The SPI2-encoded SseA chaperone has discrete domains required for SseB stabilization and export, and binds within the C-terminus of SseB and SseD

Microbiology ◽  
2004 ◽  
Vol 150 (7) ◽  
pp. 2055-2068 ◽  
Author(s):  
Daniel V. Zurawski ◽  
Murry A. Stein
Keyword(s):  
Type Iii Secretion ◽  
Coiled Coil ◽  
Amphipathic Helix ◽  
Yeast Two Hybrid ◽  
C Terminus ◽  
N Terminus ◽  
Domain Mapping ◽  
Self Association ◽  
Discrete Domains ◽  
Two Hybrid

SseA, a key Salmonella virulence determinant, is a small, basic pI protein encoded within the Salmonella pathogenicity island 2 and serves as a type III secretion system chaperone for SseB and SseD. Both SseA partners are subunits of the surface-localized translocon module that delivers effectors into the host cell; SseB is predicted to compose the translocon sheath and SseD is a putative translocon pore subunit. In this study, SseA molecular interactions with its partners were characterized further. Yeast two-hybrid screens indicate that SseA binding requires a C-terminal domain within both partners. An additional central domain within SseD was found to influence binding. The SseA-binding region within SseB was found to encompass a predicted amphipathic helix of a type participating in coiled-coil interactions that are implicated in the assembly of translocon sheaths. Deletions that impinge upon this putative coiled-coiled domain prevent SseA binding, suggesting that SseA occupies a portion of the coiled-coil. SseA occupancy of this motif is envisioned to be sufficient to prevent premature SseB self-association inside bacteria. Domain mapping on the chaperone was also performed. A deletion of the SseA N-terminus, or site-directed mutations within this region, allowed stabilization of SseB, but its export was disrupted. Therefore, the N-terminus of SseA provides a function that is essential for SseB export, but dispensable for partner binding and stabilization.

scholarly journals P1 ParB Domain Structure Includes Two Independent Multimerization Domains

1999 ◽  
Vol 181 (19) ◽  
pp. 5898-5908 ◽  
Author(s):  
Jennifer A. Surtees ◽  
Barbara E. Funnell
Keyword(s):  
Amino Acids ◽  
Domain Structure ◽  
Cross Linking ◽  
Yeast Two Hybrid ◽  
Yeast Two Hybrid System ◽  
C Terminus ◽  
Self Association ◽  
Two Hybrid ◽  
Chemical Cross Linking

ABSTRACT ParB is one of two P1-encoded proteins that are required for active partition of the P1 prophage in Escherichia coli. To probe the native domain structure of ParB, we performed limited proteolytic digestions of full-length ParB, as well as of several N-terminal and C-terminal deletion fragments of ParB. The C-terminal 140 amino acids of ParB form a very trypsin-resistant domain. In contrast, the N terminus is more susceptible to proteolysis, suggesting that it forms a less stably folded domain or domains. Because native ParB is a dimer in solution, we analyzed the ability of ParB fragments to dimerize, using both the yeast two-hybrid system and in vitro chemical cross-linking of purified proteins. These studies revealed that the C-terminal 59 amino acids of ParB, a region within the protease-resistant domain, are sufficient for dimerization. Cross-linking and yeast two-hybrid experiments also revealed the presence of a second self-association domain within the N-terminal half of ParB. The cross-linking data also suggest that the C terminus is inhibitory to multimerization through the N-terminal domain in vitro. We propose that the two multimerization domains play distinct roles in partition complex formation.

HZwint-1, a novel human kinetochore component that interacts with HZW10

2000 ◽  
Vol 113 (11) ◽  
pp. 1939-1950 ◽  
Author(s):  
D.A. Starr ◽  
R. Saffery ◽  
Z. Li ◽  
A.E. Simpson ◽  
K.H. Choo ◽  
...  
Keyword(s):  
Hela Cells ◽  
Coiled Coil ◽  
Yeast Two Hybrid ◽  
Interacting Protein ◽  
Dicentric Chromosomes ◽  
Centromere Function ◽  
Coiled Coil Domain ◽  
Gfp Fluorescence ◽  
Two Hybrid ◽  
Active Centromere

