From the
Department of Chemistry, University of
Illinois at Chicago, Chicago, Illinois 60607, the
Medical Research Council Laboratory of Molecular
Biology, Hills Road, Cambridge CB2 2QH, United Kingdom, the
||Department of Microbiology and Immunology, Weill
Medical College of Cornell University, New York, New York 10021, and the
**Howard Hughes Medical Institute and Department of
Cell Biology, Yale University School of Medicine, New Haven, Connecticut
06510
Received for publication, March 20, 2003 , and in revised form, May 8, 2003.
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ABSTRACT |
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INTRODUCTION |
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The epsin N-terminal homology
(ENTH) domain
(913)
is a highly conserved domain of 140 amino acids that has been identified
in all epsins and binds PtdIns(4,5)P2 with high affinity and
specificity
(68).
The ENTH domain is made of a superhelix of 7
-helices with an eighth
-helix misaligned with the superhelical axis, which is structurally
similar to the superhelical VHS domain
(14). The AP180/CALM and
HIP1/HIP1R protein families contain an N-terminal domain that is homologous to
the ENTH domain and binds PtdIns(4,5)P2
(13). X-ray structural
analysis of the AP180 N-terminal
homology (ANTH) domains of AP180/CALM
(7) showed that the ANTH domain
is extended by one or more
-helices when compared with the ENTH
domain.
Both the AP180 ANTH domain and the epsin ENTH domain bind
PtdIns(4,5)P2 with high specificity, but structurally in a
different manner. The ANTH domain binds PtdIns(4,5)P2 via
solvent-exposed Lys and His side chains on one side of the domain, and only
the lipid headgroup is contacted by the protein
(7). The ENTH domain binds
PtdIns(4,5)P2 in a pocket and makes extensive contacts with both
the headgroup and glycerol backbone
(8). On binding the
PtdIns(4,5)P2 headgroup, residues 315 of the ENTH domain
adopt an -helical structure which makes up one side of the
PtdIns(4,5)P2-binding pocket and provides ionic interactions. The
outer surface of this amphipathic helix, termed helix 0, was proposed to lie
in the plane of the lipid bilayer with the Leu6, Met10,
and Ile13 residues buried into the hydrophobic phase. This
insertion of helix 0 was proposed to displace the lipid headgroups thus
driving membrane curvature (8).
A number of mutations of the hydrophobic residues were used to advance this
hypothesis; however, biophysical evidence for membrane insertion of the epsin
ENTH domain has not been shown.
In this study, we investigated the effect of PtdIns(4,5)P2 on the membrane binding of the epsin ENTH and the AP180 ANTH domains by surface plasmon resonance (SPR) and monolayer analyses. Results from these measurements as well as the calculation of their electrostatic potential in the absence and presence of PtdIns(4,5)P2 indicate that the ENTH and ANTH domains have distinctly different modes of membrane interaction.
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EXPERIMENTAL PROCEDURES |
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Mutagenesis and Protein ExpressionThe R114A mutant of epsin
ENTH was prepared by the overlap extension polymerase chain reaction method
(16), and all other mutants
were prepared as described previously
(8). Each construct was
subcloned into the pGEX-4T-1 vector containing a N-terminal glutathione
S-transferase fusion and transformed into Escherichia coli
DH5 cells for plasmid isolation. After verifying the DNA sequence, the
plasmid was transformed into E. coli BL21 cells for protein
expression. One liter of 2x YT medium (16 g of tryptone, 10 g of yeast
extract, and 5 g of NaCl in 1 liter of H2O) containing 100 µg/ml
ampicillin was inoculated with BL21 cells harboring each construct and grown
at 37 °C until absorbance at 600 nm reached 0.4. At this time, 20 mg of
isopropyl-1-thio-
-D-galactopyranoside was added, and cells
were then incubated at 25 °C for 16 h.
