View metadata, citation and similar papers at core.ac.uk brought to you by CORE

provided by Elsevier - Publisher Connector Cell, Vol. 101, 259–270, April 28, 2000, Copyright 2000 by Cell Press Structure of the ERM Reveals the FERM Domain Fold Masked by an Extended Binding Tail Domain

Matthew A. Pearson,*§ David Reczek,* surface structures (Berryman et al., 1993). Despite their Anthony Bretscher,*‡ and P. Andrew Karplus*†‡ distinct names, their functional similarities are such that *Department of Molecular Biology and Genetics the ERM can be considered as isoforms of a Cornell University single key protein. Indeed, Drosophila and C. elegans Ithaca, New York 14853 each have only one type of ERM protein. The absolute † Department of Biochemistry and Biophysics requirement for at least one ERM protein in the formation Oregon State University of cell surface structures is best shown by antisense Corvallis, Oregon 97331 experiments in cultured cells (Takeuchi et al., 1994). The recent report that a moesin knockout mouse has no phenotype (Doi et al., 1999) is consistent with the func- Summary tional similarities of ERM proteins, in that the remaining and could substitute for moesin. The ezrin-radixin-moesin (ERM) protein family link ac- The ERM proteins are different from most FERM con- tin filaments of cell surface structures to the plasma taining proteins in that, as shown in Figure 1B, their membrane, using a C-terminal F-actin binding seg- activity is regulated by intramolecular masking of pro- ment and an N-terminal FERM domain, a common tein–protein interaction sites (reviewed in Bretscher, membrane binding module. ERM proteins are regu- 1999). Such conformational regulation is poorly under- lated by an intramolecular association of the FERM stood but may have wide generality as it has been pro- and C-terminal tail domains that masks their binding posed for several proteins, with another cytoskeletal sites. The crystal structure of a dormant moesin example being vinculin (Johnson and Craig, 1994, 1995; Bakolista et al., 1999). For ERM proteins, the polypeptide FERM/tail complex reveals that the FERM domain has can be divided into three regions: an 300 residue N-ter- ف /three compact lobes including an integrated PTB/PH minal FERM domain, an 200 residue central ␣ domain ف EVH1 fold, with the C-terminal segment bound as an that has a heptad hydrophobicity pattern characteristic extended peptide masking a large surface of the FERM of coiled–coil proteins, and an 100 residue C-terminal ف domain. This extended binding mode suggests a novel tail domain. ERM proteins are isolated as monomers mechanism for how different signals could produce and homo- or heterodimers in which the FERM domain varying levels of activation. Sequence conservation is bound tightly to the tail domain (Gary and Bretscher, suggests a similar regulation of the tumor suppressor 1995) (Figure 1). Such molecules are dormant, because . the FERM/tail interaction mutually masks the binding sites for other molecules (Gary and Bretscher, 1995; Introduction Takahashi et al., 1997; Reczek and Bretscher, 1998). For –membrane crosslinking, the dormant FERM domains (band four-point-one, ezrin, radixin, molecule becomes activated and the FERM domain at- moesin homology domains) are widespread protein taches to the membrane by binding specific membrane -amino acids in length that are involved proteins (see Figure 1B legend), while the last 34 resi 300ف modules of in localizing proteins to the plasma membrane (Chisti dues of the tail bind actin filaments (Turunen et al., 1994; et al., 1998). The FERM domain defines members of Pestonjamasp et al., 1995). Two pathways are believed the band 4.1 superfamily, which includes cytoskeletal to contribute in vivo and in vitro to activation: phosphor- proteins such as erythrocyte band 4.1, , and the ylation of the tail at Thr558 (human moesin numbering ezrin-radixin-moesin (ERM) protein family, as well as is used throughout) (Nakamura et al., 1995; Matsui et several tyrosine and phosphatases and the tu- al., 1998; Pietromonaco et al., 1998) and exposure to mor suppressor protein, merlin (Figure 1A). The ERM polyphosphatidyl inositides (Niggli et al., 1995, Hirao et proteins have been a subject of particular interest be- al., 1996; Huang et al., 1999; Matsui et al., 1999; Naka- cause of their direct involvement in the dynamics of mura et al., 1999). Phosphorylation at Thr558 weakens interaction between the plasma membrane and the actin the FERM/tail interaction (Matsui et al., 1998) and, in cytoskeleton. Specifically, they function as conforma- the presence of phospholipids, unmasks the membrane tionally regulated membrane–cytoskeletal crosslinkers protein and F-actin binding sites (Simons et al., 1998). in actin-rich cell surface structures, especially microvilli, In addition, Thr558Asp mutants of moesin appear to be microspikes, and membrane ruffles (Bretscher, 1999). at least partially activated (Oshiro et al., 1998; Huang et Ezrin (Bretscher, 1983; Gould et al., 1986; Pakkanen al., 1999). Aside from binding to membranes, the acti- and Vaheri, 1989), radixin (Tsukita et al., 1989; Funayama vated FERM domain of ERM proteins can also bind the et al., 1991), and moesin (Lankes and Furthmayr, 1991) guanine dissociation inhibitor of Rho GTPase sequence identity) that are (RhoGDI), which suggests that in addition to functioning %75ف) are close homologs all localized near the plasma membrane in actin-rich cell as a crosslinker, ERM proteins may influence Rho signal- ing pathways (Takahashi et al., 1997). Merlin, the product of the neurofibromatosis type 2 ‡ To whom correspondence should be addressed (e-mail: apb5@ cornell.edu [A. B.]; [email protected] [P. A. K.]). tumor suppressor gene (Gusella et al., 1999), is unique § Present address: Laboratory of Molecular Biology, NIDDK, Na- in that its FERM domain has a very high 60% identity tional Institutes of Health, Bethesda, MD 20892. with the ERM proteins. Merlin’s function is still poorly Cell 260