HZwint-1 (Human ZW10 interacting protein-1) was identified in a yeast two hybrid screen for proteins that interact with HZW10. HZwint-1 cDNA encodes a 43 kDa protein predicted to contain an extended coiled-coil domain. Immunofluorescence studies with sera raised against HZwint-1 protein revealed strong kinetochore staining in nocodazole-arrested chromosome spreads. This signal co-localizes at the kinetochore with HZW10, at a position slightly outside of the central part of the centromere as revealed by staining with a CREST serum. The kinetochore localization of HZwint-1 has been confirmed by following GFP fluorescence in HeLa cells transiently transfected with a plasmid encoding a GFP/HZwint-1 fusion protein. In cycling HeLa cells, HZwint-1 localizes to the kinetochore of prophase HeLa cells prior to HZW10 localization, and remains at the kinetochore until late in anaphase. This localization pattern, combined with the two-hybrid results, suggests that HZwint-1 may play a role in targeting HZW10 to the kinetochore at prometaphase. HZwint-1 was also found to localize to neocentromeres and to the active centromere of dicentric chromosomes. HZwint-1 thus appears to associate with all active centromeres, implying that it plays an important role in correct centromere function.

scholarly journals Genetic Analysis of theEscherichia coliFtsZ·ZipA Interaction in the Yeast Two-hybrid System

2001 ◽  
Vol 276 (15) ◽  
pp. 11980-11987 ◽  
Author(s):  
Steven A. Haney ◽  
Elizabeth Glasfeld ◽  
Cynthia Hale ◽  
David Keeney ◽  
Zhizhen He ◽  
...  
Keyword(s):  
Hybrid System ◽  
Enzyme Linked Immunosorbent Assay ◽  
Wild Type ◽  
Yeast Two Hybrid ◽  
Conserved Sequence ◽  
Yeast Two Hybrid System ◽  
C Terminus ◽  
Two Hybrid

The recruitment of ZipA to the septum by FtsZ is an early, essential step in cell division inEscherichia coli. We have used polymerase chain reaction-mediated random mutagenesis in the yeast two-hybrid system to analyze this interaction and have identified residues within a highly conserved sequence at the C terminus of FtsZ as the ZipA binding site. A search for suppressors of a mutation that causes a loss of interaction (ftsZD373G) identified eight different changes at two residues within this sequence.In vitro, wild type FtsZ interacted with ZipA with a high affinity in an enzyme-linked immunosorbent assay, whereas FtsZD373Gfailed to interact. Two mutant proteins examined restored this interaction significantly.In vivo, the alleles tested are significantly more toxic than the wild typeftsZand cannot complement a deletion. We have shown that a fusion, which encodes the last 70 residues of FtsZ in the two-hybrid system, is sufficient for the interaction with FtsA and ZipA. However, when the wild type sequence is compared with one that encodes FtsZD373G, no interaction was seen with either protein. Mutations surrounding Asp-373 differentially affected the interactions of FtsZ with ZipA and FtsA, indicating that these proteins bind the C terminus of FtsZ differently.

scholarly journals Tubulin Folding Cofactor TBCB is a Target of the Salmonella Effector Protein SseK1

2020 ◽  
Vol 21 (9) ◽  
pp. 3193 ◽  
Author(s):  
Juan Luis Araujo-Garrido ◽  
Fernando Baisón-Olmo ◽  
Joaquín Bernal-Bayard ◽  
Francisco Romero ◽  
Francisco Ramos-Morales
Keyword(s):  
Host Cell ◽  
Type Iii Secretion ◽  
Salmonella Enterica Serovar Typhimurium ◽  
Yeast Two Hybrid ◽  
Microtubule Network ◽  
Binding Partner ◽  
Tubulin Binding ◽  
Serovar Typhimurium ◽  
Novel Target ◽  
Two Hybrid

Salmonella enterica serovar Typhimurium is a human and animal pathogen that uses type III secretion system effectors to manipulate the host cell and fulfill infection. SseK1 is a Salmonella effector with glycosyltransferase activity. We carried out a yeast two-hybrid screen and have identified tubulin-binding cofactor B (TBCB) as a new binding partner for this effector. SseK1 catalyzed the addition of N-acetylglucosamine to arginine on TBCB, and its expression promoted the stabilization of the microtubule cytoskeleton of HEK293T cells. The conserved Asp-x-Asp (DxD) motif that is essential for the activity of SseK1 was required for the binding and modification of TBCB and for the effect on the cytoskeleton. Our study has identified a novel target for SseK1 and suggests that this effector may have a role in the manipulation of the host cell microtubule network to provide a safe niche for this pathogen.

scholarly journals The Complex Interplay between the Neck and Hinge Domains in Kinesin-1 Dimerization and Motor Activity

2005 ◽  
Vol 16 (8) ◽  
pp. 3529-3537 ◽  
Author(s):  
Friederike Bathe ◽  
Katrin Hahlen ◽  
Renate Dombi ◽  
Lucia Driller ◽  
Manfred Schliwa ◽  
...  
Keyword(s):  
Disulfide Bonds ◽  
Coiled Coil ◽  
Proximal Part ◽  
Tail Domain ◽  
Yeast Two Hybrid ◽  
Complex Interplay ◽  
Hybrid Data ◽  
Neck Domain ◽  
Two Hybrid ◽  
Complex Manner

