TheRACBindingDomain/IRSp53-MIMHomologyDomain ... · NOVEMBER 17, 2006•VOLUME 281•NUMBER 46...

The RAC Binding Domain/IRSp53-MIM Homology Domainof IRSp53 Induces RAC-dependent Membrane Deformation*

Received for publication, July 18, 2006, and in revised form, September 25, 2006 Published, JBC Papers in Press, September 25, 2006, DOI 10.1074/jbc.M606814200

Shiro Suetsugu‡, Kazutaka Murayama§1, Ayako Sakamoto§, Kyoko Hanawa-Suetsugu§, Azusa Seto§,Tsukasa Oikawa‡, Chiemi Mishima§, Mikako Shirouzu§, Tadaomi Takenawa‡2, and Shigeyuki Yokoyama§¶3

From the ‡Department of Biochemistry, Institute of Medical Science, the University of Tokyo, Shirokanedai, Minato-ku,Tokyo 108-8639, §RIKEN Genomic Sciences Center, Suehiro-cho, Tsurumi, Yokohama 223-0045, and the ¶Department ofBiophysics and Biochemistry, Graduate School of Science, the University of Tokyo, Hongo, Bunkyo-ku, Tokyo 113-0033, Japan

The concave surface of the crescent-shaped Bin-amphiphy-sin-Rvs (BAR) domain is postulated to bind to the cell mem-brane to induce membrane deformation of a specific curva-ture. The Rac binding (RCB) domain/IRSp53-MIM homologydomain (IMD) has a dimeric structure that is similar to thestructure of the BAR domain; however, the RCB domain/IMDhas a “zeppelin-shaped” dimer. Interestingly, the RCBdomain/IMD of IRSp53 possesses Rac binding, membranebinding, and actin filament binding abilities. Here we reportthat the RCB domain/IMD of IRSp53 induces membrane defor-mation independent of the actin filaments in a Rac-dependentmanner. In contrast to the BAR domain, the RCB domain/IMDdid not cause long tubulation of the artificial liposomes; how-ever, the Rac binding domain caused the formation of smallbuds on the liposomal surface.When expressed in cells, the Racbinding domain induced outward protrusion of the plasmamembrane in a direction opposite to that induced by the BARdomain. Mapping of the amino acids responsible for membranedeformation suggests that the convex surface of the Rac bindingdomain binds to the membrane in a Rac-dependent manner,which may explain the mechanism of the membrane deforma-tion induced by the RCB domain/IMD.

IRSp53 is an adaptor protein with anN-terminal Rac binding(RCB)4 domain (residues 1–228) and an Src homology 3domain. RNA interference of IRSp53 is reported to decrease

lamellipodia formation (1). However, membrane protrusionwith very dim or absent phalloidin (a reagent that stains actinfilament) staining has been observed in the IRSp53-overex-pressing cells (2, 3). This observation suggested that IRSp53might affect membrane organization independent of the actinfilaments.The RCB domain of IRSp53 shares its homology with the

MIM (missing in metastasis) protein, and this domain is alsonamed the IRSp53/MIM homology domain (IMD) (residues1–250 of IRSp53). Activated Rac binds to the RCB domain ofIRSp53 (4, 5). In contrast, the IMD of MIM is reported to bindto the inactive form of Rac (6).In addition to the Rac binding (RCB) activity, the RCB

domain/IMD of IRSp53 or MIM possesses the actin filamentbundling activity (7). Overexpression of IMD is shown to causeactin filament-containing protrusions; however, some of theprotrusions were branched and lacked the phalloidin staining(7).Recently, Millard et al. (8) reported the crystal structure of

the IMD fragment (residues 1–250); this fragment is a “zeppe-lin-shaped” dimer of a bundle of three extended�-helices and ashorter C-terminal helix. They found that the basic residues atthe extreme ends of the dimer are important for the actin fila-ment bundling activity. The substitution of the basic residueswithGlu is reported to reduce the actin binding activity by 50%.The IMD structure suggested that the bundle of three

extended �-helices is structurally similar to the Bin/amphiphy-sin/Rvs (BAR) domain. This domain has a curved structure, andits concave surface is postulated to bind to the cell membrane.The expressed amphiphysin BARdomainwas targeted to intra-cellular vesicles, and it caused membrane tubulation (9–11).The purified BARdomain protein induced liposome tubulationin vitro (9). Thus, the BAR domain is a membrane-binding andtubulation module. In the BAR domain, the concave surfaceand the basic residues at the extreme ends are involved in thebinding of the BAR domain to the membrane.Therefore, the structural similarity and presence of protru-

sions without the actin filaments suggest that RCB domain/IMD is a membrane-binding module as well as an actin fila-ment-binding module.In this study, the crystal structure of the RCB domain (resi-

* This work was supported by the RIKEN Structural Genomics/Proteomics Ini-tiative, the National Project on Protein Structural and Functional Analyses,Ministry of Education, Culture, Sports, Science and Technology (to S. Y.), bygrants-in-aid for scientific research on priority areas from the Ministry ofEducation, Culture, Sports, and Technology of Japan, and from the JapanScience and Technology Corp. (to T. T. and S. S.). The costs of publication ofthis article were defrayed in part by the payment of page charges. Thisarticle must therefore be hereby marked “advertisement” in accordancewith 18 U.S.C. Section 1734 solely to indicate this fact.

The atomic coordinates and structure factors (code 1WDZ) have been depositedin the Protein Data Bank, Research Collaboratory for Structural Bioinformat-ics, Rutgers University, New Brunswick, NJ (http://www.rcsb.org/).