Figure 1. Representative FERM Domain– Containing Proteins and a Model for the Con- formational Masking of ERM Proteins (A) The proteins of the band 4.1 superfamily contain FERM domains (cyan) in combination with other types of domains, including PDZ, tyrosine phosphatase, SH2-like, tyrosine ki- nase, -like, head, and PH do- mains (Girault et al., 1999). The ERM proteins, and possibly merlin, have a C-terminal tail domain (red) that can mask binding sites on the FERM domain. (B) The dormant ERM molecule (left) has bind- ing sites masked by an intramolecular inter- action. Due to this association, the FERM and tail domains of ERMs have also been called by the functionally descriptive names the N- and C-terminal ERM-association domains (N-ERMAD and C-ERMAD) (Gary and Bret- scher, 1995). After activation (right), the pro- tein is able to link actin filaments that become bound to the tail (red) to membrane proteins (integral or peripheral) that become bound to the FERM domain (cyan). The role of the putative coiled-coil domain (A) is unknown. Activation by phosphorylation at Thr558 is shown as the best characterized factor that affects activation. Although multiple ERM proteins may combine in series to form ex- tended crosslinks, a single crosslinking mole- cule is shown. Known membrane-associated ligands for the ERM–FERM domains include the integral membrane proteins CD44, CD43 (Tsukita et al., 1994; Yonemura et al., 1998), and intercellular adhesion molecules (Hel- ander et al., 1996; Serrador et al., 1997; Heiska et al., 1998), and the peripheral mem- brane proteins ERM-binding phosphoprotein 50 (EBP50) (Reczek et al., 1997) and ex- changer 3 kinase Aregulatory protein (E3KARP) (Reczek and Bretscher, 1998; Yun et al., 1998). The central ␣ domain can bind the regulatory subunit of cAMP-dependent protein kinase, and help localize this kinase to the plasma membrane (Dransfield et al., 1997). understood, but it is clearly distinct from ERM proteins, prototype for all FERM domains, advancing studies of as its function cannot be substituted by ERM proteins, this large family of membrane-associated proteins. and it does not have a C-terminal actin binding site (Huang et al., 1998). Although a functionally important Results and Discussion head-to-tail interaction similar to that of the ERM family members has been identified in merlin isoform I (Sher- Overall Structure man et al., 1997; Huang et al., 1998; Gronholm et al., Attempts to crystallize full-length dormant ezrin or moe- 1999), the low level of sequence identity between the sin were unsuccessful, so we set out to crystallize a C-terminal domains has made it unclear whether the complex of the FERM and C-terminal tail domains. The merlin interaction is similar in detail to that in ERM pro- human moesin FERM (residues 1–297) and tail (residues teins. 467–577) domains were each expressed as selenomethi- Here, we present the crystal structure of the noncova- onine (Se-Met) derivatives, and the complex was formed lent 1:1 complex of the recombinant FERM and tail do- in vitro and crystallized. Crystals of the complex dif- mains of human moesin. The structure reveals that the fracted X-rays to 1.9 A˚ resolution, and the structure was tail contacts the compact FERM domain as an extended solved by multiple-wavelength anomalous diffraction peptide inhibitor, allowing new insight into the confor- (MAD) (Table 1). In the crystal, the moesin FERM/tail mational events involved in ERM activation and mem- complex is present as a domain swapped dimer of com- brane–cytoskeleton crosslinking. The structure also in- plexes with residues 502–577 of the tail binding to one dicates that similarities between ERM proteins and FERM domain, and residues 488–494 crossing over to merlin extend beyond the FERM domain and permits interact with a second FERM domain (Figure 2). Because most of the oncogenic of merlin to be mapped the protein exists in solution as a 1:1 complex, the ob- onto this structure. Finally, the structure of moesin is a served arrangement is a crystallization artifact, and here Structure and Masking of Human Moesin 261

Table 1. MAD Phasing and Refinement Statistics MAD Data Collection (2.3 A˚ ) MAD Structure Factor Ratios

a a Wavelength (A˚ ) Completeness (%) Rmeas ␭1 ␭2 ␭3

␭1 ϭ 0.9797 89 (84) 0.065 (0.292) 0.058 0.038 0.055 (0.042)

␭2 ϭ 0.9795 89 (85) 0.067 (0.317) 0.069 0.060 (0.042)

␭3 ϭ 0.9643 89 (86) 0.068 (0.359) 0.069 (0.046) High Resolution Data Collection and Refinement (1.9 A˚ ) Data Quality

a a a Reflections (N) Completeness (%) Redundancy Rmeas 71,584 95 (92) 3.6 (3.7) 0.090 (0.445) Refinement Resolution range 20–1.9 A˚ Working set No. reflections 66,990 R 0.227 Test set No. reflections 3568 R 0.287 Bulk solvent k ϭ 0.335 B ϭ 31.3 A˚ 2 2 2 2 Anisotropic overall BB11 ϭϪ3.64 A˚ , B22 ϭϪ1.04 A˚ , B33 ϭ 4.68 A˚ Model ideality rms bonds 0.016 A˚ rms angles 1.7Њ rms B, bonded atoms 4.8 A˚ 2 a Values in parentheses correspond to the highest resolution bin. we describe the structure derived by mapping the ob- for phosphotyrosine binding (PTB), pleckstrin homology served interactions onto a single complex (Figure 3). (PH), and Enabled/VASP Homology 1 (EVH1) domains The FERM domain is composed of three structural (Forman-Kay and Pawson, 1999). These domains are modules (F1, F2, and F3) that together form a compact often present in and cytoskeletal proteins, clover-shaped structure (Figure 3). F1 (residues 4–82) where they bind peptide and/or phospholipid ligands. contains a 5-stranded mixed ␤ sheet packed against an The unexpected presence of a PH/PTB-like domain inte-

␣ helix with a short 310 helix prior to the start of strand grated into the FERM domain is reminiscent of an unrec- 3. F2 (residues 96–195) is composed of five ␣ helices, ognized SH2 domain that combines with other domains with an excursion of 36 residues between helices B and to form an amino-terminal module of the Cbl adaptor C that contains a long loop and a short ␣ helix. F3 protein (Meng et al., 1999), and serves as a reminder (residues 204–297) consists of a sandwich of two orthog- that even well worked out sequence fingerprints are not onal antiparallel ␤ sheets followed by a long helix, with powerful enough to recognize all homologs.

80ف a turn of 310 helix in the loop connecting the two sheets. In contrast with the expected globularity of an The center of the clover is filled largely by the 13 residue residue domain, the C-terminal tail adopts an extended, linker between F1 and F2 (which includes a short ␣ helix) meandering structure (Figure 3) that efficiently blocks a and the 8 residue linker between F2 and F3. The three- large area of the FERM surface. It is highly unusual for lobed nature and secondary structure of the moesin a polypeptide this large to bind to another protein in FERM domain indicate that the structural predictions such an extended manner. The first ordered residues of Turunen et al. (1998) were reasonably accurate, but (488–494) of the tail extend the second ␤ sheet of the contradict the prediction made by hydrophobic cluster F3 module by forming an antiparallel ␤ strand alongside analysis that the FERM domain consists of a duplication strand 5 of F3. Residues 495–501 form a poorly ordered of two 140 residue domains (Girault et al., 1998). connection to residues 502–577 that fold into four major Each of the three FERM lobes is surprisingly similar ␣ helices (designated A through D) and two short helices in structure to proteins whose sequences are not recog- that extend across the surface of domains F2 and F3. nizably similar to FERM domains. F1 is very similar to Because the tail domain makes few internal tertiary inter- the structure of (Vijay-Kumar et al., 1987), a actions, the structure it adopts in complex with the fold that is found in several proteins of dissimilar se- FERM domain is unlikely to be stably adopted by an quence and function. F2 is similar to the structure of isolated tail domain. In support of this prediction, the acyl-CoA binding protein (Kragelund et al., 1993), which FERM domain and the FERM/tail complex are relatively binds acyl-CoAs of various acyl chain lengths. F3 shares stable to proteolysis in crude bacterial extracts, whereas the fold of an adaptable ligand binding module seen the free tail is very sensitive (data not shown). In total, Cell 262

