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Membrane recognition by
phospholipid-binding domains
Mark A. Lemmon
Abstract | Many different globular domains bind to the surfaces of cellular membranes, or to
specific phospholipid components in these membranes, and this binding is often tightly
regulated. Examples include pleckstrin homology and C2 domains, which are among the
largest domain families in the human proteome. Crystal structures, binding studies and
analyses of subcellular localization have provided much insight into how members of this
diverse group of domains bind to membranes, what features they recognize and how binding
is controlled. A full appreciation of these processes is crucial for understanding how protein
localization and membrane topography and trafficking are regulated in cells.
Inner leaflet
A lipid layer that faces the
inside of the cell.
Stereospecificity
Specific recognition of a
particular stereoisomer in a
binding reaction.
Amphiphilicity
Possession of both
hydrophobic and hydrophilic
regions.
Department of
Biochemistry and Biophysics,
University of Pennsylvania
School of Medicine,
809C Stellar-Chance
Laboratories,
422 Curie Boulevard,
Philadelphia, Pennsylvania
19104‑6059, USA.
e‑mail: [email protected].
upenn.edu
doi:10.1038/nrm2328
Association of proteins with the surface of intracellular
membranes is essential for a wide variety of cellular
functions — from signalling and trafficking to maintaining cell structure — and involves an ever-growing
array of lipid-binding domains. For example, membrane
anchoring (and dynamics) of the cytoskeleton requires
the direct interaction of lipid-binding domains with the
membrane surface1,2. Moreover, upon stimulation of
cell surface receptors, numerous signalling proteins are
transiently recruited to specific locations in plasma
(and other) membranes, where they exert their functions (such as lipid modification or activation of small
GTPases) or become effectively co-localized with partners in a signal-transduction pathway2,3. Some cellular
compartments are ‘marked’ by the presence of specific
lipids, and recognition of these lipids is required for
the intracellular trafficking machinery to discern one
intracellular organelle from another4.
Despite common themes, including the almost
exclusive use of acidic phospholipids as binding targets,
the domains and proteins that bind membrane surfaces
vary widely in their binding mechanisms, which allows
differences to be exploited for distinct modes of control.
In this review, I discuss how different lipid-binding
domains associate with membranes, illustrating where
possible both the similarities and distinctions between
domains and how their individual properties are ideally
suited for their particular cellular function. To be able to
include some detail, I focus only on domains that have
well defined globular structures and on those that bind
their target lipids in a membrane context. Readers are
referred to excellent recent reviews5,6 for a discussion of
membrane association by unstructured clusters of basic
nature reviews | molecular cell biology
and hydrophobic residues, which share some functions
with the domains that are discussed here.
Targets of phospholipid-binding domains
The main acidic phospholipids in mammalian cell membranes are phosphatidylserine, phosphatidic acid and
phosphatidylinositol. In addition, a small proportion of
the membrane phosphatidylinositol is phosphorylated
at the 3‑, 4‑ and/or 5‑positions to generate phospho­
inositides. The approximate ratios of the different acidic
phospholipids in mammalian cells are listed in BOX 1,
although precise levels vary considerably depending
on the cell type and growth conditions. The phospho­
inositides are always very minor species; indeed, in the
inner leaflet of the plasma membrane of normal cells,
phosphatidylinositol‑(4,5)-bisphosphate (PtdIns(4,5)P2)
is estimated to account for only 0.5–1.0% of phospho­lipid
molecules, whereas phosphatidylserine accounts for
~25–35%5. However, phosphoinositides have a disproportionately major role in directing the membrane association of phospholipid-binding domains, as discussed
below, and their levels are acutely regulated2,7.
Diverse phospholipid-binding domains
At least 10 different globular domain types bind phospho­
lipids at the membrane surface (TABLE 1). The inter­actions
of these domains with the membrane surface fall into
two broad classes: some are highly specific and involve
stereospecific recognition of particular membrane
components; others are non-specific and involve attraction
to a general physical property of the membrane (such as
charge, amphiphilicity and curvature). These two extremes
are spanned by the domains listed in TABLE 1. Indeed,
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Box 1 | Relative levels of acidic phospholipids in mammalian cells
The main acidic phospholipids in mammalian cells are phosphatidylserine, phosphatidic
acid and phosphatidylinositol, which constitute approximately 8.5%, 1.5% and 1.0%,
respectively, of total lipid (by weight) in erythrocytes105. The phosphoinositides are
much less abundant, and their approximate relative levels have been estimated by
Stephens et al.7 (see table). Estimated fold increases in the levels of each phosphoinositide
in response to selected treatments are also shown. Phosphatidylinositol‑(3,4,5)trisphosphate (PtdIns(3,4,5)P3) and PtdIns(3,4)P2 levels are robustly (but transiently over
10–30 minutes) elevated when phosphoinositide 3‑kinases (PI3Ks) are activated by
agonists for many cell-surface receptors106. The levels of PtdIns(4,5)P2 fall slightly
following its conversion by phospholipase‑C-mediated hydrolysis (to generate inositol
trisphosphate (Ins(1,4,5)P3) and diacylglycerol) and PI3K-mediated phosphorylation.
The fall in PtdIns4P levels primarily reflects its 5‑phosphorylation to replenish pools of
PtdIns(4,5)P2. PtdIns5P levels were shown to be elevated in platelets following thrombin
treatment107, in response to various stresses and during the cell cycle108. PtdIns3P levels
seem to be relatively constant, although PtdIns3P is restricted primarily to endosomes
and multivesicular bodies. Finally, PtdIns(3,5)P2 is implicated in several cellular
processes58 and its levels are elevated in response to cellular stress. Note that PtdIns5P,
PtdIns(3,4)P2 and PtdIns(3,4,5)P3 have not been detected in Saccharomyces cerevisiae.
However, PtdIns(3,4,5)P3 (but not PtdIns(3,4)P2 or PtdIns5P) has been detected in
Schizosaccharomyces pombe109.
Lipid
Relative level (%)*‡
Fold increase on stimulation
Phosphatidylserine
8.5
1
Phosphatidic acid
1.5
1
Phosphatidylinositol
1.0
1
PtdIns3P
0.002
1§
PtdIns4P
0.05
0.7||
PtdIns5P
0.002
3–20¶
PtdIns(4,5)P2
0.05
0.7||
PtdIns(3,4)P2
0.0001
10||
PtdIns(3,5)P2
0.0001
2–30#
*Phosphoinositide values are taken from Stephens et al.7. ‡Relative levels of total
phosphatidylserine, phosphatidylinositol and phosphatidic acid reflect human erythrocyte values
from Tanford105. §Note that acute insulin-induced PtdIns3P production has also been reported110.
||
Estimated changes upon ligand stimulation of neutrophils106. ¶In response to thrombin
stimulation, cellular stress and during the cell cycle108. PtdIns5P has not been described in
yeasts111. #PtdIns(3,5)P2 levels increase by up to 30-fold in yeast and 2–6-fold in plant and animal
cells in response to hyperosmotic stress58.
Second messengers
Molecules that act in a cell to promote responses to
extracellular stimuli.
Phorbol esters
Polycyclic esters that are
isolated from croton oil. The
most common are phorbol‑12myristate‑13-acetate and
12‑O-tetradecanoyl-phorbol‑ 13-acetate. These are both
potent carcinogens or tumour
promoters because they mimic
diacylglycerol and thereby
irreversibly activate protein
kinase C.
they are even spanned within certain domain classes.
