Crystal Structure of Rab11 in Complex with Rab11 Family Interacting Protein 2

William N. Jagoe,1 Andrew J. Lindsay,2 restricted to epithelial cells (Goldenring et al., 1993). Randy J. Read,3 Airlie J. McCoy,3 The Rab11 proteins regulate the endosomal recycling Mary W. McCaffrey,2 and Amir R. Khan1,* transport of vesicular cargo containing transferrin recep- 1 School of Biochemistry and Immunology tors (Ullrich et al., 1996; Wilcke et al., 2000; Prekeris et al., Trinity College 2000; Lindsay and McCaffrey, 2002), the chemokine Dublin 2 receptor CXCR2 (Fan et al., 2004), and polymeric IgA Ireland receptors (Wang et al., 2000). More recently, Rab25 has 2 Molecular Cell Biology Laboratory been shown to determine the aggressiveness of breast Department of Biochemistry and ovarian cancers, and its expression has been linked Biosciences Institute to tumorigenesis (Cheng et al., 2004, 2006). University College Cork The biological effects of Rabs are elicited by their GTP Cork bound conformation through interactions with effector Ireland proteins. The structures of Rabs have a common G pro- 3 Department of Haematology tein fold with a central six-stranded (mixed) b sheet University of Cambridge flanked by a helices on both sides. The nucleotide state Cambridge Institute for Medical Research of Rabs affects the local conformation of a pair of highly Wellcome Trust/MRC Building conserved regions termed switch 1 and switch 2 (Vetter Hills Road and Wittinghofer, 2001). The crystal structures of Rab3- Cambridge, CB2 2XY rabphilin, Rab4-rabenosyn5, Rab5-rabaptin5, Rab7-RILP, United Kingdom and Rab22-rabenosyn5 have been determined (Oster- meier and Brunger, 1999; Zhu et al., 2004; Wu et al., 2005a; Eathiraj et al., 2005), and both switch 1 and switch Summary 2 have well-defined electron density in these structures. Switch 1 and/or switch 2 contribute to interactions with The small GTPase Rab11 regulates the recycling of the Rab binding domain (RBD) of effectors, which are endosomes to the plasma membrane via interactions generally a-helical in conformation (Kawasaki et al., with the Rab11 family of interacting proteins (FIPs). 2005). However, the orientation of the helices, the oligo- FIPs contain a highly conserved Rab binding domain meric states of effectors, and detailed interactions with (RBD) at their C termini whose structure is unknown. Rabs are unique in all of these complexes. An emerging Here, we have determined the crystal structure of the theme in Rab recognition is the exploitation of noncon- RBD of FIP2 in complex with Rab11(GTP) by single served residues combined with structural diversity in wavelength anomalous diffraction methods. The over- conserved regions (switch 1, switch 2, interswitch) to all structure is a heterotetramer with dyad symmetry, achieve selective Rab binding by effectors (Pfeffer, arranged as a Rab11-(FIP2)2-Rab11 complex. FIP2 2005; Eathiraj et al., 2005; Merithew et al., 2001). forms a central a-helical coiled coil, with both helices In recent years, a novel set of effectors termed the contributing to the Rab11 binding patch on equivalent Rab11 family of interacting proteins (hereafter abbrevi- and opposite sides of the homodimer. Switch 1 of ated as FIPs) that contain a highly conserved C-terminal Rab11 is embedded between the two helices, while RBD has been identified (Prekeris et al., 2000, 2001; switch 2 remains flexible and is peripherally associ- Hales et al., 2001; Lindsay and McCaffrey, 2004b; Lind- ated with the effector. The complex reveals the struc- say et al., 2002). In contrast to their RBDs, FIPs are di- tural basis for Rab11 recognition by FIPs and suggests verse in sequence length and composition toward their the molecular mechanisms underlying endocytic recy- N termini, presumably a feature that underpins their spe- cling pathways. cific roles in Rab11-mediated vesicle trafficking. Rip11, FIP2, and RCP all contain C2 domains toward their N Introduction termini and are categorized as class I FIPs. They are predominantly localized to the endocytic recycling com- The Rab family of small GTPases, which contains nearly partment (ERC), and their C2 domains have recently 70 proteins, constitutes the largest member of the Ras been observed to participate in this localization through superfamily (Bock et al., 2001; Pfeffer, 2005). Rabs are interactions with lipid bilayers enriched in anionic phos- anchored to lipid bilayers via C-terminal prenylation pholipids (Lindsay and McCaffrey, 2004a). The class II sites at cysteine residues, and they regulate various as- FIPs, FIP3 and FIP4, possess an ERM (ezrin-radixin- pects of membrane dynamics, including organelle struc- moesin) domain, EF hands, and a proline-rich region, ture, vesicle motility, docking, and fusion (Zerial and and they are found in the ERC, the trans-Golgi network, McBride, 2001). The Rab11 subfamily comprises three and centrosomes. Class II proteins interact with ADP ri- isoforms—Rab11a, Rab11b, and Rab25. Rab11a and bosylation factor (ARF) GTPases (Hickson et al., 2003), Rab11b are found in most tissue (Goldenring et al., allowing for potential crosstalk between the two signal- 1996; Lapierre et al., 2003), while Rab25 expression is ing pathways. Recent studies identified a role for FIP3 and FIP4 in endosomal trafficking to the cleavage furrow during cytokinesis via interactions with Rab11 and Arf6 *Correspondence: [email protected] (Horgan et al., 2004; Fielding et al., 2005). FIP1 lacks Structure 1274

both a C2 domain and EF hands, and it belongs to nei- Table 1. Data Collection and Reﬁnement Statistics ther the class I nor the class II family (Wallace et al., 2002; Hales et al., 2001). Orthorhombic Trigonal

