Structural Basis for the Interaction of the Golgi-Associated Retrograde Protein Complex with the T-SNARE Syntaxin 6

Structure Short Article

Structural Basis for the Interaction of the Golgi-Associated Retrograde Protein Complex with the t-SNARE Syntaxin 6

Guillermo Abascal-Palacios,1,4 Christina Schindler,2,4 Adriana L. Rojas,1 Juan S. Bonifacino,2,* and Aitor Hierro1,3,* 1Structural Biology Unit, CIC bioGUNE, Bizkaia Technology Park, 48160 Derio, Spain 2Cell Biology and Metabolism Program, Eunice Kennedy Shriver National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, MD 20892, USA 3IKERBASQUE, Basque Foundation for Science, 48011 Bilbao, Spain 4These authors contributed equally to this work *Correspondence: [email protected] (J.S.B.), [email protected] (A.H.) http://dx.doi.org/10.1016/j.str.2013.06.025

SUMMARY (1) capture of transport vesicles in the vicinity of the target organ- elle, and (2) regulation of the fusion event through actions on the The Golgi-Associated Retrograde Protein (GARP) SNAREs. The Golgi-Associated Retrograde Protein (GARP) (also complex is a tethering factor involved in the fusion known as VFT) complex promotes fusion of retrograde transport of endosome-derived transport vesicles to the vesicles derived from endosomes with the trans-Golgi network trans-Golgi network through interaction with compo- (TGN), a critical step for transmembrane proteins that cycle nents of the Syntaxin 6/Syntaxin 16/Vti1a/VAMP4 between these organelles (Bonifacino and Hierro, 2011). GARP SNARE complex. The mechanisms by which GARP is conserved from yeast to humans and consists of four subunits named Vps51 (Ang2 in humans), Vps52, Vps53, and Vps54 (Con- and other tethering factors engage the SNARE fusion ibear and Stevens, 2000; Siniossoglou and Pelham, 2001, 2002; machinery are poorly understood. Herein, we report Conibear et al., 2003; Liewen et al., 2005; Pe´ rez-Victoria et al., the structural basis for the interaction of the human 2010b; Luo et al., 2011). The subunits of GARP are structurally Ang2 subunit of GARP with the Syntaxin 6 and the related to each other as well as to subunits of other multisubu- closely related Syntaxin 10. The crystal structure of nit-tethering complexes such as the exocyst, conserved oligo- the Syntaxin 6 Habc domain in complex with a pep- meric Golgi (COG), and Dsl1 complexes (Pe´ rez-Victoria et al., tide from the N terminus of Ang2 shows a binding 2010a; Vasan et al., 2010). Most of these subunits share a struc- mode in which a dityrosine motif of Ang2 interacts ture consisting of tandem helical bundle domains, for which the with a highly conserved groove in Syntaxin 6. complexes are collectively referred to as Complexes Associated Structure-based mutational analyses validate the with Tethering Containing Helical Rods (CATCHR) (Yu and crystal structure and support the phylogenetic Hughson, 2010). GARP interacts with components of the Syntaxin (Stx) 6/Stx conservation of this interaction. 16/Vti1a/VAMP4 SNARE complex involved in fusion of retrograde transport carriers with the TGN (Mallard et al., 2002; INTRODUCTION Pe´ rez-Victoria and Bonifacino, 2009; Pe´ rez-Victoria et al., 2010b). However, the mechanisms by which tethering com- Transport of macromolecular cargo between organelles of the plexes function in vesicle fusion are poorly understood, particu- endomembrane system occurs by budding of vesicles from a larly with regard to their roles in SNARE complex assembly. donor compartment followed by fusion of the vesicles with an Herein, we present a biochemical and X-ray crystallographic acceptor compartment (Bonifacino and Glick, 2004). Budding analysis of the Ang2 interaction with Stx6 and the closely related most often involves protein coats that select specific cargos Stx10. We show a significantly different binding mode from that while promoting vesicle formation. On the other end, fusion is reported for the yeast Vps51-Tlg1 interaction (Fridmann-Sirkis mediated by a protein ensemble comprising small GTPases et al., 2006). Mutational analyses confirm the validity of this of the Rab, Arl, or Rho families, tethering factors, soluble binding mode in vitro and in vivo and support its phylogenetic N-ethylmaleimide-sensitive factor attachment protein receptors conservation in most eukaryotes (SNAREs) on both the vesicles (v-SNAREs) and the target organ- elle (t-SNAREs), and Sec1-Munc18 (SM) proteins (Su¨ dhof and RESULTS Rothman, 2009; Yu and Hughson, 2010). The specificity of different vesicular transport steps is determined by the use of A 50 Amino Acid N-Terminal Segment from Ang2 distinct sets of budding and fusion proteins. Interacts with the Habc Domain from Stx6 and Stx10 Tethering factors are long coiled-coil proteins or multisubunit In previous work, we found an interaction between the Ang2 sub- complexes that are recruited to membranes by small GTPases unit of human GARP and the TGN t-SNARE Stx6 (Pe´ rez-Victoria (Yu and Hughson, 2010). They have at least two functions: et al., 2010b)(Figure 1A). To analyze further the specificity of this

