeleton.     

Human Ca2+ receptor cysteine-rich domain. Analysis of function of mutant and chimeric receptors

The 612-residue extracellular domain of the human Ca(2+) receptor (hCaR) has been speculated to consist of a Venus's-flytrap domain (VFT) and a cysteine-rich domain. We studied the function of the hCaR Cys-rich domain by using mutagenesis and chimera approaches. A chimeric hCaR with the sequence from residues 540-601 replaced by the corresponding sequence from the Fugu CaR remained fully functional. Another chimeric hCaR with the same region of sequence replaced by the corresponding sequence from metabotropic glutamate receptor subtype 1 (mGluR1) still was activated by extracellular Ca(2+) ([Ca(2+)](o)), but its function was severely compromised. Chimeric receptors with the hCaR VFT and mGluR1 seven-transmembrane domain plus C-tail domain retained good response to [Ca(2+)](o) whether the Cys-rich domain was from hCaR or from mGluR1. Mutant hCaR with the Cys-rich domain deleted failed to respond to [Ca(2+)](o), although it was expressed at the cell surface and capable of dimerization. Our results indicate that the hCaR Cys-rich domain plays a critical role in signal transmission from VFT to seven-transmembrane domain. This domain tolerates a significant degree of amino acid substitution and may not be directly involved in the binding of [Ca(2+)](o).     

Sequence-specific DNA recognition by the myb-like domain of the human telomere binding protein TRF1: a model for the protein-DNA complex.

Telomeres consist of tandem arrays of short G-rich sequence motifs packaged by specific DNA binding proteins. In humans the double-stranded telomeric TTAGGG repeats are specifically bound by TRF1 and TRF2. Although telomere binding proteins from evolutionarily distant species are not sequence homologues, they share a Myb-like DNA binding motif. Here we have used gel retardation, primer extension and DNase I footprinting analyses to define the binding site of the isolated Myb-like domain of TRF1 and present a three-dimensional model for its interaction with human telomeric DNA. Our results suggest that the Myb-like domain of TRF1 recognizes a binding site centred on the sequence GGGTTA and that its DNA binding mode is similar to that of the homeodomain-like motifs of the yeast telomere binding protein RAP1. The implications of these findings for recognition of telomeric DNA in general are discussed.     

Pex13p: docking or cargo handling protein?

The Src homology 3 (SH3) domain-containing peroxisomal membrane protein Pex13p is an essential component of the import machinery for matrix proteins and forms a binding site for the peroxisomal targeting type I (PTS1) receptor Pex5p. The interaction between these two proteins can be described as novel in several ways. In the yeasts Saccharomyces cerevisiae and Pichia pastoris, the SH3 domain itself is responsible for the interaction but not via the typical P-x-x-P motifs that are common to SH3 ligands as Pex5p lacks such a motif. Instead, a region of Pex5p containing a W-x-x-x-F/Y motif is crucial for this binding. In mammals, again W-x-x-x-F/Y motifs appear to be important for the interaction but the SH3 domain seems not to be the site for Pex5p binding, this being located in the N-terminus of Pex13p. Despite these differences in the details of the Pex13p-Pex5p interaction, the association of the two proteins is a crucial step in Pex5p-mediated protein import into peroxisomes in both yeasts and mammals.     

1H and 15N NMR resonance assignments and secondary structure of titin type I domains

Titin/connectin is a giant muscle protein with a highly modular architecture consisting ofmultiple repeats of two sequence motifs, named type I and type II. Type I modules have beensuggested to be intracellular members of the fibronectin type III (Fn3) domain family. Alongthe titin sequence they are exclusively present in the region of the molecule located in thesarcomere A-band. This region has been shown to interact with myosin and C-protein. Oneof the most noticeable features of type I modules is that they are particularly rich insemiconserved prolines, since these residues account for about 8% of their sequence. We havedetermined the secondary structure of a representative type I domain (A71) by 15N and 1HNMR. We show that the type I domains of titin have the Fn3 fold as proposed, consisting ofa three- and a four-stranded -sheet. When the two sheets are placed on top of each other toform the -sandwich characteristic of the Fn3 fold, 8 out of 10 prolines are found on the sameside of the molecule and form an exposed hydrophobic patch. This suggests that thesemiconserved prolines might be relevant for the function of type I modules, providing asurface for binding to other A-band proteins. The secondary structure of A71 was structurallyaligned to other extracellular Fn3 modules of known 3D structure. The alignment shows thattitin type I modules have closest similarity to the first Fn3 domain of Drosophila neuroglian.     

