Contrasting Membrane Interaction Mechanisms of AP180 N-terminal Homology (ANTH) and Epsin N-terminal Homology (ENTH) Domains

Epsin and AP180/CALM are endocytotic accessory proteins that have been implicated in the formation of clathrin-coated pits. Both proteins have phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2)-binding domains in their N termini, but these domains are structurally and functionally different. To understand the basis of their distinct properties, we measured the PtdIns(4,5)P2-dependent membrane binding of the epsin N-terminal homology (ENTH) domain and the AP180 N-terminal homology (ANTH) domain by means of surface plasmon resonance and monolayer penetration techniques and also calculated the effect of PtdIns(4,5)P2 on the electrostatic potential of these domains. PtdIns(4,5)P2 enhances the electrostatic membrane association of both domains; however, PtdIns(4,5)P2 binding exerts distinct effects on their membrane dissociation. Specifically, PtdIns(4,5)P2 induces the membrane penetration of the N-terminal α-helix of the ENTH domain, which slows the membrane dissociation of the domain and triggers the membrane deformation. These results provide the biophysical explanation for the membrane bending activity of epsin and its ENTH domain. Epsin and AP180/CALM are endocytotic accessory proteins that have been implicated in the formation of clathrin-coated pits. Both proteins have phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2)-binding domains in their N termini, but these domains are structurally and functionally different. To understand the basis of their distinct properties, we measured the PtdIns(4,5)P2-dependent membrane binding of the epsin N-terminal homology (ENTH) domain and the AP180 N-terminal homology (ANTH) domain by means of surface plasmon resonance and monolayer penetration techniques and also calculated the effect of PtdIns(4,5)P2 on the electrostatic potential of these domains. PtdIns(4,5)P2 enhances the electrostatic membrane association of both domains; however, PtdIns(4,5)P2 binding exerts distinct effects on their membrane dissociation. Specifically, PtdIns(4,5)P2 induces the membrane penetration of the N-terminal α-helix of the ENTH domain, which slows the membrane dissociation of the domain and triggers the membrane deformation. These results provide the biophysical explanation for the membrane bending activity of epsin and its ENTH domain. Clathrin-mediated endocytosis is an energetically demanding process that is necessary for a diverse number of cellular processes, such as nutrient uptake, removal of receptors from the cell surface, and synaptic vesicle recycling (1Marsh M. McMahon H.T. Science. 1999; 285: 215-220Crossref PubMed Scopus (481) Google Scholar, 2Schmid S.L. Annu. Rev. Biochem. 1997; 66: 511-548Crossref PubMed Scopus (674) Google Scholar, 3Brodsky F.M. Chen C.Y. Knuehl C. Towler M.C. Wakeham D.E. Annu. Rev. Cell Dev. Biol. 2001; 17: 517-568Crossref PubMed Scopus (538) Google Scholar). This process involves cooperation of clathrin, the AP-2 adaptor complex and a number of other accessory proteins, including epsin. The process of endocytosis occurs through four general steps including bud initiation, bud formation, bud constriction, and vesicle scission (2Schmid S.L. Annu. Rev. Biochem. 1997; 66: 511-548Crossref PubMed Scopus (674) Google Scholar, 3Brodsky F.M. Chen C.Y. Knuehl C. Towler M.C. Wakeham D.E. Annu. Rev. Cell Dev. Biol. 2001; 17: 517-568Crossref PubMed Scopus (538) Google Scholar). Initiation of the bud site is thought to occur by the membrane association of AP180 and epsin as well as the adaptor complex AP-2. These proteins recruit clathrin to the membrane. The bud then forms and constricts, while the activities of amphiphysin and endophilin remodel the membrane at the bud neck. Finally, the GTPase activity of dynamin is required for the release of the vesicle into the cytoplasm (4Hinshaw J.E. Annu. Rev. Cell Dev. Biol. 2000; 16: 483-519Crossref PubMed Scopus (584) Google Scholar). Recently, the importance of phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2) 1The 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. in clathrin-mediated endocytosis has come to light (5Cremona O. De Camilli P. J. Cell Sci. 2001; 114: 1041-1052Crossref PubMed Google Scholar). In particular, epsin and AP180, which are necessary for the formation of clathrin-coated vesicles, bind PtdIns(4,5)P2 through their conserved N-terminal domains (6Itoh T. Koshiba S. Kigawa T. Kikuchi A. Yokoyama S. Takenawa T. Science. 