Browse
Submit Data
Databases
API
Help

Dataset Information

15 Views

0 Connections

0 Citations

0 Reanalyses

0 Downloads

Omics score: 0

Structural Organization and Dynamics of Homodimeric Cytohesin Family Arf GTPase Exchange Factors in Solution and on Membranes.

ABSTRACT: Membrane dynamic processes require Arf GTPase activation by guanine nucleotide exchange factors (GEFs) with a Sec7 domain. Cytohesin family Arf GEFs function in signaling and cell migration through Arf GTPase activation on the plasma membrane and endosomes. In this study, the structural organization of two cytohesins (Grp1 and ARNO) was investigated in solution by size exclusion-small angle X-ray scattering and negative stain-electron microscopy and on membranes by dynamic light scattering, hydrogen-deuterium exchange-mass spectrometry and guanosine diphosphate (GDP)/guanosine triphosphate (GTP) exchange assays. The results suggest that cytohesins form elongated dimers with a central coiled coil and membrane-binding pleckstrin-homology (PH) domains at opposite ends. The dimers display significant conformational heterogeneity, with a preference for compact to intermediate conformations. Phosphoinositide-dependent membrane recruitment is mediated by one PH domain at a time and alters the conformational dynamics to prime allosteric activation by Arf-GTP. A structural model for membrane targeting and allosteric activation of full-length cytohesin dimers is discussed.

SUBMITTER: Das S

PROVIDER: S-EPMC6948192 | biostudies-literature |

REPOSITORIES: biostudies-literature

ACCESS DATA

Json Xml

Similar Datasets

Structural Dynamics Control Allosteric Activation of Cytohesin Family Arf GTPase Exchange Factors.

Project description:Membrane dynamic processes including vesicle biogenesis depend on Arf guanosine triphosphatase (GTPase) activation by guanine nucleotide exchange factors (GEFs) containing a catalytic Sec7 domain and a membrane-targeting module such as a pleckstrin homology (PH) domain. The catalytic output of cytohesin family Arf GEFs is controlled by autoinhibitory interactions that impede accessibility of the exchange site in the Sec7 domain. These restraints can be relieved through activator Arf-GTP binding to an allosteric site comprising the PH domain and proximal autoinhibitory elements (Sec7-PH linker and C-terminal helix). Small-angle X-ray scattering and negative-stain electron microscopy were used to investigate the structural organization and conformational dynamics of cytohesin-3 (Grp1) in autoinhibited and active states. The results support a model in which hinge dynamics in the autoinhibited state expose the activator site for Arf-GTP binding, while subsequent C-terminal helix unlatching and repositioning unleash conformational entropy in the Sec7-PH linker to drive exposure of the exchange site.

| S-EPMC5752578 | biostudies-literature

Structural basis for membrane recruitment and allosteric activation of cytohesin family Arf GTPase exchange factors.

Project description:Membrane recruitment of cytohesin family Arf guanine nucleotide exchange factors depends on interactions with phosphoinositides and active Arf GTPases that, in turn, relieve autoinhibition of the catalytic Sec7 domain through an unknown structural mechanism. Here, we show that Arf6-GTP relieves autoinhibition by binding to an allosteric site that includes the autoinhibitory elements in addition to the PH domain. The crystal structure of a cytohesin-3 construct encompassing the allosteric site in complex with the head group of phosphatidyl inositol 3,4,5-trisphosphate and N-terminally truncated Arf6-GTP reveals a large conformational rearrangement, whereby autoinhibition can be relieved by competitive sequestration of the autoinhibitory elements in grooves at the Arf6/PH domain interface. Disposition of the known membrane targeting determinants on a common surface is compatible with multivalent membrane docking and subsequent activation of Arf substrates, suggesting a plausible model through which membrane recruitment and allosteric activation could be structurally integrated.

| S-EPMC3761562 | biostudies-literature

Structural basis and mechanism of autoregulation in 3-phosphoinositide-dependent Grp1 family Arf GTPase exchange factors.