1 Present address: Tohoku University Biomedical Engineering Research Orga-nization, Aoba-ku, Sendai 980-8575, Japan.

2 To whom correspondence may be addressed. Tel.: 81-3-5449-5410; E-mail:[email protected].

3 To whom correspondence may be addressed. Tel.: 81-45-503-9149; Fax:81-45-503-9195; E-mail: [email protected].

4 The abbreviations used are: RCB, Rac binding domain; BAR, Bin-amphiphy-sin-Rvs; GTP�S, guanosine 5�-3-O-(thio)triphosphate; ELISA, enzyme-linked immunosorbent assay; GST, glutathione S-transferase; BPM, basicpatch mutant; GFP, green fluorescent protein; PE, phosphatidylethanol-

amine; PC, phosphatidylcholine; PIP3, phosphatidylinositol triphosphate;PS, phosphatidylserine; IMD, IRSp53-MIM homology domain; GMPPNP,guanyl-5�-yl imidodiphosphate; DiI, DiIC18(3).

THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 281, NO. 46, pp. 35347–35358, November 17, 2006© 2006 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in the U.S.A.

NOVEMBER 17, 2006 • VOLUME 281 • NUMBER 46 JOURNAL OF BIOLOGICAL CHEMISTRY 35347

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dues 1–228) was determined; the three-helix bundle dimer wasformed in the absence of helix 4 of the IMD (residues 1–250),demonstrating that the overall structure of the RCB domain/IMD was similar to that of the BAR domain. The RCB domainexpression in the cells caused membrane protrusions thatlacked phalloidin staining in a Rac-dependent manner. More-over, in the presence of Rac, the isolated RCB domain induceddeformation of the artificial liposomes in a manner that wasdifferent from that induced by the BAR domain. Mapping ofthe residues responsible for membrane deformation sug-gests the involvement of the convex surface of the RCBdomain in the binding of the RCB domain to the membrane.These findings indicate that the RCB domain is a Rac-de-pendent membrane tubulation module as well as an actinfilament-bundling module.

EXPERIMENTAL PROCEDURES

Purification—The human IRSp53-RCB domain (residues1–228) was expressed as an N-terminal GST fusion protein inEscherichia coli BL21 (DE3) cells. The fragment encoding theRCB domain was excised from full-length IRSp53 by BamHIand StuI digestions. This fragmentwas inserted into the BamHIand SmaI sites of pGEX 4T-1 (Amersham Biosciences). TheBamHI site is followed by ACC (Thr) and then the first aminoacid residue. The protein was purified on a glutathione affinitycolumn, and the GST tag was cleaved with thrombin. Afterion-exchange chromatography, gel filtration was conducted.Proteinwas concentrated to 54.6mg/ml in 20mMHepes buffer,pH 7.6, 0.1 mM EDTA, 1 mM MgCl2, 100 mM KCl, and 2 mM

dithiothreitol. The final sample included genetically manipu-lated fragments (residues�2–0 and 229–239). IRSp53 and Racwere expressed in a baculovirus system and purified asdescribed previously (1).Crystallization and Data Collection—The IRSp53-RCB do-

main was crystallized by the hanging drop vapor diffusionmethod. The protein solution (8.7 mg/ml) was mixed with anequal volume of reservoir solution, including 90 mM Tris-HClbuffer, pH 8.5, 27% PEG4000, 190mM sodium acetate, and 3.5%2-methyl-2,4-pentanediol. A mercury derivative was obtainedby soaking the crystals in 5mM ethylmercurithiosalicylic acid inthe reservoir solution for 10 min. X-ray diffraction data werecollected under cryogenic conditions (100 K). Crystals wereflash-frozen after an incubation in reservoir buffer with a cryo-protectant (30% ethylene glycol). Initial x-ray data collectionsfor the native (data set II) and mercury derivative crystals wereconducted with in-house CuK� radiation (� � 1.5418 Å), usingan R axis IV�� RIGAKU Image Plate detector mounted on aRigaku FR-D rotation anode x-ray generator. High resolutionnative data (data set I) were collected at SPring-8 (Harima,Japan) on beamline BL44B2with aMar Research CCDdetector(Table 1). All x-ray diffraction data were integrated and scaledwith the use of the HKL2000 package (12).Phase Determination and Structure Refinement—The crystal

structure of the RCB domain was determined by single isomor-phous replacement, including anomalous scattering. Twomer-cury sites were found with SOLVE (13). The initial phases wereextended to the native data set I with RESOLVE (13).

The calculated electron density was of sufficient quality totrace the main chains. The model was built manually by O (14)and was refined with the use of the CNS program package (15).All refinement steps were monitored with the free R-factor,based on 10% of the x-ray data. After a simulated annealingprotocol, the structurewas refined by atom-positional and tem-perature factor refinement, as well as manually. The figureswere produced with PyMol. Surface electrostatic potentialswere prepared with GRASP (16).ELISALipid BindingAssay—Lipid vesicles (phosphatidyleth-

anolamine (PE)/phosphatidylcholine (PC) � 1:1, total 2 �g),containing 10% (by weight) of phosphatidylinositol triphos-phate (PIP3) resuspended in 100% ethanol were coated onto96-well plates (Immulon 2HB; Thermo Labsystems) and air-dried at room temperature. The wells were then blocked with5% bovine serum albumin in phosphate-buffered saline, beforeincubation with the appropriate glutathione S-transferase(GST) fusion protein (1.0 �g ml�1) for 45 min. The wells werewashedwith 0.05%Tween 20 in phosphate-buffered saline, andglutathione-conjugated peroxidase (1.0 �g ml�1, Sigma) wasoverlaid and detected by a reaction with ortho-phenylenedia-mine. Absorbancewasmeasured at 492 nmwith an ELISAplatereader (Bio-Rad).Pulldown Assay for Rac-RCB Domain Association—Rac pro-

tein was purified from E. coli, and was loaded with GTP�S asdescribed previously (17). RCB or IMD domain was expressedas anN-terminal GST fusion protein. Equal amounts of variousRCB or IMD GST fusion proteins were immobilized on beads,and binding to GTP�S-loaded Rac (2 �M) was examined. Afterwashing, boundRacwas analyzed byWestern blotting and den-sitometry. The amount of boundRacwas expressed as arbitraryunits.Actin Filament Sedimentation Assay—Binding of filamen-

tous (F)-actin to the RCB domain of IRSp53 was analyzed byco-sedimentation assay. A globular (G)-actin solution was firstclarified by ultracentrifugation at 100,000 � g for 20 min, and