2001; 291: 1047-1051Crossref PubMed Scopus (388) Google Scholar, 7Ford M.G. Pearse B.M. Higgins M.K. Vallis Y. Owen D.J. Gibson A. Hopkins C.R. Evans P.R. McMahon H.T. Science. 2001; 291: 1051-1055Crossref PubMed Scopus (605) Google Scholar, 8Ford M.G. Mills I.G. Peter B.J. Vallis Y. Praefcke G.J. Evans P.R. McMahon H.T. Nature. 2002; 419: 361-366Crossref PubMed Scopus (795) Google Scholar). The epsin N-terminal homology (ENTH) domain (9Itoh T. Takenawa T. Cell Signal. 2002; 14: 733-743Crossref PubMed Scopus (117) Google Scholar, 10Hurley J.H. Wendland B. Cell. 2002; 111: 143-146Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar, 11Nossal R. Zimmerberg J. Curr. Biol. 2002; 12: R770-R772Abstract Full Text Full Text PDF PubMed Scopus (25) Google Scholar, 12Kay B.K. Yamabhai M. Wendland B. Emr S.D. Protein Sci. 1999; 8: 435-438Crossref PubMed Scopus (102) Google Scholar, 13De Camilli P. Chen H. Hyman J. Panepucci E. Bateman A. Brunger A.T. FEBS Lett. 2002; 513: 11-18Crossref PubMed Scopus (123) Google Scholar) 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 (6Itoh T. Koshiba S. Kigawa T. Kikuchi A. Yokoyama S. Takenawa T. Science. 2001; 291: 1047-1051Crossref PubMed Scopus (388) Google Scholar, 7Ford M.G. Pearse B.M. Higgins M.K. Vallis Y. Owen D.J. Gibson A. Hopkins C.R. Evans P.R. McMahon H.T. Science. 2001; 291: 1051-1055Crossref PubMed Scopus (605) Google Scholar, 8Ford M.G. Mills I.G. Peter B.J. Vallis Y. Praefcke G.J. Evans P.R. McMahon H.T. Nature. 2002; 419: 361-366Crossref PubMed Scopus (795) Google Scholar). The ENTH domain is made of a superhelix of 7 α-helices with an eighth α-helix misaligned with the superhelical axis, which is structurally similar to the superhelical VHS domain (14Lohi O. Poussu A. Mao Y. Quiocho F. Lehto V.P. FEBS Lett. 2002; 513: 19-23Crossref PubMed Scopus (61) Google Scholar). 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 (13De Camilli P. Chen H. Hyman J. Panepucci E. Bateman A. Brunger A.T. FEBS Lett. 2002; 513: 11-18Crossref PubMed Scopus (123) Google Scholar). X-ray structural analysis of the AP180 N-terminal homology (ANTH) domains of AP180/CALM (7Ford M.G. Pearse B.M. Higgins M.K. Vallis Y. Owen D.J. Gibson A. Hopkins C.R. Evans P.R. McMahon H.T. Science. 2001; 291: 1051-1055Crossref PubMed Scopus (605) Google Scholar) showed that the ANTH domain is extended by one or more α-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 (7Ford M.G. Pearse B.M. Higgins M.K. Vallis Y. Owen D.J. Gibson A. Hopkins C.R. Evans P.R. McMahon H.T. Science. 2001; 291: 1051-1055Crossref PubMed Scopus (605) Google Scholar). The ENTH domain binds PtdIns(4,5)P2 in a pocket and makes extensive contacts with both the headgroup and glycerol backbone (8Ford M.G. Mills I.G. Peter B.J. Vallis Y. Praefcke G.J. Evans P.R. McMahon H.T. Nature. 2002; 419: 361-366Crossref PubMed Scopus (795) Google Scholar). On binding the PtdIns(4,5)P2 headgroup, residues 3–15 of the ENTH domain adopt an α-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 (8Ford M.G. Mills I.G. Peter B.J. Vallis Y. Praefcke G.J. Evans P.R. McMahon H.T. Nature. 2002; 419: 361-366Crossref PubMed Scopus (795) Google Scholar). 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. Materials—1-Palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), 1-palmitoyl-2-oleoyl-sn-glycero-phosphoethanolamine (POPE), and 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoserine (POPS) were from Avanti Polar Lipids (Alabaster, AL). 1,2-Dipalmitoyl derivatives of phosphatidylinositol 3,5-bisphosphate, phosphatidylinositol 3,4,5-triphosphate, and PtdIns(4,5)P2 were a kind gift from Dr. Karol Bruzik. Phospholipid concentrations were determined by phosphate analysis (15Kates M. Techniques of Lipidology. 