Domain Exchange Research Articles

CD96 Interaction with CD155 via Its First Ig-like Domain Is Modulated by Alternative Splicing or Mutations in Distal Ig-like Domains

The adhesion receptor CD96 (TACTILE) is a transmembrane glycoprotein possessing three extracellular immunoglobulin-like domains. Among peripheral blood cells, CD96 is expressed on T cells as well as NK cells and a subpopulation of B cells. A possible function of this receptor in NK cell-mediated killing activities was suggested recently. Moreover, CD96 was described as a tumor marker for T-cell acute lymphoblastic leukemia and acute myeloid leukemia. CD96 binds to CD155 (poliovirus receptor) and nectin-1, an adhesion receptor related to CD155. Here we report that human but not mouse CD96 is expressed in two splice variants possessing either an I-like (variant 1) or V-like (variant 2) second domain. With the notable exception of an AML tumor sample, variant 2 predominates in all the CD96-expressing cell types and tissues examined. Using chimeric human/murine CD96 receptors, we show that the interaction with its ligands is mediated via the outermost V-like domain. In contrast to mouse, however, the binding of human CD96 to CD155 is sensitive to the characteristics of the two downstream domains. This is illustrated by a significantly weaker CD96/CD155 interaction mediated by variant 1 when compared with variant 2. Moreover, recent evidence suggested that mutations in human CD96 correlate with the occurrence of a rare form of trigonocephaly. One such mutation causing a single amino acid exchange in the third domain of human CD96 decreased the capacity of both variants to bind to CD155 considerably, suggesting that a CD96-driven adhesion to CD155 may be crucial in developmental processes.

Phase-Field Simulation of Ferroelectric Domain Microstructure Changes in BaTiO<SUB>3</SUB>

In this article, we focus on the ferroelectric domain microstructure changes followed by the structural phase transition from cubic to tetragonal phase in BaTiO3, and its morphological developments are simulated on the basis of the phase-field method. The circuit structure of polarization moments of ferroelectric domains including twin defects is simulated, and the domain morphology is controlled by both the electric dipole-dipole interaction among polarization moments and the elastic interaction among domains with different tetragonal distortion. The ferroelectric domain exchange induced by external electric field is also simulated, then the dielectric property, i.e., the polarization hysteresis curve, is calculated by integrating all the x components of polarization moment vector over the microstructure. Furthermore, the simulation of the reversible ferroelectric domain switching, which is a new phenomenon recently discovered by Ren, is also simulated as an advanced application of the present simulation model. [doi:10.2320/matertrans.MC200806]

Genetic Hypervariability in Two Distinct Deuterostome Telomerase Reverse Transcriptase Genes and their Early Embryonic Functions

Functional proteins of complex eukaryotes within the same species are rather invariant. A single catalytic component of telomerase TERT is essential for an active telomerase complex that maintains telomeres. Surprisingly, we have identified two paralogous SpTERT-L and SpTERT-S genes with novel domains in Strongylocentrotus purpuratus (purple sea urchin). The SpTERT-S and SpTERT-L genes were differentially expressed throughout embryogenesis. An unusual germline nucleotide substitution and amino acid variation was evident in these TERTs. The hypervariability of SpTERT-S haplotypes among different individuals reached unprecedented levels of pi > 0.2 in exon 11 region. The majority of nucleotide changes observed led to nonsynonymous substitutions creating novel amino acids and motifs, suggesting unusual positive selection and rapid evolution. The majority of these variations were in domains involved in binding of SpTERT to its RNA component. Despite hypervariability at protein level, SpTERT-S conferred telomerase activity, and its suppression during early embryogenesis led to arrest at late mesenchymal blastula. Domain exchange and embryo rescue experiments suggested that SpTERT may have evolved functions unrelated to classic telomerase activity. We suggest that telomerase has a specific and direct function that is essential for integration of early polarity signals that lead to gastrulation. Identification of these unique hypervariable telomerases also suggests presence of a diversity generation mechanism that inculcates hypervariable telomerases and telomere lengths in germline.

