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Signaling and Subcellular Targeting by Membrane-Binding Domains

and .

Laboratory of Molecular Biology, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland 20892-0580; e-mail:

Reproduced from Annu. Rev. Biophys. Biomol. Struct. 2000. 29:49-79.

Protein kinase C homology-1 and -2, FYVE, and pleckstrin homology domains are ubiquitous in eukaryotic signal transduction and membrane-trafficking proteins. These domains regulate subcellular localization and protein function by binding to lipid ligands embedded in cell membranes. Structural and biochemical analysis of these domains has shown that their molecular mechanisms of membrane binding depend on a combination of specific and nonspecific interactions with membrane lipids. In vivo studies of green fluorescent protein fusions have highlighted the key roles of these domains in regulating protein localization to plasma and internal membranes in cells.

Perspectives and Overview

Subcellular targeting of proteins is a fundamental control mechanism in eukaryotic cells. Localization to different cell compartments is often brought about by protein-protein interaction domains (70, 100). Another major class of subcellular targeting domains binds specifically to lipid ligands in cell membranes. The best known members of this group are the protein kinase C (PKC) homology-1 (C1) (54, 111) and -2 (C2) domains (89, 110), the pleckstrin homology (PH) domain (8, 34, 73, 109), and the FYVE domain (34, 42, 141). Although some C1, C2, and PH domains interact with proteins in addition to—or instead of—lipids, their best known roles are in lipid binding. This review emphasizes the membrane-binding mechanisms of these domains and their role in cell signaling.

These are exciting times for research on signal transduction domains. Studies of green fluorescent protein fusions with signaling proteins are yielding quantitative kinetic information in living cells. The three-dimensional structures of the C1, C2, FYVE, and PH domains have all been solved at high resolution by X-ray crystallography, and they have also been studied by nuclear magnetic resonance (NMR) and electron paramagnetic resonance (EPR). Site-directed mutagenesis, fluorescence, and surface pressure studies have made critical contributions to understanding how these proteins interact with membranes. Databases such as SMART (115) and Pfam (6; provide the most comprehensive census yet of signal-transducing domains. With the rapid growth of interest in membrane targeting as a mechanism for signal transduction, these developments are due for review.

C1 Domains

The C1 domain is a compact zinc-containing motif of ~50 amino acid residues, formerly known as a “cysteine-rich” domain (Figure 1a). The C1 domain was discovered as a conserved region responsible for the allosteric activation of PKC isozymes (PKCs) by diacylglycerol and phorbol esters. C1 domains are now known to occur not only in PKCs but in >200 different proteins in the nonredundant sequence databases [these numbers, obtained from the SMART database (115), are higher than quoted elsewhere owing both to new discoveries and to the inclusion of orthologs]. Some of these proteins, including PKCs, the chimaerins, Unc-13 (54, 111), and RasGRP (24, 111), are effectors of diacylglycerol. However, many of the known C1 domains do not bind diacylglycerol. This group of C1 domains is referred to as “atypical,” and they are implicated in interactions with small G-proteins and membrane lipids other than diacylglycerol.

Figure 1. a.

Figure 1

a. Alignment of C1 domains. Zn2+-liganding residues are shown in bold. Membrane-interacting and diacylglycerol-binding-site residues are boxed. Vav and Raf represent atypical C1 domains that do not bind diacylglycerol and lack the crucial boxed residues. (more...)

Structure of the C1 Domain

C1 domains contain two small β sheets and a short C-terminal α-helix that are built around two 3-Cys-1-His Zn2+-binding clusters (52,146; Figure 2a). The Zn2+ ions are an integral part of the structure. The diacylglycerol- and phorbol ester-binding site is formed at one tip of the domain, where part of the second β sheet unzips. The linked ring structures of phorbol are inserted lengthwise into the narrow groove at the tip of the C1 domain. The 3- and 20-oxygens of phorbol interact with main-chain groups exposed by unzipping of two β strands (Figure 1b). One of the acyl group oxygens and the 3-hydroxyl of diacylglycerol are believed to occupy the same sites, whereas it is less clear how the second acyl group oxygen interacts.

Figure 2. Membrane-docked structures of (a) PKCδ-CIB phorbol ester complex with the myristoyl tail modeled (146), (b ) cPLA2-C2 Ca 2+ complex (102), (c) Vps27p-FYVE with PI3P modeled (85), and (d) PLC δ1-PH complex with Ins (1) P3, with dimyristoyl group modeled (31).

Figure 2

Membrane-docked structures of (a) PKCδ-CIB phorbol ester complex with the myristoyl tail modeled (146), (b ) cPLA2-C2 Ca 2+ complex (102), (c) Vps27p-FYVE with PI3P modeled (85), and (d) PLC δ1-PH complex with Ins (1) P3, with dimyristoyl (more...)

Figure 1. b.

Figure 1

b. Schematic of the typical C1 phorbol ester-binding site (modified from Reference ).

Diacylglycerol-Promoted Membrane Association

One entire end of the C1 domain surrounding the binding groove is almost completely hydrophobic (Figure 2a). The region adjoins a basic ring that circumscribes the midsection of the domain surface. NMR studies in short-chain lipid micelles (145) and surface pressure analysis of C1 domain mutants of PKCα (84) confirmed the prediction that the hydrophobic region penetrates into the membrane interior while the basic ring contacts the membrane surface (146). There is an exceptionally strong synergism between diacylglycerol or phorbol ester binding and membrane binding (86), and the presence of diacylglycerol or phorbol ester is required for targeting of C1 domains to membranes. The monomeric phorbol ester head group binds 104-fold more weakly than tetradecanoyl phorbol acetate presented in mixed micelles (63). The synergistic binding is explained by the two types of binding surfaces: a stereospecific diacylglycerol-phorbol ester-binding site in a groove surrounded by a nonstereospecific membrane-binding site. Binding of either diacylglycerol or bulk membrane to its site alone leaves interactions with other sites unsatisfied; hence simultaneous binding is favored.