The degree (and type) of specificity in phospholipid
binding has important functional implications. Naturally,
a domain that recognizes only the most general properties of a membrane could associate with all intra­cellular
membranes all of the time. Some temporal (but not necessarily spatial) specificity can be introduced if membrane
association requires the presence of a soluble second
messenger such as Ca2+. Some spatial (and temporal)
specificity can also be introduced if the phospholipidbinding domain has a preference for membrane surfaces
with high curvature. Both of these mechanisms are used
in biology, but the most specific cases involve domains
that selectively recognize rare membrane components that
are restricted in their location, time of synthesis or both.
Among phospholipids, the phosphoinositides represent
the best examples of acutely regulated binding targets
(BOX 1), and all phosphoinositides except PtdIns4P and
PtdIns5P have one or more well characterized specific
binding domains.
100 | february 2008 | volume 9
Target-specific binding domains
The first domains that were shown to recognize membranes in a target-specific manner were C1 (defined
below) 8 and pleckstrin homology (PH) 9 domains.
C1 domains are considered ‘honorary’ phospholipidbinding domains here, as their physiological binding
target (diacylglycerol (DAG)) is one phosphate group
short of qualifying as a phospholipid.
Fused to green fluorescent protein (GFP), the
target-specific domains described in this section provide a set of convenient ‘probes’ for monitoring intra­
cellular localization (FIG. 1) and levels of their specific
targets10,11.
C1 domains. C1 domains are named after ‘conserved
region-1’ from protein kinase C (PKC), and were
identified almost 20 years ago as the binding sites that
are responsible for PKC activation by phorbol esters
and DAG8. They are zinc-finger domains of ~50 amino
acids that contain the signature motif HX12CX2CX13–14
CX2CX4HX2CX7C (in which C is cysteine, H is histidine and X is any residue). Typical C1 domains all bind
DAG and are found in PKC isoforms and DAG kinases8.
Atypical C1 domains do not bind phorbol esters or
DAG, and their function is not clear.
A crystal structure of the (typical) C1B domain from
PKCδ (which contains two C1 domains: C1A and C1B)
has provided important insights into how this domain
associates with membranes12. A band of hydrophobic
side chains encircles the DAG/phorbol ester-binding
site and penetrates the apolar milieu of the membrane
(FIG. 2). C1 domains bind 10–80 fold more strongly to
phorbol esters that are embedded in phosphatidylserine
membranes than to the same free phorbol esters13. This
difference reflects the contribution of multiple driving
forces to membrane association by C1 domains: that
is, direct (1:1) interaction with the target ligand (DAG
or phorbol ester), membrane partitioning of hydrophobic side chains, plus electrostatic attraction of key
basic residues in the C1 domain to phosphatidylserine
headgroups. This combination of driving forces is
required for membrane targeting in vivo, with the acute
generation of DAG functioning as the trigger10.
Phosphoinositide-specific PH domains. The name ‘plecks­
trin homology’ reflects the identification of a ~100amino-acid region of sequence homology that occurs
twice in pleckstrin (the major PKC substrate in platelets), and in numerous other proteins with membraneassociated functions9. The N‑terminal PH domain
from pleckstrin was found to bind phosphoinositides14,
and studies of the PH domain from phospholipase Cδ1
(PLCδ1) provided the first evidence for stereospecific recognition of a phosphoinositide headgroup by a protein
domain15,16. The PLCδ1 PH domain (PLCδ–PH) binds
strongly to either PtdIns(4,5)P2 or to its isolated headgroup, d‑myo-inositol-1,4,5-trisphosphate (Ins(1,4,5)P3).
In contrast to the C1 domain, PLCδ–PH actually binds
more strongly to isolated Ins(1,4,5)P3 than to the headgroup of membrane-embedded PtdIns(4,5)P2. Moreover,
Ins(1,4,5)P3 efficiently displaces PtdIns(4,5)P2 from the
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Table 1 | Occurrence of selected phospholipid-binding domains in commonly studied organisms
Domain*
Humans
Mus musculus
Caenorhabditis elegans
Drosophila melanogaster
Saccharomyces cerevisiae
PH
303 (258)
284 (241)
79 (71)
81 (73)
32 (29)
PKC C2‡
200 (125)
200 (126)
70 (48)
62 (43)
22 (11)
C1
79 (58)
77 (57)
48 (34)
38 (28)
2 (1)
PX
35 (35)
39 (39)
11 (11)
14 (14)
15 (15)
FYVE
27 (26)
28 (27)
17 (17)
12 (12)
6 (5)
Discoidin C2‡§
24 (18)
24 (18)
3 (3)
7 (5)
-
GRAM
18 (15)
19 (15)
5 (4)
5 (4)
7 (6)
F-BAR||
14 (14)
17 (17)
3 (3)
4 (4)
4 (4)
Annexin
56 (13)
51 (12)
15 (4)
18 (6)
-
Gla§
13 (13)
17 (17)
-
-
-
N-BAR
9 (9)
12 (12)
5 (5)
3 (3)
2 (2)
ENTH/ANTH
9 (9)
7 (7)
5 (5)
3 (3)
8 (8)
The number of examples of each domain (with the number of proteins represented in parentheses) was obtained using the SMART database116 (see Further information)
in Genomic Mode in July 2007. The number of examples of each domain quoted here is directly comparable with those quoted for various modular signalling domains by
Bhattacharyya et al.117. *It should be noted that these domains are identified solely by sequence homology. Their function as phospholipid-binding domains may not be
fully conserved across (or within) species. ‡Note that PKC-class and discoidin-class C2 domains are not related. §Discoidin C2 domains and Gla domains are the only
extracellular phospholipid-binding domains listed. All others are intracellular. ||Termed FCH domain in the SMART database. ANTH, AP180 N‑terminal homology; BAR,
Bin, amphiphysin and Rvs; C1, conserved region-1; C2, conserved region-2; ENTH, epsin N‑terminal homology; FYVE, Fab1, YOTB, Vac1, EEA1; Gla, γ‑carboxyglutamate
rich; GRAM, glucosyltransferases, Rab-like GTPase activators and myotubularins; PH, pleckstrin homology; PKC, protein kinase C; PX, Phox homology.
Zinc finger
A small structural motif that is
found in many proteins,
including phospholipid-binding
proteins, DNA-binding proteins
and ubiquitin ligases. Zinc
fingers are characterized by
particular sequences of
cysteines and histidines that
coordinate bound Zn2+ ions.
The bound Zn2+ ions are
structurally crucial, and their
ability to nucleate the protein
structure obviates the need for
a hydrophobic core.
Guanine nucleotideexchange factor
A protein that facilitates the
exchange of GDP for GTP in the
nucleotide-binding pocket of a
GTP-binding protein.
Agammaglobulinaemia
A disorder that is caused by an
inability to make mature B cells
and, as a result, antibodies.
X‑linked agammaglobulinaemia
can arise from mutations in the
PH domain of Bruton’s tyrosine
kinase (BTK) that block the
ability of BTK to respond to
phosphoinositide 3‑kinase
signalling. Activation of BTK is
crucial for B‑cell maturation.
PLCδ–PH-binding site, which leads to the dissociation
of the PH domain from membranes when Ins(1,4,5)P3
is generated in cells17 or added in vitro18.
A crystal structure19 of PLCδ–PH bound to Ins(1,4,5)P3
provided a clear view of how this PH domain recognizes the pattern of phosphate groups that is specific
to PtdIns(4,5)P2. This structure also indicated that
membrane binding by PLCδ–PH need not involve significant membrane penetration. There is no equivalent
of the band of hydrophobic side chains that surrounds
the C1 domain ligand-binding site, although PLCδ–PH
does show membrane-insertion activity under some
circumstances20.
