scholarly journals The endosome fusion regulator early-endosomal autoantigen 1 (EEA1) is a dimer

1999 ◽  
Vol 338 (2) ◽  
pp. 539-543 ◽  
Author(s):  
Judy CALLAGHAN ◽  
Anne SIMONSEN ◽  
Jean-Michel GAULLIER ◽  
Ban-Hock TOH ◽  
Harald STENMARK
Keyword(s):  
Protein Interactions ◽  
Quaternary Structure ◽  
Gradient Centrifugation ◽  
Active Form ◽  
Coiled Coil ◽  
Membrane Lipid ◽  
Small Gtpase ◽  
C Terminus ◽  
Early Endosomes ◽  
Heptad Repeats

EEA1, an early-endosomal protein originally identified as an autoantigen, is essential for endocytic membrane fusion. It interacts with early endosomes via binding to the membrane lipid phosphatidylinositol 3-phosphate (PtdIns3P) and the active form of the small GTPase Rab5. Most of the EEA1 sequence contains heptad repeats characteristic of proteins involved in coiled-coil protein–protein interactions. Here we have investigated the ability of EEA1 to self-interact. Crosslinking of cytosolic and recombinant EEA1 resulted in the disappearance of the 180-kDa monomer in SDS/PAGE and the strong appearance of a ∼ 350-kDa crosslinked product. Glycerol gradient centrifugation experiments indicated that native EEA1 had the same hydrodynamic properties as the ∼ 350-kDa crosslinked complex. Two-hybrid analysis indicated that N- and C-terminal fragments of EEA1 can interact with themselves, but not with each other, suggesting that EEA1 forms parallel coiled-coil dimers. The ability of the C-terminus of EEA1 to dimerize correlates with its ability to bind to Rab5 and early endosomes, whereas its binding to PtdIns3P is independent of dimerization. These data enable us to propose a model for the quaternary structure of EEA1.

scholarly journals Divisome under Construction: Distinct Domains of the Small Membrane Protein FtsB Are Necessary for Interaction with Multiple Cell Division Proteins

2009 ◽  
Vol 191 (8) ◽  
pp. 2815-2825 ◽  
Author(s):  
Mark D. Gonzalez ◽  
Jon Beckwith
Keyword(s):  
Cell Division ◽  
Membrane Proteins ◽  
Protein Interactions ◽  
Cell Envelope ◽  
Coiled Coil ◽  
Main Function ◽  
Protein Protein Interactions ◽  
Molecular Scaffold ◽  
C Terminus ◽  
Under Construction

ABSTRACT Cell division in bacteria requires the coordinated action of a set of proteins, the divisome, for proper constriction of the cell envelope. Multiple protein-protein interactions are required for assembly of a stable divisome. Within the Escherichia coli divisome is a conserved subcomplex of inner membrane proteins, the FtsB/FtsL/FtsQ complex, which is necessary for linking the upstream division proteins, which are predominantly cytoplasmic, with the downstream division proteins, which are predominantly periplasmic. FtsB and FtsL are small bitopic membrane proteins with predicted coiled-coil motifs, which themselves form a stable subcomplex that can recruit downstream division proteins independently of FtsQ; however, the details of how FtsB and FtsL interact together and with other proteins remain to be characterized. Despite the small size of FtsB, we identified separate interaction domains of FtsB that are required for interaction with FtsL and FtsQ. The N-terminal half of FtsB is necessary for interaction with FtsL and sufficient, when in complex with FtsL, for recruitment of downstream division proteins, while a portion of the FtsB C terminus is necessary for interaction with FtsQ. These properties of FtsB support the proposal that its main function is as part of a molecular scaffold to allow for proper formation of the divisome.

scholarly journals Exocyst Requirement for Endocytic Traffic Directed Toward the Apical and Basolateral Poles of Polarized MDCK Cells

2007 ◽  
Vol 18 (10) ◽  
pp. 3978-3992 ◽  
Author(s):  
Asli Oztan ◽  
Mark Silvis ◽  
Ora A. Weisz ◽  
Neil A. Bradbury ◽  
Shu-Chan Hsu ◽  
...  
Keyword(s):  
Mdck Cells ◽  
Small Gtpase ◽  
Junctional Complex ◽  
Recycling Endosomes ◽  
Permeabilized Cells ◽  
Exocyst Complex ◽  
C Terminus ◽  
Streptolysin O ◽  
Early Endosomes ◽  
Endocytic Traffic

