Multivalent mechanism of membrane insertion by the FYVE domain

Targeting of a wide variety of proteins to membranes involves specific recognition of phospholipid head groups and insertion into lipid bilayers. For example, proteins that contain FYVE domains are recruited to endosomes through interaction with phosphatidylinositol 3-phosphate (PtdIns(3)P). However, the structural mechanism of membrane docking and insertion by this domain remains unclear. Here, the depth and angle of micelle insertion and the lipid binding properties of the FYVE domain of early endosome antigen 1 are estimated by NMR spectroscopy. Spin label probes incorporated into micelles identify a hydrophobic protuberance that inserts into the micelle core and is surrounded by interfacially active polar residues. A novel proxyl PtdIns(3)P derivative is developed to map the position of the phosphoinositide acyl chains, which are found to align with the membrane insertion element. Dual engagement of the FYVE domain with PtdIns(3)P and dodecylphosphocholine micelles yields a 6-fold enhancement of affinity. The additional interaction of phosphatidylserine with a conserved basic site of the protein further amplifies the micelle binding affinity and dramatically alters the angle of insertion. Thus, the FYVE domain is targeted to endosomes through the synergistic action of stereospecific PtdIns(3)P head group ligation, hydrophobic insertion and electrostatic interactions with acidic phospholipids.     

Phosphatidylinositol 3-phosphate induces the membrane penetration of the FYVE domains of Vps27p and Hrs

The FYVE domain mediates the recruitment of proteins involved in membrane trafficking and cell signaling to phosphatidylinositol 3-phosphate (PtdIns(3)P)-containing membranes. To elucidate the mechanism by which the FYVE domain interacts with PtdIns(3)P-containing membranes, we measured the membrane binding of the FYVE domains of yeast Vps27p and Drosophila hepatocyte growth factor-regulated tyrosine kinase substrate and their mutants by surface plasmon resonance and monolayer penetration analyses. These measurements as well as electrostatic potential calculation show that PtdIns(3)P specifically induces the membrane penetration of the FYVE domains and increases their membrane residence time by decreasing the positive charge surrounding the hydrophobic tip of the domain and causing local conformational changes. Mutations of hydrophobic residues located close to the PtdIns(3)P-binding pocket or an Arg residue directly involved in PtdIns(3)P binding abrogated the penetration of the FYVE domains into the monolayer, the packing density of which is comparable with that of biological membranes and large unilamellar vesicles. Based on these results, we propose a mechanism of the membrane binding of the FYVE domain in which the domain first binds to the PtdIns(3)P-containing membrane by specific PtdIns(3)P binding and nonspecific electrostatic interactions, which is then followed by the PtdIns(3)P-induced partial membrane penetration of the domain.     

Phosphatidylinositol 3-phosphate recognition and membrane docking by the FYVE domain

The FYVE domain is a small zinc binding module that recognizes phosphatidylinositol 3-phosphate [PtdIns(3)P], a phospholipid enriched in membranes of early endosomes and other endocytic vesicles. It is usually present as a single module or rarely as a tandem repeat in eukaryotic proteins involved in a variety of biological processes including endo- and exocytosis, membrane trafficking and phosphoinositide metabolism. A number of FYVE domain-containing proteins are recruited to endocytic membranes through the specific interaction of their FYVE domains with PtdIns(3)P. Structures and PtdIns(3)P binding modes of several FYVE domains have recently been characterized, shedding light on the molecular basis underlying multiple cellular functions of these proteins. Here, structural and functional aspects and the current mechanism of the multivalent membrane anchoring by monomeric or dimeric FYVE domain are reviewed. This mechanism involves stereospecific recognition of PtdIns(3)P that is facilitated by non-specific electrostatic contacts and modulated by the histidine switch, and is accompanied by hydrophobic insertion. Contributions of each component to the FYVE domain specificity and affinity for PtdIns(3)P-containing membranes are discussed.     