PH domains are well known effectors of the lipid
second messengers PtdIns(3,4,5)P3 and PtdIns(3,4)P2,
which are generated transiently upon activation of
almost all cell surface receptors21. A small subclass of PH
domains, including those from Bruton’s tyrosine kinase
(BTK)22,23, general receptor for phosphoinositides‑1
(GRP1)24 and protein kinase B (PKB; also known as
AKT)25, recognize one or both of these second messengers with remarkable specificity and affinity. These PH
domains represent classic examples of signal-regulated
membrane-targeting modules. In each case, the isolated
PH domain (as a GFP fusion protein) is predominantly
cytosolic in unstimulated cells, but undergoes a dramatic
transient relocalization to the plasma membrane on signal-dependent activation of phosphoinositide 3‑kinase
(PI3K)11,26. The selectivity and affinity characteristics of
these PH domains allow them to target only membranes
that contain PtdIns(3,4,5)P3 and/or PtdIns(3,4)P2, despite
PtdIns(4,5)P2 being present at 10–20-fold higher levels
(BOX 1). This highly specific recognition of PI3K products is
responsible for signal-dependent membrane recruitment
(and activation) of kinases such as BTK and PKB/AKT,
nature reviews | molecular cell biology
guanine nucleotide-exchange factors (GEFs) such as GRP1,
and other key signalling molecules21. Amino-acid subs­
titutions in PH domains that abolish PtdIns(3,4,5)P3
binding cause severe signalling defects, as seen in
X‑linked agammaglobulinaemia when the BTK PH domain
is mutated27. Conversely, mutations that promote constitutive association of certain PH domains with the plasma
membrane can cause cancer, as was recently reported for
PKB/AKT28. Intriguing studies29 have also indicated that
certain PtdIns(3,4,5)P3-specific PH domains might be
positively regulated by the soluble headgroup inositol‑
(1,3,4,5)-tetrakisphosphate (Ins(1,3,4,5)P4), although
mechanistic details remain unclear.
Crystal structures have provided insight into phos­
pho­inositide recognition by PH domains30. All highaffinity, stereospecific PH domains share a similar
phospho­inositide-binding site. PH domains have a
7‑stran­ded β‑sandwich structure, shown in FIG. 2, and the
β1–β2 loop between the first two β‑strands functions as
a ‘platform’ for the interaction with the phosphoinositide
headgroup. This loop lines a deep binding pocket and
contains the sequence motif 31 KXn(K/R)XR, in which the
basic side chains (from K (lysine) and R (arginine)) form
most phosphate-group interactions. Basic (and other) side
chains from elsewhere in the domain make additional
contacts that define the preferred inositol ring orientation
and phosphorylation pattern. Distinct phosphoinositidebinding specificities are largely attributable to variations
on the same theme, and robust rules have been defined for
predicting ligand preference on the basis of sequence30,32.
In addition to this ‘canonical’ mode of phospho­
inositide recognition by PH domains, several PH
domains, including those from the GEF TIAM1 and from
the Rho GTPase-activating protein‑9 (ARHGAP9), achieve
modestly specific and high-affinity phosphoinositide
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PX domains
FYVE domains
PH domains
O
O
P
HO
O–
O
O
O
P
HO
O–
O
PtdIns3P 5-K
OH
OH
P
3
ns
dI
Pt
-K
Pt
dI
ns
3
K
4-
OH
PtdIns(3,4)P2
PtdIns(3,4,5)P3
OH
PtdIns
C1 domains
?
O
O
P
HO
O–
O
HO
PtdIns5P 4-K
OH
P
OH
PtdIns5P
O
O
P
HO
O–
O
EN
K
5 OH
PHD fingers
P
P
K
5-
6
OH
P
4P
3 4
O
O
P
HO
O–
O
ns
O
K
HO
1
PtdIns4P
5-
2
SHIPs
P
ns
HO
O
–
OH
OH
P
P
O
P
PtdIns(3,4)P2 5-K
PI3
O
OH
OH
HO
O
O
O
P
HO
O–
O
PI3K
dI
Pt
OH
O
O
O
P
HO
O–
O
PT
O
dI
Pt
DAG
O
PtdIns(3,5)P2
P
O
P
OH
PtdIns3P
O
OH
P
OH
PtdIns 4-K
PROPPINs
PH domains
OH
HO
P
P
PtdIns(4,5)P2
Figure 1 | Domains that bind specific lipid targets. The structures and interconversion reactions are shown for all
phosphoinositides that are found in mammalian cells. The phosphoinositide kinases that catalyse the addition of phosphate
Reviews | Molecular Cell Biology
groups to the 3‑, 4‑ and/or 5‑positions are shown, as are the lipid phosphatases PTENNature
(phosphatase
and tensin homologue
on chromosome 10) and SHIP (SH2-containing inositol 5′-phosphatase). The phospholipid-binding domains that recognize
specific phosphoinositides (and diacylglycerol (DAG)) are shown. Only β‑propellers that bind phosphoinositides
(PROPPINs) recognize phosphatidylinositol‑(3,5)-bisphosphate (PtdIns(3,5)P2). Only pleckstrin homology (PH) domains
recognize PtdIns(4,5)P2, PtdIns(3,4,5)P3 or PtdIns(3,4)P2 with high specificity. All ‘Fab1, YOTB, Vac1, EEA1’ (FYVE) domains
bind PtdIns3P, as do nearly all Phox-homology (PX) domains (although there are a few mammalian exceptions that
reportedly bind PtdIns(4,5)P2 or PtdIns(3,4)P2). Whether PtdIns4P-specific domains exist remains unclear37,104,112, although
certain PH domains have been reported to prefer this lipid113. A split PH domain from VPS36 binds PtdIns3P36. The status of
plant homeodomain (PHD) fingers as putative PtdIns5P effectors114 is not clear. PX, Phox homology.
GTPase-activating proteins
(GAPs). Proteins that stimulate
the intrinsic ability of a GTPase
to hydrolyse GTP to GDP.
GAPs negatively regulate
GTPases by converting them
from active states (GTP bound)
to inactive states (GDP bound).
Split PH domain
A pleckstrin homology (PH)
domain with an interrupted
sequence. Regions of
polypeptide that are well
separated in the primary
sequence of a protein can
interact with one another to
form a globular PH domain
fold. The interruptions are
usually in the flexible loops of
the PH domain and can
harbour other domains.
binding through a distinct site33. This ‘non-canonical’
site is related to the binding site for Ins(1,4,5)P3 in the
spectrin PH domain34 and lies on the opposite side of the
β1–β2 loop from the canonical site (FIG. 2). Intriguingly,
a similar site is thought to be responsible for the binding
of PtdIns3P and other phosphoinositides to the ‘split PH
domain’ that is found in the GLUE (GRAM-like ubiquitinbinding in Eap45) domain of VPS36 (a component of
the endosomal sorting complex required for transport
(ESCRT) II complex)35,36. These findings imply that at
least one additional class of phosphoinositide-binding
PH domain beyond that exemplified by those present
in PLCδ1, BTK, GRP1 and PKB/AKT has yet to be fully
characterized.
Genome-wide studies 37 have shown that most
Saccharomyces cerevisiae PH domains do not bind
strongly or specifically to phosphoinositides. Of the
~234 PH domains in the human proteome (TABLE 1),
only ~10% are known to bind strongly and specifically
102 | february 2008 | volume 9
to phospho­inositides. Although several PH domains are
well known (and well understood) as phosphoinositidespecific membrane-targeting domains, these constitute
only a small minority of a large and poorly understood
class of domains. The functions of the rest remain
unclear, and other ligands are being sought38.
FYVE domains. All ‘Fab1, YOTB, Vac1, EEA1’ (FYVE)
domains specifically recognize PtdIns3P, which is primarily found in endosomes, multivesicular bodies and
phagosomes39. Like C1 domains, FYVE domains are
zinc fingers. They contain 60–70 amino acids, comprising two β‑hairpins and a small C‑terminal α‑helix
that are held together by two tetrahedrally coordinated
Zn2+ ions40. A conserved basic motif (RR/KHHCR) in
the first β‑strand contributes to a shallow, positively
charged binding pocket for PtdIns3P, and is responsible
for all but two of the direct hydrogen bonds that exist
between FYVE domains and PtdIns3P39,41.