The coiled-coil domain is required for HS1 to bind to F-actin and activate Arp2/3 complex

HS1 (hematopoietic lineage cell-specific protein 1), a substrate of protein tyrosine kinases in lymphocytes, binds to F-actin, and promotes Arp2/3 complex-mediated actin polymerization. However, the mechanism for the interaction between HS1 and F-actin has not yet been fully characterized. HS1 contains 3.5 tandem repeats, a coiled-coil region, and an SH3 domain at the C terminus. Unlike cortactin, which is closely related to HS1 and requires absolutely the repeat domain for F-actin binding, an HS1 mutant with deletion of the repeat domain maintains a significant F-actin binding activity. On the other hand, deletion of the coiled-coil region abolished the ability of HS1 to bind to actin filaments and to activate the Arp2/3 complex for actin nucleation and actin branching. Furthermore, a peptide containing the coiled-coil sequence only was sufficient for F-actin binding. Within cells overexpressing green fluorescent protein-tagged HS1 proteins, wild type HS1 co-localizes with cortical F-actin at the cell leading edge, whereas mutants with deletion of either the coiled-coil region or the repeat domain diffuse in the cytoplasm. Immunoprecipitation analysis reveals that the coiled-coil deletion mutant binds poorly to F-actin, whereas the mutant without the repeat domain fails to bind to both Arp2/3 complex and F-actin. These data suggest that the HS1 coiled-coil region acts synergistically with the repeat domain in the modulation of the Arp2/3 complex-mediated actin polymerization.     

X11L2, a new member of the X11 protein family, interacts with Alzheimer's beta-amyloid precursor protein

We screened proteins for interaction with Alzheimer's beta-amyloid precursor protein (APP) and cloned a new member of the X11 protein family, X11L2. The PID/PTB element of X11L2 protein interacted with the intracellular domain of APP by GST binding assay, and in vivo interaction was confirmed by coimmunoprecipitation from cell extracts overexpressing APP and HA-tagged X11L2. This gene encoded 575 amino acids and the deduced amino acid sequence was highly homologous to rat Mint3. Three protein-protein interaction domains, a PID/PTB and two PDZ elements, were conserved among the X11 protein family, and the N-terminal region of X11L2 protein had several putative SH3 binding motifs, PXXP. Unlike other members of the X11 protein family, X11L2 mRNA was expressed in various tissues.     

Human U4/U6 snRNP recycling factor p110: mutational analysis reveals the function of the tetratricopeptide repeat domain in recycling

After each spliceosome cycle, the U4 and U6 snRNAs are released separately and are recycled to the functional U4/U6 snRNP, requiring in the mammalian system the U6-specific RNA binding protein p110 (SART3). Its domain structure is made up of an extensive N-terminal domain with at least seven tetratricopeptide repeat (TPR) motifs, followed by two RNA recognition motifs (RRMs) and a highly conserved C-terminal sequence of 10 amino acids. Here we demonstrate under in vitro recycling conditions that U6-p110 is an essential splicing factor. Recycling activity requires both the RRMs and the TPR domain but not the highly conserved C-terminal sequence. For U6-specific RNA binding, the two RRMs with some flanking regions are sufficient. Yeast two-hybrid assays reveal that p110 interacts through its TPR domain with the U4/U6-specific 90K protein, indicating a specific role of the TPR domain in spliceosome recycling. On the 90K protein, a short internal region (amino acids 416 to 550) suffices for the interaction with p110. Together, these data suggest a model whereby p110 brings together U4 and U6 snRNAs through both RNA-protein and protein-protein interactions.     