A high-resolution solution structure of a trypanosomatid FYVE domain

FYVE domain proteins play key roles in regulating membrane traffic in eukaryotic cells. The FYVE domain displays a remarkable specificity for the head group of the target lipid, phosphatidylinositol 3-phosphate (PtdIns[3]P). We have identified five putative FYVE domain proteins in the genome of the protozoan parasite Leishmania major, three of which are predicted to contain a functional PtdIns(3)P-binding site. The FYVE domain of one of these proteins, LmFYVE-1, bound PtdIns(3)P in liposome-binding assays and targeted GFP to acidified late endosomes/lysosomes in mammalian cells. The high-resolution solution structure of its N-terminal FYVE domain (LmFYVE-1[1-79]) was solved by nuclear magnetic resonance. Functionally significant clusters of residues of the LmFYVE-1 domain involved in PtdIns(3)P binding and dependence on low pH for tight binding were identified. This structure is the first trypanosomatid membrane trafficking protein to be determined and has been refined to high precision and accuracy using residual dipolar couplings.     

Phosphatidylinositol 3-phosphate-interacting domains in PIKfyve. Binding specificity and role in PIKfyve. Endomenbrane localization.

PIKfyve is a phosphatidylinositol (PtdIns) 3-phosphate (P)-metabolizing enzyme, which, in addition to a C-terminally positioned catalytic domain, harbors several evolutionarily conserved domains, including a FYVE finger. The FYVE finger domains are thought to direct the protein localization to intracellular membrane PtdIns 3-P. Recent studies with several FYVE domain proteins challenge this general concept. Here we have examined the binding of PIKfyve's FYVE domain to PtdIns 3-P in vitro and in vivo and a plausible contribution of this binding mechanism for the intracellular localization of the full-length protein. We document now a specific and high affinity interaction of a recombinantly produced PIKfyve FYVE domain peptide fragment with PtdIns 3-P-containing liposomes that requires the presence of the conservative core of basic residues within the FYVE domain. PIKfyve localization to membranes of the late endocytic pathway was found to be absolutely dependent on the presence of an intact FYVE finger. Cell treatment with PI 3-kinase inhibitor wortmannin dissociated endosome-bound PIKfyve, indicating that the protein targeted the membrane PtdIns 3-P. An enzymatically inactive peptide fragment of the PIKfyve catalytic domain was found to also specifically bind to PtdIns 3-P-containing liposomes, with residue Lys-1999 being critical in the interaction. This binding, however, was of relatively low affinity and, in the cellular context, was found ineffective in directing the molecule to PtdIns 3-P-enriched endosomes. Collectively, these results demonstrate that interaction of the FYVE domain with PtdIns 3-P is absolutely necessary for PIKfyve targeting to the membranes of the late endocytic pathway and determine PIKfyve as a downstream effector of PtdIns 3-P.     

FYVE finger proteins as effectors of phosphatidylinositol 3-phosphate.

Phosphatidylinositol 3-phosphate (PtdIns(3)P), generated via the phosphorylation of phosphatidylinositol by phosphatidylinositol 3-kinase (PI 3-kinase), plays an essential role in intracellular membrane traffic. The underlying mechanism is still not understood in detail, but the recent identification of the FYVE finger as a protein domain that binds specifically to PtdIns(3)P provides a number of potential effectors for PtdIns(3)P. The FYVE finger (named after the first letter of the four proteins containing it; Fab1p, YOTB, Vac1p and EEA1) is a double-zinc binding domain that is conserved in more than 30 proteins from yeast to mammals. It is found in several proteins involved in intracellular traffic, and FYVE finger mutations that affect zinc binding are associated with the loss of function of several of these proteins. The interaction of FYVE fingers with PtdIns(3)P may serve three alternative functions: First, to recruit cytosolic FYVE finger proteins to PtdIns(3)P-containing membranes (in concert with accessory molecules); second, to enrich for membrane bound FYVE finger proteins into PtdIns(3)P containing microdomains within the membrane; and third, to modulate the activity of membrane bound FYVE finger proteins.     