The octameric exocyst complex is associated with the junctional complex and recycling endosomes and is proposed to selectively tether cargo vesicles directed toward the basolateral surface of polarized Madin-Darby canine kidney (MDCK) cells. We observed that the exocyst subunits Sec6, Sec8, and Exo70 were localized to early endosomes, transferrin-positive common recycling endosomes, and Rab11a-positive apical recycling endosomes of polarized MDCK cells. Consistent with its localization to multiple populations of endosomes, addition of function-blocking Sec8 antibodies to streptolysin-O–permeabilized cells revealed exocyst requirements for several endocytic pathways including basolateral recycling, apical recycling, and basolateral-to-apical transcytosis. The latter was selectively dependent on interactions between the small GTPase Rab11a and Sec15A and was inhibited by expression of the C-terminus of Sec15A or down-regulation of Sec15A expression using shRNA. These results indicate that the exocyst complex may be a multipurpose regulator of endocytic traffic directed toward both poles of polarized epithelial cells and that transcytotic traffic is likely to require Rab11a-dependent recruitment and modulation of exocyst function, likely through interactions with Sec15A.

scholarly journals The Dense-Core Vesicle Maturation Protein CCCP-1 Binds RAB-2 and Membranes through its C-terminal Domain

2017 ◽  
Author(s):  
Jérôme Cattin-Ortolá ◽  
Irini Topalidou ◽  
Annie Dosey ◽  
Alexey J. Merz ◽  
Michael Ailion
Keyword(s):  
Structure Function ◽  
Function Analysis ◽  
Coiled Coil ◽  
Lipid Binding ◽  
Small Gtpase ◽  
Dense Core ◽  
Binding Motif ◽  
Dense Core Vesicle ◽  
Terminal Domain ◽  
C Terminus

AbstractDense-core vesicles (DCVs) are secretory organelles that store and release modulatory neurotransmitters from neurons and endocrine cells. Recently, the conserved coiled-coil protein CCCP-1 was identified as a component of the DCV biogenesis pathway in the nematode C. elegans. CCCP-1 binds the small GTPase RAB-2 and colocalizes with it at the trans-Golgi. Here we report a structure-function analysis of CCCP-1 to identify domains of the protein important for its localization, binding to RAB-2, and function in DCV biogenesis. We find that the CCCP-1 C-terminal domain (CC3) has multiple activities. CC3 is necessary and sufficient for CCCP-1 localization and for binding to RAB-2, and is required for the function of CCCP-1 in DCV biogenesis. Additionally, CCCP-1 binds membranes directly through its CC3 domain, indicating that CC3 may comprise a previously uncharacterized lipid-binding motif. We conclude that CCCP-1 is a coiled-coil protein that binds an activated Rab and localizes to the Golgi via its C-terminus, properties similar to members of the golgin family of proteins. CCCP-1 also shares biophysical features with golgins; it has an elongated shape and forms oligomers.Synopsis statementCCCP-1 is a coiled-coil protein important for dense-core vesicle (DCV) biogenesis. A structure-function analysis of CCCP-1 shows that its C-terminal domain is required for (1) localization to membrane compartments near the trans-Golgi, (2) binding to activated RAB-2, (3) function in DCV biogenesis, and (4) direct binding to membranes. CCCP-1 has an elongated shape and forms oligomers. These findings suggest that CCCP-1 resembles members of the golgin family of proteins that act as membrane tethers.

scholarly journals Domains of alpha-SNAP required for the stimulation of exocytosis and for N-ethylmalemide-sensitive fusion protein (NSF) binding and activation.