Cells were harvested for 10 min at 4,000 x g, and the resulting pellet was resuspended in 10 ml of 20 mM Tris-HCl, pH 8.0, containing 0.16 M NaCl, 50 µM phenylmethylsulfonyl fluoride, and 0.1% Triton X-100. The solution was then sonicated for 8 min using a 30-s sonication followed by 30-s cooling on ice. This was followed by centrifugation at 48,000 x g to separate the soluble and insoluble fractions. The supernatant was filtered into a 50-ml tube, and 1 ml of GST·TagTM resin (Novagen, Madison, WI) was added. The mixture was incubated on ice with gentle stirring (80 rpm) for 30 min. After this time, the mixture was poured onto a column, which was washed with 20 ml of 20 mM Tris-HCl, pH 8.0, containing 0.16 M NaCl. The column was then sealed and 1 ml of 20 mM Tris-HCl, pH 8.0, containing 0.16 M NaCl and 0.25 mM CaCl2 along with 2 units of thrombin were added to cleave the glutathione S-transferase tag. After 6 h at 25 °C, the protein was eluted from the column in 5 fractions using 1 ml of 20 mM Tris, pH 8.0, containing 0.16 M NaCl. Purity was checked on an 18% polyacrylamide gel, and samples were pooled and concentrated to 1 ml. Protein concentration was then determined using the BCA method (Pierce). The AP180 ANTH domain was purified as described previously (8).
Monolayer Penetration ExperimentsThe penetration of ENTH
and ANTH domains into the phospholipid monolayer was measured at 23 °C by
monitoring the change in surface pressure () at constant surface area
using a circular Teflon trough (4-cm diameter and 1-cm depth) and Wilhelmy
plate connected to a Cahn microbalance as described previously
(17). A lipid monolayer
containing various combinations of phospholipids was spread onto the subphase
composed of 10 mM HEPES, pH 7.4, containing 0.16 M NaCl
until the desired initial surface pressure (
0) was reached.
After the signal stabilized (
5 min), 50 µg of proteins were injected
to the subphase through the hole in the wall of the trough, and the change in
surface pressure (
) was monitored for 45 min while stirring the
subphase at 60 rpm. Typically, the
value reached a maximum after
20 min. It has been shown empirically that
caused by protein is
mainly due to the penetration of the protein into the lipid monolayer. For
example, in the case of the C2 domain of group IVA cytosolic phospholipase
A2, excellent agreement was found between large
caused
by several residues in the calcium-binding loops
(17) and their actual membrane
penetration measured by fluorescence
(18) and electron spin
resonance studies (19,
20). The maximal
value depended on the protein concentration and reached a saturation value
(e.g. [epsin ENTH]
3.0 µg/ml); therefore, protein
concentration in the subphase was maintained above such values to ensure that
the observed
represented a maximum value. The resulting
was plotted versus
0 from which the
critical surface pressure (
c) was determined as the x
intercept (21,
22).
Kinetic and Equilibrium SPR ExperimentsAll SPR binding measurements were performed at 23 °C. The coating of the L1 sensor chip has been described in detail previously (23, 24). The sensor chip surface was washed and then coated by injecting 90 µl of vesicles containing various phospholipids (see "Results") at 5 µl/min to give a response of 6,000 resonance units. Similarly, a control surface was coated with vesicles, typically without phosphoinositide of interest, to give the same resonance unit response as the active binding surface. Under our experimental conditions, no binding was detected to this control surface beyond the refractive index change for the ENTH and ANTH domains. Each lipid layer was stabilized by injecting 10 µl of 50 mM NaOH three times at 100 µl/min. Typically, no decrease in lipid signal was seen after the first injection. Kinetic SPR measurements were done at the flow rate of 30 µl/min. 30 µl of protein in 10 mM HEPES, pH 7.4, containing 0.16 M KCl was injected to give an association time of 60 s, while the dissociation was monitored for 400 s at which time the protein had completely dissociated. The lipid surface was washed with 10 µl of 50 mM NaOH before the next protein injection.