The FYVE domain of early endosome antigen 1 is required for both phosphatidylinositol 3-phosphate and Rab5 binding. Critical role of this dual interaction for endosomal localization

Early endosome antigen 1 (EEA1) is 170-kDa polypeptide required for endosome fusion. EEA1 binds to both phosphtidylinositol 3-phosphate (PtdIns3P) and to Rab5-GTP in vitro, but the functional role of this dual interaction at the endosomal membrane is unclear. Here we have determined the structural features in EEA1 required for binding to these ligands. We have found that the FYVE domain is critical for both PtdIns3P and Rab5 binding. Whereas PtdIns3P binding only required the FYVE domain, Rab5 binding additionally required a 30-amino acid region directly adjacent to the FYVE domain. Microinjection of glutathione S-transferase fusion constructs into Cos cells revealed that the FYVE domain alone is insufficient for localization to cellular membranes; the upstream 30-amino acid region required for Rab5 binding must also be present for endosomal binding. The importance of Rab5 in membrane binding of EEA1 is underscored by the finding that the increased expression of wild-type Rab5 increases endosomal binding of EEA1 and decreases its dependence on PtdIns3P. Thus, the levels of Rab5 are rate-limiting for the recruitment of EEA1 to endosome membranes. PtdIns3P may play a role in modulating the Rab5 EEA1 interaction.     

Molecular mechanism of membrane docking by the Vam7p PX domain

The Vam7p t-SNARE is an essential component of the vacuole fusion machinery that mediates membrane trafficking and protein sorting in yeast. Vam7p is recruited to vacuoles by its N-terminal PX domain that specifically recognizes PtdIns(3)P in the bilayers, however the precise mechanism of membrane anchoring remains unclear. Here we describe a molecular basis for membrane targeting and penetration by the Vam7p PX domain based on structural and quantitative analysis of its interactions with lipids and micelles. Our results derived from in vitro binding measurements using NMR, monolayer surface tension experiments and mutagenesis reveal a multivalent membrane docking mechanism involving specific PtdIns(3)P recognition that is facilitated by electrostatic interactions and accompanying hydrophobic insertion. Both the hydrophobic and electrostatic components enhance the Vam7p PX domain association with PtdIns(3)P-containing membranes. The inserting Val(70), Leu(71), and Trp(75) residues located next to the PtdIns(3)P binding pocket are surrounded by a basic patch, which is involved in nonspecific electrostatic contacts with acidic lipids, such as PtdSer. Substitution of the insertion residues significantly reduces the binding and penetrating power of the Vam7p PX domain and leads to cytoplasmic redistribution of the EGFP-tagged protein. The affinities of the PX domain for PtdIns(3)P and other lipids reveal a remarkable synergy within the multivalent complex that stably anchors Vam7p at the vacuolar membrane.     

Molecular mechanism of membrane targeting by the GRP1 PH domain

The general receptor for phosphoinositides isoform 1 (GRP1) is recruited to the plasma membrane in response to activation of phosphoinositide 3-kinases and accumulation of phosphatidylinositol 3,4,5-trisphosphate [PtdIns(3,4,5)P(3)]. GRP1's pleckstrin homology (PH) domain recognizes PtdIns(3,4,5)P(3) with high specificity and affinity, however, the precise mechanism of its association with membranes remains unclear. Here, we detail the molecular basis of membrane anchoring by the GRP1 PH domain. Our data reveal a multivalent membrane docking involving PtdIns(3,4,5)P(3) binding, regulated by pH and facilitated by electrostatic interactions with other anionic lipids. The specific recognition of PtdIns(3,4,5)P(3) triggers insertion of the GRP1 PH domain into membranes. An acidic environment enhances PtdIns(3,4,5)P(3) binding and increases membrane penetration as demonstrated by NMR and monolayer surface tension and surface plasmon resonance experiments. The GRP1 PH domain displays a 28 nM affinity for POPC/1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine/PtdIns(3,4,5)P(3) vesicles at pH 6.0, but binds 22-fold weaker at pH 8.0. The pH sensitivity is attributed in part to the His355 residue, protonation of which is required for the robust interaction with PtdIns(3,4,5)P(3) and significant membrane penetration, as illustrated by mutagenesis data. The binding affinity of the GRP1 PH domain for PtdIns(3,4,5)P(3)-containing vesicles is further amplified (by approximately 6-fold) by nonspecific electrostatic interactions with phosphatidylserine/phosphatidylinositol. Together, our results provide new insight into the multivalent mechanism of the membrane targeting and regulation of the GRP1 PH domain.     