Figure 1. The Habc Domains of Stx6 and Stx10 Bind a 50 Amino Acid N-Terminal Segment from Ang2 (A) Schematic representation of human Ang2 and human Stx6 is shown. Amino acid numbers are indicated. CC, coiled coil; SNARE, SNARE domain; TMD, transmembrane domain. (B) Y2H analysis of the interaction of SNARE cytosolic domains fused to GAL4-AD with Vps53, Ang2, and Ang2 (1–299) fused to GAL4-BD is presented. The SV40 large T antigen (TAg) and p53 were used as controls. Growth in the absence of histidine (ÀHis) is indicative of interactions. 3-amino-1,2,4-triazole (3-AT) was used to sup- press self-activation. (C) Y2H analysis of the effect of truncations of the Stx6 cytosolic domain on interaction with Ang2 and Ang2 (1–299) is shown. (D) In vitro assay of Ang2 (1–299) binding to cytosolic portions of SNAREs is presented. GST-tagged SNAREs or GST as a control was prebound to glutathione Sepharose and incubated with deter- gent extracts from HeLa cells transiently transfected with a plasmid encoding Ang2 1–299 OSF. Bound proteins were subjected to SDS-PAGE and immunoblotting with an antibody to the FLAG epitope. To estimate the amounts of GST-protein fusions, immunoblots were stained with Ponceau S. A sample of the total extract was included as input control. The positions of molecular mass markers (in kDa) are indicated on the left. (E) Y2H analysis of the interaction of the Habc domains of Stx6 and Stx10 with Ang2 and Ang2 (1–299) is shown. (F) Y2H mapping of the Ang2 binding site to the ﬁrst 50 residues of Ang2 is presented.

full-length Ang2 and Ang2 1–299, in contrast to the Stx6 SNARE domain (Stx6 176–238), which did not interact with either Ang2 construct (Figure 1C). GST pull-down assays confirmed that interaction, we performed yeast two-hybrid (Y2H) analysis of the both the Stx6 Habc (Stx6 1–103) and cytosolic (Stx6 1–238) binding of Ang2 to the cytosolic domains of several SNAREs that domains bound the N-terminal region of Ang2 (Ang2 1–299) function at the TGN and endosomes. We found that full-length tagged with FLAG-One-STrEP (OSF) (Figure 1D), whereas the Ang2 interacted with the cytosolic domains of Stx6 (Stx6 1– Stx6 linker (Stx6 103–175) and SNARE domain (Stx6 176–238), 238) and the paralogous (61% amino acid sequence identity) the Stx8 cytosolic domain (Stx8 1–218) and Vamp4 cytosolic Stx10 (Stx10 1–232) and more weakly with the cytoplasmic domain (Vamp4 1–114), did not bind (Figure 1D). Y2H analysis domain of the v-SNARE Vamp4 (Vamp4 1–114) (Figure 1B). We also showed interaction of the Stx10 cytosolic (Stx10 1–232) did not detect interaction of Ang2 with the cytosolic domain of and Habc (Stx10 1–103) domains with both full-length Ang2 the more distantly related (31% amino acid sequence identity) and Ang2 1–299 (Figure 1E). Further Y2H assays identified aa Stx8 (Stx8 1–218) (Figure 1B). Further Y2H analysis showed 1–50 from Ang2 as the minimal segment capable of interacting that the N-terminal region of Ang2 (residues 1–299) (Figure 1A), with the Habc domains from both Stx6 (Stx6 1–103) and Stx10 which mediates assembly with the other subunits of GARP (Stx10 1–103) (Figure 1F). These experiments thus demonstrated (Pe´ rez-Victoria et al., 2010b), is sufficient for interaction with a specific interaction between a 50 amino acid N-terminal Stx6 and Stx10 (Figure 1B). segment from Ang2 and the Habc domains from Stx6 and Stx10. Both Stx6 and Stx10 belong to the Stx family of Q-SNAREs (Fasshauer et al., 1998) and possess an N-terminal regulatory A Dityrosine Motif in the N-Terminal Region of Ang2 domain named Habc (Fernandez et al., 1998), followed by a Is Required for Binding to the Habc Domain of Stx6 linker region, a fusogenic SNARE domain, and a C-terminal and Stx10 transmembrane anchor (Figure 1A). Using the Y2H system, we Inspection of the 50 N-terminal amino acids from human Ang2 in found that the Stx6 Habc domain (Stx6 1–103) bound to both comparison to those from other species revealed a highly