Project description:Arf GTPases regulate membrane trafficking and actin dynamics. Grp1, ARNO, and Cytohesin-1 comprise a family of phosphoinositide-dependent Arf GTPase exchange factors with a Sec7-pleckstrin homology (PH) domain tandem. Here, we report that the exchange activity of the Sec7 domain is potently autoinhibited by conserved elements proximal to the PH domain. The crystal structure of the Grp1 Sec7-PH tandem reveals a pseudosubstrate mechanism of autoinhibition in which the linker region between domains and a C-terminal amphipathic helix physically block the docking sites for the switch regions of Arf GTPases. Mutations within either element result in partial or complete activation. Critical determinants of autoinhibition also contribute to insulin-stimulated plasma membrane recruitment. Autoinhibition can be largely reversed by binding of active Arf6 to Grp1 and by phosphorylation of tandem PKC sites in Cytohesin-1. These observations suggest that Grp1 family GEFs are autoregulated by mechanisms that depend on plasma membrane recruitment for activation.

| S-EPMC2156038 | biostudies-literature

Structural insights into a new homodimeric self-activated GTPase family.

Project description:The human XAB1/MBDin GTPase and its close homologues form one of the ten phylogenetically distinct families of the SIMIBI (after signal recognition particle, MinD and BioD) class of phosphate-binding loop NTPases. The genomic context and the partners identified for the archaeal and eukaryotic homologues indicate that they are involved in genome maintenance--DNA repair or replication. The crystal structure of PAB0955 from Pyrococcus abyssi shows that, unlike other SIMIBI class G proteins, these highly conserved GTPases are homodimeric, regardless of the presence of nucleotides. The nucleotide-binding site of PAB0955 is rather rigid and its conformation is closest to that of the activated SRP G domain. One insertion to the G domain bears a strictly conserved GPN motif, which is part of the catalytic site of the other monomer and stabilizes the phosphate ion formed. Owing to this unique functional feature, we propose to call this family as GPN-loop GTPase.

| S-EPMC2002535 | biostudies-literature

Solution structure of the cytohesin-1 (B2-1) Sec7 domain and its interaction with the GTPase ADP ribosylation factor 1.

Project description:Cytohesin-1 (B2-1) is a guanine nucleotide exchange factor for human ADP ribosylation factor (Arf) GTPases, which are important for vesicular protein trafficking and coatamer assembly in the cell. Cytohesin-1 also has been reported to promote cellular adhesion via binding to the beta2 integrin cytoplasmic domain. The solution structure of the Sec7 domain of cytohesin-1, which is responsible for both the protein's guanine nucleotide exchange factor function and beta2 integrin binding, was determined by NMR spectroscopy. The structure consists of 10 alpha-helices that form a unique tertiary fold. The binding between the Sec7 domain and a soluble, truncated version of human Arf-1 was investigated by examining 1H-15N and 1H-13C chemical shift changes between the native protein and the Sec7/Arf-1 complex. We show that the binding to Arf-1 occurs through a large surface on the C-terminal subdomain that is composed of both hydrophobic and polar residues. Structure-based mutational analysis of the cytohesin-1 Sec7 domain has been used to identify residues important for binding to Arf and for mediating nucleotide exchange. Investigations into the interaction between the Sec7 domain and the beta2 integrin cytoplasmic domain suggest that the two proteins do not interact in the solution phase.

| S-EPMC20903 | biostudies-literature

A Eukaryote-Wide Perspective on the Diversity and Evolution of the ARF GTPase Protein Family.