A highly conserved glutamic acid in ALFY inhibits membrane binding to aid in aggregate clearance

Autophagy‐linked FYVE protein (ALFY) is a large, multidomain protein involved in the degradation of protein aggregates by selective autophagy. The C‐terminal FYVE domain of ALFY has been shown to bind phosphatidylinositol 3‐phosphate (PI(3)P); however, ALFY only partially colocalizes with other FYVE domains in cells. Thus, we asked if the FYVE domain of ALFY has distinct membrane binding properties compared to other FYVE domains and whether these properties might affect its function in vivo. We found that the FYVE domain of ALFY binds weakly to PI(3)P containing membranes in vitro. This weak binding is the result of a highly conserved glutamic acid within the membrane insertion loop in the FYVE domain of ALFY that is not present in any other human FYVE domain. In addition, not only does this glutamic acid reduce binding to membranes in vitro and inhibits its targeting to membranes in vivo, but it is also important for the ability of ALFY to clear protein aggregates.     

Mechanistic similarities in docking of the FYVE and PX domains to phosphatidylinositol 3-phosphate containing membranes

Phosphatidylinositol 3-phosphate [PtdIns(3)P], a phospholipid produced by PI 3-kinases in early endosomes and multivesicular bodies, often serves as a marker of endosomal membranes. PtdIns(3)P recruits and activates effector proteins containing the FYVE or PX domain and therefore regulates a variety of biological processes including endo- and exocytosis, membrane trafficking, protein sorting, signal transduction and cytoskeletal rearrangement. Structures and PtdIns(3)P binding modes of several FYVE and PX domains have recently been characterized, unveiling the molecular basis underlying multiple cellular functions of these proteins. Here, structural and functional aspects and current mechanisms of the multivalent membrane anchoring by the FYVE and PX domains are reviewed and compared.     

Interaction of the EEA1 FYVE finger with phosphatidylinositol 3-phosphate and early endosomes. Role of conserved residues

FYVE zinc finger domains, which are conserved in multiple proteins from yeast to man, interact specifically with the membrane lipid phosphatidylinositol 3-phosphate (PtdIns(3)P). Here we have investigated the structural requirements for the interaction of the FYVE finger of the early endosome antigen EEA1 with PtdIns(3)P and early endosomes. The binding of the FYVE finger to PtdIns(3)P is Zn(2+)-dependent, and Zn(2+) could not be replaced by any other bivalent cations tested. By surface plasmon resonance, the wild-type FYVE finger was found to bind to PtdIns(3)P with an apparent K(D) of about 50 nm and a 1:1 stoichiometry. Mutagenesis of cysteines involved in Zn(2+) coordination, basic residues thought to be directly involved in ligand binding and other conserved residues, resulted in a 6- to >100-fold decreased affinity for PtdIns(3)P. A mutation in the putative PtdIns(3)P-binding pocket, R1375A, may prove particularly informative, because it led to a strongly decreased affinity for PtdIns(3)P without affecting the FYVE three-dimensional structure, as measured by fluorescence spectroscopy. Whereas the C terminus of EEA1 localizes to early endosomes when expressed in mammalian cells, all the FYVE mutants with reduced affinity for PtdIns(3)P were found to be largely cytosolic. Furthermore, whereas expression of the wild-type EEA1 C terminus interferes with early endosome morphology, the point mutants were without detectable effect. These results support recently proposed models for the ligand binding of the FYVE domain and indicate that PtdIns(3)P binding is crucial for the localization and function of EEA1.     