Kinesin-1 dimerizes via the coiled-coil neck domain. In contrast to animal kinesins, neck dimerization of the fungal kinesin-1 NcKin requires additional residues from the hinge. Using chimeric constructs containing or lacking fungal-specific elements, the proximal part of the hinge was shown to stabilize the neck coiled-coil conformation in a complex manner. The conserved fungal kinesin hinge residue W384 caused neck coiled-coil formation in a chimeric NcKin construct, including parts of the human kinesin-1 stalk. The stabilizing effect was retained in a NcKinW384F mutant, suggesting important π -stacking interactions. Without the stalk, W384 was not sufficient to induce coiled-coil formation, indicating that W384 is part of a cluster of several residues required for neck coiled-coil folding. A W384-less chimera of NcKin and human kinesin possessed a non–coiled-coil neck conformation and showed inhibited activity that could be reactivated when artificial interstrand disulfide bonds were used to stabilize the neck coiled-coil conformation. On the basis of yeast two-hybrid data, we propose that the proximal hinge can bind kinesin's cargo-free tail domain and causes inactivation of kinesin by disrupting the neck coiled-coil conformation.

scholarly journals IE1 and hr facilitate the localization of Bombyx mori nucleopolyhedrovirus ORF8 to specific nuclear sites

2005 ◽  
Vol 86 (11) ◽  
pp. 3031-3038 ◽  
Author(s):  
WonKyung Kang ◽  
Noriko Imai ◽  
Yu Kawasaki ◽  
Toshihiro Nagamine ◽  
Shogo Matsumoto
Keyword(s):  
Bombyx Mori ◽  
Fluorescent Protein ◽  
Coiled Coil ◽  
Terminal Region ◽  
Mutant Protein ◽  
Yeast Two Hybrid ◽  
Focus Formation ◽  
Coiled Coil Domain ◽  
Nuclear Foci ◽  
Two Hybrid

The Bombyx mori nucleopolyhedrovirus (BmNPV) ORF8 protein has previously been reported to colocalize with IE1 to specific nuclear sites during infection. Transient expression of green fluorescent protein (GFP)-fused ORF8 showed the protein to have cytoplasmic localization, but following BmNPV infection the protein formed foci, suggesting that ORF8 requires some other viral factor(s) for this. Therefore, interacting factors were looked for using the yeast two-hybrid system and IE1 was identified. We mapped the interacting region of ORF8 using a yeast two-hybrid assay. An N-terminal region (residues 1–110) containing a predicted coiled-coil domain interacted with IE1, while a truncated N-terminal region (residues 1–78) that lacks this domain did not. In addition, a protein with a complete deletion of the N-terminal region failed to interact with IE1. These results suggest that the ORF8 N-terminal region containing the coiled-coil domain is required for the interaction with IE1. Next, whether IE1 plays a role in ORF8 localization was investigated. In the presence of IE1, GFP-ORF8 localized to the nucleus. In addition, cotransfection with a plasmid expressing IE1 and a plasmid containing the hr3 element resulted in nuclear foci formation. A GFP-fused ORF8 mutant protein containing the coiled-coil domain, previously shown to interact with IE1, also formed nuclear foci in the presence of IE1 and hr3. However, ORF8 mutant proteins that did not interact with IE1 failed to form nuclear foci. In contrast to wild-type IE1, focus formation was not observed for an IE1 mutant protein that was deficient in hr binding. These results suggest that IE1 and hr facilitate the localization of BmNPV ORF8 to specific nuclear sites.

scholarly journals PDZD8 Is a Novel Gag-Interacting Factor That Promotes Retroviral Infection

2010 ◽  
Vol 84 (17) ◽  
pp. 8990-8995 ◽  
Author(s):  
Matthew S. Henning ◽  
Scott G. Morham ◽  
Stephen P. Goff ◽  
Mojgan H. Naghavi
Keyword(s):  
Viral Entry ◽  
Coiled Coil ◽  
Yeast Two Hybrid ◽  
Retroviral Infection ◽  
Gag Protein ◽  
Cellular Factors ◽  
Immunodeficiency Virus ◽  
Two Hybrid ◽  
Hiv 1

ABSTRACT In a yeast two-hybrid screen for cellular factors that could interact with human immunodeficiency virus type 1 (HIV-1) Gag protein, we identified PDZD8 and confirmed the interaction by coimmunoprecipitation (co-IP). PDZD8 overexpression promoted the initiation of reverse transcription and increased infection by pseudotyped retroviruses independent of the route of viral entry, while transient knockdown of endogenous levels decreased HIV-1 infection. A mutant of PDZD8 lacking a predicted coiled-coil domain in its Gag-interacting region failed to bind Gag and promote HIV-1 infection, identifying the domain of PDZD8 required for mediating these effects. As such, we identify PDZD8 as a novel positive mediator of retroviral infection.