1996 ◽  
Vol 7 (5) ◽  
pp. 693-701 ◽  
Author(s):  
R J Barnard ◽  
A Morgan ◽  
R D Burgoyne
Keyword(s):  
Membrane Proteins ◽  
Fusion Protein ◽  
Conformational Changes ◽  
Protein Interactions ◽  
Atp Hydrolysis ◽  
Coiled Coil ◽  
Protein Protein Interactions ◽  
Permeabilized Cells ◽  
C Terminus ◽  
Adrenal Chromaffin

The binding of alpha-SNAP to the membrane proteins syntaxin, SNAP-25, and synaptobrevin leads to the recruitment of the N-ethylmaleimide-sensitive fusion protein (NSF). ATP hydrolysis by NSF has been suggested to drive conformational changes in one or more of these membrane proteins that are essential for regulated exocytosis. Functional evidence for a role of alpha-SNAP in exocytosis in adrenal chromaffin cells comes from the ability of this protein to stimulate Ca(2+)-dependent exocytosis in digitonin-permeabilized cells. Here we examine the effect of a series of deletion mutants of alpha-SNAP on exocytosis, and on the ability of alpha-SNAP to interact with NSF, to define essential domains involved in protein-protein interactions in exocytosis. Deletion of extreme N- or C-terminal regions of alpha-SNAP produced proteins unable to bind to syntaxin or to stimulate exocytosis, suggesting that these domains participate in essential interactions. Deletion of C-terminal residues abolished the ability of alpha-SNAP to bind NSF. In contrast, deletion of up to 120 N-terminal residues did not prevent the binding of NSF to immobilized alpha-SNAP and such mutants were also able to stimulate the ATPase activity of NSF. These results suggest that the C-terminus, but not the N-terminus, of alpha-SNAP is crucial for interactions with NSF. The involvement of the C-terminus of alpha-SNAP, which contains a predicted coiled-coil domain, in the binding of both syntaxin and NSF would place the latter two proteins in proximity in a ternary complex whereupon the energy derived from ATP hydrolysis by NSF could induce a conformational change in syntaxin required for exocytosis to proceed.

mARVCF cellular localisation and binding to cadherins is influenced by the cellular context but not by alternative splicing

2001 ◽  
Vol 114 (21) ◽  
pp. 3873-3884 ◽  
Author(s):  
Zoe Waibler ◽  
Annette Schäfer ◽  
Anna Starzinski-Powitz
Keyword(s):  
Alternative Splicing ◽  
Protein Interactions ◽  
Coiled Coil ◽  
Distinct Pattern ◽  
Subcellular Localisation ◽  
Protein Protein Interactions ◽  
Protein Protein Interaction ◽  
C Terminus ◽  
Coiled Coil Region ◽  
Cellular Context

ARVCF, a member of the catenin family, is thought to contribute to the morphoregulatory function of the cadherin-catenin complex. Recently, we reported the isolation and characterisation of murine ARVCF (mARVCF), particularly its interaction with M-cadherin. Here, we describe the identification of novel mARVCF isoforms that arise by alternative splicing. At the N-terminus, alternative splicing results in the inclusion or omission of a coiled-coil region probably important for protein-protein interactions. At the C-terminus, four isoforms also differ by domains potentially important for selective protein-protein interaction. The eight putative mARVCF isoforms were expressed as EGFP-fusion proteins in six different cell lines that exhibit a distinct pattern of cadherins. Apparently, binding of the mARVCF isoforms to M-, N-, or E-cadherin is generally unaffected by their altered N- and C-termini, as revealed by the MOM recruitment assay. However, mARVCF isoforms reproducibly exhibit differential localisation in distinct cellular environments. For example, mARVCF isoforms are unable to colocalise with N-cadherin in EJ28 carcinoma cells but do so in HeLa cells. Our results suggest that the subcellular localisation of mARVCF may be determined not only by the presence or absence of an appropriate interaction partner, in this case cadherins, but also by the cellular context.

