Two auxin-responsive domains interact positively to induce expression of the early indoleacetic acid-inducible gene PS-IAA4/5.

The plant growth hormone indole-3-acetic acid (IAA) transcriptionally activates expression of several genes in plants. We have previously identified a 164-bp promoter region (-318 to -154) in the PS-IAA4/5 gene that confers IAA inducibility. Linker-scanning mutagenesis across the region has identified two positive domains: domain A (48 bp; -203 to -156) and domain B (44 bp; -299 to -256), responsible for transcriptional activation of PS-IAA4/5 by IAA. Domain A contains the highly conserved sequence 5'-TGTCCCAT-3' found among various IAA-inducible genes and behaves as the major auxin-responsive element. Domain B functions as an enhancer element which may also contain a less efficient auxin-responsive element. The two domains act cooperatively to stimulate transcription; however, tetramerization of domain A or B compensates for the loss of A or B function. The two domains can also mediate IAA-induced transcription from the heterologous cauliflower mosaic virus 35S promoter (-73 to +1). In vivo competition experiments with icosamers of domain A or B show that the domains interact specifically and with different affinities to low abundance, positive transcription factor(s). A model for transcriptional activation of PS-IAA4/5 by IAA is discussed.

The Development of Cell Processes Induced by tau Protein Requires Phosphorylation of Serine 262 and 356 in the Repeat Domain and Is Inhibited by Phosphorylation in the Proline-rich Domains

The differentiation of neurons and the outgrowth of neurites depends on microtubule-associated proteins such as tau protein. To study this process, we have used the model of Sf9 cells, which allows efficient transfection with microtubule-associated proteins (via baculovirus vectors) and observation of the resulting neurite-like extensions. We compared the phosphorylation of tau23 (the embryonic form of human tau) with mutants in which critical phosphorylation sites were deleted by mutating Ser or Thr residues into Ala. One can broadly distinguish two types of sites, the KXGS motifs in the repeats (which regulate the affinity of tau to microtubules) and the SP or TP motifs in the domains flanking the repeats (which contain epitopes for antibodies diagnostic of Alzheimer’s disease). Here we report that both types of sites can be phosphorylated by endogenous kinases of Sf9 cells, and that the phosphorylation pattern of the transfected tau is very similar to that of neurons, showing that Sf9 cells can be regarded as an approximate model for the neuronal balance between kinases and phosphatases. We show that mutations in the repeat domain and in the flanking domains have opposite effects. Mutations of KXGS motifs in the repeats (Ser262, 324, and 356) strongly inhibit the outgrowth of cell extensions induced by tau, even though this type of phosphorylation accounts for only a minor fraction of the total phosphate. This argues that the temporary detachment of tau from microtubules (by phosphorylation at KXGS motifs) is a necessary condition for establishing cell polarity at a critical point in space or time. Conversely, the phosphorylation at SP or TP motifs represents the majority of phosphate (>80%); mutations in these motifs cause an increase in cell extensions, indicating that this type of phosphorylation retards the differentiation of the cells.

Mutations in Drosophila Enabled and Rescue by Human Vasodilator-stimulated Phosphoprotein (VASP) Indicate Important Functional Roles for Ena/VASP Homology Domain 1 (EVH1) and EVH2 Domains

Drosophila Enabled (Ena) was initially identified as a dominant genetic suppressor of mutations in the Abelson tyrosine kinase and, more recently, as a member of the Ena/human vasodilator-stimulated phosphoprotein (VASP) family of proteins. We have used genetic, biochemical, and cell biological approaches to demonstrate the functional relationship between Ena and human VASP. In addition, we have defined the roles of Ena domains identified as essential for its activity in vivo. We have demonstrated that VASP rescues the embryonic lethality associated with loss of Ena function in Drosophila and have shown that Ena, like VASP, is associated with actin filaments and focal adhesions when expressed in cultured cells. To define sequences that are central to Ena function, we have characterized the molecular lesions present in two lethal ena mutant alleles that affected the Ena/VASP homology domain 1 (EVH1) and EVH2. A missense mutation that resulted in an amino acid substitution in the EVH1 domain eliminated in vitro binding of Ena to the cytoskeletal protein zyxin, a previously reported binding partner of VASP. A nonsense mutation that resulted in a C-terminally truncated Ena protein lacking the EVH2 domain failed to form multimeric complexes and exhibited reduced binding to zyxin and the Abelson Src homology 3 domain. Our analysis demonstrates that Ena and VASP are functionally homologous and defines the conserved EVH1 and EVH2 domains as central to the physiological activity of Ena.