TABLE 1Data collection and refinement statistics

Native I Native II Hg derivativeSpace group P21 P21 P21Cell dimensions, Åa 60.833 60.845 60.743b 69.258 69.783 69.635c 68.715 67.954 67.997�,° 110.124 110.183 109.967

Resolution range, Å 50 to 2.63 50 to 3.00 1950.3.20Wavelength, Å 1 1.5418 1.5418No. of references 66,002 40,479 66,009No. of unique references 15,858 10,720 8937Completeness (last shell), % 98.6 (98.2) 98.5 (97.6) 99.2 (98.6)Rsyma (last shell), % 5.9 (30.3) 8.0 (27.2) 11.7 (33.8)I/I (�) 26.1 (5.5) 16.4 (5.4) 16.8 (8.4)Resolution range, Å 41.9 to 2.63No. of unique refs. 15,846Rwb; Rfree, % 23.5; 29.7No. of protein atoms 3644No. of water molecules 70Root mean square deviationBonds, ° 0.007Angle, Å 1.1

Mean B factor 51aRsym � �h�i �Ii(h) � �I(h)��/�h�iIi(h).bRw � � �Fo��Fc�/��Fo�, where Fo and Fc are the observed and calculated structurefactor amplitudes.

Membrane Deformation by the RCB Domain

35348 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 281 • NUMBER 46 • NOVEMBER 17, 2006

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the G-actin was polymerized to F-actin for 1 h at room temper-ature. Various GST-RCB domain solutions (2 �M in 10 mMHepes, pH 7.9, 100 mM KCl, 2 mMMgCl2, 5 mM EGTA, and 0.1mM CaCl2) were also clarified by ultracentrifugation. F-actin (2�M) and RCB domain solutions were mixed and incubated atroom temperature for 1 h, and the mixture was centrifuged at100,000 � g for 20 min. Supernatants and pellets (equal vol-umes)were then analyzed by SDS-PAGE followedby densitom-etry. Percentages of protein amount in the precipitates areshown with standard deviations.Liposome Co-sedimentation Assay—Synthetic liposomes

contained PC/PE/PIP3 � 48:48:4 (weight ratio) or PC/PE/PS/PIP3 � 48:48:12:4. Liposomes were resuspended at a concen-tration of 1 mM test lipid in 50 mM Hepes, pH 7.9, and 150 mMNaCl and were subjected to five cycles of freezing (liquid N2)and thawing (37 °C water bath). RCB and/or Rac, at the indi-cated concentrations, and liposomes (0.5 mM for PC) weremixed in 10mMHepes, pH 7.9, 100mMKCl, 2mMMgCl2, 5mMEGTA, and 0.1 mM CaCl2, and the mixture was incubated at4 °C for 60 min. After centrifugation at 25,000 rpm for 20 min,vesicle pellets were suspended in SDS sample buffer and frac-tionated by SDS-PAGE. Bound protein was identified by Coo-massie Brilliant Blue staining or Western blotting and quanti-fied by densitometry with ImageJ software. Liposomeconcentrations were calculated by dividing the total concentra-

tion in half, because 50% of the totalwas assumed to be on the insidelayer of the vesicles. TheKd value forRCB-liposome association was cal-culated from the amount of co-pre-cipitated protein by curve fitting ofthe equilibrium response.Dual Polarization Interferome-

try—The interactions between RCBand small GTPases were investi-gated with an AnaLight Bio200interferometer (Farfield Sensors).GST-RCB was coated onto the sur-face of different channels of anamine sensor chip, according to themanufacturer’s instructions, withBS3 chemical linkers. The bufferconsisted of 10 mM Hepes, pH 7.5,150 mM NaCl, and 3 mM MgCl2.Tag-free Rac and Cdc42 proteinspurified from E. coli were eachloaded with GTP�S or GDP andinjected at different concentrationsin the same buffer (associationphase). Binding was examined atfive or more different protein con-centrations, and Kd (�kd/ka) valueswere calculated from curve fitting.The observed association rates (kon)over the initial 60 s of associationwere plotted (Fig. 4C). The slopeand intercept yielded ka and kd val-ues, respectively.

Electron Microscopy—RCB domain was expressed as anN-terminal GST fusion protein in E. coli. IRSp53 and Rac pro-teins were expressed in a baculovirus system and were purifiedas described previously (1). Electron microscopy was per-formed as described previously (18, 19). Liposomes (Folch frac-tion 1; Sigma) (0.1 mg/ml final concentration) were incubatedat 37 °C for 15 min in buffer XB (10 mMHepes, pH 7.9, 100 mMKCl, 5 mM EGTA, 0.1 mM CaCl2, 2 mM MgCl2) with RCBdomain or IRSp53 (0.5�M) andGTP�S-loaded Rac (0.5�M). Atthe end of the incubation, aliquots were adsorbed onto collodi-on-coated copper electron microscopy grids (Nissin EM) for 1min at room temperature, washed in 0.1 M Hepes, pH 7.4,stained in 1–2% uranyl acetate for 30 s, blotted, and thenallowed to air dry.Time-lapse Microscopy of Liposome Deformation—Time-

lapse analysis of liposome deformation was performed asdescribed previously (20). To adhere liposomes onto glass cov-erslips, two 1-�l droplets of lipid solution (10mg/ml Folch frac-tion 1 (Sigma) in chloroform) were spotted onto each coverslipand allowed to dry under vacuum for at least 1 h. Lipids werethen prehydrated in 50 �l of XB� 1mg/ml bovine serum albu-min for 20–30 min at 37 °C in a small chamber built by placingthe coverslip over a glass slide with strips of double-sided tapeas spacers. Twenty microliters of protein solution (1 mg/ml)was then injected between the glass pieces. Time-lapse images

FIGURE 1. Amino acid sequence alignment among the RCB domains and IMDs. Identical and highly con-served residues are shown in black. Conserved residues among IRSp53/IRTKS/FLJ22582 are shown in gray.Columns indicate helices in the RCB domain. The residues for the binding of the RCB domain to lipids, Rac, andactin filaments are labeled by their amino acid numbers.