involves the binding of the C-terminal helix D of the tail in a groove on the surface between the two sheets of the F3 ␤ sandwich. The side chains of Phe574 and Met577 bind in a hydrophobic pocket formed by resi- dues Leu216, Ile227, Lys237, Ile238, and Phe267, and the terminal carboxylate group hydrogen bonds with residues Asn210 and Ser214. The burial of Met577 at the interface explains the observation that the FERM/ tail interaction is largely disrupted in ERM mutants trun- cated at residue 575 (Gary and Bretscher, 1995; Reczek and Bretscher, 1998). With respect to its position on F3, this binding site is analogous to the sites of inositol phosphate binding to PH domains (Ferguson et al., 1995; Hyvo¨ nen et al., 1995). Also, as seen in the PH domains, a positively charged loop between strands 1 and 2 of F3 participates in the binding. Interestingly, moesin is the first structural example of a PH/PTB/EVH1 fold that has a substrate (the C-terminal tail domain) bound at both the canonical PTB and PH binding sites simultane- ously, underscoring the flexibility of this fold as a ligand binding module and confirming the expectation of Forman-Kay and Pawson (1999). In terms of electrostatics, the interface includes a re- Figure 2. The FERM/Tail Monomer Structure Is Derived from the Dimer Seen in the Crystal gion of negative charge on the surfaces of FERM lobes Residues 495–501 adopt a poorly ordered but visible extended seg- F1 and F2 that packs against an area of positive charge ment connecting the two FERM/tail complexes in the asymmetric on the surface of the tail (Figure 5). In addition, lobe F3 unit. However, this must be a crystallization artifact, because dy- contains a region of positive charge noted above that namic light scattering shows that the complex is monomeric in interacts with the largely neutral helix D of the tail. Aside solution (data not shown). Also, such a dimeric arrangement is not from the FERM/tail-interface, the exposed surfaces of possible for intact moesin, both because it exists as a monomer both the domains are dominated by regions of positive and because the poorly ordered connecting segment is stabilized charge (not shown). by an H bond between the C-terminal carboxylate of Pro297 with the amide nitrogen of residue 499, and this carboxylate does not exist in full-length moesin. As indicated in the Figure by the dashed Conformational Activation black line, the residues 494 and 502 bound to a single FERM domain Activation of ERM proteins involves weakening the -A˚ ) and could easily be linked in a single FERM/tail interaction so as to unmask the membrane 15ف) are in close proximity complex. Figures 2, 3A, 3B, 6, and 7 were generated with MOLSCRIPT protein and actin binding sites (Figure 1B). The remark- (Kraulis, 1991) and Raster3D (Merritt and Bacon, 1997). able distributed nature of the tail means that its high affinity derives from the binding affinities of the five parts of the extended structure whose interactions are largely the FERM/tail interactions bury a remarkable 2700 A˚ 2 of independent of each other: strand 1 and helices A, B, the FERM surface and 2950 A˚ 2 (36%) of the tail surface. C, and D. Even if all individual affinities are moderate, The near perfect sequence conservation among inter- the net affinity can be very high because of the chelate face residues (Figure 4) provides a strong argument that effect (Fersht, 1999). In other words, the pieces are all this structure represents the relevant domain complex connected to one another and the cost of losing overall present in the dormant ERM molecules. translational and rotational entropy during binding is only paid once. Biophysical studies of the thermody- The FERM/Tail Interface namics and dynamics of this novel inhibitory mode will In addition to several interaction regions of lower com- be required to provide quantitative insight into activa- plementarity where pockets of water molecules mediate tion. However, qualitatively, one clear advantage this the interaction, there are three regions of tight contact binding mode has over a single globular domain that that we describe here. The first is the interaction be- binds as a unit is that, here, multiple independent signals tween strand 1 of the tail and lobe F3, where, in addition (interactions) can contribute to achieve differing levels to the main chain hydrogen bonding that extends the ␤ of activation by competing with or weakening different sheet, the side chain of Leu494 is buried in a hydropho- parts of the interaction surface. bic pocket formed by residues Ile245, Ile248, and Phosphorylation of a specific C-terminal threonine His288. Interestingly, the position of this tail domain contributes to the activation of ERM proteins (Matsui et strand matches well with that observed for peptides al., 1998). In moesin, this residue, Thr558, is located on bound to PTB domains, with the Leu494 bound in a helix C of the tail, at an edge of the interface where it pocket equivalent to that occupied by a hydrophobic is both in contact with the FERM domain and exposed residue found upstream of the phosphotyrosine in PTB to solvent (Figures 5 and 6). The structure shows that substrates (Borg and Margolis, 1998). The second region phosphorylation at this position will weaken the helix of tight contact is between the hydrophobic face of tail C/FERM interaction due to both electrostatic and steric helix A and hydrophobic residues on helices B and D of effects. In terms of electrostatics, Thr558 is in a posi- F2 (see Figure 4 for exact residues). The third interaction tively charged surface, and is positioned opposite to Structure and Masking of Human Moesin 263

Figure 3. The Overall Structure of the FERM/Tail Complex (A) A stereo ribbon diagram of the complex. Visible are the three-leaf clover domain structure of the FERM domain (cyan) and the extended conformation of the tail domain (red). Sidechains packing in this well-packed central region of the clover include Phe84, Tyr85, Pro86, Phe102, Gln105, Val106, Ile115, Ala195, Gln196, Leu198, Tyr201, Gly202, Leu283, and Leu290. 90Њ around the horizontal axis from the view in (A), to highlight the conformation of theف B) A stereo view of the complex, after rotating) C-terminal tail (red). (C) The topologies of the three FERM lobes and the tail domain are shown schematically with helices represented by boxes and strands by arrows. Helices shorter than six residues are considered as extended turns and are not given a letter designation. Secondary structure was assigned using DSSP (Kabsch and Sander, 1983). Cell 264

Figure 4. Sequence Conservation for ERMs and Related Proteins Sequences for (A) the F1, F2, and F3 lobes of the FERM domains of human moesin (Swissprot P26038), ezrin (P15311), radixin (P35241), merlin (P35240), and band 4.1 (P11171) and (B) the C-terminal tails of human moesin, ezrin, radixin, and merlin were aligned with CLUSTALW (Thompson et al., 1994) and manually adjusted. Secondary structure elements are indicated above the sequences and every tenth moesin residue has a dot. Residues highlighted in cyan in the moesin sequence are involved in the FERM/tail interface (defined as residues having Structure and Masking of Human Moesin 265