Intramolecular Activation Mechanism of the Dictyostelium LRRK2 Homolog Roco Protein GbpC

GbpC is a large multidomain protein involved in cGMP-mediated chemotaxis in the cellular slime mold Dictyostelium discoideum. GbpC belongs to the Roco family of proteins that often share a central core region, consisting of leucine-rich repeats, a Ras domain (Roc), a Cor domain, and a MAPKKKinase domain. In addition to this core, GbpC contains a RasGEF domain and two cGMP-binding domains. Here, we report on an intramolecular signaling cascade of GbpC. In vitro, the RasGEF domain of GbpC specifically accelerates the GDP/GTP exchange of the Roc domain. Moreover, cGMP binding to GbpC strongly stimulates the binding of GbpC to GTP-agarose, suggesting cGMP-stimulated GDP/GTP exchange at the Roc domain. The function of the protein in vivo was investigated by rescue analysis of the chemotactic defect of gbpC null cells. Mutants that lack a functional guanine exchange factor (GEF), Roc, or kinase domain are inactive in vivo. Together, the results suggest a four-step intramolecular activation mechanism of the Roco protein GbpC: cGMP binding to the cyclic nucleotide-binding domains, activation of the GEF domain, GDP/GTP exchange of Roc, and activation of the MAPKKK domain.

Aberrant intracellular trafficking of a variant B glycosyltransferase

Little is known about the mechanism by which amino acid polymorphisms outside the catalytically active cleft of ABO glycosyltransferases cause weak ABO phenotypes. Extensive ABO phenotyping and genotyping were performed to classify the blood of a healthy blood group O donor with weak isoagglutinins. ABO antigen and glycosyltransferase expression profiles were then studied in eukaryotic transfection experiments, and the topology of ABO glycosyltransferase was analyzed. The donor's red blood cells were retyped as A(weak), and his serum contained weakly reactive anti-A and anti-B. Sequence analysis revealed two novel ABO alleles. A donor splice-site mutation detected at the exon 6/intron 6 junction of an ABO*A101 allele was predicted to result in skipping exon 6 in the mRNA. The other haplotype displayed a single 688G>C substitution predicting a Gly230Arg exchange in the catalytic domain in an otherwise normal ABO*B101 allele. The transfection studies revealed very weak expression of B antigen by the novel ABO*B allele. According to the topologic analysis, steric hindrance due to the Gly230Arg exchange may cause conformational changes in the variant B transferase. Compared to the wild-type B transferase, the transfected cells exhibited lower-level protein expression and intracellular dislocation. This study provides first evidence that aberrant trafficking of variant ABO transferases may be involved in the formation of weak ABO phenotypes.

Component and protein domain exchange analysis of a thermoresponsive, two-component regulatory system of Pseudomonas syringae

Two closely related phytopathogenic bacterial strains, Pseudomonas syringae pv. glycinea PG4180 and P. syringae pv. tomato DC3000, produce the chlorosis-inducing phytotoxin coronatine (COR) in a remarkably divergent manner. PG4180 produces COR at the virulence-promoting temperature of 18 degrees C, but not at 28 degrees C. In contrast, temperature has no effect on COR synthesis in DC3000. A modified two-component system consisting of the histidine protein kinase (HPK), CorS, the response regulator (RR), CorR, and a third component, CorP, governs COR biosynthesis in both strains. A plasmid-based component and domain swapping approach was used to introduce different combinations of RRs, HPKs and hybrid HPKs into corS mutants of both strains. Subsequently, expression levels of the COR biosynthetic cma operon were determined using RNA dot-blot analysis, suggesting that CorRSP of PG4180 mediates a thermoresponsive phenotype dependent on the genomic background of each strain. The reciprocal experiment demonstrated a loss of temperature dependence in the corS mutant of PG4180. The presence of corR from PG4180 led to more pronounced cma expression in DC3000 and was associated with thermoresponsiveness, while corS of PG4180 did not mediate a temperature-dependent phenotype in the DC3000 mutant containing native corR and corP. These findings were substantiated by RT-PCR experiments. The C-terminal domain of CorS of PG4180 mediated thermosensing, while the N terminus did not respond to temperature changes, suggesting cytosolic perception of the temperature signal.