Figure 2. A Dityrosine Motif in Ang2 Is Required for Binding to the Habc Domain of Stx6 and Stx10 (A) Alignment of N-terminal segments of Ang2/Vps51 from different species using ClustalW is shown. (B) Y2H analysis of the interaction of Stx6 and Stx10 fragments with full-length Ang2, Ang2 1–69, and Ang2 1–69 with a deletion of aa 2–23 (Ang2 1–69 D2–23) and Ang2 with the conserved dityrosine motif mutated to alanine (Ang2 1–69 YY/AA) is presented. (C and D) In vitro binding of Ang2 1–299 OSF (C) or Ang2 1–299 OSF YY/AA (D) expressed in HeLa cells to GST-SNAREs is illustrated. The experiment was performed as described in the legend to Figure 1D. (E) Strep-Tactin (STREP) pull-down assay of endogenous SNAREs with different Ang2 OSF constructs expressed by transfection in HeLa cells is shown. Endogenous Stx6 and Vti1A SNAREs as well as clathrin heavy chain (Chc) and actin (loading controls) were detected by immunoblotting. Ct, control. The positions of molecular mass markers (in kDa) are indicated on the left of (C)–(E). conserved tyrosine pair: Tyr40 and Tyr41 (Figure 2A). Additional N-terminal segment (Figure 2A). Indeed, using the Y2H system, amino acids ﬂanking the two aromatic amino acids also show a we found that mutation of Tyr40 and Tyr41 to alanine (YY/AA signiﬁcant degree of conservation within an otherwise variable mutant) completely abolished the interaction of a human Ang2

Table 1. Data Collection and Structure Reﬁnement Parameters endogenous Vps52 (Figure 2E). From these experiments, we concluded that a dityrosine motif within the 50 N-terminal amino Data Collection acids from Ang2 is required for interaction with the Stx6 and Wavelength 0.9334 Stx10 Habc domains in vitro and in vivo. Number of images 132 Unit cell parameters Structure of an Ang2 Peptide Bound to Stx6-Habc a 41.64 A˚ To elucidate the structural basis for the Ang2-Stx6 interaction, b 45.66 A˚ we determined the crystal structure of the Ang2 peptide c 58.34 A˚ 33AHGMLKLYYGLSEGEAA49 in complex with the Stx6 Habc a 90 domain. The structure was solved by molecular replacement using the coordinates of Stx6-Habc (Protein data Bank [PDB] b 90 1LVF) (Misura et al., 2002) as the search model and reﬁned to g 90 ˚ 1.8 A resolution with Rfactor and Rfree values of 14.9 and 19.3, Space group P21 respectively (Table 1). The difference Fourier map showed clear Resolution (A˚ ) 50.0–1.80 (1.86–1.80) electron density for a portion of the Ang2 peptide comprising

Rmerge (%) 4.8 (35.8) residues 33AHGMLKLYYGLS44 (Figure 3A). The remaining resi- Completeness (%) 97.2 (91.0) dues could not be modeled onto the electron density and Multiplicity (%) 2.7 (2.1) were therefore deemed to be disordered. Overall, the Ang2 I/s (%) 20.09 (2.15) peptide forms a short a helix that lies between a helices a and b at the N-terminal side of Stx6 (Figure 3B). The C terminus of Alpha_twin 0.49 the Ang2 peptide, to which the rest of the GARP complex is Number of reﬂections (observed) 19,877 bound, and the C terminus of Stx6, which contains the mem- Structure Reﬁnement brane-anchoring domain, extend in opposite directions (Fig- R factor (%) 14.95 ure 3B), consistent with a possible role of this interaction in