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were obtained with a phase contrast microscope (Zeiss)equipped with a camera (CCD-782-Y/HS; Princeton Instru-ments). �40 oil-immersion objective (NA � 1.30 FLUAR;Zeiss) was used.Cell Culture, Transfection, and Microscopy—COS-7 cells

were cultured as described previously (21). Transfections wereperformed with Lipofectamine 2000 (Invitrogen) according tothe manufacturer’s instructions. RCB domain was taggedN-terminally with a brighter variant of GFP (Venus) (22) andwas expressed under the control of the human cytomegalovirusimmediate early promoter. DNA encoding constitutively activeRac (G12Vmutant) was inserted into the pEF-BOS vector witha Myc tag (4). After fixation, permeabilization, and blocking ofcells, cells were stained with an anti-GFP antibody (MBL) oranti-Myc antibody (9E10; Sigma) followed by an Alexa-488- orAlexa-633-conjugated secondary antibody (Molecular Probes).Actin filaments were visualized by rhodamine-conjugatedphalloidin (Molecular Probes). Latrunculin B (1 �g/ml) wastreated for 30 min before fixation. For membrane staining,membrane-staining dye (DiI (DiIC18(3)); Molecular Probes)was incubated with cells before fixation, and then cells wereobserved without permeabilization and antibody staining. Flu-orescent imageswere taken through amicroscope (Nikon)witha confocal microscopy system (Radience 2000; Bio-Rad) atroom temperature. �60 oil immersion objective NA � 1.40(Nikon) was used. Images were assembled with Adobe Photo-shop. In each plate, photographs were cropped, and each fluo-rochrome was adjusted identically for brightness and contrastto represent the observed images. Time-lapse analysis was per-formed with total-internal-reflection microscopy system(Olympus) with �100 oil immersion objective NA � 1.45(Olympus) and MetaMorph software.Coordinates—Atomic coordinates of the IRSp53 RCB

domain have been deposited in the Protein Data Bank (acces-sion code 1WDZ).

RESULTS AND DISCUSSION

Structure of the RCBDomain—The three-dimensional struc-ture of the RCB domain (amino acid residues 1–228) (Fig. 1) ofIRSp53 was determined by single isomorphous replacementwith the anomalous scatteringmethod. TheRCBdomain struc-ture had an asymmetric unit that included two subunits, A andB; this unit formed an elongated homodimer with a length of180 Å (Fig. 2A). With the exception of the subunit B residues150–158, which could not be identified because of a disorder,polypeptide chains were traced for both the subunits in an elec-tron density map. The subunits were related by a local 2-foldrotation axis and were superimposed with a root mean squaredeviation of 0.497 Å (mean value for the 222 common C-�

atoms). Each subunit consisted of six �-helices, namely �1–�6(Fig. 1), that are arranged as an antiparallel helix bundle. Thehelix bundle structure of the IRSp53 RCB domain was almostidentical to the corresponding portion of the reported structureof the IRSp53 IMD, which is 22 residues longer than the RCBdomain (8). These additional IMD residues form a short helix(helix 4) that interacts with the other three helices in the IMDstructure (8). Despite the absence of this short helix, the RCBdomain formed a bundle structure, suggesting that the shorthelix does not contribute to bundle formation. Thus, the RCBdomain is an independent entity with a structure and surfaceelectrostatic properties similar to that of the BAR domain (Fig.1 and Fig. 2, A and B).Membrane-binding Sites of the RCB Domain—The RCB

domain and full-length IRSp53 bind to lipids presumably viaelectrostatic interactionswith little selectivity for specific phos-phoinositides (1). To determine the lipid-binding surface of theRCB domain dimer, we constructed a series of Glu and/or Alasubstitutions for 20 Lys orArg residues and tested its lipid bind-ing ability by ELISA (Fig. 2C) (23). At the ends of the dimer,there is a basic patch consisting of Lys-142, Lys-143, Arg-145,Lys-146, and Lys-147. A basic patchmutant (BPM), in which allthese five residues were replaced by Ala, did not bind to nega-tively charged phosphoinositides (Fig. 2C); the structure of theRCB domain was not destroyed, as revealed by the circulardichroism spectrum (data not shown). Next, we replaced two ofthe basic amino acids in the basic patch according to their sidechain orientations. The replacement of Lys-142 andArg-145 byeitherAla (K142A/R145A) orGlu (K142E/R145E) did not affectthe RCB domain-lipid binding. In contrast, the K143A/K147Aand K143E/K147E mutations dramatically reduced the RCBdomain-lipid binding (Fig. 2C), whereas the K143E mutationdid not affect this binding. Therefore, Lys-147 was determinedto be the most important residue in the basic patch.Arg-128 and Lys-130 are located close to each other near the

basic patch. The RCB domain-lipid binding was significantlydecreased by the R128E/K130E mutations. Furthermore, theK108E and K171A mutants showed decreased lipid binding,whereas Glu substitutions for Arg-29, Lys-40, Lys-58, Lys-70,Lys-121, Lys-136, Lys-152, Lys-156, Lys-160, and Arg-192 didnot (Fig. 2C). Therefore, the RCB domain-lipid bindinginvolves a limited region; among the basic residues tested, Lys-108 (�4), Lys-130 (�5), Lys-147 (�5–�6 loop), and Lys-171 (�6)were determined to be particularly important for this binding(Fig. 1 and Fig. 2D). With the exception of Lys-147, these Lysresidues are located in a straight line along the straight helixbundle (Fig. 2D, front view). Lys-147 is located on the flexible�5–�6 loop and can relocate its side chain to position itself on

