scholarly journals N-terminal 45 amino acids of DEF6 are necessary and sufficient to spontaneously colocalise with DCP1 in P-bodies

2019 ◽  
Author(s):  
Huaitao Cheng ◽  
Maha Alsayegh ◽  
Fred Sablitzky
Keyword(s):  
Amino Acids ◽  
Rho Gtpases ◽  
Immunological Synapse ◽  
Coiled Coil ◽  
Nucleotide Exchange ◽  
P Bodies ◽  
Mrna Metabolism ◽  
Coiled Coil Domain ◽  
Nucleotide Exchange Factor ◽  
Ef Hand

ABSTRACTDEF6 (Differentially Expressed in FDCP 6, also known as IBP and/or SLAT) is critical for the development of autoimmune disease and cancer. In T cells, DEF6 participates in TCR-mediated signalling determining T helper cell-mediated immune responses. In addition, DEF6 acts as a guanine nucleotide exchange factor (GEF) for Rho GTPases facilitating F-actin assembly and stabilisation of the immunological synapse (IS). However, DEF6 is also a component of mRNA processing bodies (P-bodies) linking it to mRNA metabolism. Including DEF6, more than 34 proteins have been shown to localise in P-bodies many of which contain a coiled coil domain, a super-secondary structure likely to facilitate interaction between these proteins. Accordingly, we suggested that the coiled coil domain in the C-terminal end of DEF6 was mediating P-body localisation of DEF6 under cellular stress conditions. However, a comprehensive analysis of wild type and mutant DEF6 proteins expressed in COS7 cells revealed that the coiled coil domain is dispensable for P-body colocalisation. Instead, we show here that the N-terminal 45 amino acids of DEF6 that contain one Ca2+-binding EF hand motif are sufficient to target DEF6 to P-bodies whereas the N-terminal 30 amino acids containing a disrupted EF hand motif are insufficient.

scholarly journals The coiled coil domain of DEF6 facilitates formation of large vesicle-like, cytoplasmic aggregates that trap the P-body marker DCP1 and exhibit prion-like features

2019 ◽  
Author(s):  
Huaitao Cheng ◽  
Fred Sablitzky
Keyword(s):  
Conformational Change ◽  
Rho Gtpases ◽  
Immunological Synapse ◽  
Coiled Coil ◽  
Nucleotide Exchange ◽  
Ph Domain ◽  
Mutant Proteins ◽  
P Bodies ◽  
Coiled Coil Domain ◽  
Large Vesicle

ABSTRACTDEF6, also known as SLAT and IBP, is critical for the development of autoimmune disease and cancer. In T cells, DEF6 participates in TCR-mediated signalling determining T helper cell-mediated immune responses. In addition, DEF6 acts as a guanine nucleotide exchange factor for Rho GTPases facilitating F-actin assembly and stabilisation of the immunological synapse. However, DEF6 is also a component of mRNA processing bodies (P-bodies) linking it to mRNA metabolism. DEF6 can adopt multiple conformations that result in different cellular localisations and functions. Post translational modifications such as phosphorylation result in conformational change liberating functional domains that are masked in the native stage of DEF6. ITK phosphorylation of Try210/222 liberates the N-terminal end and to a certain extend also the C-terminal coiled coil domain of DEF6 resulting in P-body colocalisation. In fact, the N-terminal 45 amino acids of DEF6 that encode a Ca2+-binding EF hand are sufficient to target P-bodies. Mutant proteins that unleashed the C-terminal coiled coil domain of DEF6 spontaneously aggregated forming large vesicle-like, cytoplasmic structures. These aggregates trapped proteins such as the P-body component DCP1 altering its cytoplasmic localisation. However, cellular stress reversed aggregate formation in mutant DEF6 proteins that contained ITAM and PH domain in conjunction with the coiled coil domain resulting in colocalisation with DCP1. Furthermore, coiled coil-mediated aggregates appeared to function like prions enforcing conformational change onto wild type DEF6 protein.

N-terminal targeting of guanine nucleotide exchange factors (GEF) for ADP ribosylation factors (ARF) to the Golgi

2000 ◽  
Vol 113 (11) ◽  
pp. 1883-1889 ◽  
Author(s):  
S.Y. Lee ◽  
B. Pohajdak
Keyword(s):  
Coiled Coil ◽  
Guanine Nucleotide ◽  
Nucleotide Exchange ◽  
Ph Domain ◽  
Adp Ribosylation ◽  
Guanine Nucleotide Exchange ◽  
Coiled Coil Domain ◽  
Nucleotide Exchange Factor ◽  
N Terminus ◽  
Point Mutagenesis