Project description:The evolution of eukaryotic cellular complexity is interwoven with the extensive diversification of many protein families. One key family is the ARF GTPases that act in eukaryote-specific processes, including membrane traffic, tubulin assembly, actin dynamics, and cilia-related functions. Unfortunately, our understanding of the evolution of this family is limited. Sampling an extensive set of available genome and transcriptome sequences, we have assembled a data set of over 2,000 manually curated ARF family genes from 114 eukaryotic species, including many deeply diverged protist lineages, and carried out comprehensive molecular phylogenetic analyses. These reconstructed as many as 16 ARF family members present in the last eukaryotic common ancestor, nearly doubling the previously inferred ancient system complexity. Evidence for the wide occurrence and ancestral origin of Arf6, Arl13, and Arl16 is presented for the first time. Moreover, Arl17, Arl18, and SarB, newly described here, are absent from well-studied model organisms and as a result their function(s) remain unknown. Analyses of our data set revealed a previously unsuspected diversity of membrane association modes and domain architectures within the ARF family. We detail the step-wise expansion of the ARF family in the metazoan lineage, including discovery of several new animal-specific family members. Delving back to its earliest evolution in eukaryotes, the resolved relationship observed between the ARF family paralogs sets boundaries for scenarios of vesicle coat origins during eukaryogenesis. Altogether, our work fundamentally broadens the understanding of the diversity and evolution of a protein family underpinning the structural and functional complexity of the eukaryote cells.

| S-EPMC8358228 | biostudies-literature

The ARF guanine nucleotide exchange factor GBF1 is targeted to Golgi membranes through a PIP-binding domain.

Project description:ADP-ribosylation factors (ARF) GTPases are activated by guanine nucleotide exchange factors (GEFs) to support cellular homeostasis. Key to understanding spatio-temporal regulation of ARF signaling is the mechanism of GEF recruitment to membranes. Small GEFs are recruited through phosphoinositide (PIP) binding by a pleckstrin homology (PH) domain downstream from the catalytic Sec7 domain (Sec7d). The large GEFs lack PH domains, and their recruitment mechanisms are poorly understood. We probed Golgi recruitment of GBF1, a GEF catalyzing ARF activation required for Golgi homeostasis. We show that the homology downstream of Sec7d-1 (HDS1) regulates Golgi recruitment of GBF1. We document that GBF1 binds phosphoinositides, preferentially PI3P, PI4P and PI(4,5)P2, and that lipid binding requires the HDS1 domain. Mutations within HDS1 that reduce GBF1 binding to specific PIPs in vitro inhibit GBF1 targeting to Golgi membranes in cells. Our data imply that HDS1 and PH domains are functionally analogous in that each uses lipid-based membrane information to regulate GEF recruitment. Lipid-based recruitment of GBF1 extends the paradigm of lipid regulation to small and large GEFs and suggests that lipid-based mechanisms evolved early during GEF diversification. This article has an associated First Person interview with the first author of the paper.

| S-EPMC5826047 | biostudies-literature

Structural organization and dynamics of FCHo2 docking on membranes.

Project description:Clathrin-mediated endocytosis (CME) is a central trafficking pathway in eukaryotic cells regulated by phosphoinositides. The plasma membrane phosphatidylinositol-4,5-bisphosphate (PI(4,5)P2) plays an instrumental role in driving CME initiation. The F-BAR domain-only protein 1 and 2 complex (FCHo1/2) is among the early proteins that reach the plasma membrane, but the exact mechanisms triggering its recruitment remain elusive. Here, we show the molecular dynamics of FCHo2 self-assembly on membranes by combining minimal reconstituted in vitro and cellular systems. Our results indicate that PI(4,5)P2 domains assist FCHo2 docking at specific membrane regions, where it self-assembles into ring-like-shaped protein patches. We show that the binding of FCHo2 on cellular membranes promotes PI(4,5)P2 clustering at the boundary of cargo receptors and that this accumulation enhances clathrin assembly. Thus, our results provide a mechanistic framework that could explain the recruitment of early PI(4,5)P2-interacting proteins at endocytic sites.

| S-EPMC8798043 | biostudies-literature

Specificity and membrane partitioning of Grsp1 signaling complexes with Grp1 family Arf exchange factors.