Locking in alternate conformations of the integrin αLβ2 I domain with disulfide bonds reveals functional relationships among integrin domains

We used integrin αLβ2 heterodimers containing I domains locked open (active) or closed (inactive) with disulfide bonds to investigate regulatory interactions among domains in integrins. mAbs to the αL I domain and β2 I-like domain inhibit adhesion of wild-type αLβ2 to intercellular adhesion molecule-1. However, with αLβ2 containing a locked open I domain, mAbs to the I domain were subdivided into subsets (i) that did not inhibit, and thus appear to inhibit by favoring the closed conformation, and (ii) that did inhibit, and thus appear to bind to the ligand binding site. Furthermore, αLβ2 containing a locked open I domain was completely resistant to inhibition by mAbs to the β2 I-like domain, but became fully susceptible to inhibition after disulfide reduction with DTT. This finding suggests that the I-like domain indirectly contributes to ligand binding by regulating opening of the I domain in wild-type αLβ2. Conversely, locking the I domain closed partially restrained conformational change of the I-like domain by Mn2+, as measured with mAb m24, which we map here to the β2 I-like domain. By contrast, locking the I domain closed or open did not affect constitutive or Mn2+-induced exposure of the KIM127 epitope in the β2 stalk region. Furthermore, locked open I domains, in αLβ2 complexes or expressed in isolation on the cell surface, bound to intercellular adhesion molecule-1 equivalently in Mg2+ and Mn2+. These results suggest that Mn2+ activates αLβ2 by binding to a site other than the I domain, most likely the I-like domain of β2.

Elongation factor TFIIS contains three structural domains: solution structure of domain II.

Transcription elongation by RNA polymerase II is regulated by the general elongation factor TFIIS. This factor stimulates RNA polymerase II to transcribe through regions of DNA that promote the formation of stalled ternary complexes. Limited proteolytic digestion showed that yeast TFIIS is composed of three structural domains, termed I, II, and III. The two C-terminal domains (II and III) are required for transcription activity. The structure of domain III has been solved previously by using NMR spectroscopy. Here, we report the NMR-derived structure of domain II: a three-helix bundle built around a hydrophobic core composed largely of three tyrosines protruding from one face of the C-terminal helix. The arrangement of known inactivating mutations of TFIIS suggests that two surfaces of domain II are critical for transcription activity.

Identifying functional domains within terpene cyclases using a domain-swapping strategy.

Cyclic terpenes and terpenoids are found throughout nature. They comprise an especially important class of compounds from plants that mediate plant- environment interactions, and they serve as pharmaceutical agents with antimicrobial and anti-tumor activities. Molecular comparisons of several terpene cyclases, the key enzymes responsible for the multistep cyclization of C10, C15, and C20 allylic diphosphate substrates, have revealed a striking level of sequence similarity and conservation of exon position and size within the genes. Functional domains responsible for a terminal enzymatic step were identified by swapping regions approximating exons between a Nicotiana tabacum 5-epi-aristolochene synthase (TEAS) gene and a Hyoscyamus muticus vetispiradiene synthase (HVS) gene and by characterization of the resulting chimeric enzymes expressed in bacteria. While exon 4 of the TEAS gene conferred specificity for the predominant reaction products of the tobacco enzyme, exon 6 of the HVS gene conferred specificity for the predominant reaction products of the Hyoscyamus enzyme. Combining these two functional domains of the TEAS and HVS genes resulted in a novel enzyme capable of synthesizing reaction products reflective of both parent enzymes. The relative ratio of the TEAS and HVS reaction products was also influenced by the source of exon 5 present in the new chimeric enzymes. The association of catalytic activities with conserved but separate exonic domains suggests a general means for generating additional novel terpene cyclases.