C1 domains from PKCγ can translocate from the cytosol to the plasma membrane within a few seconds after addition of diacylglycerol (95). Free fatty acids can stimulate PKC translocation to a variety of different cell compartments depending on the isozyme (117), yet specific binding to PKC C1 and C2 domains has not been documented. Free fatty acids could modulate the nonspecific interactions of PKC-C1 or PKC-C2 with membranes.

Predicting C1 Domain Function from Sequence

The structure of the C1-phorbol ester complex and its nonspecific membrane-binding surface depend on the conservation of a number of amino acids in the group of “typical” C1 domains that do bind diacylglycerol and phorbol ester (Figure 1a). The typical C1 domains conserve both the large hydrophobic residues that form the nonspecific binding surface and three structural residues (a Pro, Gly, and Gln) involved in the stabilization of the binding groove (64, 146). Counting from the first conserved His, the consensus motif for the typical C1 domains begins at Pro-11:PXArCX2CX2Hy3GX0–1HyX2QG, where X is any amino acid; Ar is Phe, Trp, or Tyr; Hy is any hydrophobic residue; and residues involved in groove formation or membrane penetration are shown in bold (64, 146). This signature is inconsistent with the properties of synthetic peptide models for PKC C1 domains (58), although the motif has been largely successful in predicting the properties of naturally occurring and recombinant C1 domain-containing proteins. For example, this motif is present in the most recently discovered C1 domain, that of RasGRP (24). RasGRP is targeted to cell membranes in response to diacylglycerol via its C1 domain (131), revealing a new pathway from diacylglycerol to Ras signaling.

Atypical C1 domains occur in two large groups of proteins: the diacylglycerol kinases (DAGKs) (132) and effectors and regulators of small G-proteins. The function of the DAGK C1 domains is mysterious. None of these kinases is known to bind phorbol ester, and the C1 domains of DAGKα are dispensable for catalytic activity (113, 132). The atypical C1 domain of Raf is involved in allosteric regulation of this protein kinase by activated Ras, although the primary binding site for Ras lies elsewhere, on the Raf-RBD domain. Several regions on the surface of the Raf-C1structure (87) appear to be involved in autoinhibitory interactions in the inactive conformation of Raf (18, 19). At least one epitope, comprising Lys-144 and Leu-160 of c-Raf-1, overlaps with the phorbol ester-binding site on the typical C1 domains and probably has direct interactions with Ras (19).

Multi-C1-Domain Proteins: the Contribution of Context

In most of the PKCs and DAGKs, C1 domains occur in pairs. The function of individual C1 domains depends on their context in the larger protein, as illustrated by the interdependent allosteric activation of PKC by various lipids (93, 119). The diacylglycerol-binding sites on the C1 domains of PKCγ are obstructed in the inactive cytosolic form of the enzyme, as judged by translocation kinetics in vivo (94). Diacylglycerol binding to the C1 domain is believed to be coupled to a large-scale conformational change that alters the interactions of the C1 domains with the kinase catalytic domains, thereby allosterically activating the enzyme (53, 93, 94). PKCδ and PKD/PKCμ both contain two C1 domains, C1A and C1B. For these isozymes, the C1B contributes to phorbol ester-stimulated translocation by an order of magnitude more than the C1A (57, 128). It remains to be seen whether other PKCs follow this pattern.

C2 Domains

C2 domains are ~120-residue domains that were originally discovered as a conserved sequence motif in the Ca2+-dependent PKCs. There are now ~600 C2 domains identified in >400 different proteins (see above regarding numbers taken from the SMART database). Much of the intense interest in these domains arises from the roles of C2 domain proteins not only in signal transduction, but also in inflammation, synaptic vesicle trafficking and fusion, and many other cell processes (89, 110). Many, but not all, C2 domains bind phospholipid membranes in the presence of Ca2+. Some C2 domains bind membranes constitutively and do not bind Ca2+ at all. Other C2 domains bind proteins instead of membranes, using both Ca2+-dependent and -independent mechanisms. Still other C2 domains bind soluble inositol polyphosphates, usually in a Ca2+-independent manner.

Structure of the C2 Domain

Structures of five different C2 domains are now known: the C2A domain of synaptotagmin I (SytI) (126) and the C2 domains of PKC-β(126) and PKC-δ (98) and of phospholipases A2 (cPLA2) (21, 102, 144) and C-δ1 (PLCδ1) (27, 44). The structure of the C2 domain is a β sandwich related to the immunoglobulin fold (45). Two permutations of the C2 fold occur, known as types I (S-type) and II (P-type), in which the sequence starts at a position in the β sheet offset by a single strand in one as compared with the other (Figure 3a). The Ca2+-binding sites are formed by three loops at one tip of the structure. The loops, known as the Ca2+-binding regions (CBRs), correspond structurally to the antigen-binding complementarity-determining regions of antibody Fabs. In addition to forming the Ca2+-binding sites of the Ca2+-dependent class of C2 domains, the CBRs are involved in phospholipid specificity and probably in other ligand-binding interactions.

Figure 3. a.

Figure 3

a. Structure-based alignment of C2 domains. Membrane-binding residues and specific Ca2+-binding residues are boxed. Residues that bind Ca2+ through the backbone are not indicated. The Ca2+ sites in which the ligands participate are marked by Roman numerals. (more...)

Ca2+-Binding Sites

Ca2+-binding affinities are strongly dependent on the presence of phospholipid or other ligands. cPLA2-C2 binds two Ca2+ in the presence or absence of membrane (Figure 3b) (92, 102, 144). SytI-C2A and PKCβ-C2 bind three ions, although binding to the third site is immeasurably weak in the absence of an exogenous acidic ligand (125, 134). PLCδ1-C2 binds three ions in the absence of ligands (28, 46). A hypothetical fourth site exists on PLCδ1-C2, corresponding to the very low-affinity third site on SytI, but this has not been confirmed. The individual Ca2+ sites within a C2 domain have distinct functions in binding and enzyme activation (7, 83).