C-Terminal Signals Regulate Targeting of Glycosylphosphatidylinositol-Anchored Proteins to the Cell Wall or Plasma Membrane in Candida albicans

Fungal glycosylphosphatidylinositol (GPI)-anchored proteins localize to the plasma membrane (PM), cell wall (CW), or both. To study signals that regulate PM versus CW targeting in Candida albicans, we (i) fused the N and/or C termini of the GPI CW protein Hwp1p and the GPI PM protein Ecm331p to green fluorescent protein (GFP) and (ii) expressed and localized the resulting fusions. Forty-seven amino acids from the C terminus of Hwp1p were sufficient to target GFP to the CW, and 66 amino acids from the C terminus of Ecm331p were sufficient to target GFP to the PM. Truncation and mutagenesis studies showed that G390 was the omega cleavage site in Ecm331p. Domain exchange and mutagenesis studies showed that (i) the 5 amino acids immediately N-terminal to the omega sites (the omega - 5 to omega - 1 amino acids) played key roles in targeting to the PM or CW; (ii) KK and FE residues at positions omega - 1 and omega - 2, respectively, targeted to the PM and CW; and (iii) a loss of I at position omega - 5 increased PM retention. Small fluorescent reporters can be used to study the peptide signals that regulate PM versus CW targeting of GPI proteins and may be useful for identifying proteins that interact with key targeting signals.

The Structural Determinants that Lead to the Formation of Particular Oligomeric Structures in the Pancreatic-Type Ribonuclease Family

Pancreatic-type ribonucleases are a family of RNA degrading enzymes that share different degrees of sequence identity but a very similar 3D-structure. The prototype of this family is bovine pancreatic ribonuclease or ribonuclease A. This enzyme has been the object of landmark work on the folding, stability, protein chemistry, catalysis, enzyme-substrate interaction and molecular evolution. In the recent years, the interest in the study of pancreatic-type ribonucleases has increased due to the involvement of some members of this family in special biological functions. In addition, dimeric and also higher oligomeric structures can be attained by the members of this family. The oligomers described structurally to date are mainly formed by 3D-domain swapping, a process which consists of the exchange of identical domains (i.e. identical structural elements, usually the N- and C-termini) between the subunits and is considered to be a mechanism for amyloid-type aggregate formation. This review compares the dimeric and oligomeric structures of different members of the pancreatic-type ribonuclease family which are able to acquire these structures, namely, bovine seminal ribonuclease, ribonuclease A and its human counterpart, human pancreatic ribonuclease. A specific focus is placed on what is known about the structural determinants that lead to the acquisition of a particular oligomeric structure and on the proposed mechanism of 3D-swapping.

The Catalytic and Lectin Domains of UDP-GalNAc:Polypeptide α-N-Acetylgalactosaminyltransferase Function in Concert to Direct Glycosylation Site Selection

UDP-GalNAc:polypeptide alpha-N-Acetylgalactosaminyltransferases (ppGalNAcTs), a family (EC 2.4.1.41) of enzymes that initiate mucin-type O-glycosylation, are structurally composed of a catalytic domain and a lectin domain. Previous studies have suggested that the lectin domain modulates the glycosylation of glycopeptide substrates and may underlie the strict glycopeptide specificity of some isoforms (ppGalNAcT-7 and -10). Using a set of synthetic peptides and glycopeptides based upon the sequence of the mucin, MUC5AC, we have examined the activity and glycosylation site preference of lectin domain deletion and exchange constructs of the peptide/glycopeptide transferase ppGalNAcT-2 (hT2) and the glycopeptide transferase ppGalNAcT-10 (hT10). We demonstrate that the lectin domain of hT2 directs glycosylation site selection for glycopeptide substrates. Pre-steady-state kinetic measurements show that this effect is attributable to two mechanisms, either lectin domain-aided substrate binding or lectin domain-aided product release following glycosylation. We find that glycosylation of peptide substrates by hT10 requires binding of existing GalNAcs on the substrate to either its catalytic or lectin domain, thereby resulting in its apparent strict glycopeptide specificity. These results highlight the existence of two modes of site selection used by these ppGalNAcTs: local sequence recognition by the catalytic domain and the concerted recognition of distal sites of prior glycosylation together with local sequence binding mediated, respectively, by the lectin and catalytic domains. The latter mode may facilitate the glycosylation of serine or threonine residues, which occur in sequence contexts that would not be efficiently glycosylated by the catalytic domain alone. Local sequence recognition by the catalytic domain differs between hT2 and hT10 in that hT10 requires a pre-existing GalNAc residue while hT2 does not.