Figure 5. Electrostatic Features of the FERM/Tail Interface The molecular surfaces of the domains reveal the electrostatic potential (from negative [red] to positive [blue]) at the interface. The region containing the C-terminal F-actin binding site is outlined, and the position of Thr558 is marked with an asterisk. Molecular surfaces in Figures 4C and 5 and the electrostatic potential were calculated using GRASP (Honig and Nicholls, 1995). The interface surfaces for residues 488–494 of the tail and their docking site are not visible. the heart of a negatively charged surface of the FERM complex. The intimate involvement of these residues in domain (Figure 5), where the introduction of the nega- the FERM/tail interface provides a simple direct mecha- tively charged phosphoryl group would have a strong nism for the masking of the actin binding site. Three detrimental effect. A of Thr558 to Asp that arguments indicate that the structure seen here does mimics this charge change is weakly activating (Oshiro not provide a model for the conformation that these et al., 1998; Huang et al., 1999), indicating that the elec- residues will adopt when they bind to actin. First, phos- trostatic change is important. In terms of sterics, the phorylation of Thr558 does not block actin binding (Mat- side chains surrounding Thr558 approach it closely sui et al., 1998), but, as discussed above, it must cause enough that there is not sufficient room for a phosphoryl at least some rearrangement of the tail. Second, this group (Figure 6), so that some structural rearrangement region of the tail makes so few intramolecular tertiary must occur, and, given the proximity of these residues interactions that its conformation is clearly heavily dic- to the interface, any perturbations will clearly affect the tated by docking onto the FERM domain. And third, the stability of the complex. last 26 residues of the tail is the only part of the domain The contribution of anionic phospholipid vesicles to which has high sequence conservation of residues not activation is less clear, but they bind to both the intact buried in the FERM/tail interface (Figure 4C). This would dormant protein and to the isolated FERM domain of only be expected if actin binding places additional con- ERM proteins (Niggli et al., 1995). Given the highly posi- straints on the evolution of these residues, and suggests tively charged surface of lobe F3, we speculate that that these “back-side” residues are more involved in the negatively charged phospholipids would have the actin binding than they are in the FERM/tail interaction. highest affinity for this part of the interface, and might The observation that the FERM domain of ERM pro- compete with and weaken the binding of helix D to the teins binds to several membrane proteins (see Figure FERM domain. 1B) and to phospholipids has led to the expectation that it has multiple binding sites. Although no experimental Potential Binding Sites on Activated ERM Molecules data exist to specifically locate these binding sites on The portion of the tail responsible for F-actin binding the FERM structure, considerations of residue conser- has been mapped to its last 34 residues (Turunen et al., vation and of homology provide guides for future studies 1994), which form tail domain helices B, C, and D in the aimed at dissecting these binding functions. The first

Ͼ10 A˚ 2 more side chain surface area buried in the complex than in the isolated domains). Residues colored magenta in the merlin sequence are those that are either invariant or conserved as hydrophobic in ERMs and merlin, and residues indicated in red below the merlin sequence are single site missense mutations in merlin that are associated with human tumors (Turunen et al., 1998). The phosphorylation site at Thr558 is indicated with an asterisk. The six most well conserved residues among all FERM domains (Girault et al., 1999) are outlined with boxes and highlighted in yellow. (C) Molecular surfaces, after opening the complex in Figure 3B like a book, colored to highlight those residues that are invariant or conserved as hydrophobic in human ERMs (green and magenta surfaces), or human ERM proteins and merlin (magenta surface only). Quantitative analysis of the enhanced conservation of the interface residues in merlin is as follows: for the FERM domain, the identity levels among ERM proteins and merlin are 60% for interface residues versus only 36% for other surface residues; for the C-terminal tails, the corresponding numbers are 28% versus 7%. The labeled magenta surface on the backside of the FERM domain (lobe F1) indicates sequence conservation among ERMs and merlin in a position that is analogous to a protein binding site on the ubiquitin-like domains of Raf kinase and elongin B (see Figure 6). Cell 266

Figure 6. Stereodiagram of the Region Surrounding Thr558 Thr558 is in van der Waals contact with FERM residues Thr235 and Pro236, and is accessible to solvent from the right between the side chains of Arg553, Lys557, and Gln561. The electron density (from an NCS averaged map calculated with coefficients 2FO Ϫ 2FC, ␣C) is contoured at 2.2 ϫ␳rms. A phosphoryl group modeled onto Thr558 collides with surrounding atoms from Arg553 and Gln561. guide is the high conservation of surface residues not seen in Figure 4C, strong conservation implicates two involved in the interface, as these residues are likely patches on the backside of the ERM FERM domain, a to have a functional importance. As an example, the large one on lobe F1 and a smaller one at the edge of conservation of noninterface residues in the last 26 resi- lobe F2. The second guide is the similarity of lobes F1, dues of the tail discussed above would have provided F2, and especially F3 to known protein modules. The an accurate indication of the location of an interaction binding sites on these analogous modules are worth site even in the absence of biochemical studies. As documenting because if they represent true homologies then positions of binding sites may be conserved de- spite the high degree of sequence divergence. Figure 7 illustrates the five such sites that can be mapped onto FERM domain. Among these, sites 1, 4, and 5 seem most worthy of attention because site 1 corresponds to the large conserved patch on domain F1 (Figure 4C), and sites 4 and 5 are both making direct interactions with the tail and are thus masked in the complex.

FERM/Tail Interaction in the Tumor Suppressor Merlin The ERM homolog merlin exists as two alternatively spliced isoforms each containing an N-terminal domain sequence identity with the FERM of %60ف that shares the ERMs (Rouleau et al., 1993; Trofatter et al., 1993; Gusella et al., 1999). In contrast, the C-terminal 100 Figure 7. Potential FERM Domain Interaction Sites identity %20ف amino acids of merlin isoform I have only The positions of five binding sites on proteins that are structurally with the ERM tail, while isoform II has a truncated C similar to the three FERM lobes are indicated: (1) by analogy with the protein interaction site seen for elongin B (Stebbins et al., 1999) terminus. The present structural data reveal the en- and the Ras binding domain of Raf kinase (Nassar et al., 1995); (2) hanced conservation of residues that lie on the moesin by analogy with the lipid binding site of the acyl CoA binding protein FERM/tail interface (Figure 4), providing strong evidence (Kragelund et al., 1993), but this site is blocked by the loop connect- that the interaction in merlin is equivalent. A remarkable ing the F2 helices C and D; (3) by analogy with the proline-containing 81% of the residues that are invariant between the ERM peptide binding site on EVH1 domains (Prehoda, at al., 1999; Fed- tails and merlin lie on the interface. Interestingly, the erov et al., 1999); (4) by analogy with the canonical peptide binding site of PTB domains (Zhou et al., 1996); and (5) by analogy with the poor sequence conservation among the last 34 residues inositol phosphate binding sites on PH domains (Ferguson et al., between ERMs and merlin (except for those involved in 1995; Hyvo¨ nen et al., 1995). the interface) suggests that the FERM/tail interaction is Structure and Masking of Human Moesin 267