Rfree (%) 19.34 tethering of apposed membranes. The contact region in the

Percentage of reﬂections (Rfree %) 5 Stx6 Habc domain corresponds to a noticeably hydrophobic Number of water molecules 510 groove that is highly conserved as compared to other parts of Rmsd values the surface (Figure 3C; Figure S1 available online). In agreement with the biochemical data described above, Ang2 is anchored Bond lengths (A˚ ) 0.005 to this groove by the burial of Tyr40 and Tyr41. The linchpin of Bond angles () 0.835 this interaction is a network of hydrogen bonds between Ramachandran statistics (%) Tyr41Ang2, Asp5Stx6 and Arg88 Stx6 (Figure 3D). The orientation Most favored 98.3 of the Asp5Stx6 carboxyl group toward the hydrogen-bonding Additional allowed 1.7 network is favored by the relative rigidity of the following

Generously allowed 0 Pro6Stx6. Similarly, Arg88Stx6 stacks against Phe7Stx6, holding its guanidinium group toward the hydrogen-bonding network

(Figure 3D). The proximal Phe78Stx6 further stabilizes the burial N-terminal fragment (Ang2 1–69) with both the cytosolic domains of Tyr40Ang2 and Tyr41Ang2. More peripherally, Leu37Ang2 is and Habc domains from Stx6 and Stx10 (Figure 2B). In contrast, positioned in a shallow hydrophobic cavity formed by Pro6Stx6, deletion of a variable segment comprising aa 2–23 (Figure 2A) Val10Stx6, and the b carbon of Asp63Stx6 (Figure 3E). This hydro- had no effect on the interactions (Figure 2B). GST pull-down phobic interaction, together with a hydrogen bond between assays also showed a requirement of the dityrosine motif for His34Ang2 and Asp63Stx6, contributes to anchor the rest of the interaction of Ang2 1–299-OSF with the cytosolic domains of Ang2 a-helical segment to the Hab groove of Stx6. Thus, the Stx6 and Stx10 and the Habc domain of Stx6 in vitro (Figures high-resolution crystal structure identiﬁes the most critical 2C and 2D). elements of the Stx6-Ang2 interaction. To determine if these interactions are required for association of Ang2 with endogenous SNAREs in vivo, we transfected HeLa Stx6-Ang2 and Tlg1-Vps51 Exhibit Distinct cells with constructs encoding OSF-tagged Ang2 or Ang2 1–299 Crystallographic Interactions in both their wild-type and YY/AA mutant versions. We per- The closest homolog of human Stx6 in yeast is the t-SNARE formed a Strep-Tactin pull-down, followed by immunoblotting Tlg1. Indeed, the crystal structure of the Tlg1 Habc domain for Stx6, Vti1a, and several control proteins (Figure 2E). We in complex with a peptide derived from yeast Vps51 (residues observed that wild-type Ang2 and Ang2 1–299 interacted with 16–30) shows an association along the same Hab groove Stx6 and Vti1a, whereas the YY/AA mutant counterparts did (Figure 3F) and with the Vps51 peptide in a similar helical not interact (Figure 2E). The coisolation of Vti1a with Stx6 sug- conformation (Fridmann-Sirkis et al., 2006). However, despite gested that Ang2 can bind via the dityrosine motif to Stx6 in the apparent parallels between Stx6-Ang2 and Tlg1-Vps51 complex with a cognate SNARE. As a control, we showed that interactions, there are important differences in their crystallo- mutation of the dityrosine motif had no effect on the incorpora- graphic binding mode. In fact, the residues Phe27Vps51 and tion of Ang2 into the GARP complex, as evidenced by the Tyr28Vps51, which are equivalent to Tyr40Ang2 and Tyr41Ang2, coisolation of the wild-type and mutant Ang2 species with are positioned outside the Hab groove in the Tlg1-Vps51

Figure 3. Structure of a Human Ang2 Pep- tide Bound to the Human Stx6 Habc Domain