2nd Ed. Elsevier Science Publishers B. V., Amsterdam1986: 114-115Google Scholar). Restriction endonucleases and other enzymes for molecular biology were from New England Biolabs (Beverly, MA). CHAPS and octyl glucoside were from Sigma and Fisher Scientific, respectively. The liposofast microextruder and 100-nm polycarbonate filters were from Avestin (Ottawa, Ontario). Pioneer L1 sensor chip was from Biacore AB and Protein of epsin ENTH was by the PubMed Scopus Google and all other were as (8Ford M.G. Mills I.G. Peter B.J. Vallis Y. Praefcke G.J. Evans P.R. McMahon H.T. Nature. 2002; 419: 361-366Crossref PubMed Scopus (795) Google Scholar). was into the a N-terminal and into for the the was into E. for protein of of of and of in of was with and at at this of was and were then at for were for at and the was in of and The was then for a by on This was by at to the and The was into a and of was The was on with for this the was a which was with of The was then and of and with of were to the at the protein was from the in of was on an and were and to Protein was then determined the The AP180 ANTH domain was as (8Ford M.G. Mills I.G. Peter B.J. Vallis Y. Praefcke G.J. Evans P.R. McMahon H.T. Nature. 2002; 419: 361-366Crossref PubMed Scopus (795) Google Scholar). penetration of ENTH and ANTH domains into the monolayer was measured at by the in surface at surface a and and to a as M. J. Biol. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar). A lipid monolayer of was the of the surface was the of proteins were to the through the in the of the and the in surface was for while the at the a has been that by protein is to the penetration of the protein into the lipid in the of the domain of was by residues in the M. J. Biol. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar) and their membrane penetration measured by PubMed Scopus Google Scholar) and resonance A. R. Sci. S. A. 1999; PubMed Scopus Google Scholar, 2002; PubMed Scopus Google Scholar). The on the protein and a protein in the was such to that the a The was from which the surface was determined as the R. F. Scopus Google Scholar, Biochem. 2001; PubMed Scopus Google Scholar). and binding measurements were at The of the L1 sensor chip has been in J. Biol. 2001; Full Text Full Text PDF PubMed Scopus Google Scholar, 2001; PubMed Scopus Google Scholar). The sensor chip surface was and then by of at to a of resonance a surface was with vesicles, of to the resonance as the binding binding was to this surface the for the ENTH and ANTH domains. lipid was by of at in lipid was the measurements were at the of of protein in was to an association of while the dissociation was for at which the protein The lipid surface was with of the protein were for different concentrations of protein a of of the was for by the surface from The association and dissociation of all were to a binding protein site on the as 2001; PubMed Scopus Google Scholar, Biochem. J. 2001; PubMed Scopus Google Scholar, F. J. Biol. 2002; Full Text Full Text PDF PubMed Scopus Google Scholar). 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Rev. 1997; PubMed Scopus Google Scholar) was not a in as in to not of association and dissociation. and were to the of the binding was to a were determined from these the was to to for the association which in resonance to were then protein concentrations and the was determined by a analysis of the binding an is a by both and analysis for epsin ENTH and AP180 ANTH were in not thus the that in analysis is in of not the of but the of on the site on the vesicle is of is the dissociation in of of lipid Biochem. 2001; PubMed Scopus Google Scholar). to in of the of on the sensor only was determined in analysis and the affinity was calculated as a of that are similar for and This is on that which are to Biochem. 2001; PubMed Scopus Google were similar for all proteins in which the sensor chip was with the of electrostatic of ENTH and ANTH domains with and inositol were calculated with a of the and in the A. B. PubMed Scopus Google as B. A. Science. PubMed Scopus Google Scholar). In the of the electrostatic are by in with and and respectively. The electrostatic used from the B. R. B. S. M. J. Scopus Google Scholar) and from the of epsin ENTH S. Kigawa T. Kikuchi A. Yokoyama S. J. 2001; Scopus Google epsin ENTH (8Ford M.G. Mills I.G. Peter B.J. Vallis Y. Praefcke G.J. Evans P.R. McMahon H.T. Nature. 2002; 419: 361-366Crossref PubMed Scopus (795) Google and ANTH (7Ford M.G. Pearse B.M. Higgins M.K. Vallis Y. Owen D.J. Gibson A. Hopkins C.R. Evans P.R. McMahon H.T. Science. 