After sensorgrams were obtained for five different concentrations of each
protein within a 10-fold range of Kd, each of the
sensorgrams was corrected for refractive index change by subtracting the
control surface response from it. The association and dissociation phases of
all sensorgrams were globally fit to a 1:1 Langmuir binding model: protein +
(protein-binding site on the vesicle) (complex) using BIAevalutation
3.0 software (Biacore) as described previously
(2426).
The dissociation constant (Kd) was then
calculated from the equation, Kd =
kd/ka. Mass
transport (27,
28) was not a limiting factor
in our experiments, as change in flow rate (5 µl/min to 60 µl/min) did
not affect kinetics of association and dissociation. After curve fitting,
residual plots and
2 values were checked to verify the
validity of the binding model. Each data set was repeated three times to
calculate a standard deviation. Also, Kd values
were separately determined from equilibrium SPR measurements. For these
measurements, the flow rate was reduced to 2 µl/min to allow sufficient
time for the association phase, which in turn allows resonance unit values to
reach saturating response values (Req).
Req values were then plotted versus protein
concentrations (C), and the Kd value was
determined by a non-linear least squares analysis of the binding isotherm
using an equation, Req = Rmax/(1 +
Kd/C), where Rmax is
a maximal Req value. Kd
values obtained by both kinetic and equilibrium analysis for epsin ENTH and
AP180 ANTH were in agreement (data not shown), thus validating the kinetic
analysis.
It should be noted that in our SPR analysis Kd is defined in terms of not the molarity of phospholipids but the molarity of protein-binding sites on the vesicle. Thus, if each protein-binding site on the vesicle is composed of n lipids, nKd is the dissociation constant in terms of molarity of lipid monomer (22). Due to difficulty involved in accurate determination of the concentration of lipids coated on the sensor chip, only Kd was determined in our SPR analysis and the relative affinity was calculated as a ratio of Kd values assuming that n values are similar for wild type and mutants. This assumption is based on our finding that Rmax values, which are proportional to [total lipid]/n (22), were similar (within 10%) for all proteins under our experimental conditions in which the sensor chip was coated with the same amount of lipids.
Electrostatic Potential ComputationThe electrostatic potentials of ENTH and ANTH domains with and without inositol 1,4,5-triphosphate (Ins(1,4,5)P3) were calculated with a modified version of the program DelPhi and visualized in the program GRASP (29), as described previously (30). In the panels of Fig. 3, the electrostatic potentials are represented by two-dimensional equipotential contours in 0.1 M KCl, with red and blue indicating negative and positive potentials, respectively. The electrostatic calculations performed used partial charges taken from the CHARMM27 (31) force field and spatial coordinates taken from the structures of free epsin ENTH (1INZ [PDB] ) (32), Ins(1,4,5)P3-bound epsin ENTH (1H0A [PDB] ) (8), and CALM ANTH (1HFA [PDB] ) (7).
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RESULTS |
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Surface Plasmon Resonance Measurements of Membrane Binding of ENTH and ANTH DomainsThe lipid headgroup specificity of the epsin ENTH domain and the AP180 ANTH domain has been determined using soluble inositol lipids. For instance, the epsin ENTH domain binds monomeric Ins(1,4,5)P3 (Kd = 3.6 µM) 33 times more strongly than monomeric inositol 1,3,5-triphosphate (8). Also, the epsin ENTH domain has 8-fold higher affinity for Ins(1,4,5)P3 than the AP180 ANTH domain (8). However, their specificity and affinity for membrane-incorporated phosphoinositides has not been fully investigated. We first measured the affinity of the epsin ENTH domain for various phosphoinositide-containing vesicles (i.e. POPC/POPE/phosphoinositide (77:20:3)) using POPC/POPE (80:20) vesicles as a control. As listed in Table I, the epsin ENTH domain showed high affinity for POPC/POPE/PtdIns(4,5)P2 (77:20:3) vesicles (Kd = 23 ± 7 nM). It should be noted that for our SPR vesicle binding analysis Kd is defined as the dissociation constant for vesicle binding (not for phosphoinositide binding) in terms of the molarity of protein-binding sites on the vesicle, which is composed of n lipids (22) (see "Experimental Procedures"). Thus, the large difference between the Kd for monomeric Ins(1,4,5)P3 and the Kd for POPC/POPE/PtdIns(4,5)P2 (77:20:3) vesicles does not reflect the different affinities of the domain for monomeric and membrane-incorporated PtdIns(4,5)P2. When compared with POPC/POPE/PtdIns(4,5)P2 (77:20:3) vesicles, the epsin ENTH domain had much lower affinity for POPC/POPE/phosphatidylinositol 3,4-bisphosphate (77:20:3) and POPC/POPE/phosphatidylinositol 3,4,5-trisphosphate (77:20:3) vesicles. Lower estimates of Kd for these vesicles assessed from the protein concentrations employed for the SPR measurements were 10 µM, indicating that the epsin ENTH domain prefers membrane-incorporated PtdIns(4,5)P2 to other phosphoinositides by more than two orders of magnitude.