The multidomain protein Trio binds the LAR transmembrane tyrosine phosphatase, contains a protein kinase domain, and has separate rac-specific and rho-specific guanine nucleotide exchange factor domains.

rho-like GTP binding proteins play an essential role in regulating cell growth and actin polymerization. These molecular switches are positively regulated by guanine nucleotide exchange factors (GEFs) that promote the exchange of GDP for GTP. Using the interaction-trap assay to identify candidate proteins that bind the cytoplasmic region of the LAR transmembrane protein tyrosine phosphatase (PT-Pase), we isolated a cDNA encoding a 2861-amino acid protein termed Trio that contains three enzyme domains: two functional GEF domains and a protein serine/threonine kinase (PSK) domain. One of the Trio GEF domains (Trio GEF-D1) has rac-specific GEF activity, while the other Trio GEF domain (Trio GEF-D2) has rho-specific activity. The C-terminal PSK domain is adjacent to an Ig-like domain and is most similar to calcium/calmodulin-dependent kinases, such as smooth muscle myosin light chain kinase which similarly contains associated Ig-like domains. Near the N terminus, Trio has four spectrin-like repeats that may play a role in intracellular targeting. Northern blot analysis indicates that Trio has a broad tissue distribution. Trio appears to be phosphorylated only on serine residues, suggesting that Trio is not a LAR substrate, but rather that it forms a complex with LAR. As the LAR PTPase localizes to the ends of focal adhesions, we propose that LAR and the Trio GEF/PSK may orchestrate cell-matrix and cytoskeletal rearrangements necessary for cell migration.

Disease specificity of kinase domains: the src-encoded catalytic domain converts erbB into a sarcoma oncogene.

src and erbB are two tyrosine kinase-encoding oncogenes carried by retroviruses, which have distinct disease specificities. The former induces predominantly sarcomas, and the latter, leukemias. Src and ErbB have similar catalytic domains but have very different regulatory domains. A wealth of information exists concerning how different regulatory domains [Src homology 2 (SH2) and SH3 domains and autophosphorylation sites] control substrate and disease specificities. Whether the catalytic domain helps determine these specificities remains to be explored. Here we show that the Src catalytic domain is enzymatically active when substituted into the ErbB backbone and interacts with the ErbB regulatory domain. This ErbB/Src chimera displays autophosphorylation and substrate phosphorylation patterns different from those of both Src and ErbB. Neither SH2 and SH3 nor autophosphorylation sites are required for the Src catalytic domain to exert its fibroblast transforming ability. Most significantly, the catalytic domain can convert erbB from a leukemogenic oncogene into a sarcomagenic oncogene, suggesting that the leukemogenic determinants in part reside within the ErbB catalytic domain.

Genetic analysis of the mouse X inactivation center defines an 80-kb multifunction domain

Dosage compensation in mammals occurs by X inactivation, a silencing mechanism regulated in cis by the X inactivation center (Xic). In response to developmental cues, the Xic orchestrates events of X inactivation, including chromosome counting and choice, initiation, spread, and establishment of silencing. It remains unclear what elements make up the Xic. We previously showed that the Xic is contained within a 450-kb sequence that includes Xist, an RNA-encoding gene required for X inactivation. To characterize the Xic further, we performed deletional analysis across the 450-kb region by yeast-artificial-chromosome fragmentation and phage P1 cloning. We tested Xic deletions for cis inactivation potential by using a transgene (Tg)-based approach and found that an 80-kb subregion also enacted somatic X inactivation on autosomes. Xist RNA coated the autosome but skipped the Xic Tg, raising the possibility that X chromosome domains escape inactivation by excluding Xist RNA binding. The autosomes became late-replicating and hypoacetylated on histone H4. A deletion of the Xist 5′ sequence resulted in the loss of somatic X inactivation without abolishing Xist expression in undifferentiated cells. Thus, Xist expression in undifferentiated cells can be separated genetically from somatic silencing. Analysis of multiple Xic constructs and insertion sites indicated that long-range Xic effects can be generalized to different autosomes, thereby supporting the feasibility of a Tg-based approach for studying X inactivation.