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PKCδ C1
PLCδ1 PH
p40phox PX
Zn2+
Zn2+
P4
P5
Hydrophobic
band
ESCRT
(Endosomal sorting complex
required for transport). The
multiprotein ESCRT machinery
(ESCRT‑I, -II and -III) promotes
inward vesiculation at the
limiting membrane of the
sorting endosome and selects
cargo proteins for delivery to
the intralumenal vesicles of
multivesicular bodies.
Multivesicular bodies
Endosomal intermediates in
which small membrane vesicles
are enclosed in a limiting
membrane. The internal
vesicles are thought to form by
invagination and budding from
the limiting membrane.
Phagosomes
Membrane-bound vesicles that
contain microorganisms or
particulate material from the
extracellular environment.
Avidity
The overall measure of binding
between a multivalent ligand
and its receptors, which reflects
the combined strength of
multiple binding sites. Avidity
was originally defined for
antibodies, for which it refers to
the overall strength of binding
between multivalent antigens
and antibodies.
Coiled-coil domain
A protein structural domain
that often mediates subunit
oligomerization. Coiled coils
contain between two and five
α-helices that twist around
each other to form a supercoil.
Sorting nexin
Also known as SNX proteins.
These proteins are
characterized by the presence
of Phox-homology (PX)
domains and play roles in
endosomal cargo sorting as
well as other functions.
P3
P1
P1
Ins(1,4,5)P3
Phorbol
ester
(EEA1 FYVE)2
di-C4
PtdIns3P
ARHGAP9 PH
Zn2+
P3
P1
Ins(1,3)P2
Membrane
insertion
P3
P1
Ins(1,3)P2
Proposed
second anionbinding site
P4
Membrane
insertion
Membrane
p47phox PX
Zn2+
Endosomes
Vesicles that are formed by
invagination of the plasma
membrane.
Membrane
insertion
P5
Ins(1,4,5)P3
P1
di-C4
PtdIns3P
(modelled)
Membrane
Figure 2 | Structures of target-specific phospholipid-binding domains. The protein
kinase
Cδ (PKCδ)
C1 domain
Nature
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| Molecular
Cell Biology
(Protein Data Bank (PDB) code 1PTR) was solved as a complex with phorbol‑1,3-acetate12. The two Zn2+ ions are labelled,
as is the ‘hydrophobic band’ of residues that is thought to penetrate the membrane surface. The likely position of the
membrane is approximated by the shaded bar. Two pleckstrin homology (PH) domains are shown. One is the
phospholipase Cδ1 (PLCδ1) PH domain (PDB code 1MAI), which is bound to inositol‑(1,4,5)-trisphosphate (Ins(1,4,5)P3)
through the ‘canonical’ binding site19. The other is the ARHGAP9 PH domain (PDB code 2P0D), which binds Ins(1,4,5)P3
through the spectrin-like ‘non-canonical’ site33. Two Phox-homology (PX) domains are also shown. One is from p40phox,
with bound dibutanoylphosphatidylinositol 3‑phosphate (PtdIns3P) (PDB code 1H6H), which shows the
phosphoinositide-binding site and membrane insertion loop52. The other is from p47phox (PDB code 1O7K), and shows
PtdIns3P modelled into the phosphoinositide-binding site, plus a sulphate ion bound at the putative second anionbinding site, which is proposed to interact with phosphatidic acid64. A truncated ‘Fab1, YOTB, Vac1, EEA1’ (FYVE) domain
dimer from early endosome antigen‑1 (EEA1)41 bound to Ins(1,3)P2 is also shown (PDB code 1JOC), illustrating headgroup
binding, membrane insertion and dimerization. Structurally crucial Zn2+ ions are marked. Yellow side chains are those
that are likely to penetrate the membrane.
FYVE domains bind much more strongly to membrane-embedded PtdIns3P than to the isolated headgroup (Ins(1,3)P2) or short-chain phosphoinositide39,41,
and in this sense are more similar to C1 domains than
to PH domains of the PLCδ–PH class. Like C1 domains,
FYVE domains gain additional binding energy from
both membrane insertion and delocalized electrostatic
attraction39,42,43. However, even the cooperation of these
modes of interaction does not seem to be sufficient for
in vivo targeting of some FYVE domains to PtdIns3Pcontaining endosomal membranes44. Endosomal targeting of most FYVE domains is inefficient unless the
FYVE domain is also dimerized to allow multivalent
(increased avidity) binding to multiple PtdIns3P mole­
cules in the same membrane44,45. As shown in FIG. 2, the
FYVE domain from early endosome antigen‑1 (EEA1)
is preceded by a coiled-coil domain that drives its dimer­
ization and bivalent binding to PtdIns3P-containing
membranes41.
nature reviews | molecular cell biology
PX domains. A region of 130 amino acids of sequence hom­
ology that is found in components of the phagocyte NADPH
oxidase (phox) complex, termed the Phox-homo­logy
or PX domain46, was identified as a PtdIns3P-binding
domain47 in 2001. Most PX domains are found in sorting
nexin (SNX) proteins48, which are important in membrane
trafficking. Although all S. cerevisiae PX domains bind selectively to PtdIns3P, only 4 (of 15) bind with high affinity49.
In mammals, there are also examples of PX domains that
prefer PtdIns(3,4)P2 or PtdIns(4,5)P2 (Refs 50,51), but
selectivity is not strong in these cases and the preferred
ligand for most PX domains appears to be PtdIns3P48.
Structural studies52 indicate that the PX domains
(FIG. 2) employ the membrane-association mechanism
that is used by C1 and FYVE domains — with combined
headgroup binding, electrostatic attraction and membrane insertion — rather than relying primarily on headgroup interactions as PH domains do. Again, cooperation
of several driving forces is required for high-affinity
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Table 2 | Phospholipid-binding domains at a glance
Domain
Typical size
(amino acids)
Structure
Preferred target*
Membrane
insertion?
Ca2+
required?
Dimerization
required?
Refs
C1
~50
Zn2+ finger
DAG, phorbol esters
Yes
No
No
8
PKC C2‡
~130
β-sandwich
PtdSer (and others)
Yes
Yes
No
66
PH
~125
β-sandwich
Phosphoinositides, quite
diverse, some highly specific
Some reported20
No
Some examples
FYVE
60–70
Zn2+ finger
PtdIns3P
Yes
No
Most cases
39
PX
~130
α+β structure
PtdIns3P (a few bind other
phosphoinositides)
Yes
No
Most cases
48
PROPPIN
~500
β-propeller
PtdIns(3,5)P2 (PtdIns3P also in
some cases)
Unknown
No
No
58
Gla
~45
α-helical (requires
Ca2+ to fold)
PtdSer
Yes
Yes
No
62
Annexin
~310
α-helical array
Acidic phospholipids
Unknown
Yes
No
71
Discoidin
C2‡
~160
β-sandwich
PtdSer (specific)
Yes
No
No
118
ENTH
~150
α-helical solenoid
PtdIns(4,5)P2 (some
promiscuity)
Yes
No
No
77
ANTH
~280
α-helical solenoid
Phosphoinositides, relatively
little specificity
No
No
Yes
77
BAR
~240
Extended α‑helical
bundle
Acidic phospholipids (via
N‑terminal helix)
Yes
No
Yes
77
F-BAR
~320
Extended α‑helical
bundle
Acidic phospholipids
Unknown
No
Yes
91, 92
IMD
~250
Extended α‑helical
bundle
Acidic phospholipids,
especially phosphoinositides
Unknown
No
Yes
97
9
*It is important to note that functional similarity across a domain class cannot be assumed. For example, up to 80% of pleckstrin homology (PH) domains may not
bind phosphoinositides37. Some examples of each domain class may not bind phospholipids at all. ‡Note that the name C2 for these two classes of domain is entirely
coincidental. ANTH, AP180 N‑terminal homology; BAR, Bin, amphiphysin and Rvs; C1, conserved region-1; C2, conserved region-2; DAG, diacylglycerol; ENTH,
epsin N‑terminal homology; FYVE, Fab1, YOTB, Vac1, EEA1; Gla, γ‑carboxyglutamate-rich; GRAM, glucosyltransferases, Rab-like GTPase activators and
myotubularins; IMD, IRSp53/missing-in-metastasis; PH, pleckstrin homology; PKC, protein kinase C; PROPPIN, β‑propeller that binds phosphoinositides; PtdIns3P,
phosphatidylinositol-3-phosphate; PtdSer, phosphatidylserine; PX, Phox homology.