2001; 291: 1051-1055Crossref PubMed Scopus (605) Google Scholar). of ENTH and ANTH have that the membrane penetration of F. J. Biol. 2002; Full Text Full Text PDF PubMed Scopus Google Scholar) and domains A. J. Biol. Full Text Full Text PDF PubMed Scopus Google Scholar). To PtdIns(4,5)P2 also the membrane penetration of ENTH and ANTH we measured the of epsin ENTH and AP180 ANTH domains with in the presence and absence of Phospholipid at the as a highly to the membrane of protein Biochem. 2001; PubMed Scopus Google Scholar, PubMed Scopus Google Scholar, M. PubMed Scopus Google Scholar, M. J. Biol. Full Text Full Text PDF PubMed Scopus Google Scholar). In these or of a surface were at and the in surface was the of the protein into the In is to of the monolayer and an of the the surface which an of of a monolayer that a protein into R. F. Scopus Google Scholar, Biochem. 2001; PubMed Scopus Google Scholar). the surface of cell and has been to in the of A. PubMed Scopus Google Scholar, B. PubMed Scopus Google Scholar, PubMed Scopus Google for a protein to these bilayer its that the monolayer both epsin ENTH and AP180 ANTH domains have with of and respectively. the of PtdIns(4,5)P2 to the monolayer the of the epsin ENTH domain to that PtdIns(4,5)P2 penetration of the domain into cell and The of PtdIns(4,5)P2 was by a effect of phosphatidylinositol on the for the epsin ENTH domain. phosphatidylinositol a effect not the of PtdIns(4,5)P2 into the monolayer not the of the AP180 ANTH domain, that PtdIns(4,5)P2 not the membrane penetration of the AP180 ANTH domain. This also that the ANTH domain lipid including cell in the presence of of of ENTH and ANTH lipid headgroup specificity of the epsin ENTH domain and the AP180 ANTH domain has been determined inositol the epsin ENTH domain binds more inositol (8Ford M.G. Mills I.G. Peter B.J. Vallis Y. Praefcke G.J. Evans P.R. McMahon H.T. Nature. 2002; 419: 361-366Crossref PubMed Scopus (795) Google Scholar). the epsin ENTH domain has affinity for the AP180 ANTH domain (8Ford M.G. Mills I.G. Peter B.J. Vallis Y. Praefcke G.J. Evans P.R. McMahon H.T. Nature. 2002; 419: 361-366Crossref PubMed Scopus (795) Google Scholar). their specificity and affinity for has not been measured the affinity of the epsin ENTH domain for as a in the epsin ENTH domain showed high affinity for 7 that for vesicle binding analysis is as the dissociation for vesicle binding for in of the of on the which is of Biochem. 2001; PubMed Scopus Google Scholar) the the for and the for not the different of the domain for and compared with vesicles, the epsin ENTH domain affinity for and of for these from the protein concentrations for the measurements were that the epsin ENTH domain PtdIns(4,5)P2 to other by more of for and epsin ENTH determined from in in to binding with in to binding in a The AP180 ANTH domain also high specificity for the however, the AP180 ANTH domain showed affinity the epsin ENTH domain. This in is to a a and a for the ENTH domain. The membrane association of proteins by electrostatic 2001; PubMed Scopus Google and the a for the ENTH domain is with its more electrostatic the PtdIns(4,5)P2-binding site when compared with the ANTH domain (6Itoh T. Koshiba S. Kigawa T. Kikuchi A. Yokoyama S. Takenawa T. Science. 2001; 291: 1047-1051Crossref PubMed Scopus (388) Google Scholar, 7Ford M.G. Pearse B.M. Higgins M.K. Vallis Y. Owen D.J. Gibson A. Hopkins C.R. Evans P.R. McMahon H.T. Science. 2001; 291: 1051-1055Crossref PubMed Scopus (605) Google Scholar, 8Ford M.G. Mills I.G. Peter B.J. Vallis Y. Praefcke G.J. Evans P.R. McMahon H.T. Nature. 2002; 419: 361-366Crossref PubMed Scopus (795) Google Scholar) also On the other the membrane dissociation of proteins by and membrane penetration of hydrophobic residues 2001; PubMed Scopus Google Scholar). the of the epsin ENTH domain in to the AP180 ANTH domain is with monolayer penetration that the more into the To that PtdIns(4,5)P2 has effects on the membrane binding of the ENTH and ANTH we measured the vesicle binding of these domains as a of PtdIns(4,5)P2 and the PtdIns(4,5)P2 in the was from to the a and for the epsin ENTH domain and the AP180 ANTH domain, respectively. the in PtdIns(4,5)P2 effect on the of the ANTH domain but a the for epsin This