FIGURE 2. Mapping of the RCB domain residues responsible for binding of the RCB domain to lipids, Rac, and actin filaments. A, ribbon diagrams of theRCB domain and the IMD. Monomers A and B are colored cyan and light green, respectively. Helix 4 in the IMD is shown in orange. Black ellipse and line representthe local 2-fold symmetry. B, surface electrostatic potentials of the RCB domain and the arfaptin and amphiphysin BAR domains. Positively charged regions areshown in blue, and negatively charged regions are shown in red. C, lipid binding ability of various RCB domain mutants as determined by ELISA. E, Rac bindingability of various mutants of the RCB domain and the MIM IMD domain. G, actin filament binding ability of various RCB domain mutants. Error bars represent theS.D. of at least three independent experiments. Statistical significance was analyzed by Student’s t test. D, F, and H, molecular surface representations of the RCBdomain of IRSp53. Amino acids used for the mutation analysis of the RCB domain-lipid (D), RCB domain-Rac (F), and RCB domain-actin binding activities (H) areshown in red (reduced activity) and green (no change). Each figure includes a front view (perpendicular to the noncrystallographic 2-fold axis) and a side view(along the noncrystallographic 2-fold axis).



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the straight line (Fig. 1 and Fig. 2D). Each protomer of thedimeric helix bundle possesses this “lipid-binding line”; the twolines are located symmetrically on opposite sides of the dimer.In the “front” viewof the helix bundle (Fig. 2D,upper panel), thelipid-binding line on the right is visible, whereas that on the leftis located on the opposite side of the dimer (invisible in thispanel). If the dimer is sufficiently stable and does not bend, onlyone of the lipid-binding lines is used when the dimer binds tothe membrane surface.Interestingly, the corresponding region of MIM (1–238

amino acids) had less lipid binding ability (Fig. 2C). Consis-tently, the Lys-108, Lys-147, and Lys-171 are occupied withneutral amino acids in MIM (Fig. 1).

Based on the binding curve, the Kd value between the RCBdomain and phosphatidylserine (PS)-containing liposome wasestimated to be 3 �M (Fig. 3A).Rac-binding Sites of the RCB Domain—In contrast to the

binding of IRSp53 to the GTP-loaded Rac, MIM was reportedto bind to the GDP-loaded Rac (4, 6). Therefore, we performedpulldown assays to test the Rac binding ability of the RCBdomain mutants (Q23E, R11E/Q23E, K40E, K58E, K70E,K121E, R128E/K130E, K142E/R145E, K143E, K143E/K147E,and BPM), in which the conserved residues (underlined) orthe nonconserved residues between IRSp53 and MIM werereplaced (Fig. 1). Arg-11 was considered to contribute to Racbinding because the RCB domain mutations of this residue

FIGURE 3. Binding of Rac and lipids to the RCB domain. A, liposome binding to the RCB domain. Liposomes (PC/PE/PIP3 � 48:48:4 or PC/PE/PS/PIP3 �48:48:12:4) were incubated with the RCB domain at the indicated concentrations. Proteins that co-sedimented with liposomes were visualized by Westernblotting using an anti-IRSp53 antibody. Bound proteins were quantified by densitometry. B, association of the RCB domain with Rac. Upper panel, plots of konagainst GTP�S-loaded Rac or Cdc42 concentration. The kon values were obtained during the initial 60 s of association. Lower table, table showing the Kd valuesbetween RCB and Rac or Cdc42. Apparent association (kass (ka)) and dissociation (kdiss (kd)) rate constants were determined from the slope and the intercept(kon � ka � C � kd), yielding the Kd value (� kd/ka). r represents the correlation coefficient for kon � ka � C � kd. C and D, liposome binding by the RCB domainin the presence of Rac. C, PC/PE/PS/PIP3 or PC/PE liposomes were incubated with the RCB domain protein (150 nM) and baculovirus-expressed Rac (1.5 �M). Lane1, wild type (WT) or mutants of RCB domain protein were incubated with liposome followed by incubation with Rac. Lane 2, the proteins were incubated withRac followed by incubation with liposome. Lane 3, the proteins were incubated alone followed by incubation with liposome. The proteins (ppt) co-precipitatedwith liposome and proteins in the supernatant (sup) were examined by Coomassie Brilliant Blue staining. D, PC/PE/PS/PIP3 liposomes were incubated with theRCB domain protein (150 nM) and baculovirus-expressed or E. coli-expressed GTP�S-loaded Rac (1.5 �M). The proteins (ppt) co-precipitated with liposome andproteins in the supernatant (sup) were examined by Western blotting.