B2-1 (cytohesin-1) is a member of a group of proteins (including ARNO and ARNO3) that are all of similar size and domain composition. The three proteins contain an N-terminal coiled-coil domain, followed by a Sec7 and a pleckstrin homology (PH) domain. While it is well established that the Sec7 domain functions as a guanine nucleotide exchange factor (GEF) for ADP-ribosylation factors (ARFs) and the PH domain anchors the proteins to membrane phosphoinositols, the function of the N-terminal domain is unknown. Here we show that the N terminus of B2-1 (residues 1–54) is necessary and sufficient to target the protein to the Golgi. The Sec7+PH domains of B2-1 (residues 55–398) are not sufficient for Golgi localization. Further deletion analysis and point mutagenesis indicate that the coiled-coil domain within the N terminus is responsible for Golgi targeting. Furthermore, ARNO and ARNO3 N termini also have the same capability of targeting to the Golgi. We conclude that the N-terminal, (α)-helical, coiled-coil domain is used to target this family of proteins to the Golgi complex.

scholarly journals A stutter in the coiled coil domain of E.coli co-chaperone GrpE connects structure with function

2020 ◽  
Author(s):  
Tulsi Upadhyay ◽  
Vaibhav V Karekar ◽  
Ishu Saraogi
Keyword(s):  
Heat Stress ◽  
Coiled Coil ◽  
Bacterial Survival ◽  
Nucleotide Exchange ◽  
E Coli ◽  
Coiled Coil Domain ◽  
Nucleotide Exchange Factor ◽  
First Time

AbstractIn bacteria, the co-chaperone GrpE acts as a nucleotide exchange factor and plays an important role in controlling the chaperone cycle of DnaK. The functional form of GrpE is an asymmetric dimer, consisting of a long non-ideal coiled-coil. During heat stress, this region partially unfolds and prevents DnaK nucleotide exchange, ultimately ceasing the chaperone cycle. In this study, we elucidate the role of thermal unfolding of the coiled-coil domain of E. coli GrpE in regulating its co-chaperonic activity. The presence of a stutter disrupts the regular heptad arrangement typically found in an ideal coiled coil resulting in structural distortion. Introduction of hydrophobic residues at the stutter altered the structural stability of the coiled-coil. Using an in vitro FRET assay, we show for the first time that the enhanced stability of GrpE resulted in an increased affinity for DnaK. However, the mutants were defective in in vitro functional assays, and were unable to support bacterial growth at heat shock temperature in a grpE-deleted E. coli strain. This work provides valuable insights into the functional role of a stutter in the GrpE coiled-coil, and its role in regulating the DnaK-chaperone cycle for bacterial survival during heat stress. More generally, our findings illustrate how a sequence specific stutter in a coiled-coil domain regulates the structure function trade-off in proteins.

scholarly journals Roles of RabGEF1/Rabex-5 domains in regulating FcϵRI surface expression and FcϵRI-dependent responses in mast cells

Blood ◽  
2007 ◽  
Vol 109 (12) ◽  
pp. 5308-5317 ◽  
Author(s):  
Janet Kalesnikoff ◽  
Eon J. Rios ◽  
Ching-Cheng Chen ◽  
M. Alejandro Barbieri ◽  
Mindy Tsai ◽  
...  
Keyword(s):  
Mast Cells ◽  
Cell Activation ◽  
Coiled Coil ◽  
Surface Expression ◽  
Endocytic Pathway ◽  
Receptor Internalization ◽  
Nucleotide Exchange ◽  
Coiled Coil Domain ◽  
Nucleotide Exchange Factor ◽  
Ubiquitin Ligase Activity

Abstract RabGEF1/Rabex-5, a guanine nucleotide exchange factor (GEF) for the endocytic pathway regulator, Rab5, contains a Vps9 domain, an A20-like zinc finger (ZnF) domain, and a coiled coil domain. To investigate the importance of these domains in regulating receptor internalization and cell activation, we lentivirally delivered RabGEF1 mutants into RabGEF1-deficient (−/−) mast cells and examined FcϵRI-dependent responses. Wild-type RabGEF1 expression corrected phenotypic abnormalities in −/− mast cells, including decreased basal FcϵRI expression, slowed FcϵRI internalization, elevated IgE + Ag–induced degranulation and IL-6 production, and the decreased ability of −/− cytosol to support endosome fusion. We showed that RabGEF1's ZnF domain has ubiquitin ligase activity. Moreover, the coiled coil domain of RabGEF1 is required for Rabaptin-5 binding and for maintaining basal levels of Rabaptin-5 and surface FcϵRI. However, mutants lacking either of these domains normalized phenotypic abnormalities in IgE + antigen–activated −/− mast cells. By contrast, correction of these −/− phenotypes required a functional Vps9 domain. Thus, FcϵRI-mediated mast cell functional activation is dependent on RabGEF1's GEF activity.