Here, we have determined the structure of Rab11 with Space group P212121 P3121 the RBD of FIP2 in two different crystal forms. FIP2 is a Cell dimensions (A˚ ) 64.4, 91.1, 113.1 64.7, 64.7, 112.4 512 residue protein that contains a C2 domain at the N Wavelength (A˚ , Se peak) 0.97865 0.98175 ˚ terminus (residues 1–129), a myosin Vb binding region Resolution (A) 50–2.44 50–2.47 Completeness (%) 99.7 (98.5) 99.8 (100) (129–290), and an RBD at the C terminus (477–496), Rmerge (%) 10.2 (42.1) 6.7 (29.7) previously predicted to form an amphipathic a helix I/s(I) all data 8.8 16.3 (15.9) (Wallace et al., 2002; Lindsay and McCaffrey, 2004b; I/s(I) > 3 (% of data) 51.5 66.3 Junutula et al., 2004; Meyers and Prekeris, 2002). FIP2 Redundancy 13 (10) 16 (10) protein has been found to be essential for the recycling Refinement Statistics of vesicles bearing the chemokine receptor CXCR2 back Resolution (A˚ ) 50–2.44 50–2.47 to the plasma membrane (Fan et al., 2004). A ternary Number of reflections 25,444 10,206 complex of Rab11-FIP2-myosin Vb may provide the Models Rab11 Rab11 link between endosomes and the cytoskeleton to regu- (Glu7–Tyr173) (Asp6–Tyr173) late the delivery of vesicular cargo to the plasma mem- FIP2 FIP2 brane. This interaction would form the molecular basis (Arg468–Pro502) (Gly447–Ser503) for recruitment of leukocytes to the site of inflammation. FIP2 (Leu466–Pro502) The crystal structure of the Rab11-FIP2 complex reveals Rwork/Rfree (%) 22.6/27.9 19.6/25.3 that both a helices of the central helical dimer of FIP2 High-resolution shell (27.4/29.7) (22.2/33.0) contribute to the Rab11 binding patch, thus forming a Number of nonhydrogen 3,407 2,011 2-fold symmetric Rab11-(FIP2)2-Rab11 complex. Switch atoms 1 is embedded between the two a helices of FIP2, while Number of proteins 3,300 1827 switch 2 retains significant flexibility and reveals unprec- Number of GTP, ions 34 39 solute edented conformational changes from its unbound Number of waters 78 155 (GTP) conformation, as well as between the two crystal Average B factor (A˚ 2) forms in the Rab11-FIP2 complex. At the C-terminal Protein 39.2 47.6 half of the RBD, the a helix terminates and the polypep- Backbone 38.6 43.9 Side chain 40.2 47.1 tide adopts a 310 helix and a short b strand conformation that is perpendicular to the a helix and that packs against GTP 34.6 36.5 Mg2+ 29.3 36.3 b2 of Rab11. Finally, in a trigonal crystal form, the a-heli- Water 38.2 51.9 cal portion of the RBD is extended 20 residues further Rms deviations in the N-terminal direction relative to orthorhombic Bond lengths (A˚ ) 0.012 0.016 crystals. The conformational heterogeneity observed in Bond angles () 1.35 1.83 Rab11 and FIP2 likely reflects the dynamic nature of Coordinate error (ESU) ˚ ˚ Rab-effector association in cells, and it provides insight Based on Rfree 0.27 A 0.30 A into the molecular basis for endosomal trafficking Values in parentheses correspond to the statistics for the highest- pathways. resolution shell; orthorhombic = 2.53–2.44 A˚ , trigonal = 2.56–2.47 A˚ .

Results and Discussion Rab11 and one FIP2 molecule. In the P212121 space group, the symmetry is broken and the helical axis be- Overview of Rab11-FIP2 Crystals comes a noncrystallographic dyad (180.0) that lies 4 Rab11a (1–173) and FIP2 (410–512) were coexpressed away from the crystallographic c axis. Thus, the asym- in E. coli and purified as a complex. Rab11 contained metric unit consists of two molecules each of Rab11 the Q70L substitution to favor the GTP form, and the and FIP2 in the orthorhombic space group. The amino subsequent structure revealed that endogenous GTP acid segments of Rab11 and FIP2 included in the refined had been incorporated during expression and purifica- models are indicated in Table 1. Since a significant portion. Light scattering coupled with gel filtration showed tion of the expressed FIP2 polypeptide (410–446 in trigo- that FIP2 alone was dimeric (Figure S1; see the Supple- nal; 410–468 in orthorhombic) was not seen in electron mental Data available with this article online), and the density maps (see Experimental Procedures), we per- mass of the Rab11-FIP2 complex was consistent with formed N-terminal amino acid sequencing of dissolved two molecules of Rab11 and two molecules of FIP2 crystals to confirm that these regions were indeed pres- (Jagoe et al., 2006). Crystals of the complex appeared ent and not proteolyzed during purification and crystalli- in orthorhombic and trigonal forms under similar growth zation (data not shown). conditions and had related cell parameters (Table 1). As the complex has 2-fold symmetry, subsequent The overall complex is organized as a Rab11-(FIP2)2- descriptions of the structure will address one of the pro- Rab11 oligomer in both crystals, and the centrally lo- tomers of Rab11 and its interface with the FIP2 homo- cated FIP2 in the crystals is a parallel, helical coiled coil dimer. Where necessary, the FIP2 protomers will be dis- that recruits Rab11 on both sides in a symmetric fashion tinguished by using the superscript suffixes ‘‘d’’ and ‘‘e,’’

(Figure 1). In the trigonal space group P3121, the two bearing in mind that the chains are related by a 2-fold molecules of FIP2 and Rab11 are related by the crystal- axis. Discussions of the structure of Rab11-FIP2 will lographic dyad that runs down the central axis of the be conﬁned mainly to the trigonal model since the qual- coiled-coil; thus, the asymmetric unit consists of one ity of data was superior. However, the orthorhombic Structure of Rab11-FIP2 1275

Figure 2. Stereoview of the Electron Density around a Section of the Rab11-FIP2 Interface in the Orthorhombic Space Group

The 2Fo 2 Fc map is contoured at 1.3 times the rms density. The g- phosphate of GTP is labeled, Mg2+ is a blue sphere, waters are red, and hydrogen bonds are indicated by dashed lines. The Rab binding interface is formed by residues from both a helices of the FIP2 homodimer (‘‘d’’ and ‘‘e’’ sufﬁxes in superscript notation).