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(R11E/Q23E) showed impaired binding to GTP�S-loaded Rac(Fig. 2E); Gln-23 contributed slightly to Rac binding (Fig. 2E).The basic patch of MIM is reported to be involved in Rac

binding (6). Mutations, including the Lys-143 replacement(K143E, K143E/K147E, and BPM), showed significantlydecreased Rac binding ability (Fig. 2E).Our kinetic analyses revealed that the RCB domain fragment

binds to the GTP�S-loaded Rac protein with a Kd value of3.2 �M (Fig. 3B), a value comparable with that betweenRac�GMPPNP and arfaptin (3 �M) (24). Fig. 3B shows the RCBdomain bound to theGDP-loaded Racwith aKd value of 20�M,indicating the selective binding of the RCB domain to the acti-vated Rac. In contrast, Cdc42 has a weak affinity for the RCBdomain (3, 25, 26). TheKd value betweenCdc42�GTP�S and theRCB domain was 15 �M (Fig. 3B).Next, we examined whether these Rac-binding sites on the

RCB domain were involved in the RCB domain-lipid binding.The lipid binding was not significantly affected by the R11E/Q23E, Q23E, or K143E mutations (Fig. 2C). The different sitesof lipid and Rac binding suggested that activated Rac binds tothe membrane-bound RCB domain. However, these two siteslocate closely to each other. Therefore, the space limitationmayinhibit the simultaneous binding of the RCB domain to bothlipids andRac. To address this issue, we sequentially addedwildtype or mutants of the RCB domain to lipids and Rac (Fig. 3, Cand D). Rac purified from the Sf9 cells showed lipid modificationand was bound to the membrane (data not shown). Rac purifiedfromtheSf9cells co-sedimentedwith theRCBdomain, suggestingthe simultaneous binding of the RCB domain to both Rac andlipids (Fig. 3,C andD).The liposome bindings of wild type and Rac-binding defec-

tive (R11E/Q23E) mutant of RCB were independent of theorder of the incubation of Rac and liposome. However, the lip-id-binding defective mutant of RCB (R128E/K130E) hadincreased affinity to liposome when the R128E/K130E mutantwas incubated with Rac prior to addition of liposome (Fig. 3C).In contrast, when theR128E/K130Emutantwas incubatedwithliposome prior to the Rac addition (simultaneous exposures ofRac to the RCBmutant and liposome), no increase of the affin-ity in the R128E/K130E binding to liposome was observed (Fig.3C). These results suggested that the binding of membrane tothe RCB domain is required for the association between RCBdomain and Rac on the membrane.Rac purified from E. coli did not show lipidmodification, and

it decreased the binding of the RCB domain to the membranecompared with Rac purified from the Sf9 cells, indicating thatthe lipidmodification of Rac is required for simultaneous bind-ing of RCB domain to lipids and Rac (Fig. 3D).Therefore, Rac and lipid slightly compete with each other for

binding to RCB domain. However, Rac with lipid modificationappears to bind to themembrane-boundRCBdomain, presum-ably affecting the mode of binding of the RCB domain to themembrane.Actin Filament-binding Sites of the RCBDomain—The IMDs

of IRSp53 and MIM mediate actin filament bundling (7), andthe bundling activity of the IRSp53 IMD is decreased by 50%because of simultaneous Glu substitutions for Lys-142, Lys-143, Lys-146, and Lys-147 in the basic patch (8). The shorter

IRSp53 RCB domain also shows the actin filament bindingactivity, which was decreased by 30% because of the BPM byAla substitution (Fig. 2G). The MIM-(1–228) fragment alsobound to the actin filaments (Fig. 2G).To study the RCBdomain-actin filament binding, we tested a

series of basic residue mutants (Q23E, R11E/Q23E, K108E,R128E/K130E, K130E, K136E, K142E/R145E, K143E, andK143E/K147E), includingmutants with decreased lipid bindingand/or Rac binding abilities. TheK142E/R145Emutant showednormal RCB domain-lipid binding (Fig. 2C) but decreased RCBdomain-actin filament binding (Fig. 2G). Lys-142 and Arg-145were conserved between IRSp53 andMIM (Fig. 1). In contrast,the R128E/K130E mutant showed normal binding to actin fila-ments despite decreased binding to lipids (Fig. 2,C andG). TheK143E/K147E mutant, not the K143E mutant, showed

FIGURE 4. Membrane deformation induced by the RCB domain and Rac.A–C, negative staining electron microscopy. Liposomes were incubated withthe recombinant isolated RCB domain with or without GTP�S-loaded Rac (A),full-length IRSp53 with or without GTP�S-loaded Rac (B), or isolated RCBdomain mutants with GTP�S-loaded Rac (C). A, isolated BAR domain fromamphiphysin 2 was analyzed as a control. Bar, 1 �m. D and E, time-lapseanalysis of the deformation of liposomes, which were generated on glasscoverslips, induced by the RCB domain with GTP�S-loaded Rac (D) or the CIP4EFC/F-BAR domain (E). Protein-containing solutions were added at time 0.Dashed line indicates the edges of liposomes at time 0. Bar, 10 �m.



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decreased binding to the actin filament and lipids (Fig. 2, C andG), suggesting that Lys-147 is important for binding to bothactin filaments and lipids. Thus, actin filaments bind to thebasic patch (Lys-142, Arg-145, and Lys-147) at the end of theRCB domain, whereas lipids bind to the lipid-binding line,which contained the basic residues (Fig. 2,D andH). Mutationsin the Rac-binding site (R11E/Q23E and K143E) did not affectits binding to the actin filament (Fig. 2G).Residues for Binding of the RCB Domain to Membrane, Rac,

and Actin Filaments Are Involved in the RCB Domain-inducedDeformation of Artificial Liposomes in Vitro—We next exam-ined the shapes of liposomes after their incubation with thepurified RCB domain or full-length IRSp53 in the presence orabsence of Rac. After incubation of the RCB domain with lipo-somes, a small number of bud-like liposomes formed on thesurface of relatively large liposomes (Fig. 4A). When liposomeswere incubated with the GTP�S-loaded Rac and the RCBdomain, clusters of bud-like liposomes were formed presum-ably on the existing liposomes (Fig. 4A). Budding was notobserved in response to incubationwithRac alone.As a control,the BAR domain of Bin 1/amphiphysin 2 was incubated withliposomes, and tubulation was observed, as reported previously(Fig. 4A) (9). In addition, we observed the Rac-dependent for-mation of clusters of bud-like liposomes in response to incuba-tion of liposomes with full-length IRSp53 (Fig. 4B).We then tested whether the RCB domain mutants could

induce liposomes to form clusters of buds in the presence ofRac. As expected, the lipid binding-deficient R128E/K130Emutant showed no ability to induce the formation of small budson the liposomes (Fig. 4C). Consistent with the Rac-dependentformation of small buds on the liposomes by thewild-type RCB,the Rac binding-deficient R11E/Q23E or K143E mutantsshowed greatly decreased ability to induce the formation ofsmall buds on the liposomes (Fig. 4C).Most surprisingly, the actin filament binding-deficient

K142E/R145E mutant showed decreased formation of smallbuds on the liposomes, indicating that residues involved in RCBdomain-actin filament binding contribute to membrane defor-mation after the RCB domain binds to liposomes (Fig. 4C).