Antiferromagnetic domain size and exchange bias

Using neutron diffraction, we measured the sizes of antiferromagnetic domains in three ferromagnet/antiferromagnet bilayer samples as a function of the magnitude and sign of exchange bias, temperature, and antiferromagnet composition. Neutron-scattering techniques were applied to thin films with masses less than $10\text{ }\ensuremath{\mu}\text{g}$. We found the antiferromagnetic domain size to be consistently small regardless of the exchange bias. For a Co/untwinned single crystalline antiferromagnet (AF)-fluoride bilayer, the antiferromagnetic domain size is comparable to the crystallographic domain size of the AF. For one sample the highest temperature at which the exchange bias was nonzero (i.e., the blocking temperature) was suppressed by $\ensuremath{\sim}3\text{ }\text{K}$ compared to the N\'eel temperature of the antiferromagnet.

Mutation in a Novel Connexin-like Gene (Gjf1) in the Mouse Affects Early Lens Development and Causes a Variable Small-Eye Phenotype

The purpose of the study was the characterization of the novel small-eye mutant Aey12 in the mouse. The eyes of the mutants were described morphologically and histologically and by in situ hybridization. The homozygotes were viable and fully fertile, which identifies Aey12 as a new microphthalmia phenotype in the mouse, different from Maf or Pax6 mutants. Histologic analysis indicated the presence of the lens vesicle; however, the primary fiber cells did not elongate properly. Genome-wide linkage analysis mapped the mutation to the proximal region of chromosome 10 between the markers D10Mit206 and D10Mit189. Among the positional candidate genes, one EST (expressed sequence tag), D230044M03Rik, encodes a connexin-like protein. A G-->T point mutation was identified at cDNA position 96, resulting in an R32Q amino acid exchange in a transmembrane domain. The mutation leads to a loss of an SsiI restriction site, which is present in five wild-type mouse strains (102, C3H, C57BL/6, DBA, and JF1). The gene is expressed in the posterior part of the lens vesicle, where the primary fiber elongation starts. In the mutants, the expression pattern of Pax6, Prox1, Six3, and Crygd are modified, but not the pattern of Pax2. The mutated mouse gene belongs to the family of connexin-encoding genes (gene symbols Gja-Gje). Together with its rat and human homologues, it defines a new subgroup, referred to as Gjf1. The mouse mutant described herein offers a new functional candidate gene for microphthalmia-related disorders at the corresponding locus on human chromosome 6, area q24.

Biochemical and Structural Analysis of Substrate Promiscuity in Plant Mg2+-Dependent O-Methyltransferases

Plant S-adenosyl-l-methionine-dependent class I natural product O-methyltransferases (OMTs), related to animal catechol OMTs, are dependent on bivalent cations and strictly specific for the meta position of aromatic vicinal dihydroxy groups. While the primary activity of these class I enzymes is methylation of caffeoyl coenzyme A OMTs, a distinct subset is able to methylate a wider range of substrates, characterized by the promiscuous phenylpropanoid and flavonoid OMT. The observed broad substrate specificity resides in two regions: the N-terminus and a variable insertion loop near the C-terminus, which displays the lowest degree of sequence conservation between the two subfamilies. Structural and biochemical data, based on site-directed mutagenesis and domain exchange between the two enzyme types, present evidence that only small topological changes among otherwise highly conserved 3-D structures are sufficient to differentiate between an enzymatic generalist and an enzymatic specialist in plant natural product methylation.