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The AP180 ANTH domain also had high specificity for PtdIns(4,5)P2-containing vesicles. For the same POPC/POPE/PtdIns(4,5)P2 (77:20:3) vesicles; however, the AP180 ANTH domain showed 12-fold lower affinity than the epsin ENTH domain. This difference in Kd is due to a 2.7-fold larger ka and a 4.3-fold smaller kd for the ENTH domain. The membrane association of peripheral proteins can be accelerated by long range electrostatic interactions (24), and the larger ka for the ENTH domain is consistent with its more positive electrostatic potential, especially around the PtdIns(4,5)P2-binding site when compared with the ANTH domain (68) (see also Fig. 3). On the other hand, the membrane dissociation step of peripheral proteins can be slowed by short range specific interactions and membrane penetration of hydrophobic residues (24). Thus, the smaller kd value of the epsin ENTH domain in comparison to the AP180 ANTH domain is consistent with our monolayer penetration data showing that the former penetrates more effectively into PtdIns(4,5)P2-containing monolayers than the latter. To further demonstrate that PtdIns(4,5)P2 has differential effects on the membrane binding of the ENTH and ANTH domains, we measured the vesicle binding of these domains as a function of PtdIns(4,5)P2 and KCl concentrations. When the PtdIns(4,5)P2 content in the vesicles was reduced from 3 to 0.5 mol %, the ka decreased 5.4- and 9-fold for the epsin ENTH domain and the AP180 ANTH domain, respectively. However, the reduction in PtdIns(4,5)P2 content had little effect on the kd of the ANTH domain but caused a 2-fold increase the kd for epsin ENTH. This difference again underscores the specific effect of PtdIns(4,5)P2 on the membrane penetration of the ENTH domain. Increasing the ionic strength of medium (i.e. from 0.16 to 0.5 M KCl) slowed the adsorption of the ENTH domain to POPC/POPE/PtdIns(4,5)P2 (77:20:3) vesicles by 45-fold (i.e. smaller ka) but prolonged its membrane residence by 3-fold (i.e. smaller kd). In contrast, the binding of the AP180 ANTH domain to the same vesicles was not detectable in the presence of 0.5 M KCl, again indicating that the ANTH domain interacts mainly with the membrane surface by nonspecific electrostatic interactions. Together, these results indicate that while the membrane association of both the epsin ENTH domain and the AP180 ANTH domain is driven primarily by nonspecific electrostatic interactions, the membrane dissociation of the former is uniquely governed by hydrophobic forces, due to its PtdIns(4,5)P2-induced membrane penetration.