the only common functional constraint on this region of leading to the important conclusion that the structure merlin, and is consistent with the lack of a C-terminal solved here is not only a model for the dormant ERM F-actin binding site in merlin. proteins, but a model for all FERM domains, including These observations confirm that the moesin structure those in activated ERMs. is a good model for merlin isoform I, providing a frame- In contrast to the residues internal to the FERM do- work for understanding the mutations that lead to non- main, the poor sequence conservation of residues on functional merlin and hence tumor formation. There are the FERM/tail interface rules out this type of interaction 14 single site substitution mutations that are associated in other FERM domain–containing proteins. However, with human tumors (Figure 4) (Turunen et al., 1998). Six the binding sites of other FERM domains may also be of these residues are buried in FERM domain, where regulated by peptide inhibitors, albeit through a set of they are likely to cause destabilization or misfolding. different specific interactions. Indeed, the highly effi- Interestingly, these buried mutations are at positions cient and versatile inhibition mode revealed here pro- throughout the FERM domain (three in F1, one in F2, vides a paradigm for thinking of other cytoskeletal pro- one in the linker between F2 and F3, and one in F3), teins that are regulated by masking. Furthermore, the indicating that the whole FERM unit is important for structure of the moesin FERM/tail complex presented merlin function. Of the eight other mutations, three (mer- here opens the door for refined mutagenesis experi- lin Leu535Pro, Gln538Pro, and Leu539His) correspond ments aimed at elucidating details of the structure– to tail residues in moesin (Val518, His521, and Leu522) function relations of ERM proteins, merlin, and all FERM that are on the interface, at the site of tight association domains. with lobe F2. The remaining five surface mutations are scattered over the FERM domain (two in F1, one in the Experimental Procedures linker between F1 and F2, one in F2, and one in F3) and do not cluster into a region on the surface. In addition Protein Expression and Purification to these natural mutations, mutagenesis experiments in The cDNA sequences encoding the human moesin FERM (residues 1–296) tail (residues 467–577) domains were amplified by PCR from Drosophila have been used to identify important func- clone HEBA06 (a generous gift from Dr. Stachowitz, Genzentrum, tional regions of merlin, including the “blue box” region Munich, Germany) for expression in a pQE vector (Qiagen). All re- that includes residues corresponding to 161–167 of the combinant sequences were determined to be free of PCR errors. moesin FERM domain (LaJeunesse et al., 1998). A mis- For the expression of Se-Met substituted protein, each moesin con- sense mutation (Met177Ile in Drosophila merlin) corre- struct was cotransformed with the pRep4 repressor plasmid (Qia- sponding to Gln167 in moesin resulted in merlin with gen) into the methionine auxotrophic E. coli strain B834(DE3) (Nova- gen). Frozen cells from 1 L and 3 L cultures expressing FERM and lowered activity, similar to that of an isolated merlin tail domains, respectively, were thawed and resuspended together. FERM domain. While moesin Gln167 is not on the FERM/ Cell lysis and purification of the complex were carried out by essen- tail interface, the neighboring residues 161–163 are, sug- tially the same protocol as was used for the isolated FERM domain gesting that the blue box mutation might lead to weaken- (Reczek et al., 1997). 1–2 mM DTT was added to all buffers to protect ing of the complex. In contrast to the missense mutation, the Se-Met from oxidation. deletion of residues in the blue box lead to nonfunctional merlin, which again indicates the importance of proper Structure Determination Crystals were grown at 4ЊC using the hanging drop vapor diffusion folding of the FERM domain. method with drops containing 8 ␮l of protein solution at a concentra- tion of 5 mg mlϪ1 in 10 mM HEPES (pH 7.5), 20 mM NaCl, 1 mM The FERM Domain of Other Members of the Band EDTA, and 5 mM DTT and 4 ␮l of reservoir solution consisting of 4.1 Superfamily 100 mM HEPES (pH 7.8), 100–150 mM Li2SO4, and 18%–20% PEG- Sequence conservation indicates that the structure of 3350. For data collection, crystals were transferred directly from drops to a cryobuffer consisting of the reservoir solution supple- moesin is a useful prototype for the FERM domains of mented with 10% glycerol. After a few minutes, they were flash other proteins. In fact, the six best conserved FERM frozen in an N2 stream. residues (Figure 4) (Girault et al., 1999) are buried in the MAD data were collected at the Cornell High Energy Synchrotron structure, consistent with the conservation of the fold Source (CHESS) beamline F2 using an ADSC CCD detector and throughout the band 4.1 superfamily. Although there are processed with MOSFLM (Leslie, 1993) and SCALA (Collaborative three structural domains (lobes F1, F2, and F3) making Computational Project, Number 4, 1994). The space group was origi- nally designated as P2221 with unit cell indices: a ϭ 54 A˚ ,bϭ up the FERM domain, two observations suggest that 112 A˚ , and c ϭ 153 A˚ . Normal structure factors for the anomalous the FERM domain functions as a single unit rather than scatterers were calculated by MADSYS (Hendrickson and Ogata, a collection of three separate modules. First, the linkers 1997), and Patterson correlation analysis using SHELX (Sheldrick between the three lobes are rather short (13 and 8 resi- et al., 1993) identified eight selenium positions that were used for dues), and their sequences are well-conserved in the protein phase calculation with MADSYS. The overall figure of merit ˚ ERM proteins, merlin, and band 4.1 (Figure 4). Second, was 0.444 to 2.5 A resolution. The resulting electron density map showed a clear protein–solvent boundary for two FERM/tail com- interacting side chains in this central region, including plexes in the asymmetric unit. After solvent flattening using PHASES those from the linkers and the domains, are very well (Furey and Swaminathan, 1997), one complex had much stronger conserved, and two of the aforementioned six highly electron density and its C␣ backbone was modeled. The main chain conserved FERM residues, Gln105 and Gly202, are in atoms were then built automatically using O (Jones et al., 1991). this core. Such interdomain interactions indicate that Modeling of side chains and subsequent rounds of manual fitting structural changes in one domain would impact and were carried out using CHAIN (Sack and Quiocho, 1997) and refine- ments were done in X-PLOR (Bru¨ nger, 1996). The eight selenium possibly destabilize the other domains. This conserved positions found during phasing all corresponded to methionines in central core of residues provides evidence that the this first complex. The electron density for the second molecule, FERM domain remains structurally rigid upon activation, which was related to the first by a pseudo 2-fold axis parallel to c, Cell 268

