Multivalent Mechanism of Membrane Insertion by the FYVE Domain

Abstract

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.Cellular processes including signal transduction, vesicular trafficking, and cytoskeletal rearrangement require selective recruitment of proteins to membrane surfaces. Well established mechanisms for localizing cytosolic proteins to membranes include electrostatic interactions through a basic peptide sequence, anchoring by covalently attached acyl chains, and association with the cytoplasmic domains of transmembrane proteins (reviewed in Refs. 1 and 2). (4), PDZ (5), and PTB (6) domains. Although the majority of these domains can interact with several PIs, the FYVE domain is remarkably selective for PtdIns(3)P (7-9). In addition to PI ligation, these domains often insert hydrophobic elements into the membrane bilayer, as has been demonstrated for the C2 (10), ENTH (11), FERM (12), FYVE (13), and PX (14) domains and the vinculin tail (15). Insertion into the membrane can be accompanied by interactions with multiple lipid head groups. For example, the PX domain of the p47 subunit of the NADPH oxidase binds cooperatively to PtdIns(3)P and phosphatidic acid (16), and the vinculin tail co-ligates phosphatidylinositol 4,5-bisphosphate and PtdSer (15). Although it is becoming evident that insertion of proteins into membranes is widespread, the three-dimensional orientations and quantitative binding properties remain challenging to characterize. The most common electron paramagnetic resonance and fluorescence approaches have provided important insights (17-20) but require covalent attachment of paramagnetic groups to various positions of the protein or mutations of residues. The inevitable effects of these modifications on lipi...

Structural Basis for Interactions of thePhytophthora sojaeRxLR Effector Avh5 with Phosphatidylinositol 3-Phosphate and for Host Cell Entry

“…The latter can be explained by the fact that the RFLR motif is present and may contribute to PtdIns ( for PtdIns(3)P to be 2.3 µM. This value is within the range of other PtdIns(3)P-binding protein domains such as the EEA1 FYVE and the Vam7p PX domains (131,132), but is 3-10-fold higher to that estimated for Avr1b and AvrL567 using effector binding to cells and liposome binding assays (11); this higher affinity value may be explained by the presence of additional tags in the proteins tested in these assays that may contribute to the binding (134). Nonetheless, a modest affinity of a protein to phosphoinositides, such as Avh5, may be required for its further release from intracellular membranes to target other subcellular compartments (135).…”

Regulation of ubiquitin-dependent cargo sorting by multiple endocytic adaptors at the plasma membrane

“…However, additional anionic phospholipids also participate in targeting FYVE domain-containing proteins (33). To determine whether the ESCRT-0 complex can also bind to membranes that are devoid of PI3P, we conducted a series of liposome coflotation assays.…”

The Molecular Basis of the Differential Subcellular Localization of FYVE Domains

“…Structural and biophysical studies have indicated that several factors beside PtdIns(3)P binding contribute to the membrane affinity of FYVE domains. They include nonspecific electrostatic interactions between basic protein residues and the anionic membrane surface (27)(28)(29), hydrophobic interactions achieved by the partial membrane insertion of the residues located in the loop (so-called turret loop, see Fig. 1) near the PtdIns(3)P-binding pocket (24,25,27,29,30), and FYVE domain dimerization (21,25,26).…”

“…They include nonspecific electrostatic interactions between basic protein residues and the anionic membrane surface (27-29), hydrophobic interactions achieved by the partial membrane insertion of the residues located in the loop (so-called turret loop, see Fig. 1) near the PtdIns(3)P-binding pocket (24,25,27,29,30), and FYVE domain dimerization (21,25,26). Mutational studies of many FYVE domain-containing proteins have indicated that FYVE domains play an important role in their binding to endosomes in the cell (21,31,32).…”