Figure 3. b.

Figure 3

b. Schematic of the Ca2+-binding sites in C2 domains. Site I is occupied in cPLA2 (cp) and PLCδ1 (pl); site II is occupied in all Ca2+-binding C2 domains; and sites III and IV are known or predicted to be bound in PLCδ1, SytI-C2A (sy), (more...)

Membrane-Binding Sites

There is an emerging consensus on the membrane-docking modes of Ca2+-dependent membrane-binding C2 domains (Figure 2b). The structures of the PLCδ1-C2 and cPLA2-C2 are known in the context of the larger enzyme (21, 27, 44). The presence of the phospholipase active sites in the same structure provides a powerful constraint on the orientation of the C2 domain with respect to the bilayer, which led to a detailed model of C2-membrane docking (44). The membrane-docked PLCδ1-C2 placed the CBR3 region closest to the membrane and juxtaposed the Ca2+-binding sites with the membrane surface. In this model, the concave face of the C2 structure and strand β3 in particular face the membrane surface across a distance of 5–10 Å. The overall orientation is similar for cPLA2-C2, but this C2 domain has a larger and more hydrophobic CBR1 than that of PLCδ1-C2 (21, 102, 144).

The inferences from structures have been confirmed and elaborated on by functional studies. Trp residues incorporated into the SytI-C2A and cPLA2-C2 domains as fluorescent reporters reveal membrane penetration by SytI-C2A's CBR1 (13) and both CBR1 and CBR3 of cPLA2-C2 (90, 103). NMR of SytI-C2A (12) and NMR and EPR studies of cPLA2-C2 support this picture (4, 144). Scanning mutagenesis and surface pressure measurements on PKCα (84) and cPLA2 (7) came to similar conclusions. NMR suggests that residues on the concave face of cPLA2-C2 sense an altered environment when bound to the membrane (144). However, mutagenesis of the concave faces of PKCβII-C2 (61) and cPL A2-C2 (7) show that this region does not contribute substantially to membrane binding even though it is oriented toward the membrane surface.

Phospholipid Specificity and Subcellular Localization

Most Ca2+-dependent C2 domains bind acidic phospholipids (89, 110), and PLCδ1-C2 was most recently added to this group (81). cPLA2-C2, in contrast, seems to prefer neutral membranes, especially phosphatidylcholine (PC) (91). cPLA2 also binds phosphatidylmethanol, and it has been suggested that small head groups promote binding by favoring the bilayer insertion of cPLA2-C2 (51). The differences in specificity correlate with the structures of the CBRs and with the ionic strength dependence of the interaction. The CBR3 of SytI and many other acidic phospholipid-binding C2 domains contains basic residues, whereas hydrophobic residues predominate in cPLA2. cPLA2 also contains a helix in its CBR1 that inserts part of its hydrophobic surface into the membrane (4, 7, 90, 102, 103, 144). This helix is not present in the acidic phospholipid binders. Consistent with these ideas, SytI-C2/membrane binding is attenuated by >500 mM NaCl, the signature of an electrostatic interaction, whereas cPLA2-C2 binding is not (20). An aromatic cluster specific to the cPLA2-C2 structure is predicted to form a choline head group-binding site that may explain the preference for PC over other zwitterionic lipids (144).

Subcellular localization of C2 domains correlates with their phospholipid specificity. PKCα-C2 (16) and PKCγ (94) translocate to the plasma membrane, rich in the acidic phospholipid phosphatidylserine (PS), when free [Ca2+] increases. This is vividly illustrated by the plasma membrane translocation of PKCγ coincident with Ca2+ oscillations (94). In contrast, increased cytoplasmic [Ca2+] induces intact cPLA2 and cPLA2-C2 to translocate to the PC-rich nuclear envelope and endoplasmic reticulum (43, 103).

Mechanism of Ca2+-Dependent Membrane Binding

Three mechanisms by which Ca2+ could promote membrane binding by C2 domains have been widely discussed. The first is the “Ca2+ bridge” model. The second model invokes a conformational change in which the structure of the CBRs is altered by Ca2+ binding such that their ability to bind membranes is increased. The third is the “electrostatic switch” mechanism. These three mechanisms are not necessarily mutually exclusive, nor do they exhaust the possibilities.

The Ca2+ Bridge Model

In the Ca2+ bridge model, Ca2+ ions are specifically coordinated by functional groups provided by both the C2 domain and by phospholipids. The annexins provide a precedent (127). The membrane-docked position of the C2 domain tip at the bilayer surface is consistent with a Ca2+ bridge. All efforts at forming specific Ca2+-bridged complexes between C2 domains and short-chain phospholipids have thus far disappointed. However, a structure of a Ca2+-bridged complex between the cPLA2-C2 and the sulfonate moiety of a morpholineethanesulfonic acid buffer ion has been reported (21; Figure 3b). The 10 Å between the putative choline site and Ca2+ site I suggests that a single PC molecule would be unlikely to both coordinate Ca2+ and occupy the choline pocket. The crystal structure of the PKCβ-C2 (125) reveals a Ca2+-bridged protein dimer that provides a different model for chelation in the putative phospholipid complex (Figure 3b). This model would position the phosphodiester ~8 Å nearer to the membrane center (or the protein 8 Å farther from it) than would be suggested by the cPLA2-C2 [N-morpholino]ethanesulfonic acid complex. Arguing against a bridge mechanism, cPLA2-C2 is capable of Ca2+-dependent partitioning to pure Triton micelles (20). It appears that Ca2+ bridging is an important contributing factor but cannot on its own serve as a general explanation for all Ca2+-dependent membrane binding by C2 domains.