was initially difficult to interpret. After several rounds of fitting and Bretscher, A. (1999). Regulation of cortical structure by the ezrin- simulated annealing refinements, difference Fourier maps revealed radixin-moesin protein family. Curr. Opin. Cell Biol. 11, 109–116. peaks that represented 16 selenium positions for the second mole- Bru¨ nger, A.T. (1996). X-PLOR Version 3.8, Department of Molecular cule in an orientation that suggested two overlapping half-occupied Biophysics and Biochemistry, Yale University, New Haven, CT. complexes separated by 5 A˚ in a direction perpendicular to the c Chishti, A.H., Kim, A.C., Marfatia, S.M., Lutchman, M., Hanspal M., axis. These ghost images led us to suspect that the space group Jindal, H., Liu, S.C., Low, P.S., Rouleau, G.A., Mohandas, N., et al. was misassigned, with a pseudo 21-screw axis being assigned as (1998). The FERM domain: a unique module involved in the linkage crystallographic and a crystallographic 2-fold axis being assigned of cytoplasmic proteins to membrane. Trends Biochem. Sci. 23, as pseudo. This problem was corrected by changing the space 281–282. group from P222 to the nonstandard P22 2. After the unit cell axes 1 1 Collaborative Computational Project, No. 4. (1994). The CCP4 suite: were permuted to convert the diffraction data and the model to programs for protein crystallography. Acta Crystallogr. 50, 760–763. the standard P2221 (with unit cell indices: a ϭ 54 A˚ ,bϭ 153 A˚ , c ϭ112 A˚ ), the electron density for the second complex immediately Doi, Y., Itoh, M., Yonemura, S., Ishihara, S., Takano, H., Noda, T., improved. After this problem was resolved, crystallographic refine- and Tsukita, S. (1999). Normal development of mice and unimpaired ment was continued against a more complete 1.9 A˚ resolution data cell adhesion/cell motility/actin-based cytskeleton without compen- set collected from two crystals at CHESS beamlines F1 and F2. satory up-regulation of ezrin or radixin in moesin gene knockout. J. Refinement included tight noncrystallographic symmetry restraints Biol. Chem. 274, 2315–2321. on C␣ positions. Dransfield, D.T., Bradford, A.J., Smith, J., Martin, M., Roy, C., There are two FERM/tail complexes in the asymmetric unit, with Mangeat, P.H., and Goldenring, J.R. (1997). Ezrin is a cyclic AMP- a root-mean-square (rms) deviation of 0.05 A˚ for C␣ positions. The dependent protein kinase anchoring protein. EMBO J. 16, 35–43. current model includes residues 4–297 and 488–577 for each com- Fedorov, A.A., Federov, E., Gertler, F., and Almo, S.C. (1999). Struc- plex, as well as 412 water molecules and 2 sulfate ions. Unusual ture of EVH1, a novel proline-rich ligand-binding module involved geometries in well-ordered regions of the structure include a cis- in cytoskeletal dynamics and neural function. Nat. Struct. Biol. 6, Pro at residue 75 and main chain dihedrals of φ ϭ 66Њ, ␺ϭϪ104Њ 661–666. at Asp252. Mobile segments whose conformational details are less Fersht, A. (1999). Structure and Mechanism in Protein Science: A reliable are residues 21–23, 260–263, and 495–501. Due to weak Guide to Catalysis and Protein Folding (New York: W. H. electron density, the following surface residues are modeled as Ala Freeman and Co.), pp. 345–346. in both complexes: Lys64, Asp69, Lys72, Lys139, Arg495, Asp497, Ferguson, K.M., Lemmon, M.A., Schlessinger, J., and Sigler, P.B. Met499, Glu516, and Lys517. (1995). Structure of the high affinity complex of inositol trisphos- phate with a phospholipase C pleckstrin homology domain. Cell 83, Structure Analysis 1037–1046. Structural similarities were determined using DALI (Holm and Sander, 1993). Lobe F1 was most similar to the structure of ubiquitin Forman-Kay, J.D., and Pawson, T. (1999). Diversity in protein recog- (Vijay-Kumar et al., 1987) (PDB code 1UBI; rms deviation of 1.7 A˚ nition by PTB domains. Curr. Opin. Struct. Biol. 9, 690–695. between 70 C␣ positions and Z score ϭ 10.2). Lobe F2 was most Funayama, N., Nagafuchi, A., Sato, N., Tsukita, S., and Tsukita, S. similar to the acyl-CoA binding protein (Kragelund et al., 1993) (PDB (1991). Radixin is a novel member of the band 4.1 family. J. Cell code 1ACA; rms deviation of 2.7 A˚ for 74 C␣ atoms and Z score ϭ Biol. 115, 1039–1048. 7.6). Lobe F3 was most similar to the phosphotyrosine binding do- Furey, W., and Swaminathan, S. (1997). PHASES-95: a program main of insulin substrate 1 (Zhou et al., 1996) (PDB code package for processing and analyzing diffraction data from macro- 1IRS; rms deviation of 2.2 A˚ for 91 C␣ atoms and Z score ϭ 12.1). molecules. Methods Enzymol. 277, 590–620. Accessible surface areas were calculated using the program Gary, R., and Bretscher, A. (1995). Ezrin self-association involves AREAIMOL (Collaborative Computational Project, No. 4, 1994). binding of an N-terminal domain to a normally masked C-terminal domain that includes the F-actin binding site. Mol. Biol. Cell 6, 1061– Acknowledgments 1075. Girault, J.A., Labesse, G., Mornon, J.P., and Callebaut, I. (1998). This work was supported by NIH grant GM36652, including a supple- Janus kinases and focal adhesion kinases play in the 4.1 band: A ment to specifically support structural studies. M. A. P. was sup- superfamily of band 4.1 domains important for cell structure and ported by NIH Training Grant GM08500. This work is based upon signal transduction. Molec. Med. 4, 751–769. research conducted at the Cornell High Energy Synchrotron Source Girault, J.A., Labesse, G., Mornon, J.P., and Callebaut, I. (1999). The (CHESS), which is supported by NSF award DMR-9311772, using the N-termini of FAK and JAKs contain divergent band 4.1 domains. Macromolecular Diffraction at CHESS (MacCHESS) facility, which is Trends Biochem. Sci. 24, 54–57. supported by NIH award RR-01646. We would like to thank the members of the CHESS and MacCHESS staff for their help in data Gould, K.L., Cooper, J.A., Bretscher, A., and Hunter, T. (1986). The collection and processing. We would also like to thank James Hurley protein- substrate, p81, is homologous to a chicken (NIH) and Jeffrey Roberts (Cornell) for critically reading the manu- microvillar core protein. J. Cell Biol. 102, 660–669. script. Gronholm, M., Sainio, M., Zhao, F., Heiska, L., Vaheri, A., and Carpen, O. (1999). Homotypic and heterotypic interaction of the neurofibro- Received October 22, 1999; revised March 17, 2000. matosis 2 tumor suppressor protein merlin and the ERM protein ezrin. J. Cell Sci. 112, 895–904. References Gusella, J.F., Ramesh, V., MacCollin, M., and Jacoby, L.B. (1999). Merlin: the neurofibromatosis 2 tumor suppressor. Biochim. Bio- Bakolista, C., De Pereda, J.M., Bagshaw, C.R., Critchley, D.R., and phys. Acta 1423, M29-M36. Liddington, R.C. (1999). Crystal structure of the vinculin tail suggests Heiska, L., Alfthan, K., Gronholm, M., Vilja, P., Vaheri, A., and Carpen, a pathway for activation. Cell 99, 603–613. O. (1998). Association of ezrin with intercellular adhesion molecule-1 Berryman, M., Franck, Z., and Bretscher, A. (1993). Ezrin is concen- and -2 (ICAM-1 and ICAM-2). Regulation by phosphatidylinositol trated in the apical microvilli of a wide variety of epithelial cells, 4,5-bisphosphate. J. Biol. Chem. 273, 21893–21900. whereas moesin is found primarily in endothelial cells. J. Cell Sci. Helander, T.S., Carpen, O., Turunen, O., Kovanen, P.E., Vaheri, A., 105, 1025–1043. and Timonen, T. (1996). ICAM-2 redistributed by ezrin as a target Borg, J.P., and Margolis, B. (1998). Function of PTB Domains. Curr. for killer cells. Nature 382, 265–268. Top. Microbiol. Immun. 228, 23–38. Hendrickson, W.A., and Ogata, C.M. (1997). Phase determination Bretscher, A. (1983). Purification of an 80,000-dalton protein that from multiwavelength anomalous diffraction measurements. Meth- is a component of the isolated cytoskeleton, and its ods Enzymol. 276, 494–523. localization in nonmuscle cells. J. Cell Biol. 97, 425–432. Hirao, M., Sato, N., Kondo, T., Yonemura, S., Monden, M., Sasaki, Structure and Masking of Human Moesin 269

