scholarly journals Structural characterization of the RH1-LZI tandem of JIP3/4 highlights RH1 domains as a cytoskeletal motor-binding motif

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Fernando Vilela ◽  
Christophe Velours ◽  
Mélanie Chenon ◽  
Magali Aumont-Nicaise ◽  
Valérie Campanacci ◽  
...  
Keyword(s):  
Leucine Zipper ◽  
Homology Modelling ◽  
Coiled Coil ◽  
Structural Data ◽  
Adaptor Proteins ◽  
Protein Family ◽  
Binding Motif ◽  
Rab Gtpases ◽  
Cytoskeletal Motors

Abstract JIP3 and JIP4 (JNK-interacting proteins 3 and 4) are adaptors for cargo recruitment by dynein/dynactin and kinesin1 motors. Both are dimers that are stabilised by two sections of leucine zipper coiled coils. The N-terminal Leucine Zipper I (LZI) belongs to a section that binds dynein-DLIC and kinesin1-KHC, whilst the medial Leucine Zipper II (LZII) binds dynactin-p150glued and kinesin1-KLC. Structural data is available for the LZII, but the LZI section is still uncharacterized. Here we characterize the N-terminal part of JIP3/4 which consists of an RH1 (RILP homology 1) domain followed by the LZI coiled coil using bioinformatical, biophysical and structural approaches. The RH1-LZI tandem of JIP3 associates as a high affinity homodimer exhibiting elongated alpha-helical fold. 3D homology modelling of the RH1-LZI tandem reveals that the kinesin1-KHC binding site mainly overlaps with the RH1 domain. A sequence comparison search indicates that only one other protein family has RH1 domains similar to those of JIP3/4, the RILP (Rab-interacting lysosomal protein) family which consists of adaptor proteins linking Rab GTPases to cytoskeletal motors. RILPL2 is recruited through its RH1 domain by the myosin 5a motor. Here, we showed that the RH1 domain of JIP3 also interacts with myosin 5 A in vitro, highlighting JIP3/4 as possible myosin 5a adaptors. Finally, we propose that JIP3/4 and RILP family members define a unique RH1/RH2-architecture adaptor superfamily linking cytoskeletal motors and Rab GTPases.

scholarly journals A new calcium-activated dynein adaptor protein, CRACR2a, regulates clathrin-independent endocytic traffic in T cells

2018 ◽  
Author(s):  
Yuxiao Wang ◽  
Walter Huynh ◽  
Taylor D. Skokan ◽  
Ronald D. Vale
Keyword(s):  
T Cells ◽  
Coiled Coil ◽  
Adaptor Protein ◽  
Adaptor Proteins ◽  
Cytoplasmic Dynein ◽  
Rab Gtpase ◽  
Rab Gtpases ◽  
Microtubule Organizing Center ◽  
Activated T Cells

AbstractCytoplasmic dynein is a microtubule minus-end-directed motor that transports numerous intracellular cargoes. Mammalian dynein transport is initiated by coiled-coil adaptor proteins that 1) join dynein and its co-factor dynactin into a complex capable of processive motility, and 2) interact with a cargo-bound receptor, which is frequently a Rab GTPase on an organelle. Here, we report two novel dynein adaptors, CRACR2a and Rab45, which have a coiled-coil adaptor domain, a pair of EF hands, and a Rab GTPase domain fused into a single polypeptide. We find that CRACR2a-mediated dynein-dynactin motility is activated by calcium in vitro and in cells. In activated T cells, CRACR2a localizes to clathrin-independent endosomes that require microtubule-based transport to detach from the actin cortex and travel towards the microtubule organizing center. Together these results represent the first known examples of Rab GTPases that directly act as dynein adaptors and implicate CRACR2a-dynein in regulation of endocytic trafficking in T cells.