Guidelines for the assembly of novel coiled-coil structures: α-sheets and α-cylinders

2001 ◽  
Vol 68 ◽  
pp. 111-123 ◽  
Author(s):  
John Walshaw ◽  
Jennifer M. Shipway ◽  
Derek N. Woolfson
Keyword(s):  
Protein Design ◽  
Protein Interactions ◽  
Protein Structures ◽  
Coiled Coil ◽  
Data Bank ◽  
Coiled Coils ◽  
Heptad Repeat ◽  
Protein Protein Interactions ◽  
Helical Bundles ◽  
Heptad Repeats

The coiled coil is a ubiquitous motif that guides many different protein-protein interactions. The accepted hallmark of coiled coils is a seven-residue (heptad) sequence repeat. The positions of this repeat are labelled a-b-c-d-e-f-g, with residues at a and d tending to be hydrophobic. Such sequences form amphipathic α-helices, which assemble into helical bundles via knobs-into-holes interdigitation of residues from neighbouring helices. We wrote an algorithm, SOCKET, to identify this packing in protein structures, and used this to gather a database of coiled-coil structures from the Protein Data Bank. Surprisingly, in addition to commonly accepted structures with a single, contiguous heptad repeat, we identified sequences with multiple, offset heptad repeats. These 'new' sequence patterns help to explain oligomer-state specification in coiled coils. Here we focus on the structural consequences for sequences with two heptad repeats offset by two residues, i.e. a/f′-b/g′-c/a′-d/b′-e/c′-f/d′-g/e′. This sets up two hydrophobic seams on opposite sides of the helix formed. We describe how such helices may combine to bury these hydrophobic surfaces in two different ways and form two distinct structures: open 'α-sheets' and closed 'α-cylinders'. We highlight these with descriptions of natural structures and outline possibilities for protein design.

scholarly journals AglZ Is a Filament-Forming Coiled-Coil Protein Required for Adventurous Gliding Motility of Myxococcus xanthus

2004 ◽  
Vol 186 (18) ◽  
pp. 6168-6178 ◽  
Author(s):  
Ruifeng Yang ◽  
Sarah Bartle ◽  
Rebecca Otto ◽  
Angela Stassinopoulos ◽  
Matthew Rogers ◽  
...  
Keyword(s):  
Response Regulator ◽  
Myxococcus Xanthus ◽  
Coiled Coil ◽  
Gliding Motility ◽  
Isolated Cells ◽  
Genetic Studies ◽  
C Terminus ◽  
Heptad Repeats ◽  
Two Hybrid

ABSTRACT The aglZ gene of Myxococcus xanthus was identified from a yeast two-hybrid assay in which MglA was used as bait. MglA is a 22-kDa cytoplasmic GTPase required for both adventurous and social gliding motility and sporulation. Genetic studies showed that aglZ is part of the A motility system, because disruption or deletion of aglZ abolished movement of isolated cells and aglZ sglK double mutants were nonmotile. The aglZ gene encodes a 153-kDa protein that interacts with purified MglA in vitro. The N terminus of AglZ shows similarity to the receiver domain of two-component response regulator proteins, while the C terminus contains heptad repeats characteristic of coiled-coil proteins, such as myosin. Consistent with this motif, expression of AglZ in Escherichia coli resulted in production of striated lattice structures. Similar to the myosin heavy chain, the purified C-terminal coiled-coil domain of AglZ forms filament structures in vitro.

scholarly journals The trans-Golgi network GRIP-domain proteins form α-helical homodimers

2005 ◽  
Vol 388 (3) ◽  
pp. 835-841 ◽  
Author(s):  
Michael R. LUKE ◽  
Fiona HOUGHTON ◽  
Matthew A. PERUGINI ◽  
Paul A. GLEESON
Keyword(s):  
Protein Interactions ◽  
Coiled Coil ◽  
Helical Structure ◽  
Cd Spectroscopy ◽  
Protein Protein Interactions ◽  
Yeast Two Hybrid ◽  
Trans Golgi Network ◽  
Heptad Repeats ◽  
Golgi Network ◽  
Two Hybrid