Dual Roles of the Central Domain of Colicin D tRNase in TonB-mediated Import and in Immunity

Colicin D import into Escherichia coli requires an interaction via its TonB box with the energy transducer TonB. Colicin D cytotoxicity is inhibited by specific tonB mutations, but it is restored by suppressor mutations in the TonB box. Here we report that there is a second site of interaction between TonB and colicin D, which is dependent upon a 45-amino acid region, within the uncharacterized central domain of colicin D. In addition, the 8th amino acids of colicin D (a glycine) and colicin B (a valine), adjacent to their TonB boxes, are also required for TonB recognition, suggesting that high affinity complex formation involves multiple interactions between these colicins and TonB. The central domain also contributes to the formation of the immunity complex, as well as being essential for uptake and thus killing. Colicin D is normally secreted in association with the immunity protein, and this complex involves the following two interactions: a major interaction with the C-terminal tRNase domain and a second interaction involving the central domain of colicin D and, most probably, the alpha4 helix of ImmD, which is on the opposite side of ImmD compared with the major interface. In contrast, formation of the immunity complex with the processed cytotoxic domain, the form expected to be found in the cytoplasm after colicin D uptake, requires only the major interaction. Klebicin D has, like colicin D, a ribonuclease activity toward tRNAArg and a central domain, which can form a complex with ImmD but which does not function in TonB-mediated transport.

Activation segment dimerization: a mechanism for kinase autophosphorylation of non-consensus sites

Protein kinase autophosphorylation of activation segment residues is a common regulatory mechanism in phosphorylation-dependent signalling cascades. However, the molecular mechanisms that guarantee specific and efficient phosphorylation of these sites have not been elucidated. Here, we report on three novel and diverse protein kinase structures that reveal an exchanged activation segment conformation. This dimeric arrangement results in an active kinase conformation in trans, with activation segment phosphorylation sites in close proximity to the active site of the interacting protomer. Analytical ultracentrifugation and chemical cross-linking confirmed the presence of dimers in solution. Consensus substrate sequences for each kinase showed that the identified activation segment autophosphorylation sites are non-consensus substrate sites. Based on the presented structural and functional data, a model for specific activation segment phosphorylation at non-consensus substrate sites is proposed that is likely to be common to other kinases from diverse subfamilies.

The Lipid Droplet Binding Domain of Hepatitis C Virus Core Protein Is a Major Determinant for Efficient Virus Assembly

Hepatitis C virus core protein forms the viral capsid and is targeted to lipid droplets (LDs) by its domain 2 (D2). By using a comparative analysis of two hepatitis C virus genomes (JFH1 and Jc1) differing in their level of virus production in cultured human hepatoma cells, we demonstrate that the core of the genotype 2a isolate J6 that is present in Jc1 mediates efficient assembly and release of infectious virions. Mapping studies identified a single amino acid residue in D2 as a major determinant for enhanced assembly and release of infectious Jc1 particles. Confocal microscopy analyses demonstrate that core protein in JFH1-replicating cells co-localizes perfectly with LDs and induces their accumulation in the perinuclear area, whereas no such accumulation of LDs and only a partial co-localization of core and LDs were found with the Jc1 genome. By using a fluorescence recovery after photobleaching assay, we found that green fluorescent protein-tagged D2 variants are mobile on LDs and that J6- and JFH1-D2 differ in their mobility. Taken together, our results demonstrate that the binding strength of the D2 domain of core for LDs is crucial for determining the efficiency of virus assembly.

Mapping of Equine Lentivirus Receptor 1 Residues Critical for Equine Infectious Anemia Virus Envelope Binding

The equine lentivirus receptor 1 (ELR1), a member of the tumor necrosis factor receptor (TNFR) protein family, has been identified as a functional receptor for equine infectious anemia virus (EIAV). Toward defining the functional interactions between the EIAV SU protein (gp90) and its ELR1 receptor, we mapped the gp90 binding domain of ELR1 by a combination of binding and functional assays using the EIAV SU gp90 protein and various chimeric receptor proteins derived from exchanges between the functional ELR1 and the nonbinding homolog, mouse herpesvirus entry mediator (murine HveA). Complementary exchanges of the respective cysteine-rich domains (CRD) between the ELR1 and murine HveA proteins revealed CRD1 as the predominant determinant of functional gp90 binding to ELR1 and also to a chimeric murine HveA protein expressed on the surface of transfected Cf2Th cells. Mutations of individual amino acids in the CRD1 segment of ELR1 and murine HveA indicated the Leu70 in CRD1 as essential for functional binding of EIAV gp90 and for virus infection of transduced Cf2Th cells. The specificity of the EIAV SU binding domain identified for the ELR1 receptor is fundamentally identical to that reported previously for functional binding of feline immunodeficiency virus SU to its coreceptor CD134, another TNFR protein. These results indicate unexpected common features of the specific mechanisms by which diverse lentiviruses can employ TNFR proteins as functional receptors.