membrane targeting, with the presence or absence of
PtdIns3P (or other cognate ligand) determining whether
the overall binding energy is (or is not) sufficient to drive
the domain to the membrane. A few isolated PX domains,
such as those from p40phox and SNX3 (refs 49,50,53,54),
bind PtdIns3P-containing membranes with high affinity
and can be recruited independently to target membranes.
However, most PX domains bind weakly to PtdIns3Pcontaining membranes and are only targeted effectively to
membranes when they are part of a multivalent complex.
For example, SNX1 requires dimerization for its targeting to endosomes55, and the yeast SNX proteins Vps5 and
Vps17 are only recruited to PtdIns3P-containing membranes (through their low-affinity PX domains) as part
of the oligomeric retromer56 complex, which is capable of
multivalent interactions57.
Retromer
A complex of five proteins
(Vps35, Vps26, Vps29, Vps17
and Vps5 in yeast) that is
important for recycling
transmembrane proteins from
endosomes to the trans-Golgi
network.
PROPPINs. A search for potential effectors of PtdIns(3,5)P2
(Ref. 58) in S. cerevisiae led to the identification of Atg18 as
a new member of the family of phosphoinositide-binding
proteins59. Atg18 is a 500-amino-acid β‑propeller protein
that binds PtdIns(3,5)P2 with high affinity and specificity59,
and is the prototype for the PROPPINs (β-propellers that
bind phosphoinositides58) of which three are found in
S. cerevisiae and four in humans.
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In common with FYVE and PX domains, Atg18 seems
to bind much more strongly to the membrane-embedded
phosphoinositide than to its isolated headgroup or shortchain analogues (K. Narayan and M.A.L., unpublished
observations), which implies that membrane insertion
and/or delocalized electrostatic attraction to the membrane surface is important in driving membrane association. Studies of other PROPPINs, including those from
Drosophila melanogaster (in which there are three) and
humans, indicate that some bind both PtdIns(3,5)P 2
and PtdIns3P58,60,61. No structure has yet been reported
for a PROPPIN–phosphoinositide complex.
Phosphatidic acid binding. Several proteins have been
reported to recognize phosphatidic acid, including protein kinases and phosphatases, cAMP-specific phospho­
diesterases, transcription factors and others62. It has also
been reported that both the son-of-sevenless (SOS) PH
domain63 and the p47phox PX domain64 have a second
anion-binding pocket, in addition to their phospho­
inositide-binding sites, to which phosphatidic acid is
thought to bind (FIG. 2). Other than these examples, there
is no clearly defined globular domain to which phosphatidic acid-binding activity can be ascribed. Rather,
the regions implicated in such binding tend to comprise
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S
PKCα C2
Factor V discoidin C2
Ca2+
Cationic β-groove
(second anionbinding site)
PtdSer
Membrane
Annexin A5 core
Prothrombin Gla
Ca2+
Ca2+
Ca2+
groP-Ser
Lyso-PtdSer
Membrane
Figure 3 | Structures of phosphatidylserine-binding
domains.
PKCα Cell
C2 (Protein
Nature
Reviews | The
Molecular
Biology
Data Bank (PDB) code 1DSY) and annexin A5 core (PDB code 1A8A) domains are
intracellular phosphatidylserine (PtdSer)-binding domains. The discoidin C2 and Gla
domains are extracellular. The PKCα C2 domain forms a β‑sandwich with two Ca2+ ions
that are coordinated at one corner, and make bridging interactions with the bound
dicaproyl-phosphatidylserine68. Membrane penetration is thought to occur as shown,
and the proposed cationic β‑groove, at which phosphoinositides are thought to bind C2
domains66, is shown. The annexin core from annexin A5 is shown with bound glycerophosphorylserine (groP-Ser)72. Ten coordinated Ca2+ ions form bridging interactions
between the annexin core and the membrane phospholipids. The discoidin family C2
domain from factor V (PDB code 1CZS) forms a β‑sandwich structure, but is not related
to the PKC class of C2 domains. Models for membrane binding of the discoidin C2
domain76 involve a basic patch that is formed by several lysine and arginine side chains
(shown in stick representation) and a group of aromatic and aliphatic side chains (yellow)
that are thought to insert into the membrane. The prothrombin Gla domain (PDB code
1NL2)75 contains seven Ca2+ ions that are coordinated by γ‑carboxylglutamates and that
are responsible both for stabilizing the Gla-domain structure and for bridging interactions
between the Gla domain and the bound lyso-phosphatidylserine (lyso-PtdSer).
Significant membrane penetration is also proposed.
Zwitterionic phospholipids
A phospholipid with a
headgroup that is electrically
neutral (no net charge), but
that has formal positive and
negative charges on different
groups. For example,
phosphatidylcholine has a
positively charged choline
headgroup and a negatively
charged phosphate.
Phosphatidylethanolamine and
sphingomyelin are also
zwitterionic phospholipids.
short stretches of sequence, often rich in amino acids
with basic side chains62. Neither specificity nor structural
mechanisms of binding are understood. Phosphatidic
acid binding promotes localization of some proteins to
their sites of action62. By contrast, for some proteins, such
as the yeast transcription factor Opi1, phosphatidic acid
binding seems to sequester the limited quantities of the
protein away from its site of action65. Opi1 relocates to
the nucleus from the endoplasmic reticulum in response
to decreased phosphatidic acid levels62,65.
Domains with lower target specificity
For many membrane-binding domains, the physio­
logical target is an abundant phospholipid that is
ubiquitous in cell membranes (TABLE 2). For example,
several phospho­lipid-binding domains, including C2
nature reviews | molecular cell biology
domains and annexins, interact with phosphatidyl­serine.
Moreover, many phosphoinositide-binding domains
bind similarly to all of the polyphosphoinositides. These
facts raise the question as to how specificity — temporal
or spatial — can be achieved. Domains with these nonspecific characteristics do not simply bind persistently
to any membrane that contains phosphatidylserine or
polyphosphoinositides. Rather, some of them (such as
certain C2 domains and annexins) bind phospho­lipids
only when cytosolic calcium levels are transiently
elevated. Others, such as BAR and F‑BAR domains, seem
to interact only with highly curved regions of membranes. In contrast to the mechanisms discussed for C1,
PH, PX and FYVE domains, these mechanisms allow
temporal and spatial specificity in membrane targeting
without changing the nature of the headgroup itself.
Ca2+-dependent phosphatidylserine binding: C2 domains.