FIGURE 5. Membrane protrusions induced by the RCB domain. Venus-tagged wild-type (WT) RCB domain was expressed in the COS-7 or A431 cellsor the mouse embryonic fibroblasts (MEFs). The COS-7 cells were also treatedwith latrunculin B (latB) for 30 min or co-expressed with Myc-tagged Rac1T17N (staining of Myc tag is not shown). Dimethyl sulfoxide (Me2SO) was usedas a negative control (data not shown). Cells were stained with rhodamine-

phalloidin to detect the actin filaments (A). Localization of Venus-taggedRCB after staining with anti-GFP antibody is shown (B). Enlarged view ofthe images indicated by a box in A and B are shown in A� and B�, respec-tively. Quantification of cells with microspike-like membrane protrusion isshown (C).

FIGURE 6. Time-lapse analysis of the protrusions in the A431 cells. Venus-tagged wild-type RCB domain was expressed in the A431 cells. Fluorescenceof the Venus-tagged RCB-expressing cells cultured in serum was observed bytotal internal reflection microscopy at 10-s intervals.



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Possible Membrane Deformation Induced by the RCBDomain in a Direction Opposite to That Induced by the BAR orEFC/F-BAR Domains—The deformation of the liposomes toform clusters of small buds indicates two possible mechanisms

of membrane deformation. One is the process of tubulationinduced by the RCBdomain in amanner similar to that inducedby the BAR or EFC/F-BAR domains. The other is membranedeformation induced by the RCB domain in a direction oppo-

FIGURE 7. Microspike-like membrane protrusion by the RCB domain mutants. Venus-tagged wild-type (WT) or mutants of the RCB domain were expressedin the COS-7 cells. The cells were stained with rhodamine-phalloidin to detect F-actin (A). Localization of the Venus-tagged RCB after staining with anti-GFPantibody is shown (B). The cells were stained with DiI to detect membrane (C). Localization of the Venus-tagged RCB is shown by the GFP fluorescence (D).Enlarged image is shown as in Fig. 5. Quantification of cells with microspike-like membrane protrusion is shown (E).



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site to that induced by the BAR domain. In the latter case, com-pression of the spherical liposomes is thought to result in thebudding of excess lipid bilayer by protrusion of the materialspresent inside the liposomes. Therefore, we analyzed the shapeof large liposomes in the presence of the RCB protein and Racusing light microscopy. Incubation of liposomes with the EFC/F-BAR domain was performed as a control, and it resulted inthe formation of long and thin protrusions from the edges of the

large liposomes, as reported previ-ously (20, 27) (Fig. 4E). When thelarge liposomes generated ontoglass coverslips were incubatedwiththe RCB domain and Rac, regres-sion of the edges of the liposomeswas observed (Fig. 4D). Eventually,budding at the edges of the largeliposomes was observed. Long andthin protrusions were neverobserved when large liposomeswere incubated with the RCBdomain and Rac.Protrusions Induced by Expression

of the RCB Domain in Cells—When theRCBdomain fragmentwastagged with GFP and expressed incells, we observed extensive micro-spike-like structures of GFP-RCBlocalization (Fig. 5). As reported forfull-length IRSp53 (2, 3), formation ofmicrospike-like structures withoutphalloidin staining was observed intheCOS-7 andA431 cells aswell as inthemouse embryonic fibroblasts (Fig.5). The protrusions were branched;however, these were not because ofthe retraction of the cells, as revealedby the time-lapse recording (Fig. 6).Furthermore, these structures ap-peared to lack the actin filaments,because phalloidin staining wasalmost absent even after the signalswere highly intensified in most of theprotrusions (Fig. 5). Furthermore,these structures were formed evenafter a 30-min treatment with latrun-culin B, a drug that inhibits actinpolymerization (Fig. 5). Therefore,these structures are considered torepresent protrusion of the plasmamembrane.To address the Rac dependence of

the RCB domain-induced plasmamembrane protrusion, we co-ex-pressed a dominant-negative mutantofRac (T17N)with thewild-typeRCBdomain in the COS-7 cells. Althoughsome protrusions with phalloidinstaining remained, most of the pro-

trusions were significantly decreased in the cells expressing RacT17N and the RCB domain (Fig. 5).Next, we tested various RCB mutants for their ability to

induce protrusions.Mutants deficient in the Rac binding ability(R11E/Q23E or K143E) or the MIM-(1–228) fragment did notinducemembrane protrusionwithout phalloidin staining in theCOS-7 cells (Fig. 7, A, B, and E). Instead, smaller microspikesthat contained actin filaments were formed. These results indi-