A Grp on the Hsp90 Mechanism

In a recent issue of Molecular Cell, Dollins et al. (2007) present the crystal structure of Grp94, which highlights the similarity between Grp94 and Hsp90 and provides insight into the resting state of Grp94 and potentially other Hsp90 family members.

Hierarchical Delivery of an Essential Host Colonization Factor in Enteropathogenic Escherichia coli

Many significant bacterial pathogens use a type III secretion system to inject effector proteins into host cells to disrupt specific cellular functions, enabling disease progression. The injection of these effectors into host cells is often dependent on dedicated chaperones within the bacterial cell. In this report, we demonstrate that the enteropathogenic Escherichia coli (EPEC) chaperone CesT interacts with a variety of known and putative type III effector proteins. Using pull-down and secretion assays, a degenerate CesT binding domain was identified within multiple type III effectors. Domain exchange experiments between selected type III effector proteins revealed a modular nature for the CesT binding domain, as demonstrated by secretion, chaperone binding, and infection assays. The CesT-interacting type III effector Tir, which is crucial for in vivo intestinal colonization, had to be expressed and secreted for efficient secretion of other type III effectors. In contrast, the absence of other CesT-interacting type III effectors did not abrogate effector secretion, indicating an unexpected hierarchy with respect to Tir for type III effector delivery. Coordinating the expression of other type III effectors with cesT in the absence of tir partially restored total type III effector secretion, thereby implicating CesT in secretion events. Collectively, the results suggest a coordinated mechanism involving both Tir and CesT for type III effector injection into host cells.

Magnetic domain configurations of epitaxial chromium dioxide (CrO2) nanostructures

Magnetic domain structures of submicrometric epitaxial CrO2 fabricated using selective-area growth technique were studied by magnetic force microscopy. In-plane, lamellar domain structure with fragmented walls aligned along the magnetic easy axis direction is observed, indicating the existence of a large magnetocrystalline anisotropy. A classical model for ferromagnetic materials with a uniaxial anisotropy was used to explain this domain configuration. Estimates of the domain wall energy density and exchange stiffness constant for CrO2 were obtained.

A mutation in a novel connexin‐like gene affects early lens development in the mouse

Abstract Purpose: Aim of the study was the morphological and genetical characterization of Aey12, a novel dominant mouse mutant suffering from microphthalmia, lens and corneal opacities. Methods: A genome wide linkage analysis was performed using micro‐satellite markers. The Aey12 mutation was identified by sequence analysis of positional candidate genes. Histological analysis and in‐situ hybridizations were performed at the stages E10.5 ‐ E17.5. Results: Aey12 (abnormality of the eye) is a new dominant mouse mutant, which was recovered in an ENU mutagenesis program at the GSF. Aey12 animals are characterized by small eyes with cornea opacities and lenses with cataracts and vacuoles. Both, heterozygotes and homozygotes, are viable and fully fertile. As major histological defect, the elongation of the primary lens fibres is blocked resulting in the stop of lens development at this stage. Genome‐wide linkage analysis mapped the Aey12 mutation on mouse chromosome 10 between the markers D10Mit206 and D10Mit189. Among the positional candidate genes, one EST (D230044M03Rik) encodes a connexin‐like protein, which is similar to Connexin 43.4. A G‐T point mutation was identified at cDNA position 96 resulting in an R32Q amino acid exchange in a transmembrane domain. D230044M03Rik is expressed in the posterior part of the lens vesicle exactly at the position where the primary fiber elongation starts. Furthermore, an additional expression was observed in many other tissues like brain, lung, heart, liver and kidney. Conclusions: The new mouse mutant Aey12 is characterized by an early block of the eye development caused by a point mutation in a novel connexin‐like gene.