‘‘L’’ (Figures 1 and 2). The parallel dimer of FIP2 is formed Figure 1. Ribbon Model of the Rab11-FIP2 Complex by the crystallographic 2-fold axis, and the interface (Top) Rab11 molecules are yellow and magenta, while FIP2 is col- consists mainly of hydrophobic residues that are under- ored dark pink and green. Switch 1 and switch 2 are indicated, lined in Figure 3. The side chain of Glu455 (conserved 2+ GTP is represented as a stick model, and the conserved Mg ion in all FIPs) hydrogen bonds with the backbone NH of is drawn as a sphere. The short 3 helix at the C terminus of FIP2 10 Thr452, thus capping the ﬁrst turn of the a helix. The hy- (green) is also labeled. (Bottom) View of Rab11-FIP2 rotated 90 in order to show the 2-fold b axis in the crystal. The N and C termini droxyl group of Tyr453 hydrogen bonds to the carbonyl of each FIP2 molecule hug their symmetrically oriented partner. oxygen of Leu451, and its phenyl ring forms the lid of a hydrophobic pocket together with Val456, one turn down the helical axis. In orthorhombic crystals, this region of crystal with a Rab11-(FIP2)2-Rab11 complex as the asymmetric unit allows an opportunity to analyze con- the FIP2 is disordered and is not seen in electron density formational changes that may be relevant to function, maps, and the model begins at Arg468. However, given and these differences will be discussed. that the helix-capping residues from Thr452–Glu455 are highly conserved in FIPs (Figure 3), it is likely that an elongated a helix plays a structural role in recycling Structure of the FIP2 Homodimer pathways under cellular conditions. In support of our The structure of FIP2 in trigonal crystals reveals a 40 view, trigonal crystals grew in lower salt conditions residue, gently curving a helix from residue Thr452 to (100 mM (NH4)3PO4), while higher salt levels (500 mM) residue Thr492, followed by a turn, a 310 helix, a short favored orthorhombic crystals. Several interactions in- b strand, and a loop—a shape that resembles the letter volving ionizable side chains are present in this region,

Figure 3. Sequence Alignment of FIPs and the Corresponding Secondary Structure Elements

The long a helix and the short 310 helix are represented as cylinders, and the short segment that forms parallel b sheet-like hydrogen bonds with b2 of Rab11 is represented by the arrow. The gray mask represents the extent of the RBD from the crystal structure. Asterisks highlight those residues that are unique in FIP2 and are therefore candidates for homodimer speciﬁcity. Underlined amino acids correspond to the dimer interface, and the numbering below the alignments corresponds to the sequence of FIP2. Structure 1276

though Arg497–Pro502 is also well ordered (see below), it is located in a loop region (Figure 4B) and is therefore susceptible to cleavage by trypsin. The C-terminal region of FIP2 is identical in both crystals and comprises the RBD, which runs from Glu476 to Val498 (Figure 4). The a helix terminates at a proline (Pro493d), which is conserved in all FIPs and which nucleates a 310 helix (roughly perpendicular to the a helix) and a loop that forms brief parallel b sheet-like interactions with b2 of Rab11 (see below). At this point, a second proline (Pro499d) in the cis conﬁguration reverses the polypeptide direction to orient the C terminus toward the second protomer and reinforce homodimer interactions. Pro499d is conserved only in class I FIPs and FIP1 (Figure 3), while the next residue (Tyr500d) is unique to FIP2 and contributes to the FIP2 dimer interface via a side chain hydrogen bond to Glu491e. In addition, the phenyl ring of Tyr500d lies below the guanidino group of Arg487e, suggesting cation-p interactions, while the opposite face of the ring stacks against Pro502d (Figure 4B).

Structure of the Rab11-FIP2 Complex The 23 residue RBD of FIP2 (Glu476–Val498) at the C-terminal half of the coiled coil is the most conserved in the FIP family, and it had previously been predicted to form a continuous amphipathic a helix (Lindsay and McCaf- frey, 2004b; Junutula et al., 2004; Meyers and Prekeris, 2002). However, the crystal structure of the RBD reveals

an a helix followed by a 310 helix and a b strand, roughly perpendicular to the a helical axis, that mediates both dimer formation and interactions with Rab11 (Figures 1–4). The Rab11 molecules do not interact with each other, consistent with a monomeric form of Rab11(GTP) (Pasqualato et al., 2004). The structure of the complex reveals that the binding interface is formed by both protomers in FIP2, which interact with switch 1 and switch 2, as well as b2 of Rab11. Switch 1 (Ile44–Val46) is buried between the two helices of FIP2, packing against Leu477d, Tyr480d, and Ile481d. In addition to packing, the phenolic side chain of Tyr480d makes a hydrogen Figure 4. Rab Binding Interfaces in the Complex bond with the backbone of Val46(N). Ile481 is within van der Waals distance of Gly45 (C ), and it is also in- (A) Switch 1 (yellow) interacts with both helices of FIP2 (green and a dark pink) at the ‘‘YID’’ consensus sequence. volved in intimate contacts at the homodimeric interface