Investigation of the binding geometry of a peripheral membrane protein

A growing number of modules including FYVE domains target key signaling proteins to membranes through specific recognition of lipid headgroups and hydrophobic insertion into bilayers. Despite the critical role of membrane insertion in the function of these modules, the structural mechanism of membrane docking and penetration remains unclear. In particular, the three-dimensional orientation of the inserted proteins with respect to the membrane surface is difficult to define quantitatively. Here, we determined the geometry of the micelle penetration of the early endosome antigen 1 (EEA1) FYVE domain by obtaining NMR-derived restraints that correlate with the distances between protein backbone amides and spin-labeled probes. The 5- and 14-doxyl-phosphatidylcholine spin-labels were incorporated into dodecylphosphocholine (DPC) micelles, and the reduction of amide signal intensities of the FYVE domain due to paramagnetic relaxation enhancement was measured. The vector of the FYVE domain insertion was estimated relative to the molecular axis by minimizing the paramagnetic restraints obtained in phosphatidylinositol 3-phosphate (PI3P)-enriched micelles containing only DPC or mixed with phosphatidylserine (PS). Additional distance restraints were obtained using a novel spin-label mimetic of PI(3)P that contains a nitroxyl radical near the threitol group of the lipid. Conformational changes indicative of elongation of the membrane insertion loop (MIL) were detected upon micelle interaction, in which the hydrophobic residues of the loop tend to move deeper into the nonpolar core of micelles. The micelle insertion mechanism of the FYVE domain defined in this study is consistent with mutagenesis data and chemical shift perturbations and demonstrates the advantage of using the spin-label NMR approach for investigating the binding geometry by peripheral membrane proteins.     

Phosphatidylinositol-phosphates mediate cytoskeletal reorganization during phagocytosis via a unique modular protein consisting of RhoGEF/DH and FYVE domains in the parasitic protozoon Entamoeba histolytica

To understand the roles of phosphoinositides [PtdIns] in phagocytosis of parasitic eukaryotes, we examined the interaction of phosphatidylinositol-3-phosphate [PtdIns(3)P] and putative PtdIns-P-binding proteins during phagocytosis in the enteric protozoan parasite Entamoeba histolytica. It was previously shown that phagocytosis in E. histolytica is indispensable for virulence and is inhibited by PtdIns 3-kinase inhibitors. We demonstrated by time-lapse live imaging that during the initiation of phagocytosis, the PtdIns(3)P biomarker GFP–Hrs–FYVE, was translocated to the phagocytic cup, phagosome, and to tunnel-like structures connecting the plasma membrane and phagosomes. E. histolytica possesses 12 FYVE domain-containing proteins (EhFP1-12), 11 of which also contain the RhoGEF/DH domain. Among them EhFP4 was shown to be recruited to the tunnel-like structures and to the proximal region of the phagosome. We further demonstrated that EhFP4 physically interacted with 4 of 10 predominant Rho/Rac small GTPases. Phosphoinositide binding assay showed that EhFP4 unexpectedly bound to PtdIns(4)P via the carboxyl-terminal domain and that the FYVE domain modulates the binding specificity of EhFP4 to PtdIns-P. Expression of the FYVE domain from EhFP4 inhibited phagocytosis while enhancement was observed when mammalian Hrs–FYVE domain was expressed. Altogether, we demonstrated that PtdIns(3)P, PtdIns(4)P and EhFP4 coordinately regulate phagocytosis and phagosome maturation in this parasitic eukaryote.     

Phosphoinositides Differentially Regulate Protrudin Localization through the FYVE Domain

Protrudin is a FYVE (Fab 1, YOTB, Vac 1, and EEA1) domain-containing protein involved in transport of neuronal cargoes and implicated in the onset of hereditary spastic paraplegia. Our image-based screening of the lipid binding domain library revealed novel plasma membrane localization of the FYVE domain of protrudin unlike canonical FYVE domains that are localized to early endosomes. The membrane binding study by surface plasmon resonance analysis showed that this FYVE domain preferentially binds phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P₂), phosphatidylinositol 3,4-bisphosphate (PtdIns(3,4)P₂), and phosphatidylinositol 3,4,5-trisphosphate (PtdIns(3,4,5)P₃) unlike canonical FYVE domains that specifically bind phosphatidylinositol 3-phosphate (PtdIns(3)P). Furthermore, we found that these phosphoinositides (PtdInsP) differentially regulate shuttling of protrudin between endosomes and plasma membrane via its FYVE domain. Protrudin mutants with reduced PtdInsP-binding affinity failed to promote neurite outgrowth in primary cultured hippocampal neurons. These results suggest that novel PtdInsP selectivity of the protrudin-FYVE domain is critical for its cellular localization and its role in neurite outgrowth.     