The invisibility cloak: Chitin binding protein of Verticillium nonalfalfae disguises fungus from plant chitinases

2018 ◽  
Author(s):  
Helena Volk ◽  
Kristina Marton ◽  
Marko Flajšman ◽  
Sebastjan Radišek ◽  
Ingo Hein ◽  
...  
Keyword(s):  
Fungal Infections ◽  
Binding Protein ◽  
Homology Modelling ◽  
Binding Motif ◽  
Carbohydrate Binding ◽  
Fungal Cell ◽  
Verticillium Nonalfalfae ◽  
Chitin Binding Protein ◽  
Plant Chitinases

AbstractDuring fungal infections, plant cells secrete chitinases that digest chitin in the fungal cell walls. The recognition of released chitin oligomers via lysin motif (LysM)-containing immune receptors results in the activation of defence signalling pathways. We report here that Verticillium nonalfalfae, a hemibiotrophic xylem-invading fungus, prevents this recognition process by secreting a CBM18 (carbohydrate binding motif 18)-chitin binding protein, VnaChtBP, which is transcriptionally activated specifically during the parasitic life stages. VnaChtBP is encoded by the Vna8.213 gene which is highly conserved within the species, suggesting high evolutionary stability and importance for the fungal lifestyle. In a pathogenicity assay, however, Vna8.213 knockout mutants exhibit wilting symptoms similar to the wild type fungus, suggesting that Vna8.213 activity is functionally redundant during fungal infection of hop. In binding assay, recombinant VnaChtBP binds chitin and chitin oligomers in vitro with submicromolar affinity and protects fungal hyphae from degradation by plant chitinases. Using a yeast-two-hybrid assay, homology modelling and molecular docking, we demonstrated that VnaChtBP forms dimers in the absence of ligands and that this interaction is stabilized by the binding of chitin hexamers with a similar preference in the two binding sites. Our data suggest that, in addition to chitin binding LysM (CBM50) and Avr4 (CBM14) fungal effectors, structurally unrelated CBM18 effectors have convergently evolved to prevent hydrolysis of the fungal cell wall against plant chitinases.

scholarly journals Critical Roles of Phosphorylation and Actin Binding Motifs, but Not the Central Proline-rich Region, for Ena/Vasodilator-stimulated Phosphoprotein (VASP) Function during Cell Migration

2002 ◽  
Vol 13 (7) ◽  
pp. 2533-2546 ◽  
Author(s):  
Joseph J. Loureiro ◽  
Douglas A. Rubinson ◽  
James E. Bear ◽  
Gretchen A. Baltus ◽  
Adam V. Kwiatkowski ◽  
...  
Keyword(s):  
Cell Motility ◽  
Cell Movement ◽  
Protein Family ◽  
Actin Dynamics ◽  
Actin Binding ◽  
Binding Motif ◽  
Conserved Sequence ◽  
Cellular Processes ◽  
Sequence Elements

The Ena/vasodilator-stimulated phosphoprotein (VASP) protein family is implicated in the regulation of a number of actin-based cellular processes, including lamellipodial protrusion necessary for whole cell translocation. A growing body of evidence derived largely from in vitro biochemical experiments using purified proteins, cell-free extracts, and pathogen motility has begun to suggest various mechanistic roles for Ena/VASP proteins in the control of actin dynamics. Using complementation of phenotypes in Ena/VASP-deficient cells and overexpression in normal fibroblasts, we have assayed the function of a panel of mutants in one member of this family, Mena, by mutating highly conserved sequence elements found in this protein family. Surprisingly, deletion of sites required for binding of the actin monomer-binding protein profilin, a known ligand of Ena/VASP proteins, has no effect on the ability of Mena to regulate random cell motility. Our analysis revealed two features essential for Ena/VASP function in cell movement, cyclic nucleotide-dependent kinase phosphorylation sites and an F-actin binding motif. Interestingly, expression of the C-terminal EVH2 domain alone is sufficient to complement loss of Ena/VASP function in random cell motility.

scholarly journals Loss of cargo binding in the human myosin VI deafness mutant (R1166X) leads to increased actin filament binding

2016 ◽  
Vol 473 (19) ◽  
pp. 3307-3319 ◽  
Author(s):  
Susan D. Arden ◽  
David A. Tumbarello ◽  
Tariq Butt ◽  
John Kendrick-Jones ◽  
Folma Buss
Keyword(s):  
Amino Acids ◽  
Adaptor Protein ◽  
Adaptor Proteins ◽  
Actin Binding ◽  
Binding Motif ◽  
Myosin Vi ◽  
Binding Ability ◽  
Activated State ◽  
Cargo Binding