T., Takai, Y., Tsukita, S., and Tsukita, S. (1996). Regulation mecha- Wittinghofer, A. (1995). The 2.2 A˚ crystal structure of the Ras-binding nisms of ERM (ezrin/radixin/moesin) protein/plasma membrane as- domain of the serin/threonine kinase c-Raf1 in complex with Rap1A sociation: possible involvement of phosphatidylinositol turnover and and a GTP analogue. Nature 375, 554–560. Rho-dependent signaling pathway. J. Cell Biol. 135, 37–51. Niggli, V., Andreoli, C., Roy, C., and Mangeat, P. (1995). Identification Holm, L., and Sander, C. (1993). Protein structure comparison by of a phosphatidylinositol-4,5-bisphosphate-binding domain in the alignment of distance matrices. J. Mol. Biol. 233, 123–138. N-terminal region of ezrin. FEBS Lett. 376, 172–176. Honig, B. and Nicholls, A. (1995). Classical electrostatics in biology Oshiro, N., Fukata, Y., and Kaibuchi, K. (1998). Phosphorylation of and chemistry. Science 268, 1144–1149. moesin by rho-associated kinase (Rho-kinase) plays a crucial role Huang, L., Ichimaru, E., Pestonjamasp, K., Cui, X., Nakamura, H., in the formation of microvilli-like structures. J. Biol. Chem. 273, Lo, G.Y., Lin, F.I., Luna, E.J., and Furthmayr, H. (1998). Merlin differs 34663–34666. from moesin in binding to F-actin and in its intra- and intermolecular Pakkanen, R., and Vaheri, A. (1989). Cytovillin and other microvillar interactions. Biochem. Biophys. Res. Commun. 248, 548–553. proteins of human choriocarcinoma cells. J. Cell. Biochem. 41, 1–12. Huang, L., Wong, T.Y., Lin, R.C., and Furthmayr, H. (1999). Replace- Pestonjamasp, K., Amieva, M.R., Strassel, C.P., Nauseef, W.M., ment of threonine 558, a critical site of phosphorylation of moesin Furthmayr, H., and Luna, E.J. (1995). Moesin, ezrin, and p205 are in vivo, with aspartate activates F-actin binding of moesin. Regula- actin-binding proteins associated with neutrophil plasma mem- tion by conformational change. J. Biol. Chem. 274, 12803–12810. branes. Mol. Biol. Cell 6, 247–259. Hyvo¨ nen, M., Macias, M.J., Nilges, M., Oschkinat, H., Saraste, M., Pietromonaco, S.F., Simons, P.C., Altman, A., and Elias, L. (1998). and Wilmanns, M. (1995). Structure of the binding site for inositol Protein kinase C-theta phosphorylation of moesin in the actin-bind- phosphates in a PH domain. EMBO J. 14, 4676–4685. ing sequence. J. Biol. Chem. 273, 7594–7603. Johnson, R.P., and Craig, S.W. (1994). An intramolecular association Prehoda, K.E., Lee, D.J., and Lim, W.A. (1999). Structure of the between the head and tail domains of vinculin modulates talin bind- enabled/VASP homology domain-peptide complex: a key compo- ing. J. Biol. Chem. 269, 12611–12619. nent in the spatial control of actin assembly. Cell 97, 471–480. Johnson, R.P., and Craig, S.W. (1995). F-actin binding site masked Reczek, D., Berryman, M., and Bretscher, A. (1997). Identification by the intramolecular association of vinculin head and tail domains. of EBP50: A PDZ-containing phosphoprotein that associates with Nature 373, 261–264. members of the ezrin-radixin-moesin family. J. Cell Biol. 139, Jones, T.A., Zou, J.Y., Cowan, S.W., and Kjeldgaard, M. (1991). 169–179. Improved methods for building protein models in electron density Reczek, D., and Bretscher, A. (1998). The carboxyl-terminal region maps and the location of errors in these models. Acta Crystallogr. of EBP50 binds to a site in the amino-terminal domain of ezrin that is 47, 110–119. masked in the dormant molecule. J. Biol. Chem. 273, 18452–18458. Kabsch, W., and Sander, C. (1983). Dictionary of protein secondary Rouleau, G.A., Merel, P., Lutchman, M., Sanson, M., Zucman, J., structure: pattern recognition of hydrogen-bonded and geometrical Marineau, C., Hoang-Xuan, K., Demczuk, S., Desmaze, C., Plougas- features. Biopolymers 22, 2577–2637. tel, B. et al. (1993). Alteration in a new gene encoding a putative Kragelund, B.B., Anderson, K.V., Madsen, J.C., Knudsen, J., and membrane-organizing protein causes neuro-fibromatosis type 2. Poulsen, F.M. (1993). Three-dimensional structure of the complex Nature 363, 515–521. between acyl-coenzyme A binding protein and palmitoyl-coenzyme Sack, J.S., and Quiocho, F.A. (1997). CHAIN: a crystallographic mod- A. J. Mol. Biol. 230, 1260–1277. eling program. Methods Enzymol. 277, 158–173. Kraulis, P. (1991). MOLSCRIPT: a program to produce both detailed Serrador, J.M , Alonso-Lebrero, J.L., del Pozo, M.A., Furthmayr, H., and schematic plots of protein structures. J. Appl. Crystallogr. 24, Schwartz-Albiez, R., Calvo, J., Lozano, F., and Sanchez-Madrid, F. 946–950. (1997). Moesin interacts with the cytoplasmic region of intercellular LaJeunesse, D.R., McCartney, B.M., and Fehon, R.G. (1998). Struc- adhesion molecule-3 and is redistributed to the uropod of T lympho- tural analysis of Drosophila merlin reveals functional domains impor- cytes during cell polarization. J. Cell Biol. 138, 1409–1423. tant for growth control and subcellular localization. J. Cell Biol. 141, Sheldrick, G.M., Dauter, Z., Wilson, K.S., Hope, H., and Sieker, L.C. 1589–1599. (1993). The application of direct methods and Patterson interpreta- Lankes, W.T. and, Furthmayr, H. (1991). Moesin: a member of the tion to high-resolution native protein data. Acta Crystallogr. 49, protein 4.1-talin-ezrin family of proteins. Proc. Natl. Acad. Sci. USA 18–23. 88, 8297–8301. Sherman, L., Xu, H.M., Geist, R.T., Saporito-Irwin, S., Howells, N., Leslie, A. (1993). Data Collection and Processing. In Proceedings Ponta, H., Herrlich, P., and Gutmann, D.H. (1997). Interdomain bind- of the CCP4 Study Weekend, January 29-30, 1993, compiled by L. ing mediates tumor growth suppression by the NF2 gene product. Sawyer, N. Isaac, S. Bailey, pp. 44–51. Oncogene 15, 2505–2509. Matsui, T., Maeda, M., Doi, Y., Yonemura, S., Amano, M., Kaibuchi, Simons, P.C., Pietromonaco, S.F., Reczek, D., Bretscher, A., and K., Tsukita, S., and Tsukita, S. (1998). Rho-kinase phosphorylates Elias, L. (1998). C-terminal threonine phosphorylation activates ERM COOH-terminal threonines of ezrin/radixin/moesin (ERM) proteins and proteins to link the cell’s cortical lipid bilayer to the cytoskeleton. regulates their head-to-tail association. J. Cell Biol. 140, 647–657. Biochem. Biophys. Res. Commun. 253, 561–565. Matsui, T., Yonemura, S., Tsukita, S., and Tsukita, S. (1999). Activa- Stebbins, C.E., Kaelin, W.G., Jr., and Pavletich, N.P. (1999). Structure tion of ERM proteins in vivo by Rho involves phosphatidyl-inositol of the VHL-ElonginC-ElonginB complex: implications for VHL tumor 4-phosphate 5-kinase and not ROCK kinases. Curr. Biol. 9, 1259–1262. suppressor function. Science 284, 455–461. Meng, W., Sawasdikosol, S., Burakoff, S.J., and Eck, M.J. (1999). Takahashi, K., Sasaki, T., Mammoto, A., Takaishi, K., Kameyama, Structure of the amino-terminal domain of Cbl complexed to its T., Tsukita, S., and Takai, Y. (1997). Direct interaction of the Rho binding site on ZAP-70 kinase. Nature 398, 84–90. GDP dissociation inhibitor with ezrin/radixin/moesin initiates the ac- Merritt, E.A., and Bacon, D.J. (1997). Raster3D: photorealistic molec- tivation of the Rho small G protein. J. Biol. Chem. 272, 23371–23375. ular graphics. Methods Enzymol. 277, 505–524. Takeuchi, K., Sato, N., Kasahara, H., Funayama, N., Nagafuchi, A., Nakamura, F., Amieva, M.R., and Furthmayr, H. (1995). Phosphoryla- Yonemura, S., Tsukita, S., and Tsukita, S. (1994). Perturbation of tion of threonine 558 in the carboxyl-terminal actin-binding domain cell adhesion and microvilli formation by antisense oligonucleotides of moesin by thrombin activation of human platelets. J. Biol. Chem. to ERM family members. J. Cell Biol. 125, 1371–1384. 270, 31377–31385. Thompson, J.D., Higgins, D.G., and Gibson, T.J. (1994). CLUSTAL Nakamura, F., Huang, L., Pestonjamasp, K., Luna, E.J., and W: improving the sensitivity of progressive multiple sequence align- Furthmayr, H. (1999). Regulation of F-actin binding to platelet moe- ment through sequence weighting, position-specific gap penalties sin in vitro by both phosphorylation of threonine 558 and polyphos- and weight matrix choice. Nucleic Acids Res. 22, 4673–4680. phatidylinositides. Mol. Biol. Cell 10, 2669–2685. Trofatter, J.A., MacCollin, M.M., Rutter, J.L., Murrell, J.R., Duyao, Nassar, N., Horn, G., Herrmann, C., Scherer, A., McCormick, F., and M.P., Parry, D.M., Eldridge, R., Kley, N., Menon, A.G., Pulaski, K. et Cell 270