Computer modeling of the membrane interaction of FYVE domains

FYVE domains are membrane targeting domains that are found in proteins involved in endosomal trafficking and signal transduction pathways. Most FYVE domains bind specifically to phosphatidylinositol 3-phosphate (PI(3)P), a lipid that resides mainly in endosomal membranes. Though the specific interactions between FYVE domains and the headgroup of PI(3)P have been well characterized, principally through structural studies, the available experimental structures suggest several different models for FYVE/membrane association. Thus, the manner in which FYVE domains adsorb to the membrane surface remains to be elucidated. Towards this end, recent experiments have shown that FYVE domains bind PI(3)P in the context of phospholipid bilayers and that hydrophobic residues on a conserved loop are able to penetrate the membrane interface in a PI(3)P-dependent manner.Here, the finite difference Poisson-Boltzmann (FDPB) method has been used to calculate the energetic interactions of FYVE domains with phospholipid membranes. Based on the computational analysis, it is found that (1) recruitment to membranes is facilitated by non-specific electrostatic interactions that occur between basic residues on the domains and acidic phospholipids in the membrane, (2) the energetic analysis can quantitatively differentiate among the modes of membrane association proposed by the experimentally determined structures, (3) FDPB calculations predict energetically feasible models for the membrane-associated states of FYVE domains, (4) these models are consistent with the observation that conserved hydrophobic residues insert into the membrane interface, and (5) the calculations provide a molecular model for the hydrophobic partitioning: binding of PI(3)P significantly neutralizes positive potential in the region of the hydrophobic residues, which acts as an "electrostatic switch" by reducing the energetic barrier for membrane penetration. Finally, the computational results are extended to FYVE domains of unknown structure through the construction of high quality homology models for human FYVE sequences.     

The Phosphatidylinositol 3-Phosphate Binding Protein Vac1p Interacts with a Rab GTPase and a Sec1p Homologue to Facilitate Vesicle-mediated Vacuolar Protein Sorting

Activated GTP-bound Rab proteins are thought to interact with effectors to elicit vesicle targeting and fusion events. Vesicle-associated v-SNARE and target membrane t-SNARE proteins are also involved in vesicular transport. Little is known about the functional relationship between Rabs and SNARE protein complexes. We have constructed an activated allele of VPS21, a yeast Rab protein involved in vacuolar protein sorting, and demonstrated an allele-specific interaction between Vps21p and Vac1p. Vac1p was found to bind the Sec1p homologue Vps45p. Although no association between Vps21p and Vps45p was seen, a genetic interaction between VPS21 and VPS45 was observed. Vac1p contains a zinc-binding FYVE finger that may bind phosphatidylinositol 3-phosphate [PtdIns(3)P]. In other FYVE domain proteins, this motif and PtdIns(3)P are necessary for membrane association. Vac1 proteins with mutant FYVE fingers still associated with membranes but showed vacuolar protein sorting defects and reduced interactions with Vps45p and activated Vps21p. Vac1p membrane association was not dependent on PtdIns(3)P, Pep12p, Vps21p, Vps45p, or the PtdIns 3-kinase, Vps34p. Vac1p FYVE finger mutant missorting phenotypes were suppressed by a defective allele of VPS34. These data indicate that PtdIns(3)P may perform a regulatory role, possibly involved in mediating Vac1p protein-protein interactions. We propose that activated-Vps21p interacts with its effector, Vac1p, which interacts with Vps45p to regulate the Golgi to endosome SNARE complex.     