C2 domains, named after the second homology region
in PKC, are most well known as Ca2+ sensors and as
phosphatidylserine-binding domains 66. C2 domains
from conventional PKCs are Ca2+-dependent phospho­
lipid-binding domains. It is important to stress, however,
that not all C2 domains are capable of Ca2+-dependent
membrane binding. Indeed, a significant subgroup
of C2 domains do not bind Ca2+ at all, and several are
now known to bind targets (lipid or protein) other than
phosphatidylserine66. C2 domains comprise a characteristic 8‑stranded antiparallel β‑sandwich of ~130 amino
acids, with 3 key inter-strand loops that are responsible
for binding both Ca2+ (when relevant) and membranes
(FIG. 3). In contrast to PH, PX and FYVE domains, Ca2+dependent C2 domains lack a basic binding pocket for
their negatively charged target lipid. In fact, without
bound Ca2+, the canonical C2 domain membranebinding site is often acidic. Bound Ca2+ ions confer
positive charge and effectively ‘switch’ the electrostatic
characteristics of the binding site so that it can attract
negatively charged membranes67. The bound Ca2+ ions
also form a ‘bridge’ between the C2 domain and phosphatidylserine68 (FIG. 3). A combination of these effects
explains the Ca2+ dependence of C2-domain binding to
phosphatidylserine-containing membranes, although
the precise relationship between membrane association
and cytosolic Ca2+ levels is not clear for all C2 domains.
Like other phospholipid-binding domains mentioned
above, many (but not all) C2 domains also penetrate the
membrane surface66,69.
As mentioned above, lipid selectivity is variable across
the large C2 domain family. Ca2+-dependent C2 domains
from conventional PKCs specifically recognize phosphatidylserine with high affinity. Some other Ca2+-dependent
C2 domains bind to all anionic phospholipids, and still
others prefer zwitterionic phospholipids instead. In addition,
some C2 domains reportedly bind selectively to phospho­
inositides66, although in a manner that is typically not Ca2+
dependent and that involves a second basic patch that is
present on several C2 domains70 (termed the cationic
β‑groove66) (FIG. 3). Some C2 domains may simultaneously
engage multiple membrane components with both their
Ca2+-binding loops and cationic β‑groove. The diversity in
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REVIEWS
Fibrinolysis
The proteolysis of fibrin by
plasmin in blood clots.
Prothrombin
A pro-enzyme form of
thrombin (also known as factor
II), a serine protease that is
involved in the blood
coagulation cascade by
converting fibrinogen into
insoluble fibrin.
Factors V, VII, VIII, IX and X
Coagulation factors. Factors VII,
IX and X are serine protease
pro-enzymes that are involved
in the blood coagulation
cascade. Once activated,
factors V and VIII are cofactors
for factor Xa and IXa,
respectively.
Synaptotagmin
An integral membrane protein
with two PKC-class C2 domains
that acts as a Ca2+ sensor in
Ca2+-triggered synaptic vesicle
fusion with the plasma
membrane.
binding mode, lipid selectivity and Ca2+ dependence of C2
domains raises the intriguing possibility that individual
C2 domains are differentially membrane targeted — possibly
with defined signalling consequences.
of their PKC C2 namesakes (FIG. 3), their Ca2+-independent
specific phosphatidylserine recognition is more reminiscent of the way in which PH domains recognize
phosphoinositides.
Ca2+-dependent phosphatidylserine binding: annexins.
The annexins are also important (and abundant)
intra­cellular Ca2+-dependent phospholipid-binding
domains71. They share functional characteristics with
PKC C2 domains62, but are structurally unrelated (FIG. 3).
The ~310-amino-acid membrane-binding annexin core71
contains 4 annexin repeats, each with 5 α‑helices separated by loops that coordinate Ca2+ ions72. The bound
Ca2+ ions form a ‘bridge’ between the membrane surface
and the surface of the annexin core domain (FIG. 3) and
are coordinated simultaneously by protein and lipid.
Additional direct protein–phospholipid interactions
(and membrane insertion) are also likely to contribute
to membrane binding, but the crucial bridging role of
the Ca2+ ions brings membrane association under the
tight control of Ca2+ signalling71.
Consistent with this mode of membrane binding, most
annexins interact with acidic phospholipids in general,
although some bind preferentially to phosphatidylethanolamine or phosphatidylcholine instead. Annexin A2
reportedly prefers PtdIns(4,5)P2 (Ref. 73). The 13 human
annexins have roles in exocytosis, endocytosis, regulation
of membrane and/or cytoskeleton interactions and in the
control of ion channels71. In addition to their intracellular
functions, several annexins have extra­cellular roles that
include stimulating fibrinolysis, inhibiting blood coagulation and promoting clearance of apoptotic cells (with
extracellularly exposed phosphatidylserine). Intriguingly,
annexin A1, A2 and A5 in particular are found both
extra- and intracellularly71.
Key roles for membrane topography
A recently identified set of phospholipid-binding
domains clearly function beyond simply recruiting
their host proteins to the membrane surface. These
domains, which include ENTH/ANTH, BAR and
F‑BAR domains77, have little, if any, phospholipid target
specificity, and participate in endocytosis, cytokinesis
and other processes that involve substantial membrane
deformation.
Extracellular phosphatidylserine-binding domains.
Two classes of domain interact extracellularly with
phosphatidylserine (as the most abundant acidic phospholipid) in blood coagulation74. One is the Gla (or
γ‑carboxyglutamate-rich) domain, which is found in
prothrombin as well as in factors VII, IX and X62,74. Another
is the discoidin C2 domain, which is found in factors V
and VIII and in other proteins such as lactadherin62,74.
The discoidin C2 domain is not related to the PKC C2
domain discussed above, despite the unfortunate coincidence in nomenclature and some structural resemblance
(both are β‑sandwiches; FIG. 3).
Gla domains are small (~45 amino acids) and contain
9–12 γ‑carboxylated glutamic acid residues that coordinate a linear array of Ca2+ ions (FIG. 3) in a largely helical
domain75. The bound Ca2+ ions have a crucial ‘bridging’ role in phosphatidylserine binding, as described
above for the annexins. Residues with hydrophobic side
chains in an adjacent loop also seem to insert into the
membrane (FIG. 3).
The ~160-amino-acid discoidin C2 domains do not
bind Ca2+, but inter-strand loops in the β‑sandwich
domain form a positively charged binding site for stereo­
specific recognition of phosphatidylserine76. Although
the structural architecture of these domains is reminiscent
106 | february 2008 | volume 9
ENTH and ANTH domains. ENTH domains were
named after a region of epsin N‑terminal homology77
that is shared by a family of clathrin adaptor proteins. The
ANTH (AP180 N‑terminal homology) domain is found
in the clathrin adaptor protein AP180 (Refs 78–80). Both
domains form a superhelical solenoid of α‑helices (FIG. 4a),
and a similar fold is also found in the membrane-binding N terminus of the AP2 clathrin adaptor α‑subunit81.
These superhelical folds all bind phosphoinositides, but
with relatively little stereospecificity compared with selective PH domains. PtdIns(4,5)P2 is consistently among
the preferred ligands78–80 and, as the most abundant
phosphoinositide at the plasma membrane (where these
proteins function), this is almost certainly the relevant
physiological ligand.
ANTH and ENTH domains bind differently to
PtdIns(4,5)P2. The binding site in the ANTH domain
is a surface-lying basic patch that binds PtdIns(4,5)P2
with low affinity79 (FIG. 4a). Oligomerization of AP180,
conferred by its interaction with clathrin, is thought to
allow multiple low-affinity PtdIns(4,5)P2-binding sites
to cooperate with one another in driving membrane
association of a polymeric complex. By contrast, the
PtdIns(4,5)P2-binding site on the ENTH domain lies
in a well defined pocket78,80. An additional N‑terminal
amphipathic α‑helix (absent from the ANTH domain)
becomes ordered upon binding of the ENTH domain to
PtdIns(4,5)P2 (Ref. 78). This helix contacts the lipid headgroup — conferring further stereospecificity — but also
allows the ENTH domain to insert into, and deform,
its target membrane82 (FIG. 4a). The membrane deformation that results from ENTH- (but not ANTH-) domain
binding may help promote membrane invagination at
sites of endocytosis77. In principle, any phospholipidbinding domain that penetrates the membrane surface
(and there are many) will increase the surface area of the
leaflet into which it inserts and thus promote membrane
curvature83. The ENTH domain represents an extreme
example, and one in which membrane recruitment of
a protein functions as much to modify the topography
of the membrane as to alter localization of the protein.