(B) The 310 helix and the extended region of FIP2 (green) align with b2 of FIP2 that bridge the two Rab11 molecules (Figure 4). In of Rab11. The side chain of Tyr500 is unique to FIP2 and is sand- the overall Rab11-(FIP2)2-Rab11 heterotetramer, Tyr480 wiched between Arg487 and Pro502. The backbone amide nitrogen and Ile481 form an extended hydrophobic surface that ˚ of Val46 (switch 1) is within 3.6 A of Tyr480(OH) and is represented by is essential to stabilization of the complex (see mutagen- a blue sphere. A role for the three prolines (Pro493, Pro499, and esis studies, below). Pro502) in stabilizing the conformation of FIP2 in this region is apparent in this view. Interactions between Rab11 and FIP2 in the C-terminal part of the RBD (after Leu485) are conﬁned to one of the which may be weakened upon increase in salt concen- two protomers in the FIP2 homodimer (Figure 4B). tration (Table S1). The total surface area buried by the Met489d, which is substituted by selenomethionine in FIP2 dimer in trigonal crystals is 3620 A˚ 2, and this de- the structure, is a key residue at the interface of FIP2 creases to 1945 A˚ 2 in the orthorhombic structure. Circu- and Rab11 and is conserved in all FIPs except FIP4 lar dichroism (CD) spectroscopy of truncated molecules (Leu). The side chain of Met489d resides in a hydrophobic is also consistent with the trigonal FIP2 model in solution. pocket formed by Val46 (switch 1), Trp65, Ile76, and Constructs beginning at Gln458 were poorly structured, Ala79, and the latter two residues are localized to the he- while constructs beginning at Asp443 and Ser450 re- lical part of switch 2. As previously mentioned, Pro493d stored an a helix conformation (Wei et al., 2006). More- terminates the a helix and nucleates a single-turn 310 he- over, limited proteolysis of FIP2(Asp443–Ser512) by lix (Figure 4) that packs against the a helix, forming a trypsin resulted in cleavage after Arg449 and Arg497. hydrophobic pocket involving Val488d, Ile495d, and This segment corresponds exactly to the secondary Leu496d. This pocket extends into the FIP2 dimeric inter- d structure elements in trigonal crystals (Figure 3). Al- face, and Leu496 (from the 310 helix) also packs against Structure of Rab11-FIP2 1277

Arg74, which is mostly conserved in Rabs and makes a salt bridge with Asp482 of FIP2. However, the conformations of switch 2 in most other Rabs place the guanidino group of this residue in steric conflict with the effector. Thus, apart from Rab11, the conformations of switch 2 in the GTP state of mammalian Rabs are incompatible with binding to FIP2. Subtle changes in the conformation and position of recognition elements within conserved features of Rabs (such as the switches) have previously been identified as positive and negative determinants of specificity (Eathiraj et al., 2005; Meri- thew et al., 2001). In addition to switch 2, specificity may also be imparted by Lys41 (switch 1; salt bridge to Glu476) and Thr50 (hydrogen bonds to Arg497). Upon alignment, threonine is partly conserved in Rab sequences, but only Rab11 and Rab7 have both of these residues. How- ever, the conformation of switch 1 in Rab7 is subtly different due to the presence of a tyrosine (Tyr37 in Rab7 numbering) in place of Ser40 in Rab11. In order to avoid steric clashes with the nucleotide, a backbone rotation at Tyr37–Lys38 (Rab7) results in the shift of the lysine Figure 5. Structural Basis for Rab11 Specificity Encoded by the side chain 90 away from the equivalent Lys41 in Rab11. Switch 2 Conformation The structures of Rabs in their GTP and effector bound conformation were superimposed onto Rab11-FIP2. The coordinates were taken Comparisons with Unbound Rab11(GTP) from files 1ZBD, 1Z0K, 1TU3, and 1YHN in the Protein Data Bank. and Rab11(GDP) A distinctly nonhelical conformation of switch 2 (Sw2, yellow), posi- The structure of Rab11-FIP2 reveals unprecedented tioned toward a3, is observed in Rab11. The position of switch 1 conformational changes in switch 2 of Rab11(GTP) be- (Sw1) is also shown. The salt bridge between Arg74 and Asp482 is tween the unbound and FIP2 bound forms (Figure 6). Af- shown with dashes. ter least-squares superposition, the greatest difference occurs at Tyr73, whose side chain hydroxyls reside d 19 A˚ apart. The difference is attributable to a rearrange- Met489 . The 310 helix orients the polypeptide roughly perpendicular to the central b sheet of Rab11 (Figure 4B), ment of the loop Leu70–Ser78, placing this region closer a and two backbone hydrogen bonds take place between to 3. This conformational change is necessary for bind- residues Leu496d/Val498d of FIP2 and b2 of Rab11 ing to FIP2, otherwise residues Tyr73–Arg74 would (Phe48). In addition, there are hydrogen bonds between sterically overlap with the FIP2 helix (Arg475–Asp479). the side chains of Thr50 and Arg497d in this region When comparing the conformation of Rab11(GDP) (Figure 4B). The importance of the interactions in this alongside Rab11(GTP) and Rab11(GTP)-FIP2, switch 2 a C-terminal part of the RBD is underscored by recent follows a distinct trajectory toward 3. The result is studies of the FIP2 construct Ser50–Met489, which ter- that much of the switch 2 region in the Rab11-FIP2 complex remains free and exposed on either side of minates before the 310 helix. Despite maintaining the structural integrity of the a-helical coiled coil, this trun- the symmetric dimer. cated FIP2 fragment showed no appreciable association It should be emphasized that in both trigonal and or- with Rab11, as measured by isothermal titration calorim- thorhombic crystals, switch 2 is relatively disordered etry (Wei et al., 2006). after Gly69. This glycine precedes the (presumed) cata- lytic asparagine, and it makes a hydrogen bond through its backbone amide to the g-phosphate. Conformational Structural Basis for Rab11 Specificity heterogeneity was apparent when comparing the two The molecular basis for specificity of the FIP2 dimer for crystal forms, as well as the two molecules within the Rab11 is clarified by the structure of the complex (Fig- asymmetric unit of the orthorhombic crystal form (Fig- ure 5). The conformation of switch 2 is unique among ure 6B). However, switch 2 is clearly displaced toward mammalian members of the Rab family with known a3 in all models of the Rab11-FIP2 complex. Overall, three-dimensional structures. In the complex, switch 2 these observations suggest that switch 2 retains consid- has moved away from switch 1 and toward a3, thus erable flexibility in the GTP bound form, which is thus far allowing an intimate complex to form between switch 1 unique among known structures of Rab-effector com- and the FIP2 helices. The flexibility of switch 2 and its plexes. All other Rabs display minor conformational lack of helical content was evident in the Rab11(GTP) changes of their side chains and backbones in order to structure, and Cherfils and colleagues suggested that accommodate effector binding, and these binding reac- one possible reason for this is the paucity of switch 1- tions can essentially be considered as rigid docks of switch 2 interactions in the GTP form (Pasqualato preformed switch and interswitch regions. et al., 2004). Upon molecular modeling of other Rabs in The conformation and position of switch 1 in Rab11- complex with FIP2, we have observed that switch 2 un- FIP2 also differs from those in unbound Rab11(GTP), al- dergoes steric repulsions with the FIP2 helix within the though in a more subtle manner. The Ser40 position region of F475–D482 (Figure 5). One key example is shifts toward GTP, and the side chain forms a hydrogen Structure 1278