Identification and Characterization of a FYVE Domain from the Early Diverging Eukaryote <Emphasis Type="Italic">Giardia lamblia</Emphasis>

The morphology of the endomembrane system of Giardia lamblia appears to be significantly different from higher eukaryotes. Therefore, the molecular mechanisms controlling vesicular trafficking are also likely to be altered. Since FYVE domain is a known regulator of endosomal trafficking, the authors used BLAST search to identify FYVE domain(s) in G. lamblia. A 990 amino acid long putative FYVE domain-containing ORF was identified, which contains all the conserved sequence elements in the ligand binding pocket. Phylogenetic analysis reveals that this domain is significantly diverged. The authors have shown that the corresponding gene is expressed in G. lamblia trophozoites and cysts. In spite of this phylogenetic divergence, in vitro biochemical assay indicates that this domain preferentially binds to phosphatidylinositol 3-phosphate {PtdIns(3)P}and in vivo expression of the GFP-tagged G. lamblia FYVE domain in S. cerevisiae, displays its selective localization to PtdIns(3)P-enriched endosomes. This is the first study to characterize a PtdIns(3)P effector protein in this early-diverged eukaryote.     

Membrane-binding mechanism of the EEA1 FYVE domain revealed by multi-scale molecular dynamics simulations

Early Endosomal Antigen 1 (EEA1) is a key protein in endosomal trafficking and is implicated in both autoimmune and neurological diseases. The C-terminal FYVE domain of EEA1 binds endosomal membranes, which contain phosphatidylinositol-3-phosphate (PI(3)P). Although it is known that FYVE binds PI(3)P specifically, it has not previously been described of how FYVE attaches and binds to endosomal membranes. In this study, we employed both coarse-grained (CG) and atomistic (AT) molecular dynamics (MD) simulations to determine how FYVE binds to PI(3)P-containing membranes. CG-MD showed that the dominant membrane binding mode resembles the crystal structure of EEA1 FYVE domain in complex with inositol-1,3-diphospate (PDB ID 1JOC). FYVE, which is a homodimer, binds the membrane via a hinge mechanism, where the C-terminus of one monomer first attaches to the membrane, followed by the C-terminus of the other monomer. The estimated total binding energy is ~70 kJ/mol, of which 50–60 kJ/mol stems from specific PI(3)P-interactions. By AT-MD, we could partition the binding mode into two types: (i) adhesion by electrostatic FYVE-PI(3)P interaction, and (ii) insertion of amphipathic loops. The AT simulations also demonstrated flexibility within the FYVE homodimer between the C-terminal heads and coiled-coil stem. This leads to a dynamic model whereby the 200 nm long coiled coil attached to the FYVE domain dimer can amplify local hinge-bending motions such that the Rab5-binding domain at the other end of the coiled coil can explore an area of 0.1 μm² in the search for a second endosome with which to interact.     

The PtdIns3P‐Binding Protein Phafin 2 Mediates Epidermal Growth Factor Receptor Degradation by Promoting Endosome Fusion

Phosphatidylinositol 3‐phosphate (PtdIns3P) orchestrates endosomal cargo transport, fusion and motility by recruiting FYVE or PX domain‐containing effector proteins to endosomal membranes. In an attempt to discover novel PtdIns3P effectors involved in the termination of growth factor receptor signalling, we performed an siRNA screen for epidermal growth factor (EGF) degradation, targeting FYVE and PX domain proteins in the human proteome. This screen identified several potential regulators of EGF degradation, including HRS (used as positive control), PX kinase, MTMR4 and Phafin2/PLEKHF2. As Phafin2 has not previously been shown to be required for EGF receptor (EGFR) degradation, we performed further functional studies on this protein. Loss of Phafin2 was found to decrease early endosome size, whereas overexpression of Phafin2 resulted in enlarged endosomes. Moreover, both the EGFR and the fluid‐phase marker dextran were retained in abnormally small endosomes in Phafin2‐depleted cells. In yeast two‐hybrid analysis we identified Phafin2 as a novel interactor of the endosomal‐tethering protein EEA1, and Phafin2 colocalized strongly with EEA1 in microdomains of the endosome membrane. Our results suggest that Phafin2 controls receptor trafficking and fluid‐phase transport through early endosomes by facilitating endosome fusion in concert with EEA1.