A dimeric crystal structure for the N-terminal two domains of intercellular adhesion molecule-1

The 3.0-Å structure of a 190-residue fragment of intercellular adhesion molecule-1 (ICAM-1, CD54) reveals two tandem Ig-superfamily (IgSF) domains. Each of two independent molecules dimerizes identically with a symmetry-related molecule over a hydrophobic interface on the BED sheet of domain 1, in agreement with dimerization of ICAM-1 on the cell surface. The residues that bind to the integrin LFA-1 are well oriented for bivalent binding in the dimer, with the critical Glu-34 residues pointing away from each other on the periphery. Residues that bind to rhinovirus are in the flexible BC and FG loops at the tip of domain 1, and these and the upper half of domain 1 are well exposed in the dimer for docking to virus. By contrast, a residue important for binding to Plasmodium falciparum-infected erythrocytes is in the dimer interface. The presence of A′ strands in both domains 1 and 2, conserved hydrogen bonds at domain junctions, and elaborate hydrogen bond networks around the key integrin binding residues in domain 1 make these domains suited to resist tensile forces during adhesive interactions. A subdivision of the intermediate (I) set of IgSF domains is proposed in which domain 1 of ICAM-1 and previously described I set domains belong to the I1 set and domain 2 of ICAM-1, ICAM-2, and vascular cell adhesion molecule-1 belong to the I2 set.

The structure of the two amino-terminal domains of human ICAM-1 suggests how it functions as a rhinovirus receptor and as an LFA-1 integrin ligand

The normal function of human intercellular adhesion molecule-1 (ICAM-1) is to provide adhesion between endothelial cells and leukocytes after injury or stress. ICAM-1 binds to leukocyte function-associated antigen (LFA-1) or macrophage-1 antigen (Mac-1). However, ICAM-1 is also used as a receptor by the major group of human rhinoviruses and is a catalyst for the subsequent viral uncoating during cell entry. The three-dimensional atomic structure of the two amino-terminal domains (D1 and D2) of ICAM-1 has been determined to 2.2-Å resolution and fitted into a cryoelectron microscopy reconstruction of a rhinovirus–ICAM-1 complex. Rhinovirus attachment is confined to the BC, CD, DE, and FG loops of the amino-terminal Ig-like domain (D1) at the end distal to the cellular membrane. The loops are considerably different in structure to those of human ICAM-2 or murine ICAM-1, which do not bind rhinoviruses. There are extensive charge interactions between ICAM-1 and human rhinoviruses, which are mostly conserved in both major and minor receptor groups of rhinoviruses. The interaction of ICAMs with LFA-1 is known to be mediated by a divalent cation bound to the insertion (I)-domain on the α chain of LFA-1 and the carboxyl group of a conserved glutamic acid residue on ICAMs. Domain D1 has been docked with the known structure of the I-domain. The resultant model is consistent with mutational data and provides a structural framework for the adhesion between these molecules.

The C-terminal SET domains of ALL-1 and TRITHORAX interact with the INI1 and SNR1 proteins, components of the SWI/SNF complex

The ALL-1 gene was discovered by virtue of its involvement in human acute leukemia. Its Drosophila homolog trithorax (trx) is a member of the trx-Polycomb gene family, which maintains correct spatial expression of the Antennapedia and bithorax complexes during embryogenesis. The C-terminal SET domain of ALL-1 and TRITHORAX (TRX) is a 150-aa motif, highly conserved during evolution. We performed yeast two hybrid screening of Drosophila cDNA library and detected interaction between a TRX polypeptide spanning SET and the SNR1 protein. SNR1 is a product of snr1, which is classified as a trx group gene. We found parallel interaction in yeast between the SET domain of ALL-1 and the human homolog of SNR1, INI1 (hSNF5). These results were confirmed by in vitro binding studies and by demonstrating coimmunoprecipitation of the proteins from cultured cells and/or transgenic flies. Epitope-tagged SNR1 was detected at discrete sites on larval salivary gland polytene chromosomes, and these sites colocalized with around one-half of TRX binding sites. Because SNR1 and INI1 are constituents of the SWI/SNF complex, which acts to remodel chromatin and consequently to activate transcription, the interactions we observed suggest a mechanism by which the SWI/SNF complex is recruited to ALL-1/trx targets through physical interactions between the C-terminal domains of ALL-1 and TRX and INI1/SNR1.