Type III secretion chaperones ShcS1 and ShcO1 from Pseudomonas syringae pv. tomato DC3000 bind more than one effector

Microbiology ◽  
2005 ◽  
Vol 151 (1) ◽  
pp. 269-280 ◽  
Author(s):  
Ute Kabisch ◽  
Angelika Landgraf ◽  
Jana Krause ◽  
Ulla Bonas ◽  
Jens Boch
Keyword(s):  
Pseudomonas Syringae ◽  
Type Iii Secretion ◽  
Gel Filtration ◽  
Effector Proteins ◽  
Yeast Two Hybrid ◽  
Hybrid Analysis ◽  
Tomato Dc3000 ◽  
Type Iii ◽  
Effector Genes ◽  
Two Hybrid

The hrp-type III secretion (TTS) system is a key pathogenicity factor of the plant pathogen Pseudomonas syringae pv. tomato DC3000 that translocates effector proteins into the cytosol of the eukaryotic host cell. The translocation of a subset of effectors is dependent on specific chaperones. In this study an operon encoding a TTS chaperone (ShcS1) and the truncated effector HopS1′ was characterized. Yeast two-hybrid analysis and pull-down assays demonstrated that these proteins interact. Using protein fusions to AvrRpt2 it was shown that ShcS1 facilitates the translocation of HopS1′, suggesting that ShcS1 is a TTS chaperone for HopS1′ and that amino acids 1 to 118 of HopS1′ are required for translocation. P. syringae pv. tomato DC3000 carries two shcS1 homologues, shcO1 and shcS2, which are located in different operons, and both operons include additional putative effector genes. Transcomplementation experiments showed that ShcS1 and ShcO1, but not ShcS2, can facilitate the translocation of HopS1′ : : AvrRpt2. To characterize the specificities of the putative chaperones, yeast two-hybrid interaction studies were performed between the three chaperones and putative target effectors. These experiments showed that both ShcS1 and ShcO1 bind to two different effectors, HopS1′ and HopO1-1, that share only 16 % amino acid sequence identity. Using gel filtration it was shown that ShcS1 forms homodimers, and this was confirmed by yeast two-hybrid experiments. In addition, ShcS1 is also able to form heterodimers with ShcO1. These data demonstrate that ShcS1 and ShcO1 are exceptional class IA TTS chaperones because they can bind more than one target effector.

Expression in Escherichia coli of fragments of the coiled-coil rod domain of rabbit myosin: influence of different regions of the molecule on aggregation and paracrystal formation

1991 ◽  
Vol 99 (4) ◽  
pp. 823-836
Author(s):  
S.J. Atkinson ◽  
M. Stewart
Keyword(s):  
Escherichia Coli ◽  
Ionic Strength ◽  
Coiled Coil ◽  
Periodic Variation ◽  
Heptad Repeat ◽  
C Terminus ◽  
N Terminus ◽  
Low Ionic Strength ◽  
Myosin Rod

We have expressed in Escherichia coli a cDNA clone corresponding broadly to rabbit light meromyosin (LMM) together with a number of modified polypeptides and have used this material to investigate the role of different aspects of molecular structure on the solubility properties of LMM. The expressed material was characterized biochemically and structurally to ensure that it retained the coiled-coil conformation of the native molecule. Full-length recombinant LMM retained the general solubility properties of myosin and, although soluble at high ionic strength, precipitated when the ionic strength was reduced below 0.3 M. Constructs in which the ‘skip’ residues (that disrupt the coiled-coil heptad repeat) were deleted had solubility properties indistinguishable from the wild type, which indicated that the skip residues did not play a major role in determining the molecular interactions involved in assembly. Deletions from the N terminus of LMM did not alter the solubility properties of the expressed material, but deletion of 92 residues from the C terminus caused a large increase in solubility at low ionic strength, indicating that a determinant important for interaction between LMM molecules was located in this region. The failure of deletions from the molecule's N terminus to alter its solubility radically suggested that the periodic variation of charge along the myosin rod may not be as important as proposed for determining the strength of binding between molecules and thus the solubility of myosin.

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