(A) 2Fo À Fc electron density map of Ang2 (aa 33–44) contoured at 1.1s (blue mesh) with the ﬁnal model superimposed is presented. (B) Overall structure of human Stx6 Habc domain (cyan) in complex with the Ang2 peptide (purple) in ribbon cartoon is illustrated. (C) Stx6 Habc domain shows the surface proper- ties of the contact region with the Ang2 peptide in tube drawing. The molecular surface is colored by hydrophobicity (green) or by residue conservation (red and orange denote strictly and highly conserved residues, respectively). See also Figure S1. (D) Ang2 gets anchored through the burial of Tyr40

and Tyr41 (purple) into the conserved D5PF[...]R88 motif of Stx6 (cyan). (E) Stx6 residues (light blue) that contribute to the hydrophobic pocket are shown under translucent

surface in the vicinity of Leu37Ang2 (purple). (F) Structural comparison between the human Stx6-Ang2 complex (cyan indicates Stx6, and purple shows Ang2 peptide) and the yeast Tlg1- Vps51 complex (PDB 2C5K; brown indicates Tlg1, and green shows Vps51 peptide) is presented. Note that an 90 clockwise rotation along the helical axis of the Vps51 peptide would place

Phe27Vps51 and Tyr28Vps51 over Tyr40Ang2 and

Tyr41Ang2. (G) Superposition of the Habc domains of Stx6 (cyan), Stx10 (PDB 4DND; pink), and Tlg1 (PDB 2C5K; brown) is illustrated in ribbon cartoon with the residues corresponding to the conserved DPF [...]R motif for each domain highlighted as stick model.

Conﬁrmation of the Stx6-Ang2 Interface by Mutational and Binding Analyses To conﬁrm that the crystallography inter-

action between the 5DPF [...]R88 signa- ture sequence of Stx6 and the N-terminal

37LxxYY41 motif of Ang2 is preserved in solution, we performed isothermal titration calorimetry (ITC). We found that the Ang2 N-terminal region (residues 1–69) binds to the Stx6 Habc domain with a

KD value of 5.6 ± 0.7 mM and a best-ﬁt structure (Figures 2A and 3F). Furthermore, Tyr28Vps51, which stoichiometry of 0.8, which we interpret as a single binding is critical for binding in vivo (Fridmann-Sirkis et al., 2006), site (Figure 4A). The structure-based mutants D5LStx6 and does not contact Tlg1. Instead, it interacts with a symmetrically R88MStx6 were designed to eliminate the central network of related molecule stabilizing the crystal lattice. Another differ- hydrogen bonds between Stx6 and Ang2, whereas the mutants ence concerns the strictly conserved 6DPF [...]R73 residues of P6AStx6 and F7LStx6 were designed to disrupt the orientation of Tlg1, which do not participate in any hydrogen bonding Asp5Stx6 and the hydrophobic contact that stabilize Arg88Stx6, network with Vps51 as the equivalent residues in the Stx6- respectively. None of the mutations signiﬁcantly altered Stx6 Ang2 structure do (Figures 3G and S1). Interestingly, a simple structure, as measured by circular dichroism (CD) (Figure S2). 90 rotation of the Vps51 peptide along its helical axis would However, every single mutation rendered Stx6 Habc unable to place Phe27Vps51 and Tyr28Vps51 at positions similar to those bind Ang21–69, consistent with the central role of the 5DPF [...] of Tyr40Ang2 and Tyr41Ang2 (Figure 3F), raising the possibility R88 binding motif in Stx6 (Figure 4A). Similarly, the single mutants that the crystallized yeast complex corresponds to an interac- L37AAng2, Y40AAng2, or Y41AAng2, designed to disrupt the tion intermediate. anchoring of Ang2 to Stx6, completely abolished the binding

Figure 4. ITC of Ang2 Interactions with Stx6 and Stx10 (A and B) The top graphs represent the differential heat released during the titration of (A) Stx6 Habc with Ang2 (1–69) or (B) Stx10 Habc with Ang2 (1–69). The bottom graphs represent the ﬁtted binding isotherms. All mutants within the conserved L37xxYY41 motif of Ang2 and the D5PF[...]R88 motif of Stx6 or Stx10 are annotated on the side of their respective titration curve. The KD and binding stoichiometries (N values) are indicated as insets. N.B., no appreciable binding. See also Figure S2.