phate oxygen as Ser42. Overall, the three serine residues that were pointing away from Rab11(GTP) are found to be involved in hydrogen bonds with the nucleotide in the Rab11-FIP2 complex (Figure 6A). The preference for Rab11(GTP) relative to the GDP form is imparted by both switch 1 and switch 2. As discussed above, the conformation of switch 2 in the GDP form is incompatible with binding to FIP2 because of steric overlap (Figure 6). There are also signiﬁcant backbone conformational changes in residues K41–V46, which favor the close packing of switch 1 between the FIP2 a helices. In particular, Ile44 of switch 1 plays a critical packing role in the complex, but in its GDP state, the position and conformation of switch 1 would place the side chain of Ile44 in steric conﬂict with the ring of Tyr480 (not shown). The equilibrium dissociation con-

stant (Kd) of FIP2 with Rab11(GDP) is 1.3 mM, and it is lowered over 30-fold (40 nM) with the GTP form of Rab11 (Junutula et al., 2004). Interestingly, the recent crystal structure of Rab11b(GDP), which is 90% identi-

cal to the Rab11a isoform, revealed a 310-helical conformation in the segment Gly69–Tyr73 (Scapin et al., 2006). The helix disappeared in active Rab11b(GTP), with switch 2 adopting a conformation identical to that of Rab11a(GTP). Overall, these findings correlate with solution studies of the two GTP bound isoforms that revealed similar binding affinities for FIP2 (Kd = 40–44 nM) and are consistent with an unusually flexible conformation for switch 2 in Rab11.

Comparisons to Other Rab-Effector Complexes The crystal structures of four effectors with their cog- nate Rabs have been determined previously (Rab3-rabphilin, [Ostermeier and Brunger, 1999]; Rab5-rabaptin5, [Zhu et al., 2004]; Rab7-RILP, [Wu et al., 2005a]; Rab4- rabenosyn5 and Rab22-rabenosyn5, [Eathiraj et al., 2005]). The structure described here resembles that of Rab5-rabaptin5 and Rab7-RILP in its overall organization as a dyad symmetric complex, in which a coiled- coil effector domain brings together two independent Rab molecules on either side. However, the Rab5- rabaptin5 interaction occurs mainly through switch 2 and Figure 6. Conformational Changes in the GTP/GDP Cycle of Rab11 interswitch regions. Also, the central axis of the rabap- (A) The structure of Rab11(GDP) is pink, Rab11(GTPgS) is blue, and tin5 coiled coil lies parallel to b2 of Rab5, whereas the Rab11(GTP) bound to FIP2 is yellow. The PDB codes for Rab11 in central axis in Rab11-FIP2 is offset by 70 relative to complex with GDP and GTP are 1OIV and 1OIW, respectively. The structure of Rab11(GppNHp), PDB code 1YZK (not shown), is similar this orientation. In contrast to FIP2 and rabaptin5, the ef- to that of Rab11(GTPgS). fector RILP is a four-helix bundle that binds extensively (B) Conformational flexibility of switch 2 in the Rab11-FIP2 complex. to segments of Rab7 that are distant from the switch and The reference structure is Rab11-FIP2 in trigonal crystals (green). interswitch regions (Wu et al., 2005a). The two molecules of Rab11 in the asymmetric unit of orthorhombic In summary, a-helical motifs constitute the RBDs in all crystals (ortho_A, ortho_B) were superposed onto the structure. Rab-effector complexes determined to date, and the in- Only the switch regions and the phosphate arm of GTP are shown for clarity. Magnesium ions are represented as spheres. terface generally consists of hydrophobic interactions. The central recognition elements are switch 1 and switch 2, but effector proteins vary with respect to the position bond with the a-phosphate oxygen. This 180 flip may and orientation of their a helices on the surface of be influenced by the adjacent salt-bridged interaction Rabs. Interestingly, a single a helix is insufficient to con- between Lys41 and Glu476. The side chain of Ser42 stitute a complete Rab binding interface in these com- (switch 1) rotates to make a hydrogen bond with the g- plexes. FIP2, rabaptin5, and RILP are all dimers, while phosphate oxygen, and, together, these interactions re- the monomeric RBD of rabenosyn5 consists of a helical sult in a more intimate association of switch 1 with the hairpin. Rabphilin contains a second interface mediated nucleotide in the FIP2 bound form of Rab11. Finally, by a zinc-containing globular domain, in addition to the a third serine residue (Ser20) undergoes a side chain ro- single long a helix that interacts with switch 1 and switch tation to make a hydrogen bond with the same g-phos- 2 (for a review, see Kawasaki et al., 2005). Structure of Rab11-FIP2 1279