Project description:The Arf exchange factor Grp1 selectively binds phosphatidylinositol 3,4,5-triphosphate [PtdIns(3,4,5)P(3)], which is required for recruitment to the plasma membrane in stimulated cells. The mechanisms for phosphoinositide recognition by the PH domain, catalysis of nucleotide exchange by the Sec7 domain, and autoinhibition by elements proximal to the PH domain are well-characterized. The N-terminal heptad repeats in Grp1 have also been shown to mediate homodimerization in vitro as well as heteromeric interactions with heptad repeats in the FERM domain-containing protein Grsp1 both in vitro and in cells [Klarlund, J. K., et al. (2001) J. Biol. Chem. 276, 40065-40070]. Here, we have characterized the oligomeric state of Grsp1 and Grp1 family proteins (Grp1, ARNO, and Cytohesin-1) as well as the oligomeric state, stoichiometry, and specificity of Grsp1 complexes with Grp1, ARNO, and Cytohesin-1. At low micromolar concentrations, Grp1 and ARNO are homodimeric whereas Cytohesin-1 and Grsp1 are monomeric. When mixed with Grsp1, Grp1 homodimers and Cytohesin-1 monomers spontaneously re-equilibrate to form heterodimers, whereas approximately 50% of ARNO remains homodimeric under the same conditions. Fluorescence resonance energy transfer experiments suggest that the Grsp1 heterodimers with Grp1 and Cytohesin-1 adopt a largely antiparallel orientation. Finally, formation of Grsp1-Grp1 heterodimers does not substantially influence the binding of Grp1 to the headgroups of PtdIns(3,4,5)P(3) or PtdIns(4,5)P(2), nor does it influence partitioning with liposomes containing PtdIns(3,4,5)P(3), PtdIns(4,5)P(2), and/or phosphatidylserine.

| S-EPMC2912425 | biostudies-literature

A PH domain in the Arf GTPase-activating protein (GAP) ARAP1 binds phosphatidylinositol 3,4,5-trisphosphate and regulates Arf GAP activity independently of recruitment to the plasma membranes.

Project description:ARAP1 is a phosphatidylinositol 3,4,5-trisphosphate (PtdIns(3,4,5)P(3))-dependent Arf GTPase-activating protein (GAP) with five PH domains that regulates endocytic trafficking of the epidermal growth factor receptor (EGFR). Two tandem PH domains are immediately N-terminal of the Arf GAP domain, and one of these fits the consensus sequence for PtdIns(3,4,5)P(3) binding. Here, we tested the hypothesis that PtdIns(3,4,5)P(3)-dependent recruitment mediated by the first PH domain of ARAP1 regulates the in vivo and in vitro function of ARAP1. We found that PH1 of ARAP1 specifically bound to PtdIns(3,4,5)P(3), but with relatively low affinity (approximately 1.6 microm), and the PH domains did not mediate PtdIns(3,4,5)P(3)-dependent recruitment to membranes in cells. However, PtdIns(3,4,5)P(3) binding to the PH domain stimulated GAP activity and was required for in vivo function of ARAP1 as a regulator of endocytic trafficking of the EGFR. Based on these results, we propose a variation on the model for the function of phosphoinositide-binding PH domains. In our model, ARAP1 is recruited to membranes independently of PtdIns(3,4,5)P(3), the subsequent production of which triggers enzymatic activity.

| S-EPMC2788858 | biostudies-literature

OmicsDI is part of the ELIXIR infrastructure

OmicsDI is an Elixir interoperability service. Learn more ›

Tweets

OmicsDI Databases

PRIDE
PeptideAtlas
MassIVE
JPOST Repository
Physiome Model Repository

EGA
EVA
ENA
LINCS
PAXDB
Cell Collective

MetaboLights
Metabolomics Workbench
MetabolomeExpress
GNPS
BioModels
FAIRDOMHub

ArrayExpress
dbGaP
ExpressionAtlas
GEO
NODE

Information

Databases
Help
API
Contact us
Code on GitHub
Terms of use
Submit Data