(Figure 4A), the latter two in agreement with the result of Y2H in the N-terminal part of Ang2 and a 5DPF[...]R88 motif in the analyses (Figure 2). These results conﬁrmed that the crystallo- N-terminal Habc domain of Stx6 and Stx10. Both motifs (Fig- graphic interaction and solution binding correspond to the ure S1) are conserved in most eukaryotic orthologs of these pro- same molecular association. teins, indicating that this interaction is evolutionarily conserved. Indeed, an analogous interaction has been reported for the Ang2 Displays the Same Binding Mode for Stx6 and S. cerevisiae orthologs Vps51 and Tlg1 (Fridmann-Sirkis et al., Stx10 2006). Also in this case, mutation of a diaromatic motif (Phe27

The 5DPF [...]R88 motif in Stx6 is also present at an equivalent po- and Tyr28) in a Vps51 peptide abolishes binding to the Tlg1 sition in Stx10 (Figures 3G and S1). Furthermore, our Y2H and GST Habc domain in surface plasmon resonance and Y2H assays pull-down analyses showed that Ang2 interacts equally well with (Fridmann-Sirkis et al., 2006). However, in the published crystal

Stx6 and Stx10 (Figures 1 and 2). Thus, to evaluate whether structure, Phe27Vps51 and Tyr28Vps51 do not contact the Tlg1 Ang2 employs the same binding mode for Stx6 and Stx10, we Habc domain to which the rest of the peptide is bound but point conducted an identical ITC analysis with Stx10. As expected, outward from the Hab groove (Fridmann-Sirkis et al., 2006). A

Ang21–69 exhibited a comparable affinity (KD = 6.5 ± 0.8 mM) and structure like the one presented here would explain the require- stoichiometry (n = 0.7) for binding to the Stx10 Habc domain (Fig- ment of Phe27Vps51 and Tyr28Vps51 for Vps51 binding to Tlg1 as ure 4B). In addition, the equivalent mutations in the 5DPF[...]R88 well. We propose that the interaction details are conserved in motif of Stx10 or in the 37LxxYY41 motif of Ang2 resulted in the most eukaryotes and that the published S. cerevisiae structure complete loss of interaction between both proteins (Figure 4B). may correspond to a binding intermediate rather than the final These results strongly support a common recognition mechanism state of the complex. Disruption of the Vps51-Tlg1 interaction for Ang2 and the t-SNARES Stx6 and Stx10. Furthermore, they was not found to have any effect on GARP-dependent pro- imply that interaction of Ang2 with Stx6 and Stx10 cannot be cesses such as retrograde transport of the Sso1 and Snc1 simultaneous, suggesting that GARP may participate in more SNAREs and maintenance of vacuolar integrity in S. cerevisiae than one SNARE-mediated event at the TGN. (Fridmann-Sirkis et al., 2006). Likewise, using RNAi knock- down-rescue experiments in HeLa cells, we found that mutation DISCUSSION of the Ang2 dityrosine motif did not affect coprecipitation of Stx6 with cognate SNAREs nor did it inhibit the retrograde transport of Our biochemical and structural analyses reveal the molecular TGN46 and Shiga toxin B subunit to the TGN (data not shown). details of a highly specific interaction between a 37LxxYY41 motif Nonetheless, the evolutionary conservation of this interaction