A recently described family of TGN (trans-Golgi network) proteins, all of which contain a GRIP domain targeting sequence, has been proposed to play a role in membrane transport. On the basis of the high content of heptad repeats, GRIP domain proteins are predicted to contain extensive coiled-coil regions that have the potential to mediate protein–protein interactions. Four mammalian GRIP domain proteins have been identified which are targeted to the TGN through their GRIP domains, namely p230, golgin-97, GCC88 and GCC185. In the present study, we have investigated the ability of the four mammalian GRIP domain proteins to interact. Using a combination of immunoprecipitation experiments of epitope-tagged GRIP domain proteins, cross-linking experiments and yeast two-hybrid interactions, we have established that the GRIP proteins can self-associate to form homodimers exclusively. Two-hybrid analysis indicated that the N- and C-terminal fragments of GCC88 can interact with themselves but not with each other, suggesting that the GRIP domain proteins form parallel coiled-coil dimers. Analysis of purified recombinant golgin-97 by CD spectroscopy indicated a 67% α-helical structure, consistent with a high content of coiled-coil sequences. These results support a model for GRIP domain proteins as extended rod-like homodimeric molecules. The formation of homodimers, but not heterodimers, indicates that each of the four mammalian TGN golgins has the potential to function independently.

Current Trends in Biotherapeutic Higher Order Structure Characterization by Irreversible Covalent Footprinting Mass Spectrometry

2019 ◽  
Vol 26 (1) ◽  
pp. 35-43 ◽  
Author(s):  
Natalie K. Garcia ◽  
Galahad Deperalta ◽  
Aaron T. Wecksler
Keyword(s):  
Mass Spectrometry ◽  
Protein Structure ◽  
Protein Interactions ◽  
Quaternary Structure ◽  
Solvent Accessibility ◽  
Higher Order ◽  
Structure Characterization ◽  
Order Structure ◽  
Protein Footprinting ◽  
Wide Range

Background: Biotherapeutics, particularly monoclonal antibodies (mAbs), are a maturing class of drugs capable of treating a wide range of diseases. Therapeutic function and solutionstability are linked to the proper three-dimensional organization of the primary sequence into Higher Order Structure (HOS) as well as the timescales of protein motions (dynamics). Methods that directly monitor protein HOS and dynamics are important for mapping therapeutically relevant protein-protein interactions and assessing properly folded structures. Irreversible covalent protein footprinting Mass Spectrometry (MS) tools, such as site-specific amino acid labeling and hydroxyl radical footprinting are analytical techniques capable of monitoring the side chain solvent accessibility influenced by tertiary and quaternary structure. Here we discuss the methodology, examples of biotherapeutic applications, and the future directions of irreversible covalent protein footprinting MS in biotherapeutic research and development. Conclusion: Bottom-up mass spectrometry using irreversible labeling techniques provide valuable information for characterizing solution-phase protein structure. Examples range from epitope mapping and protein-ligand interactions, to probing challenging structures of membrane proteins. By paring these techniques with hydrogen-deuterium exchange, spectroscopic analysis, or static-phase structural data such as crystallography or electron microscopy, a comprehensive understanding of protein structure can be obtained.

scholarly journals E. coli primase and DNA polymerase III holoenzyme are able to bind concurrently to a primed template during DNA replication

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Andrea Bogutzki ◽  
Natalie Naue ◽  
Lidia Litz ◽  
Andreas Pich ◽  
Ute Curth
Keyword(s):  
Dna Replication ◽  
Dna Polymerase ◽  
Protein Interactions ◽  
Dna Polymerase Iii ◽  
Protein Protein Interactions ◽  
Single Stranded Dna ◽  
E Coli ◽  
Polymerase Iii ◽  
C Terminus ◽  
Pol Iii

Abstract During DNA replication in E. coli, a switch between DnaG primase and DNA polymerase III holoenzyme (pol III) activities has to occur every time when the synthesis of a new Okazaki fragment starts. As both primase and the χ subunit of pol III interact with the highly conserved C-terminus of single-stranded DNA-binding protein (SSB), it had been proposed that the binding of both proteins to SSB is mutually exclusive. Using a replication system containing the origin of replication of the single-stranded DNA phage G4 (G4ori) saturated with SSB, we tested whether DnaG and pol III can bind concurrently to the primed template. We found that the addition of pol III does not lead to a displacement of primase, but to the formation of higher complexes. Even pol III-mediated primer elongation by one or several DNA nucleotides does not result in the dissociation of DnaG. About 10 nucleotides have to be added in order to displace one of the two primase molecules bound to SSB-saturated G4ori. The concurrent binding of primase and pol III is highly plausible, since even the SSB tetramer situated directly next to the 3′-terminus of the primer provides four C-termini for protein-protein interactions.

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