Ca2+ Induced Conformational Changes

The structure of a truncated PLCδ1 has been determined in two different crystal forms, cubic and triclinic. In the “apo-” form of the triclinic crystal, CBR1 is almost completely invisible in electron density owing to disorder. Ca2+ analog binding in the triclinic form induces a disorder-to-order conformational change in which CBR1 adopts a well-defined conformation (44). In the cubic-crystal form, CBR1 is ordered in both apo- and bound structures (28). CBR1 in the cubic form interacts extensively with crystal packing contacts, explaining the apparent lack of a conformational change. Movement of the CBR1 in the triclinic form is much less restricted. With the exception of PLCδ1, all crystallized Ca2+-dependent C2 domains have been grown only in a single Ca2+ ligation state. SytI-C2A was crystallized as an apodomain, but it can bind one Ca2+ ion in the crystal (126). Binding of additional ions shatters the SytI-C2A crystals, strongly suggesting a conformational change. Comparison of the crystal structures of closely related apo-SytI-C2A (126) and Ca2+-saturated PKCβ-C2 (125) reveals that the CBR1 moves 1–2 Å and its mobility relative to the rest of the domain decreases fourfold, again consistent with an increase in order in the bound state. Taken together these data reveal a consistent pattern of Ca2+ effects on C2 domain conformation.

Fluorescence spectroscopy of the SytI (13) and cPLA2-C2 (92) domains indicates substantial Ca2+-induced conformational changes that extend some distance from the binding site. Chemical modification of cPLA2-C2 with TID increases several-fold on Ca2+ binding (20), consistent with a conformational change that exposes more hydrophobic surface area, although ANS binding does not increase. NMR of SytI-C2A reveals many CBR NOEs in the bound state that decrease or disappear in the apostructure, consistent with a disorder-to-order conformational change upon Ca2+ binding (116). NMR of cPLA2-C2 reveals large Ca2+-induced chemical-shift perturbations that are greatest for the CBRs but extend beyond them (144). There is now overwhelming evidence that conformational changes do occur in C2 domains when they bind Ca2+, despite statements to the contrary (110). It is still not clear how much these conformational changes contribute to Ca2+-dependent membrane binding.

Electrostatic Interactions with Membranes

The electrostatic switch model is based on the change in electrostatic potential at the tip of the C2 domain from negative to positive upon Ca2+ binding (110, 148). The electrostatic switch model is not general because it cannot explain the neutral lipid-specific C2 domains exemplified by cPLA2. There is no doubt that electrostatic interactions are involved in the membrane binding of acidic phospholipid-specific C2 domains, because the interaction can be abolished by increasing ionic strength (20, 148). The key question is whether a nonspecific electrostatic interaction is both necessary and sufficient for binding, as opposed to a necessary role for specific interactions with Ca2+ ions. A charge reversal mutant that increases the net charge in the CBRs of PKC βII-C2 by +4 severely impairs Ca2+-dependent binding but does not confer Ca2+-independent binding (26). This result rules out the electrostatic switch model as applied to the anionic lipid-binding class of C2 domains (26).

A little discussed but potentially important model invokes a decrease in Born repulsion after Ca2+ binding (D Murray, B Honig & S McLaughlin, personal communication). Born repulsion is the force that keeps ions out of the low dielectric medium of membrane and protein interiors. There is a substantial Born energy penalty for bringing ions near a low dielectric medium even if they do not enter it. By nearly neutralizing the net negative charge on the tip of the C2 domain, this penalty might be reduced. No experiments specifically designed to test this idea have been reported to date. The failure of the PKC βII-C2 charge reversal mutant (26) to bind membranes rules this mechanism out for the conventional PKC-like acidic lipid-specific C2 domains, but it has yet to be tested for cPLA2-C2. In short, no single mechanism can account for all of the Ca2+-dependent C2 domains. Conventional PKCs, SytI-C2A, and many similar proteins probably rely heavily on the Ca2+ bridge mechanism, whereas cPLA2 may depend more on the conformational change or Born mechanisms or both.

Ca2+-Independent C2 Domains

Not all C2 domains bind Ca2+ ions. Little is known of the function of these C2 domains. The Ca2+-independent C2 domains of the AplII PKC (101) and PI3K-C2β (3) bind phospholipids with low affinity and little specificity. There are enough structural data on C2 domains to predict which domains will bind and which will not. The sequences of the Ca2+-independent class show that most or all Ca2+ ligands are absent. The structure of the Ca2+-independent PKC δ-C2 confirms the expected absence of the usual acidic pocket (98). The CBRs in the PKC δ-C2 are in sharply different conformations from those in other C2 domains. This suggests that more is required to create a Ca2+-independent membrane-binding site than the mere removal of the Ca2+-binding Asp residues.

Interdomain Interactions

The structures of PLC δ1 and cPLA2 show differing degrees of interaction between C2 and the rest of the protein. The PLCδ1-C2 interacts extensively with the catalytic and EF hand domains of the enzyme, although the CBRs are not occluded (27, 44). The extensive contact surfaces of the PLCδ1-C2 suggest that it may contribute to structural stabilization. In contrast, cPLA2-C2 has almost no interactions with the catalytic domain and can pivot through an angle of ≥10°(21). Kinetics suggest that the PKCγ-C2, like the other two, is oriented in an “outside-out” manner such that its CBRs are not occluded by the rest of the protein (94). Despite the “outside-out” orientation, Ca2+ binding to particular subsites within the C2 domain appears able, directly or indirectly, to trigger activating long-range conformational changes in PKCα and in cPLA2 (7, 83).

FYVE Domains

The FYVE domains, so far identified in ~60 proteins (see above regarding numbers taken from the SMART database), are the mostly recently characterized addition to the family of membrane-binding modules. FYVE domains are ~70- to 80-residue domains containing 8 Cys or 1 His and 7 Cys residues that coordinate two Zn2+ atoms (42, 123, 141). FYVE domains are involved in endosomal localization of proteins crucial for membrane trafficking in yeast (141) and mammals (118, 123). The current fascination with FYVE domains was triggered by the 1998 discovery that effectors of class III phosphatidylinositol (PI) 3-kinases are localized by binding PI 3-phosphate (PI3P) via their FYVE domains (10, 41, 99). FYVE domains bind PI3P but not more highly phosphorylated phosphoinositides (10, 41, 99).