scholarly journals Involvement of FtsE ATPase and FtsX Extracellular Loops 1 and 2 in FtsEX-PcsB Complex Function in Cell Division of Streptococcus pneumoniae D39

mBio ◽  
2013 ◽  
Vol 4 (4) ◽  
Author(s):  
Lok-To Sham ◽  
Katelyn R. Jensen ◽  
Kevin E. Bruce ◽  
Malcolm E. Winkler
Keyword(s):  
Signal Transduction ◽  
Amino Acids ◽  
Cell Division ◽  
Streptococcus Pneumoniae ◽  
Amino Acid ◽  
Stationary Phase ◽  
Coiled Coil ◽  
Extracellular Loop ◽  
Coiled Coil Domain ◽  
Loop Domain

ABSTRACT The FtsEX protein complex has recently been proposed to play a major role in coordinating peptidoglycan (PG) remodeling by hydrolases with the division of bacterial cells. According to this model, cytoplasmic FtsE ATPase interacts with the FtsZ divisome and FtsX integral membrane protein and powers allosteric activation of an extracellular hydrolase interacting with FtsX. In the major human respiratory pathogen Streptococcus pneumoniae (pneumococcus), a large extracellular-loop domain of FtsX (ECL1FtsX) is thought to interact with the coiled-coil domain of the PcsB protein, which likely functions as a PG amidase or endopeptidase required for normal cell division. This paper provides evidence for two key tenets of this model. First, we show that FtsE protein is essential, that depletion of FtsE phenocopies cell defects caused by depletion of FtsX or PcsB, and that changes of conserved amino acids in the FtsE ATPase active site are not tolerated. Second, we show that temperature-sensitive (Ts) pcsB mutations resulting in amino acid changes in the PcsB coiled-coil domain (CCPcsB) are suppressed by ftsX mutations resulting in amino acid changes in the distal part of ECL1FtsX or in a second, small extracellular-loop domain (ECL2FtsX). Some FtsX suppressors are allele specific for changes in CCPcsB, and no FtsX suppressors were found for amino acid changes in the catalytic PcsB CHAP domain (CHAPPcsB). These results strongly support roles for both ECL1FtsX and ECL2FtsX in signal transduction to the coiled-coil domain of PcsB. Finally, we found that pcsB CC(Ts) mutants (Ts mutants carrying mutations in the region of pcsB corresponding to the coiled-coil domain) unexpectedly exhibit delayed stationary-phase autolysis at a permissive growth temperature. IMPORTANCE Little is known about how FtsX interacts with cognate PG hydrolases in any bacterium, besides that ECL1FtsX domains somehow interact with coiled-coil domains. This work used powerful genetic approaches to implicate a specific region of pneumococcal ECL1FtsX and the small ECL2FtsX in the interaction with CCPcsB. These findings identify amino acids important for in vivo signal transduction between FtsX and PcsB for the first time. This paper also supports the central hypothesis that signal transduction between pneumococcal FtsX and PcsB is linked to ATP hydrolysis by essential FtsE, which couples PG hydrolysis to cell division. The classical genetic approaches used here can be applied to dissect interactions of other integral membrane proteins involved in PG biosynthesis. Finally, delayed autolysis of the pcsB CC(Ts) mutants suggests that the FtsEX-PcsB PG hydrolase may generate a signal in the PG necessary for activation of the major LytA autolysin as pneumococcal cells enter stationary phase.

scholarly journals Sec2 protein contains a coiled-coil domain essential for vesicular transport and a dispensable carboxy terminal domain.

1990 ◽  
Vol 110 (6) ◽  
pp. 1897-1909 ◽  
Author(s):  
J Nair ◽  
H Müller ◽  
M Peterson ◽  
P Novick
Keyword(s):  
Amino Acids ◽  
Secretory Pathway ◽  
Synthetic Lethality ◽  
Coiled Coil ◽  
Protein Product ◽  
Temperature Sensitive ◽  
Vesicular Traffic ◽  
Amino Terminal ◽  
Native Molecular Mass ◽  
Coiled Coil Domain

SEC2 function is required at the post-Golgi apparatus stage of the yeast secretory pathway. The SEC2 sequence encodes a protein product of 759 amino acids containing an amino terminal region that is predicted to be in an alpha-helical, coiled-coil conformation. Two temperature-sensitive alleles, sec2-41 and sec2-59, encode proteins truncated by opal stop codons and are suppressible by an opal tRNA suppressor. Deletion analysis indicates that removal of the carboxyl terminal 251 amino acids has no apparent phenotype, while truncation of 368 amino acids causes temperature sensitivity. The amino terminal half of the protein, containing the putative coiled-coil domain, is essential at all temperatures. Sec2 protein is found predominantly in the soluble fraction and displays a native molecular mass of greater than 500 kD. All phenotypes of the temperature-sensitive sec2 alleles are partially suppressed by duplication of the SEC4 gene, but the lethality of a sec2 disruption is not suppressed. The sec2-41 mutation exhibits synthetic lethality with the same subset of the late acting sec mutants as does sec4-8 and sec15-1. The Sec2 protein may function in conjunction with the Sec4 and Sec15 proteins to control vesicular traffic.

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