suggests a critical role, perhaps in other functions ascribed to noting that Ang2 pulls down via its dityrosine motif not only GARP (Bonifacino and Hierro, 2011). Stx6 but also Vti1a (Figure 2E). Because Vti1a does not have a A search for other proteins that have an LxxYY motif near the N Habc domain, its pull-down by Ang2 is likely to depend on a terminus using the ScanProsite tool (http://prosite.expasy.org/ different interaction or to be the indirect result of SNARE com- scanprosite/) identified the sequence L49ELYY53 within the verte- plex assembly. In addition, the Vps53 and Vps54 subunits of brate Cog2 subunit of the COG complex. Like GARP, COG is a human GARP interact with the SNARE motifs of Stx6, Stx16, CATCHR-type tethering complex (Yu and Hughson, 2010; Miller and VAMP4 (Pe´ rez-Victoria and Bonifacino, 2009). All of these and Ungar, 2012) implicated in retrograde transport from endo- interactions could work together in a molecular pathway of somes to the TGN. However, Y2H and ITC experiments failed SNARE complex assembly assisted by GARP. An appealing to detect interactions between the N-terminal part of Cog2 and possibility is that the N-terminal region of Ang2 could play a the Habc domain of Stx6 (data not shown), probably due to the role in modulating the spatiotemporal segregation of Stx6 and presence of the L49ELYY53 motif in an unfavorable conforma- Stx10 during SNARE complex formation. Future studies should tional context (i.e., a predicted a helix). address how multiple interactions with GARP regulate the The DPF[...]R motif present in Stx6 and Stx10 also appears to assembly of the Stx6/Stx16/Vti1a/VAMP4 complex. be partially conserved in other Stxs such Stx1, Stx2, Stx3, Stx4, and Stx8 (Figure S3A). Indeed, the structure of Stx1A exhibits an EXPERIMENTAL PROCEDURES arginine residue stacked against a phenylalanine residue at a position comparable to that on Stx6 and Stx10 (Figure S3B). Recombinant DNAs and Y2H and Pull-Down Assays However, slight differences at the binding pocket may alter the A list of recombinant DNA constructs is shown in Table S1. For constructs generated in this study, DNA sequences were PCR amplified from human brain specificity of recognition. For example, the lack of interaction cDNA (Clontech, Mountain View) and cloned into the corresponding of Stx8 with Ang2 (Figure 1B) could be due to the stacking of argi- plasmids. Site-directed mutagenesis was performed using the QuikChange nine against tryptophan instead of phenylalanine, thus modifying Site-Directed Mutagenesis Kit (Stratagene, La Jolla). Y2H experiments were the steric and electrostatic features of the site. performed as previously described by Ohno et al. (1995) and Aguilar et al. The presence of an N-terminal Habc domain is a feature of all (2001). GST fusions to SNARE cytosolic domains were expressed in E. coli Stx-type t-SNAREs (MacDonald et al., 2010). A well-known func- BL21 Rosetta (Novagen, Madison) from pGEX-5X-1 and purified as described in Schindler and Spang (2007). GST and Strep-Tactin pull-downs were tion of Habc domains is the regulation of SNARE complex forma- performed as previously described (Pe´ rez-Victoria and Bonifacino, 2009; tion and vesicle fusion. For example, the Habc domain of Pe´ rez-Victoria et al., 2010a, 2010b). HeLa cells (ATCC, Manassas) were trans- mammalian Stx1 folds back onto the SNARE motif of the same fected using Lipofectamine 2000 (Invitrogen) according to the manufacturer’s protein to generate a closed conformation that is inactive for instructions. Cells were used 24 hr after transfection. Antibodies used have fusion (Zhou et al., 2013; Dulubova et al., 1999; Fiebig et al., been described in Pe´ rez-Victoria and Bonifacino (2009). The antibody to 1999; Lerman et al., 2000). This interaction involves binding of clathrin heavy chain was from BD Biosciences (San Diego). a helix H3 of the SNARE motif to a groove formed by a helices b and c of the Habc domain. The SM protein Munc18-1 binds Crystallization, Data Collection, and Model Building The synthetic peptide of Ang2 (aa 33–49) was obtained from GenScript USA to the closed conformation of Stx1 also through interaction (Piscataway). The peptide was resuspended in 6 M guanidine hydrochloride with the Stx1 Habc domain (Hata et al., 1993; Misura et al., and mixed in a molar ratio 2:1 with Stx6 (3–110). The mixture was then dialyzed 2000). Opening of the Stx1 structure is at least in part mediated against 150 mM NaCl, 10 mM b-ME, 50 mM Tris-HCl (pH 7.4) at 4C during by Munc13-1 (Ma et al., 2011), a large protein that is structurally 48 hr. Crystallization trials were carried out in hanging drops with the pro- homologous to CATCHR-type tethering factor subunits (Li et al., tein-peptide mixture at 20 mg/ml. After 1–2 days, large crystals appeared in 2011). This conformational change depends on interactions of 18% PEG 3350 and 100 mM ammonium dihydrogen phosphate. Crystals were flash frozen under liquid nitrogen using paraffin oil as cryoprotectant. the CATCHR-fold MUN domain of Munc13-1 with both the One data set at 1.8 A˚ resolution was collected at ESRF beamline ID14-1. Stx1 SNARE motif and Munc18-1 (Ma et al., 2011; Lerman Data reduction was carried out with the HKL2000 program (Otwinowski and et al., 2000). Several considerations suggest that this model Minor, 1997). The phases were calculated with Phaser (McCoy et al., 2007) may not apply to the Stx6-Ang2 interaction characterized in using the Stx6 structure (PDB 1LVF) as a search model. Model building and our study. First, the Stx6 Habc domain does not seem to interact refinement of the Ang2-Stx6 complex were performed using Coot (Emsley with the SNARE motif (Misura et al., 2002). Second, although the and Cowtan, 2004) and PHENIX (Adams et al., 2010). Data collection and Stx6 partner Stx16 binds the SM protein Vps45 (Dulubova et al., refinement statistics are summarized in Table 1. 2002), Stx6 itself is not known to interact with Vps45. Finally, the ITC interaction of Stx6 with Ang2 does not involve the SNARE motif Stx6 (3–110), Stx10 (1–110), Ang2 (1–69), and the corresponding mutant and CATCHR fold of these proteins (Figure 1). proteins were dialyzed overnight at 4C against 150 mM NaCl, 50 mM