FYVE Domain Structure, Ligand Binding, and Specificity

The crystal structure of the FYVE domain from Vps27p (85), a protein involved in endosomal maturation in yeast, reveals a compact core consisting of two small double-stranded β sheets and a C-terminal α-helix (Figure 2c). The structure is distantly similar to that of the C1 domain. The Zn2+-chelating Cys/His residues are located in pairs such that the first and third pairs bind one zinc atom, while the second and fourth pairs bind the other zinc atom. The surface of Vps27p-FYVE contains a relatively large basic region contributed by the conserved RKHHCR motif located near and on β1 and by a conserved arginine from the β4 strand (Figure 4a,b). Mutagenesis of the RKHHCR motif results in loss of PI3P binding (10). The sequence (R/K)(R/K)HHCR is present in all known PI3P-binding FYVE domains, although there are structurally similar domains that lack this motif (97). The basic region is divided into two subsites consisting of the first two and the last four residues of the RKHHCR motif. PI3P can be modeled so that its 1-phosphate interacts with the first two residues of the motif, while the 3-phosphate interacts with a tight pocket formed by the last three basic motif residues and the Arg contributed by β4. In support of this model, the chemical shifts of the corresponding residues in early endosomal antigen-1 (EEA-1) exhibit the largest perturbations upon titration with a water-soluble PI3P (71).

Figure 4. a.

Figure 4

a. Structure-based alignment of FYVE domains and the related rabphilin Zn2+-binding domain. Zn2+-binding residues are shown in bold. PI3P binding and membrane interacting residues are boxed. The rabphilin Zn2+-binding domain is structurally similar to (more...)

Figure 4. b.

Figure 4

b. Schematic of the PI3P-binding site of the FYVE domain. The phosphoinositide is intentionally drawn as an oversimplified achiral molecule to emphasize the pseudo-twofold relationship between PI3P and PI5P. Equivalent residues from the Vps27p (black (more...)

FYVE domains are specific for PI3P, showing negligible affinity for PI4P, polyphosphorylated phosphoinositides, or other phospholipids (10, 41, 99). Specificity is probably controlled by the distance between the two phosphate-binding subsites. This distance appears too short to tolerate binding of the 1- and 4-phosphate groups of PI4P to their respective sites simultaneously. The 3-phosphate binding pocket is too occluded to permit binding of polyphosphorylated phosphoinositides such as those that bind to PH domains. EEA1-FYVE binds to PI5P but probably too weakly to be meaningful in vivo (71). The basic phosphate-binding residues are sufficiently well conserved that it seems likely most of the as-yet-uncharacterized FYVE domains will have similar ligand specificity. It is also possible that other FYVE domains could have higher affinities for PI5P than EEA1.

FYVE Domain Binding to Membranes

The FYVE domain binds to PI3P-containing membranes such that the tip of the N-terminal loop penetrates into the bilayer (Figure 2c). This part of Vps27p-FYVE has two successive Leu residues that form an exposed hydrophobic protrusion at one end of the domain (85). The NMR resonances from the corresponding region from EEA1-FYVE disappear upon binding to PI3P-containing micelles (71), suggesting that the residues penetrate the micelle. In addition, mutation of two of these residues (Val and Thr) eliminates endosomal localization in vivo (71). This region contains two or more hydrophobic residues in most FYVE domains. The hydrophobic protrusion is also present in the FYVE-like Ze2+-binding domain of rabphilin-3A, suggesting a function for this domain in nonspecific membrane binding. In Vps27p, several lysines not involved in PI3P binding are located at the base of the protrusion (85). These lysines are poorly conserved and do not show main-chain chemical shift perturbations by micelles (71), but other sequences contain positively charged residues that map to similar parts of the domain surface. It appears likely that the hydrophobic protrusion penetrates into membranes, whereas the basic residues at the base of the protrusion interact nonspecifically with the membrane.

FYVE domain-containing proteins and FYVE domains localize to endosomal membranes containing PI3P (10, 41, 96, 99, 118, 123, 133). Endosomal localization can be blocked when PI3-kinase is inhibited with wortmannin. Although isolated FYVE domains can bind to PI3P-containing membranes, the membrane avidity of FYVE-domain containing proteins may be increased by dimerization (11, 71). EEA1-FYVE has limited ability to dimerize. However, FYVE domain-GST fusions form dimers that exhibit increased binding to PI3P-containing liposomes (71). The full-length EEA1 protein is predicted to homodimerize by forming a parallel coiled coil, so that the two C-terminal EEA1-FYVE domains are located near each other at one end of the dimer (11).

Pleckstrin Homology Domains

PH domains have been found in >500 cell regulatory proteins (see above regarding numbers taken from the SMART database). Most PH domains bind phosphoinositides, albeit with varying degrees of specificity (8, 34, 48, 73, 109). As such, they respond directly to free phosphoinositide levels regulated by phosphoinositide kinases, phosphatases, and phospholipases. The discovery over the past 3 years that signaling through PI3-kinases depends on PH domain-containing effectors has led to intense and renewed interest in these domains (34, 72). PH domains also participate in protein-protein interactions with such partners as G β γ subunits and PKC-C1 domains; this aspect of PH domains has been extensively reviewed elsewhere (8, 73, 109).