Yeast Two-Hybrid Studies and Cellular Localization of FIP2 Mutants The crystal structure of Rab11-FIP2 suggested that Tyr480 and Ile481 play an essential role in forming the interface with Rab11. Therefore, we performed site- directed mutagenesis to substitute Tyr480 with phenylalanine (Y480F) and the isoleucine at position 481 to glutamate (I481E). The effect of these amino acid substi- tutions on the subcellular localization of FIP2 was assessed. We used the yeast two-hybrid system to ex- amine the ability of these mutants to bind Rab11 and their ability to homodimerize. The nonconservative I481E mutation abolished the interaction with Rab11, while the Y480F mutation had little effect on Rab11 binding, as evidenced by the ability of yeast colonies to grow on reporter media lacking histidine (Figure 7A; see Ex- perimental Procedures). Consistent with previous studies on Rip11, neither of the FIP2 mutants affected dimerization (Figure 7B) (Junutula et al., 2004). To determine the effect of these mutations on the subcellular localization of FIP2, the wild-type and mutant proteins were expressed as green ﬂuorescent protein (GFP) fusions in HeLa cells. Western blot analysis conﬁrmed that the mutant proteins expressed well in these cells and migrated at the same molecular weight as wild-type FIP2 (Figure 7C). Upon examination by confocal microscopy, GFP-FIP2 displayed a punctate vesicular pattern, whereas both mutants were predominantly cytosolic and had some plasma membrane labeling (Figure 7D). The observed plasma membrane localization of the FIP2 mutants is likely to be mediated by the C2 domain of FIP2, and, indeed, we observed some colocalization between the mutants and an antibody that labels

PI(3,4,5)P3 (data not shown). These cellular assays demonstrate the exquisite sen- sitivity of the Tyr480/Ile481 locus and its critical role in Figure 7. Effect of Mutations on FIP2 Dimerization, Binding to vesicular localization of FIP2. The cytosolic distribution Rab11, and Cellular Localization of FIP2 mutants is similar to the phenotype seen in the (A) Yeast two-hybrid analysis of the FIP2 mutants. GAL4 activation equivalent Rip11 mutants Y628F and I629E (Junutula domain (AD) fusion constructs of FIP2 (wild-type and mutants) were cotransformed with GAL4 binding domain (BD) fusions of et al., 2004). Despite the conservative nature of the Rab11 into the L40 strain of S. cerevisiae. Transformant colonies Y480F substitution, a hydrogen bond between the phe- were spotted onto media containing histidine (His+) as a control or nolic oxygen and the backbone of Val46(N) would be media lacking histidine (His2). An interaction is indicated by growth eliminated from each of the two Rab11-RBD interfaces on His2 media. (Figure 2). Although this mutant can interact with Rab11 (B) GAL4 activation domain fusion constructs of FIP2 were cotrans- in vitro, as judged by yeast two-hydrid assays, the preformed into the L40 strain of S. cerevisiae with GAL4 binding domain fusions of FIP2. Transformant colonies were spotted onto media that sumed decrease in affinity manifests itself under cellular containing histidine (His+) as a control or media lacking histidine conditions as a phenotype that resembles mutants that (His2). An interaction is indicated by growth on His2 media. do not interact with Rab11 (Figure 7D). The correspond- (C) FIP2 wild-type and mutant proteins are expressed in HeLa cells ing mutation in Rip11(Y628F) resulted in an increase in and migrate at the same molecular weight. Lysates of HeLa cells ex- pressing GFP-FIP2, GFP-FIP2(Y480F), and GFP-FIP2(I481E) were Kd from 40 nM to 530 nM (Junutula et al., 2004). The yeast two-hybrid assays show that the FIP2(I481E) mu- separated by SDS-PAGE, transferred to nitrocellulose, and probed with anti-GFP. tant fails to interact detectably, consistent with isother- (D) The FIP2 mutants are predominantly cytosolic. HeLa cells trans- mal titration calorimetry (ITC) studies of the Rip11(I629E) fected with the indicated construct were fixed and analyzed by con- mutation (Junutula et al., 2004). The structure of Rab11- focal microscopy. FIP2 reveals the molecular basis for the severity of this mutation. Upon modeling of the I481E mutant (data not shown), the glutamate side chain would interfere with scattering coupled to gel filtration and circular dichroism the hydrophobic interface between Tyr480 and Ile44 in (CD) spectroscopy studies of our FIP2 construct switch 1 (Figure 4). (Ala410–Ser512; Figures S1 and S2) in solution have confirmed that the molecule is an a-helical dimer. Our Rab11-FIP2 and Vesicle Trafficking CD spectrum is similar to published spectra from vari- Structural and biophysical studies support a model for ous constructs (Junutula et al., 2004; Wei et al., 2006) endosomal recruitment of FIP2 in which Rab11(GTP) that encompass the entire ordered segment of FIP2 molecules bind to preformed FIP2 homodimers. Light that we observe in trigonal crystals. Upon stable Structure 1280

Figure 8. Space-Filling Models of the Com- plex Switches 1 and 2 are colored red and blue, respectively, and GTP is shown in cyan. The model reveals the approximate orientation of the complex with respect to the membrane plane. The last 35 amino acids of Rab11 lead- ing to peripheral attachment to the lipid bila- yer are shown as dotted lines. In the ‘‘top view,’’ looking down the 2-fold axis, switch 2 is available on each side for possible interactions with effectors. The slow/indirect endocytic recycling mutation S29F31 is localized to the opposite side of Rab11, adjacent to switch 1. The segment of FIP2 from Gly447 to Leu467 is shown in a ribbon model to allow unobstructed views of potential effector binding sites in the lower panel.