the effect of PtdIns(4,5)P2 on the membrane penetration of the ENTH domain. the ionic of from to the of the ENTH domain to by but its membrane by In the binding of the AP180 ANTH domain to the was not in the presence of that the ANTH domain with the membrane surface by electrostatic interactions. these results indicate that while the membrane association of both the epsin ENTH domain and the AP180 ANTH domain is by electrostatic the membrane dissociation of the is by hydrophobic to its membrane of the Epsin ENTH the membrane penetration of the ENTH domain, we the hydrophobic and residues on helix 0 to and the effects on monolayer The and bind the PtdIns(4,5)P2 headgroup with is (8Ford M.G. Mills I.G. Peter B.J. Vallis Y. Praefcke G.J. Evans P.R. McMahon H.T. Nature. 2002; 419: 361-366Crossref PubMed Scopus (795) Google Scholar). in the and mutations the monolayer penetration of the epsin ENTH domain to the the importance of the hydrophobic residues on helix 0 in membrane This in penetration was PtdIns(4,5)P2 as these mutations not the penetration into a monolayer not In the of to effect on the monolayer Finally, a of PtdIns(4,5)P2-binding residues penetration to the the that PtdIns(4,5)P2 binding is for the membrane penetration of helix The this not have as a effect as the and mutations was to to high monolayer penetration of by the of hydrophobic residues the membrane binding This is by the that the monolayer as as the monolayer then measured the vesicle binding of these by the vesicles, showed affinity the importance of PtdIns(4,5)P2 binding in the membrane affinity of the ENTH domain. that this has the monolayer the and has a electrostatic which for electrostatic vesicle binding and and the to The membrane dissociation of these is with their hydrophobic with the membrane. to a this is not of the PtdIns(4,5)P2-binding is on the side of the membrane binding surface and to electrostatic interactions. of for the in membrane binding of the epsin ENTH domain and the AP180 ANTH domain, we calculated the electrostatic potential for these domains in the absence and presence of that is a of was that PtdIns(4,5)P2 binding a of the ENTH domain the formation of helix 0 (8Ford M.G. Mills I.G. Peter B.J. Vallis Y. Praefcke G.J. Evans P.R. McMahon H.T. Nature. 2002; 419: 361-366Crossref PubMed Scopus (795) Google Scholar). is not as to which the epsin ENTH domain when contacts the membrane surface a the electrostatic potential of the ENTH domain in the absence of was calculated for the both and the in A and were the PtdIns(4,5)P2-binding pocket for both of epsin ENTH, to the presence of residues in PtdIns(4,5)P2 binding and These to the domain to the membrane surface through electrostatic which to PtdIns(4,5)P2 that the high potential of the structure also the hydrophobic residues and of helix 0, which is to an the penetration of these residues into the membrane This is the that is for their membrane penetration and and on both the protein and the membrane. the potential of the epsin ENTH domain is when PtdIns(4,5)P2 binds to the domain This that PtdIns(4,5)P2 as an electrostatic to the highly potential the hydrophobic residues on helix 0, their membrane that for the ENTH domain PtdIns(4,5)P2 a of a and the electrostatic of the ANTH domain are also with monolayer and in the ANTH domain has a electrostatic potential the ENTH domain in the absence of PtdIns(4,5)P2 which is with its The of PtdIns(4,5)P2 to the ANTH domain to a but the ENTH domain, the ANTH domain hydrophobic residues on the membrane binding surface that with the membrane. a the electrostatic by PtdIns(4,5)P2 has for the ANTH domain. electrostatic the that PtdIns(4,5)P2 in the membrane binding of ENTH and ANTH domains. Clathrin-mediated endocytosis involves a number of accessory proteins that vesicle through and M.K. McMahon H.T. Biochem. Sci. 2002; Full Text Full Text PDF PubMed Scopus Google Scholar). was thought that clathrin is to membrane evidence has that the membrane bending of these accessory proteins R. 2001; PubMed Scopus Google Scholar). these proteins, epsin (8Ford M.G. Mills I.G. Peter B.J. Vallis Y. Praefcke G.J. Evans P.R. McMahon H.T. Nature. 