Structure of the PH Domain

Structures are now known for PH domains from eight different proteins: pleckstrin (49, 143), spectrin (55, 82, 147), dynamin (23, 30, 38, 130), PLCδ1 (31), son of sevenless 1 (Sos1) (69, 151),β-adrenergic receptor kinase (βArk) (39), Bruton's tyrosine kinase (Btk) (5, 56), and insulin receptor substrate 1 (IRS-1) (22). The PH domain structure contains two orthogonal antiparallel β sheets of three and four strands (Figure 2d ). These are followed by a C-terminal α helix. The β sheets curve in a tight barrel-like conformation, while the C-terminal helix folds in to cover one end of the barrel. This fold is also found in the protein-binding phosphotyrosine binding (PTB) (25, 149, 152), enabled/VASP homology (EVH) (106), and Ran-binding (RanBD) (138) domains and as a substructure within the protein- and phospholipid-binding 4.1, ezrin, radixin, and moesin (FERM) domain (M Pearson, D Reczek, A Bretscher, PA Karplus, submitted for publication). The interstrand loops are involved in ligand binding and vary substantially in sequence and structure between PH domains. The membrane-binding face of the domain contains basic residues that assist in ligand binding.

Inositol Phosphate-Binding Subsites

The structures of complexes of PLCδ1-PH with Ins(1,4,5)P3(31) and Btk-PH with Ins(1,3,4,5)P4(5) define four different phosphate-binding subsites that participate in high-affinity specific phosphoinositide binding (Figure 5a). The general outlines of the binding site are the same for PH domains of pleckstrin (48, 49), dynamin (114, 150), Sos1 (151), and βArk (39), based on NMR chemical-shift perturbations. In both structures, the β1/β2 and β3/β4 loops of the first β sheet form most of the key interactions. In PLCδ1-PH, Ins(1,4,5)P3 is buried between the 2 loops and forms 12 hydrogen bonds to 9 different amino acids of the domain. Interactions are even more extensive in Btk-PH, involving 18 hydrogen bonds. This is consistent with the higher affinity of the latter for its cognate ligand, 40 nM (36), as compared with 210 nM for PLCδ1-PH (74).

Figure 5. a.

Figure 5

a. Structure-based alignment of PH domains (there is no structure available of the Akt-PH, which has been aligned by sequence homology). Examples of each of the four provisional PH domain groups (34) are shown. Residues that interact nonspecifically with (more...)

The 1- and 4-phosphates of Ins(1,4,5)P3 and Ins(1,3,4,5)P3 bind to equivalent subsites (denoted I and IV in Figure 5a) in PLC δ1-PH and Btk-PH. Subsite I is relatively solvent exposed and poorly defined. Subsite IV is buried and makes at least three close interactions with the 4-phosphate in both structures. Subsite IV is more positively charged in PLCδ1-PH compared with Btk-PH. There is one dramatic difference between the two structures; the inositol ring of the ligand is rotated about the axis defined by the 1- and 4-carbons of the inositol ring. Thus the 3-phosphate of Ins(1,3,4,5)P4 bound to Btk-PH occupies the subsite (III) belonging to the 5-phosphate of Ins(1,4,5)P3 bound to PLCδ1-PH. The critical Arg-28 of Btk participates in subsite III and is conserved in PLCδ1. The 5-phosphate of Ins(1,3,4,5)P4 occupies a subsite (II) that is created by a unique loop conformation in Btk-PH. Subsite II is missing in PLC δ1-PH.

Two modes of low-affinity phosphoinositide binding have been defined by the structure of the spectrin-PH-Ins(1,4,5)P3 complex (55) and by a secondary Ins(1,3,4,5)P4-binding site in the Btk gain-of-function mutant E41K (5). The Ins(1,4,5)P3 interacts with spectrin-PH via the β5/β6 loop and the opposite side of the β1/β2 loop from the PLC δ1-PH domain complex. The second Ins(1,3,4,5)P4 binds to the β3/β4 loop of Btk-PH E41K close to subsite I of the high-affinity binding site. The low-affinity sites are more solvent exposed and involve fewer contacts than those described above. Although they do not overlap, both low-affinity sites are on the membrane-binding face of the PH domain and are consistent with the overall picture of PH domain/membrane interactions inferred from other studies.

Phosphoinositide Specificity

PH domain binding to different phosphoinositide polyphosphates and inositol polyphosphates has been systematically examined (59, 62, 107), revealing a wide range of ligand affinity and specificity. Rameh et al (107, 120) subdivide PH domains into four groups, which provides a useful working classification scheme. PH domains are so divergent that sequence-based classification may not be conclusive for every case. Even classification based on in vitro function is complicated by the variety of soluble inositol polyphosphate- and phosphoinositide-binding assays in use.

Group 1

PI(3,4,5)P3-binding PH domains include those of Btk (36, 68, 107, 114), Grp1 (62, 65, 66), ARF nucleotide site opening (ARNO) (137), cytohesin-1 (88), Son-of-sevenless (Sos) (107), Tiam-1 N-terminal domain (107), Gap1IP4BP (17, 79), Gap1m (37, 80), Vav (47), and several newly identified members (59). They are highly specific for PI(3,4,5)P3, which they typically prefer over PI(4,5)P2 by ~100-fold. Their sequences have more positively charged residues (6–11 residues, including histidines) in the β1/β2 strands and loop than group 2 (Figure 5b). In Btk-PH, these additional positive residues contribute to the unique site II, which binds the 5-phosphate. Positive charges predominate in the β1/β2 loop in other group 1 PH domains, suggesting that the binding mode observed in Btk-PH may be general to this group. Subsite IV has fewer charged interactions with the 4-phosphate than in group 2, consistent with the weaker binding of group 1 PH domains to PI(4,5)P2.

Figure 5. b.

Figure 5

b. Schematic of the high-affinity phosphoinositide-binding site of PLC δ1-PH and Btk-PH. Structural elements found only in Btk are drawn in gray. Elements found in PLCδ1only or in both PH domains are drawn in black. The bound phosphoinositide (more...)