Our ﬁnding that Stx6 and Stx10 use the same binding motif to Tris-HCl (pH 7.4). For the ITC analysis, Ang2 (1–69) at 160 mM was injected interact with Ang2 emphasizes a mutually exclusive interaction into the Stx6 (3–110) or Stx10 (1–110) solution at 13 mM in aliquots of 10 ml. with the potential to participate in distinct membrane transport Measurements were carried out at 25 C on a VP-ITC Microcalorimeter events. In this regard, the transport of TGN46 and cholera toxin (MicroCal/GE Healthcare). ITC data were processed using Origin software (OriginLab). from early endosomes to the TGN requires a Stx6-containing SNARE complex, whereas MPR recycling from late endosomes ACCESSION NUMBERS to the TGN is more dependent on a Stx10-containing SNARE complex (Ganley et al., 2008). How SNAREs are enriched at The Protein Data Bank (http://www.rcsb.org/pdb) accession number for the different sites and how ternary t-SNARE complexes are preas- human Ang2 peptide bound to the human Stx6 Habc domain reported in sembled are still not well understood. In this regard, it is worth this paper is 4J2C.

SUPPLEMENTAL INFORMATION Ganley, I.G., Espinosa, E., and Pfeffer, S.R. (2008). A syntaxin 10-SNARE complex distinguishes two distinct transport routes from endosomes to the trans- Supplemental Information includes Supplemental Experimental Procedures, Golgi in human cells. J. Cell Biol. 180, 159–172. three figures, and one table and can be found with this article online at Hata, Y., Slaughter, C.A., and Su¨ dhof, T.C. (1993). Synaptic vesicle fusion http://dx.doi.org/10.1016/j.str.2013.06.025. complex contains unc-18 homologue bound to syntaxin. Nature 366, 347–351. Lerman, J.C., Robblee, J., Fairman, R., and Hughson, F.M. (2000). Structural ACKNOWLEDGMENTS analysis of the neuronal SNARE protein syntaxin-1A. Biochemistry 39, 8470– 8479. We thank A. Muga (University of the Basque Country) for help with ITC Li, W., Ma, C., Guan, R., Xu, Y., Tomchick, D.R., and Rizo, J. (2011). The crystal experiments and A. Vidaurrazaga for technical assistance. This study made structure of a Munc13 C-terminal module exhibits a remarkable similarity to use of the Proteomics Platform at CIC bioGUNE, member of CIBERehd and vesicle tethering factors. Structure 19, 1443–1455. ProteoRed-ISCIII and the European Synchrotron Radiation Facility (Grenoble) Liewen, H., Meinhold-Heerlein, I., Oliveira, V., Schwarzenbacher, R., Luo, G., under the Block Allocation Group MX1283/MX1420. This research was sup- Wadle, A., Jung, M., Pfreundschuh, M., and Stenner-Liewen, F. (2005). ported by Carlos III Health Institute grant PI11/00121, Basque Government Characterization of the human GARP (Golgi associated retrograde protein) grant PI2011-26 (to A.H.), and the intramural program of the National Institute complex. Exp. Cell Res. 306, 24–34. of Child Health and Human Development (to J.S.B.). G.A.-P. has been sup- ported by a fellowship from the Basque Government (BFI08.53). 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