Mutations in myosin VI have been associated with autosomal-recessive (DFNB37) and autosomal-dominant (DFNA22) deafness in humans. Here, we characterise an myosin VI nonsense mutation (R1166X) that was identified in a family with hereditary hearing loss in Pakistan. This mutation leads to the deletion of the C-terminal 120 amino acids of the myosin VI cargo-binding domain, which includes the WWY-binding motif for the adaptor proteins LMTK2, Tom1 as well as Dab2. Interestingly, compromising myosin VI vesicle-binding ability by expressing myosin VI with the R1166X mutation or with single point mutations in the adaptor-binding sites leads to increased F-actin binding of this myosin in vitro and in vivo. As our results highlight the importance of cargo attachment for regulating actin binding to the motor domain, we perform a detailed characterisation of adaptor protein binding and identify single amino acids within myosin VI required for binding to cargo adaptors. We not only show that the adaptor proteins can directly interact with the cargo-binding tail of myosin VI, but our in vitro studies also suggest that multiple adaptor proteins can bind simultaneously to non-overlapping sites in the myosin VI tail. In conclusion, our characterisation of the human myosin VI deafness mutant (R1166X) suggests that defects in cargo binding may leave myosin VI in a primed/activated state with an increased actin-binding ability.

scholarly journals Structure and dynamics of the DBHS protein family members SFPQ, NONO and PSPC1

2014 ◽  
Vol 70 (a1) ◽  
pp. C1655-C1655
Author(s):  
Gavin Knott ◽  
Mihwa Lee ◽  
Daniel Passon ◽  
Agata Sadowska ◽  
Archa Fox ◽  
...  
Keyword(s):  
Protein Function ◽  
Coiled Coil ◽  
Protein Family ◽  
Titration Calorimetry ◽  
Ph Homeostasis ◽  
Transcriptional Initiation ◽  
Diverse Range ◽  
Multifunctional Protein

The Drosophila behaviour/human splicing (DBHS) proteins are a family of obligatory dimeric proteins found in higher order mammals down to the simplest invertebrates. `Multifunctional protein family' essentially captures what is understood regarding DBHS protein function where they are cited to regulate transcriptional initiation, the processing and export of RNA, maintenance of genomic DNA, nuclear pH homeostasis and carcinogenesis [1]. Furthermore, with roles in binding a diverse range of RNAs and both single and double stranded DNA, it is difficult to establish a coherent picture for their nuclear activities. In humans, the family consists of three highly conserved members, namely Non-POU domain-containing octamer-binding protein (NONO/p54nrb), splicing factor proline/glutamine rich (SFPQ/PSF) and paraspeckle protein component 1 (PSPC1). The conserved DBHS region of these proteins comprises tandem RNA recognition motifs (RRMs), a NOPS domain and a C-terminal coiled-coil domain. The unique structural arrangement of these domains facilitates an intimate dimerisation interface that gives rise to a novel arrangement of RRMs [2]. Given this interface, it is not surprising that DBHS proteins likely exist as either homo- or heterodimers in vivo. Here we report the first structure of the ancestral C. elegans DBHS protein, NONO-1, refined to 2.8 Å. The structure clearly illustrates the consistent obligatory nature of DBHS dimerisation and through isothermal titration calorimetry we have demonstrated that human DBHS proteins prefer a heterodimeric state in vitro. There is a growing appreciation for the fundamental significance of DBHS proteins in human health and disease and this work highlights the critical need for a more robust assessment of in vivo DBHS function.

scholarly journals p116Rip Targets Myosin Phosphatase to the Actin Cytoskeleton and Is Essential for RhoA/ROCK-regulated Neuritogenesis

2004 ◽  
Vol 15 (12) ◽  
pp. 5516-5527 ◽  
Author(s):  
Jacqueline Mulder ◽  
Aafke Ariaens ◽  
Dick van den Boomen ◽  
Wouter H. Moolenaar
Keyword(s):  
Actin Cytoskeleton ◽  
Neurite Outgrowth ◽  
Light Chain ◽  
Leucine Zipper ◽  
Myosin Ii ◽  
Coiled Coil ◽  
Cell Spreading ◽  
Actin Binding ◽  
Myosin Phosphatase