Group 2

The members of the second group have high affinities for PI(4,5)P2 and PI(3,4,5)P2 and include PLCδ1 (15, 40, 62, 74), βArk (62, 104, 107), β-spectrin (62, 107), RasGAP (62), the N-terminal domain of pleckstrin (62, 129), DAGKδ (62, 129), oxysterol-binding protein (OSBP) (75, 107), IRS-1 (22), and others (62, 107). Group 2 domains do not discriminate substantially between PI(4,5)P2 and PI(3,4,5)P3 in vitro (62, 107). Preferential binding to PI(4,5)P2 in vivo may be more a function of the greater abundance of this lipid than discrimination against 3-phosphoinositides. PLCδ1-PH binds PI(4,5)P2 and PI(3,4,5)P2 with high affinity compared with other acidic lipids (73, 109), but other group 2 PH domains are less specific. In these group 2 domains, unlike PLCδ1-PH, strong binding to PI(4,5)P3 may depend more on the high negative charge on this lipid than on stereospecific recognition. This is consistent with the imperfect conservation of some of the key PLCδ1-PH basic side chains in group 2 domains.

Group 3

A third group, including Akt (also known as PKB) (32, 33, 59, 60, 62) and PDK1 (1, 124), binds PI(3,4)P2 as well as PI(3,4,5)P3. The only other reported member of this group is an expressed sequence tag (EST)-encoded protein of unknown function (62). Group 3 PH domains vary somewhat in their relative affinities for PI(3,4)P2 vs PI(4,5)P2 and PI(3,4,5)P3 in different reports. The β1/β2 loops of group 3 PH domains contain fewer basic residues than many of the group 1 domains, but the structural basis for specificity still is not entirely clear, pending the structure determination of a group 3 PH domain.

Group 4 and Others

Group 4 members, which include dynamin and the C-terminal PH domain of TIAM-1, exhibit relatively low binding affinity for the ligands mentioned above. The high-affinity phosphate subsites are absent or incompletely formed in these PH domains. Despite the low affinity of dynamin-PH monomers for PI(4,5)P2, the physiological importance of this interaction for endocytosis is well established. The effective affinity is bolstered by oligomerization of dynamin (67). PLC β1- and PLCβ2-PH bind nonspecifically to neutral and acidic phospholipids with low affinity (139). PLCγ -PH binds 3-phosphoinositides, including PI3P (29, 62). Neither its sequence nor its binding affinities conform to groups 1 or 3, so it may represent a new group.

Membrane-Binding Mechanisms

The positively charged face and loops of PH domains are poised to form nonspecific contacts with negatively charged phospholipids, in addition to the specific contacts already described (Figure 2d ). The importance of nonspecific contacts is highlighted by the structure of the Btk-PH E41K gain-of-function mutant (5, 77). This mutation results in constitutive activation of the protein, probably owing to persistent membrane association (76). The corresponding mutation E54K in PLCδ1 produces a similar gain in enzyme function in vitro (9). The mutation does not increase the affinity for Ins(1,3,4,5)P4 molecule to the binding pocket. In the crystal, a second Ins(1,3,4,5)P4 molecule binds to the mutated lysine on the surface of the molecule near β-strands 3 and 4; three other (native) lysine residues complete this second binding site. The mutation increases the positively charged surface on this face of the domain (5), and it may enhance membrane association of the domain nonspecifically, either by generalized binding of negative membrane surface charge or by binding of phosphoinositide lipids other than PI(3,4,5)P3(136) at the second binding site.

A loss-of-function Btk mutant, K19E, maps to the β1/β2 loop but does not directly interact with Ins(1,3,4,5)P4. This mutation does not affect specific ligand binding (5) but decreases the local positive electrostatic potential and nonspecific affinity for the membrane surface. Scanning mutagenesis of the positively charged residues in the PLCδ1-PH (142) shows that some of the surface residues are critical for membrane binding even though they are located outside the binding pocket. Their locations suggest that the β1/β2 and β3/β4 strands and loops interact substantially with the phospholipid head group region of the membrane (Figure 2d ).

Differences in affinity for soluble inositol phosphates vs the cognate membrane-bound phosphoinositides are postulated to have important regulatory consequences. PLCδ1-PH binds to PI(4,5)P2 in vesicles with micromolar affinity, but binds to the cognate Ins(1,4,5)P3 with Kd = 210 nM (74). The high binding affinity of PLC δ1-PH to Ins(1,4,5)P3 may be important in product inhibition of the enzyme (15, 74).

Localization to Cell Membranes

Stimulation of PLC causes repartitioning of green fluorescent protein-PLCδ1-PH from the plasma membrane to the cytosol (35, 50, 122, 135) concomitant with the hydrolysis of PI(4,5)P2 in the membrane and the rise in soluble Ins(1,4,5)P3 concentration. The relative contribution of the two factors to translocation is still under debate. Green fluorescent protein-PLCδ1-PH translocation has been used to visualize the coupled intracellular dynamics of Ca2+ and Ins(1,4,5)P3(50). OSBP-PH translocation to the Golgi depends on PI(4,5)P2 and at least one other unknown factor (75). Plasma membrane localization of PH domains that bind 3-phosphorylated phosphoinositides has been similarly demonstrated. Examples include the PH domains of Btk (136), ARNO (137), GAP1IP4BP(79), GAP1m(80), PDK1 (2), and Akt (140). Wortmannin inhibition of PI3K blocks plasma membrane localization of these PH domains.

Roles of PH Domains Within Larger Proteins

PI(4,5)P2 binding allosterically activates dynamin's GTPase activity (78). Since Ins(1,4,5)P3 also has this capability (114), some effects of the PH domain on the rest of the protein seem to be independent of membrane binding. Dbl homology (DH) domain-containing proteins act as guanine nucleotide exchange factors for Rho-family GTPases. The DH domains are invariably followed by a PH domain, as for Sos and Vav proteins. A crystal structure of Sos DH-PH suggests that a putative GTPase-binding site is formed by both domains; the interface includes the negatively charged side of the PH domain (121). Ligand binding to the PH domain has been proposed to allosterically modulate the nucleotide exchange activity of Sos, perhaps via ligand-induced conformational changes in the β1-β2 loop. The PH domain of Sos is not required for membrane targeting (14). By the same token, the Rac GTP exchange factor Vav is allosterically activated by PI(3,4,5)P3 (47).