Conserved bipartite motifs in yeast eIF5 and eIF2Bepsilon, GTPase-activating and GDP-GTP exchange factors in translation initiation, mediate binding to their common substrate eIF2

In the initiation phase of eukaryotic translation, eIF5 stimulates the hydrolysis of GTP bound to eIF2 in the 40S ribosomal pre-initiation complex, and the resultant GDP on eIF2 is replaced with GTP by the complex nucleotide exchange factor, eIF2B. Bipartite motifs rich in aromatic and acidic residues are conserved at the C-termini of eIF5 and the catalytic (epsilon) subunit of eIF2B. Here we show that these bipartite motifs are important for the binding of these factors, both in vitro and in vivo, to the beta subunit of their common substrate eIF2. We also find that three lysine-rich boxes in the N-terminal segment of eIF2beta mediate the binding of eIF2 to both eIF5 and eIF2B. Thus, eIF5 and eIF2Bepsilon employ the same sequence motif to facilitate interaction with the same segment of their common substrate. In agreement with this, archaea appear to lack eIF5, eIF2B and the lysine-rich binding domain for these factors in their eIF2beta homolog. The eIF5 bipartite motif is also important for its interaction with the eIF3 complex through the NIP1-encoded subunit of eIF3. Thus, the bipartite motif in eIF5 appears to be multifunctional, stimulating its recruitment to the 40S pre-initiation complex through interaction with eIF3 in addition to binding of its substrate eIF2.     

Syndapin I and endophilin I bind overlapping proline-rich regions of dynamin I: role in synaptic vesicle endocytosis

Dynamin I mediates vesicle fission during synaptic vesicle endocytosis (SVE). Its proline-rich domain (PRD) binds the Src-homology 3 (SH3) domain of a subset of proteins that can deform membranes. Syndapin I, amphiphysin I, and endophilin I are its major partners implicated in SVE. Syndapin binding is controlled by phosphorylation at Ser-774 and Ser-778 in the dynamin phospho-box. We now define syndapin and endophilin-binding sites by peptide competition and site-directed mutagenesis. Both bound the same region of the dynamin PRD and both exhibited unusual bidirectional binding modes around core PxxP motifs, unlike amphiphysin which employed a class II binding mode. Endophilin binds to tandem PxxP motifs in the sequence (778)SPTPQRRAPAVPPARPGSR(796) in dynamin, with SPTPQ being an overhang sequence. In contrast, syndapin binding involves two components in the region (772)RRSPTSSPTPQRRAPAVPPARPGSR(796). It required a single PxxP core and a non-PxxP N-terminally anchored extension which bridges the phospho-box and may contribute to binding specificity and affinity. Syndapin binding is exquisitely sensitive to the introduction of negative charges almost anywhere along this region, explaining why it is a highly tuned phospho-sensor. Over-expression of dynamin point mutants that fail to bind syndapin or endophilin inhibit SVE in cultured neurons. Due to overlapping binding sites the interactions between dynamin and syndapin or endophilin were mutually exclusive. Because syndapin acts as a phospho-sensor, this supports its role in depolarization-induced SVE at the synapse, which involves dynamin dephosphorylation. We propose syndapin and endophilin function either at different stages during SVE or in mechanistically distinct types of SVE.     

A motif conserved among the type I restriction-modification enzymes and antirestriction proteins: a possible basis for mechanism of action of plasmid-encoded antirestriction functions.

Antirestriction proteins Ard encoded by some self-transmissible plasmids specifically inhibit restriction by members of all three families of type I restriction-modification (R-M) systems in E.coli. Recently, we have identified the amino acid region, 'antirestriction' domain, that is conserved within different plasmid and phage T7-encoded antirestriction proteins and may be involved in interaction with the type I R-M systems. In this paper we demonstrate that this amino acid sequence shares considerable similarity with a well-known conserved sequence (the Argos repeat) found in the DNA sequence specificity (S) polypeptides of type I systems. We suggest that the presence of these similar motifs in restriction and antirestriction proteins may give a structural basis for their interaction and that the antirestriction action of Ard proteins may be a result of the competition between the 'antirestriction' domains of Ard proteins and the similar conserved domains of the S subunits that are believed to play a role in the subunit assembly of type I R-M systems.     