Mutational Analysis of the Epsin ENTH DomainTo investigate the membrane penetration mechanism of the ENTH domain, we mutated the hydrophobic Leu6 and Met10 residues on helix 0 to glutamine and tested the effects on monolayer penetration. The L6Q and M10Q mutants bind the PtdIns(4,5)P2 headgroup with nearly wild-type affinity, whereas liposome tubulation is abolished (8). As shown in Fig. 2, the L6Q and M10Q mutations greatly reduced the monolayer penetration of the epsin ENTH domain to the POPC/POPE/PtdIns(4,5)P2 (77:20:3) monolayer, indicating the importance of the hydrophobic residues on helix 0 in PtdIns(4,5)P2-induced membrane penetration. This reduction in penetration was PtdIns(4,5)P2 specific, as these mutations did not greatly reduce the penetration into a POPC/POPE (80:20) monolayer (data not shown). In contrast, the mutation of Arg114 to Ala had little effect on the monolayer penetration. Finally, a double-site mutation of PtdIns(4,5)P2-binding residues (R63L/H73L) caused significantly reduced penetration to the POPC/POPE/PtdIns(4,5)P2 (77:20:3) monolayer, supporting the notion that PtdIns(4,5)P2 binding is prerequisite for the membrane penetration of helix 0. The reason this mutation did not have as a dramatic effect as the L6Q and M10Q mutations was likely to be due to intrinsically high monolayer penetration power of R63L/H73L caused by the introduction of two hydrophobic residues near the membrane binding surface. This notion is supported by the finding that R63L/H73L could penetrate the POPC/POPE (80:20) monolayer almost as effectively as the POPC/POPE/PtdIns(4,5)P2 (77:20:3) monolayer (see Fig. 2).
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We then measured the vesicle binding of these mutants by the SPR analysis.
For POPC/POPE/PtdIns(4,5)P2 (77:20:3) vesicles, R63L/H73L showed
extremely low affinity (Kd > 25
µM), underscoring the importance of PtdIns(4,5)P2
binding in the overall membrane affinity of the ENTH domain. It should be
noted that although this mutant has the higher intrinsic monolayer penetrating
ability (see above), it still cannot penetrate the
PtdIns(4,5)P2containing vesicles (i.e.
c < 30 dyne/cm) and has a drastically reduced positive
electrostatic potential, which may be important for initial electrostatic
vesicle binding (see below). L6Q and M10Q had 12- and 38-fold lower affinity,
respectively, than the wild type, primarily due to larger
kd values. The faster membrane dissociation of
these mutants is consistent with their less favorable hydrophobic interaction
with the membrane. R114A had 4-fold lower Kd, due
to a smaller ka. Although this residue is not
part of the PtdIns(4,5)P2-binding site, it is on the same side of
the putative membrane binding surface (see
Fig. 4) and could contribute to
nonspecific electrostatic interactions.
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Calculation of Electrostatic PotentialTo account for the differences in PtdIns(4,5)P2-mediated membrane binding properties of the epsin ENTH domain and the AP180 ANTH domain, we calculated the electrostatic potential for these domains in the absence and presence of Ins(1,4,5)P3 that is a soluble analog of PtdIns(4,5)P2. It was shown that PtdIns(4,5)P2 binding causes a local conformation change of the ENTH domain involving the formation of helix 0 (8). However, it is not known as to which conformation the epsin ENTH domain would assume when it initially contacts the PtdIns(4,5)P2-containing membrane surface (i.e. a membrane-bound PtdIns(4,5)P2-free conformation). Thus, the electrostatic potential of the ENTH domain in the absence of Ins(1,4,5)P3 was calculated for the structures both before and after the conformational change. As shown in Fig. 3, A and B, strong positive potentials were seen near the PtdIns(4,5)P2-binding pocket for both structures of Ins(1,4,5)P3-free epsin ENTH, due to the presence of basic residues involved in PtdIns(4,5)P2 binding (i.e. Arg8, Lys11, Arg25, Arg63, Lys69, and His73). These positive potentials may initially contribute to recruiting the domain to the anionic membrane surface through nonspecific electrostatic attraction, which subsequently leads to productive PtdIns(4,5)P2 binding. Notice that the high positive potential of the induced structure (Fig. 3B) also surrounds the hydrophobic residues (i.e. Leu6 and Met10) of helix 0, which is expected to produce an energy barrier against the penetration of these residues into the low dielectric membrane interface. This is because the desolvation that is prerequisite for their membrane penetration would disrupt favorable interactions between water molecules and charged and polar groups on both the protein and the membrane. Interestingly, the positive potential of the epsin ENTH domain is dramatically reduced when PtdIns(4,5)P2 binds to the domain (Fig. 3C). This suggests that PtdIns(4,5)P2 may serve as an electrostatic switch to decrease the highly positive potential surrounding the hydrophobic residues on helix 0, thereby facilitating their membrane penetration. Thus, it would seem that for the ENTH domain PtdIns(4,5)P2 plays a dual role of inducing a conformation change and switching the electrostatic potential.