Various appendages are required for the functioning of certain PH domains. The cytohesin PH domain is followed by a 17-residue polybasic sequence, which is required for high-affinity binding to PI(3,4,5)P3 (88). The Btk PH domain is followed by a small Btk motif, which binds a single Zn2+ atom (56) and has no known role other than structural stabilization. Gβγ binds to the C-terminal helix of βArk-PH (104). The solution structure of βArk-PH reveals that this region belongs to an extension to the C-terminal helix (39), which protrudes past the core of the domain.

The activity of the Akt kinase is allosterically regulated by its PH domain, implying contacts between the PH and other domains. Phosphorylation of Akt by PDK1 is necessary for activation (1, 124). In addition to localizing Akt at the membrane, the PH domain directly regulates the susceptibility of Akt to phosphorylation. Deletion of the PH domain results in higher basal phosphorylation and activity of Akt (112). A working model postulates that the Akt-PH participates in autoinhibitory contacts with the catalytic domain of Akt that are broken upon PI(3,4)P2 binding.

Other Membrane-Binding Domains

The intensive analysis of data from genome-sequencing projects has probably left few important signaling domains undiscovered (6, 115). Of domains that have been recently identified by sequence analysis, the START domain stands out as a probable lipid-binding signaling domain (105). There are many important roles for basic and amphipathic sequences, often with covalent lipid modifications, although these sequences are not independently folded and do not qualify as domains. The catalytic domains of many enzymes involved in lipid metabolism contain membrane-interacting hydrophobic ridges and basic loops and patches that may help target them to membranes. Finally, certain SH2 and PTB domains can bind phospholipids (108, 152) in addition to their better known peptide-binding functions.

Concluding Remarks

Stereospecific and Nonspecific Interactions with Membranes

The unique interplay between specific and nonspecific interactions with membranes sets the lipid-directed class of signaling domains apart from all others. In all of the membrane-binding domains, the specific ligand-binding site is flanked by basic or hydrophobic side-chains, or both. This arrangement of specific and nonspecific binding sites has several profound consequences.

The nonspecific-interaction energy can add to the stereospecific interaction to greatly increase the net interaction energy. In practice, this can lead to ≤104-fold-higher binding affinities. In the cell, this translates into a potent membrane-targeting mechanism. In other cases, the nonspecific component may be weak or even unfavorable. Some membrane-binding domains seem to have a dual life as receptors for mutually antagonistic membrane-bound and soluble second messengers.

Nonspecific membrane interactions can, in principle, augment the stereospecificity of the specific component of the interaction. The nonspecific membrane interaction makes an additional point of contact to define stereospecific interactions. Stereospecific recognition of a chiral lipid embedded in a membrane can be achieved with only two direct contacts between the protein and the lipid, provided that the protein makes an additional contact with the membrane. The degree of exposed hydrophobic surface on the protein dictates the depth to which it can penetrate the bilayer. The locations of known specific lipid-binding sites on domain structures closely match the expected distances of the lipid head groups as measured from the center of the bilayer. The relative positioning of the specific- and nonspecific-binding sites serves as a molecular ruler that has no counterpart among domains that recognize soluble ligands.

Biological Functions for Low-Affinity and Nonspecific Interactions

Many of the interactions described for membrane-targeting domains are of relatively low affinity. This complicates the problem of sorting out physiologically important interactions from artifacts. Many weak interactions are clearly important in cells and, indeed, appear to be weak “by design”. Cooperativity can be achieved by the oligomerization of weakly interacting domains into an assembly that binds membranes strongly. Under physiological conditions, the stable interaction of many signaling proteins with membranes depends on ligand binding by two or more different domains, for instance the C1 and C2 domains of PKC. This arrangement allows proteins such as PKC to function as temporal coincidence detectors.

Targeting vs Allosteric Regulation

The current emphasis on the targeting roles of membrane-binding domains should not obscure their equally important roles in the allosteric regulation of enzymes that contain them. In an evolutionary economy, a given domain in a given protein often contributes to regulation at both levels. It is clear that many protein kinases and GTPase activating proteins are allosterically activated by engagement of their membrane-binding domains. For the best understood example, PKC, this is a multistep process in which membrane localization is necessary but not sufficient for PKC activation. In contrast, some GAPs can be activated when their PH domains bind soluble inositol phosphates, whereas GAP targeting requires a membrane-bound phosphoinositide.

Can Domain Studies Help Read Genome Sequences?

One of the great challenges to biologists in the postgenomic era will be the prediction of protein function from sequence. The concept of modular domains, developed over the past 15 years or so, is one of the most powerful tools available. The sophisticated use of domain data can contribute to predicting protein function. Clearly there is not a one-to-one correspondence between domain structure and function. Not all C1 domains bind diacylglycerol, not all C2 domains bind Ca2+, and not all PH domains bind specifically to phosphoinositides. The attribution of such functions cannot be based solely on the presence of such a domain in a protein sequence.

Fortunately, structural and functional studies have allowed sequence motifs to be discovered whereby domains can be subdivided into “flavors” with common functions. These assignments may be very reliable when the sequence of interest has high identity to that of a well-characterized domain whose structure and binding specificity are known. Prediction is much less reliable for highly divergent sequences. A complete understanding of the sequence/function relationships of domains would be most valuable. A key direction for the bioinformatics of protein domains will be to systematize and automate the process of classifying domains into functional subgroups and to increase its scope and reliability.


We thank T Balla, A Hickman, S McLaughlin, A Newton, and A Toker for comments on the manuscript. We apologize to the authors of many seminal papers, especially those predating 1994, that could not be cited for reasons of space.

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