Structure and function of the Bacillus hybrid enzyme GluXyn-1: Native-like jellyroll fold preserved after insertion of autonomous globular domain

The 1,3–1,4-β-glucanase from Bacillus macerans (wtGLU) and the 1,4-β-xylanase from Bacillus subtilis (wtXYN) are both single-domain jellyroll proteins catalyzing similar enzymatic reactions. In the fusion protein GluXyn-1, the two proteins are joined by insertion of the entire XYN domain into a surface loop of cpMAC-57, a circularly permuted variant of wtGLU. GluXyn-1 was generated by protein engineering methods, produced in Escherichia coli and shown to fold spontaneously and have both enzymatic activities at wild-type level. The crystal structure of GluXyn-1 was determined at 2.1 Å resolution and refined to R = 17.7% and R(free) = 22.4%. It shows nearly ideal, native-like folding of both protein domains and a small, but significant hinge bending between the domains. The active sites are independent and accessible explaining the observed enzymatic activity. Because in GluXyn-1 the complete XYN domain is inserted into the compact folding unit of GLU, the wild-type-like activity and tertiary structure of the latter proves that the folding process of GLU does not depend on intramolecular interactions that are short-ranged in the sequence. Insertion fusions of the GluXyn-1 type may prove to be an easy route toward more stable bifunctional proteins in which the two parts are more closely associated than in linear end-to-end protein fusions.

Defining the domains of type I collagen involved in heparin- binding and endothelial tube formation

Cell surface heparan sulfate proteoglycan (HSPG) interactions with type I collagen may be a ubiquitous cell adhesion mechanism. However, the HSPG binding sites on type I collagen are unknown. Previously we mapped heparin binding to the vicinity of the type I collagen N terminus by electron microscopy. The present study has identified type I collagen sequences used for heparin binding and endothelial cell–collagen interactions. Using affinity coelectrophoresis, we found heparin to bind as follows: to type I collagen with high affinity (Kd ≈ 150 nM); triple-helical peptides (THPs) including the basic N-terminal sequence α1(I)87–92, KGHRGF, with intermediate affinities (Kd ≈ 2 μM); and THPs including other collagenous sequences, or single-stranded sequences, negligibly (Kd ≫ 10 μM). Thus, heparin–type I collagen binding likely relies on an N-terminal basic triple-helical domain represented once within each monomer, and at multiple sites within fibrils. We next defined the features of type I collagen necessary for angiogenesis in a system in which type I collagen and heparin rapidly induce endothelial tube formation in vitro. When peptides, denatured or monomeric type I collagen, or type V collagen was substituted for type I collagen, no tubes formed. However, when peptides and type I collagen were tested together, only the most heparin-avid THPs inhibited tube formation, likely by influencing cell interactions with collagen–heparin complexes. Thus, induction of endothelial tube morphogenesis by type I collagen may depend upon its triple-helical and fibrillar conformations and on the N-terminal heparin-binding site identified here.

A model for the structure of the P domains in the subtilisin-like prohormone convertases

The proprotein convertases are a family of at least seven calcium-dependent endoproteases that process a wide variety of precursor proteins in the secretory pathway. All members of this family possess an N-terminal proregion, a subtilisin-like catalytic module, and an additional downstream well-conserved region of ≈150 amino acid residues, the P domain, which is not found in any other subtilase. The pro and catalytic domains cannot be expressed in the absence of the P domains; their thermodynamic instability may be attributable to the presence of large numbers of negatively charged Glu and Asp side chains in the substrate binding region for recognition of multibasic residue cleavage sites. Based on secondary structure predictions, we here propose that the P domains consist of 8-stranded β-barrels with well-organized inner hydrophobic cores, and therefore are independently folded components of the proprotein convertases. We hypothesize further that the P domains are integrated through strong hydrophobic interactions with the catalytic domains, conferring structural stability and regulating the properties and activity of the convertases. A molecular model of these interdomain interactions is proposed in this report.