Activation of the RhoA-Rho kinase (ROCK) pathway stimulates actomyosin-driven contractility in many cell systems, largely through ROCK-mediated inhibition of myosin II light chain phosphatase. In neuronal cells, the RhoA-ROCK-actomyosin pathway signals cell rounding, growth cone collapse, and neurite retraction; conversely, inhibition of RhoA/ROCK promotes cell spreading and neurite outgrowth. The actin-binding protein p116Rip, whose N-terminal region bundles F-actin in vitro, has been implicated in Rho-dependent neurite remodeling; however, its function is largely unknown. Here, we show that p116Rip, through its C-terminal coiled-coil domain, interacts directly with the C-terminal leucine zipper of the regulatory myosin-binding subunits of myosin II phosphatase, MBS85 and MBS130. RNA interference-induced knockdown of p116Rip inhibits cell spreading and neurite outgrowth in response to extracellular cues, without interfering with the regulation of myosin light chain phosphorylation. We conclude that p116Rip is essential for neurite outgrowth and may act as a scaffold to target the myosin phosphatase complex to the actin cytoskeleton.

scholarly journals Cloning of the Schizosaccharomyces pombe gene encoding diadenosine 5′,5′′′-P1,P4-tetraphosphate (Ap4A) asymmetrical hydrolase: sequence similarity with the histidine triad (HIT) protein family

1995 ◽  
Vol 312 (3) ◽  
pp. 925-932 ◽  
Author(s):  
Y Huang ◽  
P N Garrison ◽  
L D Barnes
Keyword(s):  
Amino Acids ◽  
Schizosaccharomyces Pombe ◽  
Sequence Similarity ◽  
Enzymic Activity ◽  
Protein Family ◽  
Partial Purification ◽  
Binding Motif ◽  
Local Similarity ◽  
Multicopy Plasmid

Diadenosine 5′,5‴-P1,P4-tetraphosphate (Ap4A) asymmetric hydrolase (EC 3.6.1.17) is a specific catabolic enzyme of Ap4A found in Schizosaccharomyces pombe. We have previously described the partial purification of Ap4A hydrolase from S. pombe [Robinson, de la Peña and Barnes (1993) Biochim. Biophys. Acta 1161, 139-148]. We determined the sequence of the N-terminal 20 amino acids of Ap4A hydrolase and designed two degenerate PCR primers based on the sequence. The 60 bp DNA fragment obtained by PCR, which is specific to Ap4A hydrolase, was used to isolate the Ap4A hydrolase gene, aph1, from S. pombe by screening a genomic DNA library in a multicopy plasmid. Ap4A hydrolase activity from the crude supernatant of a positive S. pombe transformant was about 25-fold higher than the control. There was no detectable stimulation of enzymic activity by phosphate. The aph1 gene from S. pombe contains three introns. The intron boundaries were confirmed by sequencing the cDNA of the aph1 gene from a S. pombe cDNA library. The deduced open reading frame of the aph1 gene codes for 182 amino acids. Two regions of significant local similarity were identified between the Ap4A hydrolase and the histidine triad (HIT) protein family [Séraphin (1992) DNA Sequence 3, 177-179]. HIT proteins are present in prokaryotes, yeast, plants and mammals. Their functions are unknown, except that the bovine protein inhibits protein kinase C in vitro. All four histidine residues which are conserved among the HIT proteins, including the HxHxH putative Zn(2+)-binding motif, are conserved in the Ap4A hydrolase. In addition, there are two regions of similarity between the Ap4A phosphorylases I and II from Saccharomyces cerevisiae and Ap4A hydrolase from S. pombe. These regions overlap with the HIT protein similarity regions. The aph1 gene from S. pombe is the first asymmetrical Ap4A hydrolase gene to be cloned and sequenced.

scholarly journals Engineered Single-Chain, Antiparallel, Coiled CoilMimics the MerR Metal BindingSite

2004 ◽  
Vol 186 (6) ◽  
pp. 1861-1868 ◽  
Author(s):  
Lingyun Song ◽  
Jonathan Caguiat ◽  
Zhongrui Li ◽  
Jacob Shokes ◽  
Robert A. Scott ◽  
...  
Keyword(s):  
Transcriptional Activation ◽  
Metal Binding ◽  
Coiled Coil ◽  
Mercury Resistance ◽  
Binding Motif ◽  
Single Chain ◽  
Metal Binding Domain ◽  
Α Helix