Interactions between nebulin-like motifs and thin filament regulatory proteins

Nebulin (600-900 kDa) and nebulette (107-109 kDa) are two homologous thin filament-associated proteins in skeletal and cardiac muscles, respectively. Both proteins are capped with a unique region at the amino terminus as well as a serine-rich linker domain and SH3 domains at the COOH terminus. Their significant size difference is attributed to the length of the central region wherein both proteins are primarily composed of approximately 35 amino acid repeats termed nebulin-like repeats or motifs. These motifs are marked by a conserved SXXXY sequence and high affinity binding to F-actin. To further characterize the effects that nebulin-like proteins may have on the striated muscle thin filament, we have cloned, expressed, and purified a five-motif chicken nebulette fragment and tested its interaction with the thin filament regulatory proteins. Both tropomyosin and troponin T individually bound the nebulette fragment, although the affinity of this interaction was significantly increased when tropomyosin-troponin T was tested as a binary complex. The addition of troponin I to the tropomyosin-troponin T complex decreased the binding to the nebulette fragment, indicating an involvement of the conserved T2 region of troponin T in this interaction. F-actin cosedimentation demonstrated that the nebulette fragment was able to significantly increase the affinity of the tropomyosin-troponin assembly for F-actin. The relationships provide a means for nebulin-like motifs to participate in the allosteric regulation of striated muscle contraction.     

Vacuolar H+-ATPase binding to microfilaments: regulation in response to phosphatidylinositol 3-kinase activity and detailed characterization of the actin-binding site in subunit B

Vacuolar H(+)-ATPase (V-ATPase) binds microfilaments, and that interaction may be mediated by an actin binding domain in subunit B of the enzyme. To test for possible physiologic functions of the actin binding activity of V-ATPase, early responses of resorbing osteoclasts to inhibition of phosphatidylinositol 3-kinase activity by wortmannin and LY294002 were examined. Rapid co-localization between V-ATPase and F-actin was demonstrated by immunocytochemistry, and corresponding association between V-ATPase and F-actin in immunoprecipitations and pelleting assays was detected. This response was reversed as osteoclasts recovered resorptive activity after inhibitors were removed. By expressing and characterizing fusion proteins containing segments of the actin-binding amino-terminal regions of the B subunits of V-ATPase, we mapped the actin-binding site to a 44-amino acid domain. An 11-amino acid segment with a sequence similar to the actin-binding site of human profilin I was detected within this region. 13-Mers containing these profilin-like segments bound actin in fluorescent anisotropy studies and competed with profilin for binding to actin. Using site-directed mutagenesis, the 11-amino acid profilin-like actin-binding motifs (amino acids 49-59 of B1 and 55-65 of B2) were replaced with an 11-amino acid spacer with a sequence based on the homologous sequence from subunit B of Pyrococcus horikoshii, an organism that lacks an actin cytoskeleton. These substitutions eliminated the actin-binding activity of the B subunit fusion proteins. In summary, binding between V-ATPase and F-actin in osteoclasts occurs in response to blocking phosphatidylinositol 3-kinase activity. This response was fully reversible. The actin binding activities of the B subunits of V-ATPase required 11-amino acid actin-binding motifs that are similar in sequence to the actin-binding site of mammalian profilin I.     