A recent report84 has also suggested that C2 domains
in synaptotagmin might promote membrane fusion
by inserting into — and bending — membranes in a
similar way. This effect is dependent on the presence of
multiple C2 domains.
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a
ANTH
ENTH
Membrane
insertion
P4
P4
P5
P1
Ins(1,4,5)P3
P5
P1
Membrane
b
Amphiphysin
N-BAR
~11-nm radius
Endophilin-A1
N-BAR
~14-nm radius
CIP4
F-BAR
~30-nm radius
IRSp53
IMD/I-BAR
~44-nm radius
Dynamin
A large self-assembling GTPase
that plays a crucial role in the
scission of endocytic vesicles
from the plasma membrane.
Coated pit
An invagination in the plasma
membrane, coated with
clathrin on its cytoplasmic face,
that becomes internalized and
forms a clathrin-coated
endocytic vesicle.
Nature Reviews | Molecular Cell Biology
BAR and F‑BAR domains.
Domains in the BAR (‘Bin,
amphiphysin and Rvs’) family are also thought to promote
membrane curvature77. The isolated BAR domain from
the endocytic protein amphiphysin binds and tubulates
membranes in vitro85 and shows a preference for acidic
phospholipids. With its BAR domain promoting the formation of membrane tubules, amphiphysin is thought to
cooperate with dynamin in defining the necks of coated
pits77,85. A BAR domain at the N terminus of endophilin
has similar membrane-deforming properties86.
A crystal structure of the BAR domain from
D. melanogaster amphiphysin has provided one possible
explanation for how it associates with membranes and
promotes (or senses) membrane curvature87. The BAR
domain adopts an extended coiled-coil structure and
forms a long ‘banana shaped’ dimer (FIG. 4b). Peter et al.87
propose that the entire concave face of this dimer (which
includes basic patches) abuts the membrane surface. The
BAR domain could thus impose membrane curvature,
as implied in FIG. 4b, and this could be exacerbated by
nature reviews | molecular cell biology
Figure 4 | Structures of phospholipid-binding domains
implicated in membrane curvature. a | ENTH and ANTH
domains. The epsin ENTH domain (Protein Data Bank
(PDB) code 1H0A)78 is shown on the left, and the AP180/
CALM ANTH domain is shown on the right (PDB code
1HFA)79 in an equivalent orientation. Both domains
consist of an α‑helical solenoid, which is C‑terminally
extended in the longer ANTH domain. Both domains have
bound inositol-1,4,5-trisphosphate (Ins(1,4,5)P3). In the
ANTH domain, Ins(1,4,5)P3 binds to a surface-lying basic
patch that is formed by helices 1 and 2. In the ENTH
domain complex, Ins(1,4,5)P3 binds to a different location
and makes significant interactions with an N‑terminal
helix that becomes ordered upon lipid binding (and that is
not found in the ANTH domain). This amphipathic helix
is thought to penetrate the membrane as shown, with its
hydrophobic face (side chains coloured yellow) dissolved
in the apolar membrane milieu. b | The BAR domain family.
Structures of four different BAR domain (or related)
groupings are shown to illustrate how different extended
coiled-coil bundles are proposed to ‘sense’ or promote
different degrees of membrane curvature77. At the top is
the original BAR domain structure from Drosophila
melanogaster amphiphysin (PDB code 1URU), the
‘banana-shaped’ nature of which led to the suggestion
that these domains sense curvature87. The curvature of
the dimer is such that it would follow the surface of a
vesicle with a radius of 11 nm. In addition, the N terminus
of each molecule is thought to penetrate the membrane,
presumably inducing further curvature87. The
endophilin‑A1 BAR domain (PDB code 1ZWW) is similar,
except that it has an appendage in the middle of the
concave surface115 that could reduce curvature (or insert
into the membrane). The CIP4 (Cdc42-interacting
protein‑4) F‑BAR domain is also shown (PDB code 2EFK)92.
This larger domain forms a banana shape with a smaller
degree of curvature. Finally, the IMD domain from
IRSp53/missing-in-metastasis (PDB code 1Y2O)96 may
function as an ‘inverse’ BAR (I-BAR) domain, sensing or
stabilizing a curved membrane by lying inside the bend.
Side chains that are shown in each structure represent
basic side chains that are proposed to interact with the
negatively charged membrane surface.
the insertion of an important N‑terminal amphipathic
α‑helix into the membrane. Even if the proposed electrostatic attraction of basic patches on the BAR domain
to the negatively charged membrane surface is not sufficient to induce membrane curvature, it could allow
the BAR domain to function as a curvature ‘sensor’88.
If the entire concave surface of the BAR domain must
contact the membrane surface for maximum binding
affinity (and membrane recruitment), the bananashaped dimer should bind selectively to (and thus ‘sense’)
regions of high membrane curvature87,88. The endo­philin
BAR domain includes an additional ‘appendage’ that
protrudes from the concave face of the banana-shaped
dimer (FIG. 4b) , and this might alter the preferred
curvature and/or penetrate the membrane89,90.
The extended Fer-CIP4 homology (FCH or EFC)
domain (now known as F‑BAR) is structurally related
to BAR domains91,92. F‑BAR domains are found in proteins from the PCH (Pombe Cdc15 homology) family93,
which coordinate membrane–cytoskeleton interactions.
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a
Oligomerization
Weak
Dimer
Monomer
Strong
b
Two lipids
Weak
Weak
Strong
Lipid in curved membrane
Weak
Weak
Strong
Lipid and protein
Weak
Strong
A
S
P Y
N
T
G
Weak
A
S
P Y
N
T
G
Site 1
Site 1
Site 2
c
Protein
Site 2
Figure 5 | Multidomain cooperation. a | Enhancing membrane-binding affinity by
domain oligomerization. A phospholipid-binding domain
with very
low binding
Nature Reviews
| Molecular
Cellaffinity
Biology
will associate with membranes only when it is oligomerized. The two binding sites in the
dimer (or oligomer) cooperate with one another to promote a high-avidity interaction.
b | Coincidence detection by cooperation of domains in a multidomain protein.
Consider a phospholipid-binding domain that specifically recognizes the blue lipid in
the figure, but binds too weakly to be able to drive membrane association on its own.
If this domain occurs within a protein that also has another domain with similar
characteristics (but specificity for the red lipid), then the two domains can cooperate to
bind with high avidity to membranes that contain both blue and red lipids (top panel).
Similarly, if this domain lies alongside a domain that binds to curved membranes
(middle panel), the two domains might cooperate to bind the blue lipid specifically in
regions of high curvature. Alternatively, the phospholipid-binding domain might
cooperate with a specific (but low affinity) protein-binding domain, such as an SH2
domain, to recruit the multidomain protein specifically to membranes that contain the
blue lipid and a tyrosine-phosphorylated protein (bottom panel). c | Coincidence
detection by cooperation of binding sites in a single domain. Some domains, such as
certain pleckstrin homology (PH) domains, contain binding sites for more than one
membrane component. If the two binding sites both bind different lipids, the domain
may be selectively recruited to membranes that contain both of these lipids.