Electrostatic potentials calculations of the ANTH domain are also consistent with our monolayer and SPR data. As shown in Fig. 3D, the ANTH domain has a lower positive electrostatic potential than the ENTH domain in the absence of PtdIns(4,5)P2 (Fig. 3B), which is consistent with its smaller ka. The docking of PtdIns(4,5)P2 to the ANTH domain leads to a charge neutralization (Fig. 3E), but unlike the ENTH domain, the ANTH domain lacks hydrophobic residues on the membrane binding surface that would productively interact with the membrane. As a result, the electrostatic switch by PtdIns(4,5)P2 has no functional consequences for the ANTH domain. Taken together, our electrostatic calculations supported the notion that PtdIns(4,5)P2 plays differential roles in the membrane binding of ENTH and ANTH domains.
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DISCUSSION |
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A recent study showed that the ENTH domain of epsin was as effective as the full-length protein in causing liposome tubulation, while neither AP180 nor its ANTH domain could induce the liposome tubulation independently (8). A main structural difference between the ENTH domain and the ANTH domain lies in the ligand-binding site (8). The ANTH domain does not have a well defined PtdIns(4,5)P2-binding pocket and instead uses surface exposed basic residues to coordinate the ligand. The ENTH domain also lacks a well defined ligand-binding pocket; however, for this domain the formation of the pocket is triggered by PtdIns(4,5)P2 binding. The same conformational change also aligns the hydrophobic residues, Leu6, Met10, and Ile13, toward the membrane surface. These structural differences between the two domains are translated into their distinct membrane binding properties. Our monolayer measurements clearly show that the ENTH domain can penetrate the PtdIns(4,5)P2-containing monolayer much more efficiently than the ANTH domain. Most significant, PtdIns(4,5)P2 specifically triggers the penetration of the ENTH domain, but not the ANTH domain, into the monolayer whose surface packing density recapitulates those of cell membranes and large unilamellar vesicles. Neither other phosphoinositides nor phosphatidylserine can replace the effect of PtdIns(4,5)P2 on the ENTH domain. This is similar to specific membrane penetration of FYVE domains and the PX domain of p40phox induced by phosphatidylinositol 3-phosphate (26, 33). Monolayer penetration properties of the ENTH domain mutants also verify the notion that hydrophobic residues on the same face of induced helix 0 are responsible for the membrane penetration of the ENTH domain.
Our SPR measurements and electrostatic potential calculations provide further insight into the differential PtdIns-(4,5)P2dependent membrane binding mechanisms of these two domains. The epsin ENTH domain binds the same PtdIns-(4,5)P2-containing vesicles with 12-fold greater affinity than the AP180 ANTH domain, due to faster association and slower dissociation. The faster membrane association of the epsin ENTH domain can be accounted for by its more positive electrostatic potential on its putative membrane binding surface, due to basic residues in the PtdIns(4,5)P2-binding pocket and Arg114 (see Fig. 3). The slower dissociation of epsin ENTH is consistent with the higher degree of monolayer penetration by its hydrophobic residues, including Leu6 and Met10. The 12- and 38-fold reductions in affinity by L6Q and M10Q mutations, respectively, indicate that membrane penetration of these residues contributes significantly to overall membrane binding of the ENTH domain.