formation of Rab11-(FIP2)2-Rab11, the N terminus of The eps15 homology domain (EHD) proteins EHD1 FIP2 would be oriented away from the Rab11-anchored and EHD3 bind to FIP2 via a series of ‘‘NPF’’ motifs (Nas- membrane (Figures 8 and 9). The existence of hetero- lavsky et al., 2006), the last of which is located 40 resi- dimers of FIPs in vivo has been suggested, raising the dues upstream (Asn407–Phe409) of the present model possibility of crosstalk between FIP signaling pathways of FIP2. Extension of the RBD a helix by an additional (Wallace et al., 2002; Junutula et al., 2004). However, 24 residues in the N-terminal direction may play a struc- only homodimers of FIPs are detected in vitro (Junutula tural role in the recruitment process by modulating the et al., 2004; Ducharme et al., 2005) despite the high de- position and orientation of the binding regions. Further gree of sequence and (presumably) structural conserva- toward the N terminus resides the myosin Vb tail binding tion in the RBD. An analysis of the structure suggests region of FIP2 (Arg129–Val290) (Hales et al., 2002) and that dyad-symmetric homodimers may be favored in the C2 phospholipid binding domain (Val15–Ala102) terms of affinity over heterodimers by the composition (Lindsay and McCaffrey, 2004b). Given the orientation of residues flanking the RBD (asterisks in Figure 3). of the Rab11-FIP2 complex (Figure 8), the C2 domain is more likely directed toward an opposing membrane surface, although there is sufficient intervening sequence (w345 residues) for FIP2 to reverse direction and interact with the Rab11-anchored membrane. An opposing membrane orientation of FIP2 is consistent with our previously proposed model, suggesting that C2 domains of class I FIPs target recycling vesicles to the plasma membrane (Lindsay and McCaffrey, 2004a). In addition to the role it plays in FIPs and endosomal trafficking, Rab11 is involved in related functions, including the organization of membranes for phagocytosis in macrophages (Cox et al., 2000), regulation of cellular cholesterol stores (Holtta-Vuori et al., 2002), and, more recently, targeting of vesicles to budding sites in yeast (Wu et al., 2005b). For these functions, Rab11 recruits effectors such as Rabphilin-11 (Zeng et al., 1999) and Figure 9. N-Terminal Segment of FIP2, Distal to the Rab Binding Interface Sec15 (Wu et al., 2005b) that are structurally unrelated Tyr453 forms a lid over the coiled coil, which is stabilized mainly to FIPs and that may recognize Rab11 in a distinct man- through hydrophobic contacts. However, several salt bridges and ner. Upon mutation of Rab11(Ser29) to phenylalanine hydrogen bonds also stabilize the homodimer. and subsequent trafficking assays, the authors Structure of Rab11-FIP2 1281

described a perturbation of a slow/indirect endocytic length anomalous diffraction (SAD) experiments implemented in recycling pathway relative to previously characterized PHASER (McCoy et al., 2004), by using all data from 50–2.44 A˚ . After mutants of Rab11, suggesting the presence of multiple DM, the map was of sufficient quality for automated model building by ARP-/wARP (Perrakis et al., 1999), as implemented in the CCP4 effector binding sites (Pasqualato et al., 2004). Intrigu- package (CCP4, 1994). The initial model contained 139 residues of ingly, the Ser29 locus lies exposed on equivalent and op- Rab11 (83% of the final model) and 37 residues of FIP2 (E455- posite sides of the Rab11-FIP2 complex. In conclusion, T492). Structure refinement involved the rebuilding of several loops the structure described here provides a platform for fur- (including switch 2) and extension of both polypeptides in the N and ther interactions with effectors, and it suggests a rational C termini and was performed with COOT (Emsley and Cowtan, course for future mutagenesis, structural, and cellular 2004); restrained TLS refinement was performed with Refmac5 (Mur- shudov et al., 1997). The structure of Rab11-FIP2 in the orthorhom- studies. bic crystal was determined by molecular replacement with the trigonal model. One molecule of Rab11 and one molecule of FIP2 were Experimental Procedures used as separate search models in PHASER (McCoy et al., 2005). The structure was refined with multiple rounds of Refmac5 and Protein Expression and Purification model building in COOT. Medium restraints for the structurally con- Primers containing a 50 NcoI site and a 30 EcoRI site were used to served segments of Rab11 and FIP2 were used throughout refine- PCR amplify the FIP2 cDNA corresponding to amino acid residues ment. However, map averaging did not improve electron density 410–512. Full-length FIP2 in the pTrcHisA plasmid was used as a maps, and several loop regions of Rab11 (including switch 1 and template for Taq polymerase (New England Biolabs) PCR amplifica- switch 2) and the N-terminal region of FIP2 were excluded from tion. Upstream and downstream primers were 50-gcataccatggcagc averaging; models were built and refined independently. aaaattcagggcttcaaat-30 and 50-accggaattcttaactgttagagaatttgccag ctt-30, respectively. Rab11a cDNA (coding for residues 1–173) was Site-Directed Mutagenesis similarly amplified with flanking Nco1 and EcoR1 restriction sites. Site-directed mutagenesis was performed by using the QuikChange Upstream and downstream primers were 50-aatgccatgggcacccgcga kit (Stratagene) according to the manufacturer’s instructions. cgac-gagtacgac-30 and 50-accggaattcttagtatatctctgtcagaattgtct-30, pEGFP-C1 FIP2 (Lindsay and McCaffrey, 2002) was the template, respectively. The template for PCR was full-length Rab11 in the plas- and the following sense and antisense oligonucleotides were used: mid pTrcHis, and this template contained the Q70L mutation. After FIP2(Y480F): sense 50-ACATCCGGGAACTCGAGGACTTCATCG amplification and double digestion by the restriction endonucle- ACAACCTCCTTGTAAG-30 ases, ligations were carried out with the TaKaRa ligation kit (Cam- FIP2(Y480F): antisense 50-CTTACAAGGAGGTTGTCGATGAAGT brex Corp.). FIP2 was cloned into the vector pMAL-parallel 2 (a mod- CCTCGAGTTCCCGGATGT-30 ification of pMAL-c2x containing an rTEV cleavage site) by using the FIP2(I481E): sense 50-CGGGAACTCGAGGACTACGAGGACAAC NcoI and EcoRI restriction sites and was expressed as a fusion pro- CTCCTTGTAAGG-30 tein with mannose binding protein (MBP). Rab11 was cloned into the FIP2(I481E): antisense 50-CCTTACAAGGAGGTTGTCCTCGATG pET-28b vector without an affinity tag. TCCTCGAGTTCCCG-30 The two resulting expression plasmids were cotransformed into BL21(DE3) cells. Overexpression was carried out in SeMet Media Incorporation of the mutations was confirmed by sequencing. (Molecular Dimensions) supplemented with 100 mg/ml ampicillin FIP2(Y480F) and FIP2(I481E) were digested from pEGFP-C1 with and 30 mg/ml kanamycin at 37C. Selenomethionine (100 mg/l, EcoRI and subcloned into the EcoRI site of the pGADGH yeast