ABSTRACT The repressor-activator MerR that controls transcription of the mercury resistance (mer) operon is unusual for its high sensitivity and specificity for Hg(II) in in vivo and in vitro transcriptional assays. The metal-recognition domain of MerR resides at the homodimer interface in a novel antiparallel arrangement of α-helix 5 that forms a coiled-coil motif. To facilitate the study of this novel metal binding motif, we assembled this antiparallel coiled coil into a single chain by directly fusing two copies of the 48-residue α-helix 5 of MerR. The resulting 107-residue polypeptide, called the metal binding domain (MBD), and wild-type MerR were overproduced and purified, and their metal-binding properties were determined in vivo and in vitro. In vitro MBD bound ca. 1.0 equivalent of Hg(II) per pair of binding sites, just as MerR does, and it showed only a slightly lower affinity for Hg(II) than did MerR. Extended X-ray absorption fine structure data showed that MBD has essentially the same Hg(II) coordination environment as MerR. In vivo, cells overexpressing MBD accumulated 70 to 100% more 203Hg(II) than cells bearing the vector alone, without deleterious effects on cell growth. Both MerR and MBD variously bound other thiophilic metal ions, including Cd(II), Zn(II), Pb(II), and As(III), in vitro and in vivo. We conclude that (i) it is possible to simulate in a single polypeptide chain the in vitro and in vivo metal-binding ability of dimeric, full-length MerR and (ii) MerR's specificity in transcriptional activation does not reside solely in the metal-binding step.

scholarly journals Protein flexibility is required for vesicle tethering at the Golgi

eLife ◽  
2015 ◽  
Vol 4 ◽  
Author(s):  
Pak-yan Patricia Cheung ◽  
Charles Limouse ◽  
Hideo Mabuchi ◽  
Suzanne R Pfeffer
Keyword(s):  
Coiled Coil ◽  
Rab Gtpases ◽  
Force Microscopy ◽  
Vesicle Tethering ◽  
Proximity Ligation ◽  
Atomic Force ◽  
Transport Vesicles ◽  
Late Endosomes ◽  
Specific Sequences

The Golgi is decorated with coiled-coil proteins that may extend long distances to help vesicles find their targets. GCC185 is a trans Golgi-associated protein that captures vesicles inbound from late endosomes. Although predicted to be relatively rigid and highly extended, we show that flexibility in a central region is required for GCC185’s ability to function in a vesicle tethering cycle. Proximity ligation experiments show that that GCC185’s N-and C-termini are within <40 nm of each other on the Golgi. In physiological buffers without fixatives, atomic force microscopy reveals that GCC185 is shorter than predicted, and its flexibility is due to a central bubble that represents local unwinding of specific sequences. Moreover, 85% of the N-termini are splayed, and the splayed N-terminus can capture transport vesicles in vitro. These unexpected features support a model in which GCC185 collapses onto the Golgi surface, perhaps by binding to Rab GTPases, to mediate vesicle tethering.

scholarly journals CRACR2a is a calcium-activated dynein adaptor protein that regulates endocytic traffic

2019 ◽  
Vol 218 (5) ◽  
pp. 1619-1633 ◽  
Author(s):  
Yuxiao Wang ◽  
Walter Huynh ◽  
Taylor D. Skokan ◽  
Wen Lu ◽  
Arthur Weiss ◽  
...  
Keyword(s):  
Coiled Coil ◽  
Adaptor Protein ◽  
Cell Receptor ◽  
Cytoplasmic Dynein ◽  
Receptor Activation ◽  
Rab Gtpase ◽  
Rab Gtpases ◽  
Microtubule Motor ◽  
Ef Hands

Cytoplasmic dynein is a minus end–directed microtubule motor that transports intracellular cargoes. Transport is initiated by coiled-coil adaptors that (a) join dynein and its cofactor dynactin into a motile complex and (b) interact with a cargo-bound receptor, which is frequently a Rab GTPase on an organelle. Here, we report two novel dynein adaptors, CRACR2a and Rab45, that have a coiled-coil adaptor domain, a pair of EF-hands, and a Rab GTPase fused into a single polypeptide. CRACR2a-mediated, but not Rab45-mediated, dynein motility is activated by calcium in vitro. In Jurkat T cells, elevation of intracellular calcium activates CRACR2a-mediated dynein transport. We further found that T cell receptor activation induces the formation of CRACR2a puncta at the plasma membrane, which initially associate with the actin cortex and subsequently detach and travel along microtubules, suggestive of an endocytic process. These results provide the first examples of Rab GTPases that directly act as dynein adaptors and implicate CRACR2a–dynein in calcium-regulated endocytic trafficking.

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