FIGURE 8. Model of membrane deformation induced by the IRSp53 RCB domain. A, schematic of theinteractions of the IRSp53 RCB domain to the membrane. Ellipse with dashed lines indicates the lipid-bindingline interacting with the membrane. Amino acids required for binding of the RCB domain to Rac (right) and formembrane deformation/binding to the actin filaments (left) are shown (1). Rac binding tethers one end of theRCB dimer to the membrane (2). Positive charges of the actin filament-binding sites at the other end deform themembrane by an electrostatic interaction. Dashed curved line indicates possible deformed membrane inducedby the possible convex surface of the RCB domain for lipid binding. Inset, the proposed BAR domain-membraneinteraction (9). The residues involved in the membrane deformation by the BAR domain are indicated in black(9). B, model for the BAR domain-induced membrane tubulation. The BAR and EFC/F-BAR domain proteinsdeform spherical liposomes into narrow tubules in vitro. B�, the concave surface of the BAR domain is postu-lated to surround the membrane. B, the BAR and EFC/F-BAR proteins induce inward membrane tubulation incells. C, possible model for the RCB domain-induced membrane tubulation. The RCB domain protein possiblycompresses the spherical liposome because of inward tubulation; this may be observed as buds on the surfaceof the liposomes. C�, according to the mapping of the amino acids, the membrane tubule is assumed to besupported by the convex surface of the RCB domain. C, the RCB domain induces outward membrane protru-sion in cells.



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cate that binding of the RCB domain to activated Rac is essen-tial for the extensive protrusion of the plasma membrane.We next expressed the RCB domain mutants with reduced

affinities for lipid and/or actin in cells. Protrusions were notinduced in the cells expressing the R128E/K130E mutant,which possesses decreased lipid binding ability but normalRac and actin filament binding abilities, or the K142E/R145Emutant, which possesses decreased actin filament bindingability but normal lipid and Rac binding abilities (Fig. 7, A, B,and E). Thus, all the residues that are required for the bind-ing of the RCB domain to Rac, membrane, and actin fila-ments are required for causing membrane protrusions incells.To confirm these protrusions contain cell membrane, cells

were stained with the membrane-staining dye (DiI). Theprotrusions containing RCB proteins were stained by DiI(Fig. 7, C and D). A small number of protrusions stained byDiI did not contain RCB proteins in mutant RCB-expressingcells (Fig. 7, C and D, and data not shown), indicating thespecific localization of wild-type RCB domain protein inRCB-induced protrusions.Possible Mechanism of Membrane Deformation Induced by

the RCB Domain—The shape of the liposomes in vitro did notdirectly explain the shape of cell protrusions. Therefore, itremains unclear whether the protrusion of cells was the directconsequence of the membrane deformation observed in vitro.Furthermore,we cannot completely rule out the involvement ofactin cytoskeleton in the RCB-induced protrusions because theamount of actin filaments may be too small to be detected byphalloidin staining (Figs. 6 and 7). However, the in vitromem-brane deformation and protrusion of cells suggest that thedirection of membrane deformation induced by the RCBdomain is opposite to that induced by the BAR or EFC/F-BARdomains. Mapping of the amino acids responsible for mem-brane deformation suggests the involvement of the convex sur-face of the RCB domain in the RCB domain-membrane bindingas discussed below.We postulate that the lipid-binding line on the bottom

right of the IRSp53 RCB fragment dimer is used for bindingto the membrane (Fig. 2D and Fig. 8A). The Rac-binding siteon the left is at this moment very close to the membrane, andthe other accessible RCB site on the top right is used (Fig. 2Fand Fig. 8A). If Rac binds to the Rac-binding site on the topright, then the right end of the dimer would be tethered tothe membrane via Rac. The actin filament-binding sites onthe RCB domain were also effective in inducing membranedeformation in vitro (Fig. 4). The actin filament-binding siteon the right overlaps slightly with the lipid-binding site onthe bottom right, and it might function along with the lipid-binding line on the bottom right for inducing membranedeformation. However, membrane deformation by the lipid-binding line and the actin filament-binding site on the rightis unlikely because of the close proximity of these two sites.Interestingly, the actin filament-binding site on the left thatfaces the membrane appears to bind to the membrane by theelectrostatic interaction if the association of the RCBdomain with Rac fixes the binding of the lipid-binding lineon the right to the membrane (Fig. 2H and Fig. 8A). Slight

overlap of Rac and the lipid-binding line may alter the ori-entation of the RCB domain toward the membrane to causethe membrane deformation when Rac is associated with theRCB domain. Thus, the lipid-binding line and the actin fila-ment-binding site on the RCB domain appear to present theconvex surface of the RCB domain for binding this domain tothe membrane in the presence of Rac (Fig. 8A).It is postulated that if the tubules that are formed by inward

membrane tubulation are surrounded by the concave surface ofthe BAR domain (Fig. 8B), the tubules formed by the outwardmembrane tubulation are lined by the convex surface of theRCB domain (Fig. 8C). The detailed analysis of the binding ofthe RCB domain to the membrane is required for understand-ing the mechanism of the inward and outward membraneprotrusion.

Acknowledgments—We thank Dr. Toshiki Itoh (Institute of MedicalScience, University of Tokyo) for technical support and helpfuldiscussions; Dr. Hiroyuki Fukuda (Institute of Medical Science,University of Tokyo) for tandem mass spectrometry analysis ofcrystallized protein; Dr. Atsushi Miyawaki (Brain Science Insti-tute, RIKEN) for providing the Venus vector; and Dr. NobuoKamiya, Taiji Matsu, and Dr. Hisashi Naitow for help in datacollection at the RIKEN beamline BL44B2 of SPring-8. We alsothank Dr. Kiminori Toyooka and co-workers at the RIKEN PlantScience Center for electron microscopy.

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and Shigeyuki YokoyamaAzusa Seto, Tsukasa Oikawa, Chiemi Mishima, Mikako Shirouzu, Tadaomi Takenawa

Shiro Suetsugu, Kazutaka Murayama, Ayako Sakamoto, Kyoko Hanawa-Suetsugu,RAC-dependent Membrane Deformation

The RAC Binding Domain/IRSp53-MIM Homology Domain of IRSp53 Induces

doi: 10.1074/jbc.M606814200 originally published online September 25, 20062006, 281:35347-35358.J. Biol. Chem.

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