Sigma-Aldrich) was added at the time of induction (OD600 = 0.6, two-hybrid vector. The yeast two-hybrid experiments were per- 0.5 mM IPTG), and cells were grown for an additional 3 hr, harvested formed as previously described (Lindsay and McCaffrey, 2002). by centrifugation, and stored at 220C. Frozen pellets were resus- pended in MBP extraction buffer (20 mM Tris-HCl, 200 mM NaCl, Cell Culture and Fluorescence Microscopy 5 mM MgCl2, and 10 mM b-mercaptoethanol [pH 7.8]) and sonicated HeLa cells were maintained in culture in DMEM (BioWhittaker) (2 3 1 min) at room temperature. Cell lysates were centrifuged at supplemented with 10% fetal bovine serum, 100 U/ml penicillin, 20,000 3 g to remove cell debris, and the resultant supernatant 100 mg/ml streptomycin, and 2 mM L-glutamine at 37C with 5% was applied to an amylose resin (New England Biolabs). After exten- CO2. For fluorescence microscopy, cells were grown on 10 mm sive washing with MBP extraction buffer, bound protein was eluted glass coverslips and transfected by using Effectene (Qiagen) with MBP elution buffer (extraction buffer supplemented with 10 mM according to the manufacturer’s instructions. Approximately 18 hr maltose). Eluted protein was dialyzed overnight against 10 mM Tris posttransfection, the cells were fixed with 3% paraformaldehyde (pH 8.0), 25 mM NaCl in the presence of rTEV protease (20 mg/mg and mounted on glass slides with Mowiol. Images were acquired fusion protein). Cleaved protein was loaded onto an ion-exchange on a Zeiss LSM 510 confocal microscope by using a PlanApo 633 column (MonoQ GL 5/50, GE Healthcare), and a salt gradient was 1.4 NA oil immersion objective. applied (10–500 mM NaCl) over a 20-fold excess column volume. The protein fractions corresponding to the Rab11-FIP2 complex Supplemental Data were pooled and further purified on a superdex 200 16/60 column Supplemental Data include light scattering and circular dichroism (GE Healthcare) equilibrated in column buffer (10 mM Tris-HCl, studies and are available at http://www.structure.org/cgi/content/ 100 mM NaCl, 5 mM MgCl2, 1 mM DTT [pH 8.0]). The protein peak full/14/8/1273/DC1/. was prepared for crystallization on 10 kDa cutoff concentrators (Millipore) to a final concentration of 8.25 mg/ml, as measured by Acknowledgments the Bradford dye assay (Bradford, 1976), by using bovine serum albumin as a standard. The expression and purification of FIP2 We would like to thank the European Molecular Biology Laboratory (alone) was performed in the same way as described above, but (EMBL), Grenoble, particularly Dr. Hassan Belrhali, for providing without Rab11 coexpression. support for data collection at the European Synchrotron Radiation Facility beamline EMBL-CRG BM14 under the European Commu- Crystallization and Structure Determination nity-Research Infrastructure Action FP6 Programme. This work Purified Rab11-FIP2 was crystallized by hanging drops in a 1:1 ratio was supported by a grant from Science Foundation Ireland (grant with reservoir containing 100–400 mM ammonium phosphate (pH 03/IN.3/B371) to A.R.K.; the Programme for Research in Third-Level

4.5–5.5). Trigonal crystals (P312) typically appeared in lower salt Institutions (PRTLI), administered by the Higher Education Authority concentration ranges. Data were collected on beamline BM14 (HEA), to A.R.K.; Science Foundation Ireland Investigator grants 02/ (ESRF, Grenoble) at the Se peak from a single trigonal and ortho- IN.1/B070 and 05/IN.3/B859 to M.W.M., and the Health Research rhombic crystal and were processed by using the HKL2000 package Board Career Development Grant PD/2005/05 to A.J.L. R.J.R. and (Otwinowski and Minor, 1997). Initial phases were calculated for the A.J.M. were supported by a Principal Research Fellowship awarded trigonal data set by using a novel likelihood target for single-wave- to R.J.R. by the Wellcome Trust (UK). Structure 1282

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Accession Numbers

Coordinates for the orthogonal crystal form and the trigonal crystal form have been deposited in the Protein Data Bank with accession codes 2GZD and 2GZH, respectively.