Cleavage of von Willebrand factor requires the spacer domain of the metalloprotease ADAMTS13

ADAMTS13 consists of a reprolysin-type metalloprotease domain followed by a disintegrin domain, a thrombospondin type 1 motif (TSP1), Cys-rich and spacer domains, seven more TSP1 motifs, and two CUB domains. ADAMTS13 limits platelet accumulation in microvascular thrombi by cleaving the Tyr1605-Met1606 bond in von Willebrand factor, and ADAMTS13 deficiency causes a lethal syndrome, thrombotic thrombocytopenic purpura. ADAMTS13 domains required for substrate recognition were localized by the characterization of recombinant deletion mutants. Constructs with C-terminal His6 and V5 epitopes were expressed by transient transfection of COS-7 cells or in a baculovirus system. No association with extracellular matrix or cell surface was detected for any ADAMTS13 variant by immunofluorescence microscopy or chemical modification. Both plasma and recombinant full-length ADAMTS13 cleaved von Willebrand factor subunits into two fragments of 176 kDa and 140 kDa. Recombinant ADAMTS13 was divalent metal ion-dependent and was inhibited by IgG from a patient with idiopathic thrombotic thrombocytopenic purpura. ADAMTS13 that was truncated after the metalloprotease domain, the disintegrin domain, the first TSP1 repeat, or the Cys-rich domain was not able to cleave von Willebrand factor, whereas addition of the spacer region restored protease activity. Therefore, the spacer region is necessary for normal ADAMTS13 activity toward von Willebrand factor, and the more C-terminal TSP1 and CUB domains are dispensable in vitro.     

Role of the Rab3A-binding domain in targeting of rabphilin-3A to vesicle membranes of PC12 cells.

Rab3A is a small GTPase implicated in the docking of secretory vesicles in neuroendocrine cells. A putative downstream target for Rab3A, rabphilin-3A, is located exclusively on secretory vesicle membranes. It contains near its C terminus two C2 domains that bind Ca2+ in a phospholipid-dependent manner and an N-terminal, Rab3A-binding domain that includes a Cys-rich region. We have determined that the Cys-rich domain binds two Zn2+ ions and is necessary but not sufficient for efficient binding of rabphilin to Rab3A. A minimal Rab3A-binding domain consists of residues 45 to 170 of rabphilin. HA1-tagged Rab3A and a green fluorescent protein (GFP)-rabphilin fusion were used to examine the roles of Rab3A and of rabphilin domains in the subcellular localization of these proteins. A Rab3A mutant (T54A) that does not bind rabphifin in vitro colocalized with the GFP-rabphilin fusion, indicating that Rab3A targeting is independent of its interaction with rabphilin. Deletion of the C2 domains of rabphilin reduced membrane association of GFP-rabphilin but did not cause mistargeting of the membrane-associated fraction. However, disruption of the zinc fingers, which drastically reduced Rab3A binding, did not reduce membrane association. These results suggest that the C2 domains are required for efficient membrane attachment of rabphilin in PC12 cells and that Rab3A binding may act to target the protein to the correct membrane.     

The general definition of the p97/valosin-containing protein (VCP)-interacting motif (VIM) delineates a new family of p97 cofactors

Cellular functions of the essential, ubiquitin-selective AAA ATPase p97/valosin-containing protein (VCP) are controlled by regulatory cofactors determining substrate specificity and fate. Most cofactors bind p97 through a ubiquitin regulatory X (UBX) or UBX-like domain or linear sequence motifs, including the hitherto ill defined p97/VCP-interacting motif (VIM). Here, we present the new, minimal consensus sequence RX(5)AAX(2)R as a general definition of the VIM that unites a novel family of known and putative p97 cofactors, among them UBXD1 and ZNF744/ANKZF1. We demonstrate that this minimal VIM consensus sequence is necessary and sufficient for p97 binding. Using NMR chemical shift mapping, we identified several residues of the p97 N-terminal domain (N domain) that are critical for VIM binding. Importantly, we show that cellular stress resistance conferred by the yeast VIM-containing cofactor Vms1 depends on the physical interaction between its VIM and the critical N domain residues of the yeast p97 homolog, Cdc48. Thus, the VIM-N domain interaction characterized in this study is required for the physiological function of Vms1 and most likely other members of the newly defined VIM family of cofactors.