Alternatively, if the domain binds both a particular type of lipid and a specific
membrane-association protein (a small GTPase, for example), then a protein may be
recruited selectively to membranes that contain both the lipid and the protein. This
mechanism is proposed to explain specific Golgi targeting of certain PH domains103,104.
108 | february 2008 | volume 9
The ~320-amino-acid F‑BAR domain shows some
sequence similarity to BAR domains and shares their
ability to tubulate membranes and bind acidic phospho­
lipids94,95. As shown in FIG. 4b, F‑BAR domains form
extended coiled-coil dimers that resemble longer and
less curved BAR domains, and residues implicated in
membrane binding lie on their concave surface. By analogy with the BAR domain hypothesis, F‑BAR domains
are suggested to sense or induce membrane curvature,
but they would favour a smaller degree of curvature
than favoured by amphiphysin or endophilin BAR
domains (FIG. 4b).
Another BAR-like domain is the IRSp53/missing-inmetastasis (IMD) domain, which structurally resembles
a straightened BAR domain96 (FIG. 4b). This domain
reportedly binds phosphoinositides and, intriguingly,
appears to induce evaginations (rather than invaginations) when applied to PtdIns(4,5)P2-containing membranes97. This observation is consistent with a role for
the IMD domain in filopodia formation and has led to
intriguing suggestions that it functions as an ‘inverse
BAR domain’97, inducing (or sensing) curvature of the
opposite sense (as outlined in FIG. 4b).
Cooperation of multiple interactions
Multivalent multidomain interactions. Many examples
of the domains discussed here bind phospholipids too
weakly to direct membrane association on their own,
but cooperate with other domains in the same protein
(or oligomer) to drive multivalent (high avidity) membrane binding. One example is the dimeric EEA1 FYVE
domain (FIG. 2). Another is the PH domain from the
endocytic protein dynamin. The monomeric dynamin
PH domain has a millimolar-range Kd for PtdIns(4,5)P2containing membranes, but simple dimerization brings
the apparent Kd into the micromolar range98 (FIG. 5a).
As the membrane-binding energies of the two sites in
a dimer will be additive, dimerization can in principle
reduce the apparent Kd from millimolar to nano­molar
values. Such avidity effects might ensure that only
dynamin oligomers associate with PtdIns(4,5)P 2containing membranes during endocytic vesicle scission9. Similarly, as mentioned above, Vps5 and Vps17
(which have low-affinity PX domains) are recruited
to PtdIns3P-containing membranes only as part of
the multivalent retromer complex 57. Such avidity
effects can be exploited to control membrane targeting if oligo­merization of a protein with a low-affinity
phospholipid-binding domain is tightly regulated.
Multiple domains in the same protein can also
cooperate with one another to drive membrane targeting (FIG. 5b). For example, BAR domains are frequently
found alongside other phospholipid-binding domains,
such as PH and PX domains87. A PX–BAR protein, for
example, might selectively recognize (through multidomain ‘coincidence detection’99) highly curved regions
of PtdIns3P-containing membranes. Indeed, SNX1
reportedly uses this mechanism to target high-curvature
membranes emanating from endosomes100. Coincidence
detection can also be differentially ‘tuned’, depending on
the properties of the cooperating domains. For example,
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Filopodia
Thin, transient actin
protrusions that extend out
from the cell surface and that
are formed by the elongation
of bundled actin filaments that
exist in its core.
SH2 domain
(Src homology 2). A small
protein domain (110 amino
acids) that is found in many
signalling proteins and that
recognizes phosphorylated
tyrosine residues in a particular
sequence context. SH2
domains are responsible for
recruiting downstream
signalling molecules to
activated receptor tyrosine
kinases at the cell surface.
SH3 domain
(Src homology 3). A small
protein domain (50–60 amino
acids) that recognizes prolinerich sequences that are
important for the assembly of
various different signalling
complexes.
the RASAL and CAPRI GAPs both contain a PH domain
and Ca2+-dependent C2 domains, yet differ dramatically
in how their membrane association responds to Ca2+
oscillations101. In CAPRI, PH‑domain–membrane interactions effectively dampen oscillations in C2-mediated
Ca2+-dependent recruitment. In RASAL, oscillatory C2domain-mediated interactions dominate, which results in
a very different time course of membrane recruitment.
The multiple domains that cooperate with one
another need not all be phospholipid-binding domains.
The domains described here are often found alongside
SH2, SH3 and/or other protein-binding domains102. A
phospholipid-binding domain and an SH2 domain, for
example, might specifically drive a multidomain protein
to membranes that contain both the phospho­lipid target
and a specific tyrosine-phosphorylated membrane protein (FIG. 5b). As an illustration, Ras-GAP contains two
SH2 domains, a PH domain and a C2 domain — which
allows for potentially complex signal integration with
input from up to four distinct signals.
Coincidence detection by individual domains. Some of
the domains that were described above bind simultaneously to two targets, and do not require a second domain
to function as ‘coincidence detectors’ (FIG. 5c). For example, the p47phox PX domain and the SOS PH domain
both reportedly bind phosphatidic acid in addition to
their expected phosphoinositide ligands63,64. Some C2
domains may also bind simultaneously to phosphatidylserine and other lipids66. Such domains will bind
with highest affinity to membranes that contain both of
the lipids that they recognize — this is two-lipid coincidence detection. There are also examples of phospho­
lipid-binding domains with secondary binding sites for
specific proteins. The best examples are PH domains
from the FAPP1 and oxysterol binding protein (OSBP)
family, which specifically recognize Golgi membranes in
a way that requires binding to both phosphoinositides
and Golgi-localized ARF-family small GTPases103,104.
Dual recognition of phospholipids and proteins might
be important for specifying the localization of a number
of PH and related domains, although structural details
remain poorly understood37,38,99.
Conclusions and future directions
The aim of this review is to provide an overview of phospholipid-binding domains and the diverse mechanisms
and structures that are used for specifying membrane
association. Membrane association of all these domains
uses some combination of specific headgroup recognition, delocalized electrostatic attraction to negatively
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required in the levels of those targets — are far from clear.
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Another closing thought is underlined by the question as to whether BAR domains are inducers or sensors
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all phospholipid-binding domains. We have traditionally
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binding proteins to membranes. However, the converse
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example, the polybasic myristoylated alanine-rich PKC
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it has associated with negatively charged membranes5,6.
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in membranes to which they are apposed as they are
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Acknowledgements
I thank K. Ferguson and members of the Lemmon laboratory
for comments on this review. Work in this area in my laboratory is funded by the National Institute of General Medical
Sciences (NIGMS).
DATABASES
RCSB Protein Data Bank:
http://www.rcsb.org/pdb/home/home.do
1A8A | 1CZS | 1DSY | 1H6H | 1H0A | 1HFA | 1JOC | 1MAI | 1NL2
| 1O7K | 1PTR | 1URU | 1Y2O | 1ZWW | 2EFK | 2P0D
UniProtKB: http://ca.expasy.org/sprot
AP180 | ARHGAP9 | TIAM1 | VPS36
FURTHER INFORMATION
Mark A. Lemmon’s homepage:
http://www.med.upenn.edu/camb/faculty/cbp/lemmon.html
SMART (simple modular architecture research tool):
http://smart.embl-heidelberg.de
Membrane targeting domains resource (University of
Illinois at Chicago):
http://proteomics.bioengr.uic.edu/metador/MeTaDoR.html
Structure gallery, Roger Williams laboratory web site:
http://www.mrc-lmb.cam.ac.uk/rlw/text/structuregallery.html
Harvey McMahon’s laboratory web site:
http://www.endocytosis.org
Jim Hurley’s laboratory web site:
http://www-mslmb.niddk.nih.gov/hurleygroup.html
All links are active in the online pdf
volume 9 | february 2008 | 111
© 2008 Nature Publishing Group