2002; 419: 361-366Crossref PubMed Scopus (795) Google dynamin J.E. Cell. Full Text Full Text PDF PubMed Scopus Google amphiphysin De Camilli P. Cell Biol. 1999; PubMed Scopus Google and endophilin De Camilli P. J. Cell Biol. 2001; PubMed Scopus Google Scholar) have been to to in as to all these proteins are in vesicle in and the vesicle The provides into epsin the membrane curvature through interactions. A showed that the ENTH domain of epsin was as as the protein in while AP180 its ANTH domain the (8Ford M.G. Mills I.G. Peter B.J. Vallis Y. Praefcke G.J. Evans P.R. McMahon H.T. Nature. 2002; 419: 361-366Crossref PubMed Scopus (795) Google Scholar). A structural the ENTH domain and the ANTH domain in the site (8Ford M.G. Mills I.G. Peter B.J. Vallis Y. Praefcke G.J. Evans P.R. McMahon H.T. Nature. 2002; 419: 361-366Crossref PubMed Scopus (795) Google Scholar). The ANTH domain not have a well PtdIns(4,5)P2-binding pocket and surface residues to the The ENTH domain also a well however, for this domain the formation of the pocket is by PtdIns(4,5)P2 The also the hydrophobic Leu6, Met10, and the membrane These structural the domains are into their distinct membrane binding monolayer measurements that the ENTH domain the monolayer more the ANTH domain. PtdIns(4,5)P2 triggers the penetration of the ENTH domain, but not the ANTH domain, into the monolayer surface of cell and other the effect of PtdIns(4,5)P2 on the ENTH domain. This is similar to membrane penetration of domains and the domain of by phosphatidylinositol F. J. Biol. 2002; Full Text Full Text PDF PubMed Scopus Google Scholar, A. J. Biol. Full Text Full Text PDF PubMed Scopus Google Scholar). penetration of the ENTH domain also the that hydrophobic residues on the of helix 0 are for the membrane penetration of the ENTH domain. measurements and electrostatic potential provide into the membrane binding of these domains. The epsin ENTH domain binds the with affinity the AP180 ANTH domain, to association and dissociation. The membrane association of the epsin ENTH domain for by its more electrostatic potential on its membrane binding surface, to residues in the PtdIns(4,5)P2-binding pocket and The dissociation of epsin ENTH is with the of monolayer penetration by its hydrophobic including and The and in affinity by and indicate that membrane penetration of these residues to membrane binding of the ENTH domain. of the PtdIns(4,5)P2 and the ionic of the also the different PtdIns(4,5)P2 in membrane binding of the domains. In the of the ENTH domain, PtdIns(4,5)P2 enhances a via electrostatic by membrane penetration of the hydrophobic and hydrophobic interactions. the ANTH domain, on the other PtdIns(4,5)P2 to as a the domain and the as for the of in the membrane binding of Biol. PubMed Scopus Google Scholar). This is to the presence of residues and the absence of hydrophobic residues on the PtdIns(4,5)P2 binding on these we membrane binding for the domains The epsin ENTH domain binds to by electrostatic interactions. The of is not at to that the membrane at the formation of the helix 0 and the PtdIns(4,5)P2-binding the of amphipathic at the has been well J. Biol. Full Text PDF PubMed Google Scholar, J.H. A. J. Biol. Full Text PDF PubMed Google Scholar, B. PubMed Scopus Google Scholar). The PtdIns(4,5)P2 binding then this as in the structure of the epsin complex (8Ford M.G. Mills I.G. Peter B.J. Vallis Y. Praefcke G.J. Evans P.R. McMahon H.T. Nature. 2002; 419: 361-366Crossref PubMed Scopus (795) Google the hydrophobic residues in the helix to into the membrane by the electrostatic This membrane insertion the membrane of the protein and triggers the membrane deformation. the ANTH domain, binding through electrostatic with PtdIns(4,5)P2 as a in protein and membrane structure are steps of complex and are in the vesicle results that the membrane penetration of the ENTH domain is a in the membrane This is by monolayer penetration of and that have the activity of the epsin ENTH domain. The penetration of the ENTH domain into the of the membrane the membrane which has been to for membrane Cell Biol. 2002; 12: Full Text Full Text PDF PubMed Scopus Google Scholar). are proteins, including domains and which lipid as well as or more the epsin ENTH domain the insertion of a α-helix the membrane to while insertion of a on the domain the molecular basis of this activity of the epsin ENTH domain.