al. (1993). A novel moesin-, ezrin-, radixin-like gene is a candidate for the neurofibromatosis 2 tumor suppressor. Cell 72, 791–800. Tsukita, S., Hieda, Y., and Tsukita, S. (1989). A new 82-kD barbed end-capping protein (radixin) localized in the cell-to-cell adherens junction: purification and characterization. J. Cell Biol. 108, 2369– 2382. Tsukita, S., Oishi, K., Sato, N., Sagara, J., Kawai, A., and Tsukita, S. (1994). ERM family members as molecular linkers between the cell surface glycoprotein CD44 and actin-based . J. Cell Biol. 126, 391–401. Turunen, O., Wahlstrom, T., and Vaheri, A. (1994). Ezrin has a COOH- terminal actin-binding site that is conserved in the ezrin protein family. J. Cell Biol. 126, 1445–1453. Turunen, O., Sainio, M., Jaaskelainen, J., Carpen, O., and Vaheri, A. (1998). Structure-function relationships in the ezrin family and the effect of tumor-associated point mutations in neurofibromatosis 2 protein. Biochim. Biophys. Acta 1387, 1–16. Vijay-Kumar S., Bugg, C.E., and Cook, W.J. (1987). Structure of ubiquitin refined at 1.8 A˚ resolution. J. Mol. Biol. 194, 531–544. Yonemura, S., Hirao, M., Doi, Y., Takahashi, N., Kondo, T., Tsukita, S., and Tsukita, S. (1998). Ezrin/radixin/moesin (ERM) proteins bind to a positively charged amino acid cluster in the juxta-membrane cytoplasmic domain of CD44, CD43, and ICAM-2. J. Cell Biol. 140, 885–895. Yun, C.H., Lamprecht, G., Forster, D.V., and Sidor, A. (1998). NHE3 kinase A regulatory protein E3KARP binds the epithelial brush bor- der Naϩ/Hϩ exchanger NHE3 and the cytoskeletal protein ezrin. J. Biol. Chem. 273, 25856–25863. Zhou, M.M., Huang, B., Olejniczak, E.T., Meadows, R.P., Shuker, S.B., Miyazaki, M., Trub, T., Shoelson, S.E., and Fesik, S.W. (1996). Structural basis for IL-4 receptor phosphopeptide recognition by the IRS-1 PTB domain. Nat. Struct. Biol. 3, 388–393.

Protein Data Bank ID Code

The coordinates and structure factors for the structure reported in this paper have been deposited in the Protein Data Bank under ID code 1EF1.