Variation of the PtdIns(4,5)P2 concentration and the ionic strength of the medium also underscores the different roles PtdIns(4,5)P2 play in membrane binding of the two domains. In the case of the ENTH domain, PtdIns(4,5)P2 enhances ka via electrostatic protein-PtdIns(4,5)P2 interactions, whereas it lowers kd by inducing membrane penetration of the hydrophobic residues, and thereby allowing hydrophobic interactions. For the ANTH domain, on the other hand, PtdIns(4,5)P2 would seem to simply function as a bridge between the domain and the membrane, as suggested for the role of calcium in the membrane binding of annexins (45). This is due to the presence of pre-aligned basic residues and the absence of hydrophobic residues on the PtdIns(4,5)P2 binding surface.
Based on these data, we propose differential membrane binding mechanisms for the two domains (see Fig. 4). The epsin ENTH domain initially binds to PtdIns(4,5)P2-containing anionic membranes by nonspecific electrostatic interactions. The nature of membrane-bound PtdIns(4,5)P2-free conformation is not known at present. However, it would be reasonable to assume that the initial membrane contact induces, at least partially, the formation of the helix 0 and hence the PtdIns(4,5)P2-binding pocket, since the induction of amphipathic helices at the water-membrane interface has been well documented (4648). The subsequent PtdIns(4,5)P2 binding would then lock this induced conformation, as shown in the crystal structure of the epsin ENTH-Ins(1,4,5)P3 complex (8), concomitantly inducing the hydrophobic residues in the helix to insert into the membrane by the electrostatic switch mechanism. This membrane insertion elongates the membrane residence time of the protein and triggers the membrane deformation. For the ANTH domain, membrane-protein binding takes place primarily through electrostatic interactions with PtdIns(4,5)P2 acting as a bridge. Only minor changes in protein conformation and membrane structure are expected.
Multiple steps of complex protein-protein and protein-lipid interactions
are involved in the vesicle budding. Our results strongly suggest that the
PtdIns(4,5)P2-triggered membrane penetration of the ENTH domain is
a critical step in the epsin-induced membrane curvature. This notion is
supported by much reduced monolayer penetration of L6Q and M10Q mutants that
have lost the liposome tubulation activity of the wild type epsin ENTH domain.
The penetration of the ENTH domain into the PtdIns(4,5)P2-rich
region of the plasma membrane would cause the positive membrane curvature,
which has been suggested to be essential for membrane budding
(49). Intriguingly, there are
many peripheral proteins, including phosphoinositide-specific FYVE domains and
PX domains, which penetrate lipid monolayers as well as or more effectively
than the epsin ENTH domain without inducing liposome tubulation. Perhaps the
insertion of a relatively rigid -helix distorts the membrane enough to
cause curvature, while insertion of a less ordered loop may be insufficient.
Further mechanistic studies on the membrane-ENTH domain interactions would
reveal the molecular basis of this special activity of the epsin ENTH
domain.
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FOOTNOTES |
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¶ Supported by an European Molecular Biology Long Term Postdoctoral
Fellowship.
To whom correspondence should be addressed: Dept. of Chemistry (M/C 111),
University of Illinois at Chicago, 845 West Taylor St., Chicago, IL
60607-7061. Tel.: 312-996-4883; Fax: 312-996-2183; E-mail:
wcho{at}uic.edu.
1 The abbreviations used are: PtdIns(4,5)P2, phosphatidylinositol
4,5-bisphosphate; ANTH, AP180 N-terminal homology; ENTH, epsin N-terminal
homology; Ins(1,4,5)P3, inositol 1,4,5-triphosphate; POPC,
1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine; POPE,
1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoehthanolamine; POPS,
1-palmitoyl2-oleoyl-sn-glycero-3-phosphoserine; SPR, surface plasmon
resonance; CHAPS, 3-[3-cholamidopropyl)dimethylammonio]-1-propanesulfonic
acid.
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REFERENCES |
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