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Crystal structure of human synbindin reveals two conformations of longin domain
Biochemical and Biophysical Research Communications 378 (2009) 338–343
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Biochemical and Biophysical Research Communications journal homepage: www.elsevier.com/locate/ybbrc
Crystal structure of human synbindin reveals two conformations of longin domain Shilong Fan a,b, Zhiyi Wei a,b, Hang Xu b, Weimin Gong a,b,* a b
School of Life Sciences, University of Science and Technology of China, Hefei, Anhui 230026, PR China National Key Laboratory of Macrobiomolecule, Center for Structural and Molecular Biology, Institute of Biophysics, Chinese Academy of Sciences, Beijing 100101, PR China
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Article history: Received 23 April 2008 Available online 6 May 2008
Keywords: Synbindin Crystal structure Longin domain
a b s t r a c t Transport protein particle (TRAPP) is a large multiprotein complex that involves in ER-to-Golgi and intraGolgi traffic. Synbindin, the human ortholog of yeast Trs23, is one component of the TRAPP complexes. In the hippocampal neurons the synbindin/syndecan complex is involved in synaptic membrane trafficking and thereby regulates the formation of dendritic spines. Here we present the three-dimensional structure of human synbindin, which contains a longin domain (LD) and an atypical PDZ domain (APD). In the crystal, synbindin forms a hexamer, in which the LD forms two different conformations and the APD is quite disordered. These conformational changes of synbindin suggest a possible interaction mode of the LD. Ó 2008 Elsevier Inc. All rights reserved.
The TRAPP complex is a large multi-subunit complex, which is found in both yeast and human [1,2]. BET3p, BET5p, Trs20p, Trs23p, Trs31p, Trs33p, and Trs85p are common subunits in TRAPP I and TRAPP II, and Trs65p, Trs120p, Trs130p are additional subunits of TRAPP II. The two forms of TRAPP act at different stages of membrane traffic [2]. TRAPP I binds endoplasmic-reticulum-derived vesicles to Golgi while TRAPP II is involved in transport within the Golgi. Both forms of the complex can exchange nucleotide on the Rab GTPase Ypt1p, which is consistent with the finding that TRAPP I and TRAPP II act in ER-Golgi and Golgi transport. Although the molecular mechanism governing TRAPP-mediated protein transport is still largely unknown, recent structural studies of various components of the TRAPP complex have provided much needed insights into the mode of assembly and possible action mechanism of the TRAPP complexes [3–8]. Synbindin, the human ortholog of yeast Trs23p, was first identified by a yeast two-hybrid screening using the syndecan-2 cytoplasmic domain as a bait. In hippocampal neurons, synbindin can associate with the synaptic membrane through direct interaction of its PDZ-like domain with the ‘‘EFYA” tail of Syndecan-2. It was
Abbreviations: PDZ, PSD-95/Dlg/ZO-1 homology; COG, conserved oligomeric Golgi; VFT, Vps fifty three; GARP, Golgi-associated retrograde protein ; HOPS, homotypic fusion and vacuole protein sorting; TRAPP, transport protein particle; COPII, coat protein complex II; SNARE, soluble NSF attachment receptor; LD, longin domain; APD, atypical PDZ-like domain; RMSD, root mean square deviation. * Corresponding author. Address: National Key Laboratory of Macrobiomolecule, Center for Structural and Molecular Biology, Institute of Biophysics, Chinese Academy of Sciences, Beijing 100101, PR China. Fax: +86 10 64888513. E-mail address: [email protected] (W. Gong). 0006-291X/$ - see front matter Ó 2008 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2008.04.143
further suggested that the synbindin/syndecan complex could be involved in synaptic membrane trafficking and thereby regulates the formation of dendritic spines [9]. We solved the crystal structure of synbindin at 2.8 Å resolution. Unexpectedly, we discovered that synbindin contains a longin domain split by an atypical PDZlike domain that is structurally similar to sedlin, although the two protein share very little amino acid sequence similarity. Materials and methods Protein expression and purification. The synbindin gene was amplified by PCR from a human brain library and cloned into the pET-22b vector, expressed in Escherichia coli BL21 (DE3) strain as a fusion protein containing a His6 tag at the C-terminus. Cell lysates were prepared by sonication in a buffer consisting 20 mM Tris–HCl, pH 7.5, 200 mM NaCl, and 1 mM PMSF. Synbindin was purified with Ni-affinity chromatography and further purified using a Superdex G-75 gel-filtration column (Amersham Biosciences). Selenomethionine-substituted synbindin was prepared by transforming E. coli B834 (DE3) RIL methionine auxotroph cells (Novagen) with the pET-22b vector containing the synbindin gene and growing the cells in selenomethionine-containing minimal medium. Purification of selenomethionyl synbindin was performed as described above. Crystallography. Crystals of native and selenomethionyl human synbindin were both obtained at 4 °C by the hanging-drop vapordiffusion method and drops were prepared by mixing 8 mg/ml protein with 2% MPD, 3% EG, 1% PEG 8000, and 0.1 M Hepes at pH 7.5. Data from a native crystal to 2.8 Å resolution were collected at
S. Fan et al. / Biochemical and Biophysical Research Communications 378 (2009) 338–343
100 K at a Rigaku R-AXIS IVCC imaging-plate system with a Rigaku FRE Cu rotating-anode generator. MAD (multiple-wavelength anomalous dispersion) data at three different wavelengths were collected with a selenomethionyl synbindin crystal at 100 K on beam line 3W1A of the Beijing Synchrotron Radiation Facility at Institute of High Energy Physics. All data were processed and scaled with HKL2000 [10]. Five selenium sites were located and used for phase determination at 3.5 Å by SOLVE [11] with the MAD data set, and phases were subsequently improved by density modification with RESOLVE [12]. The initial model building was performed manually with the selenium sites as references. The model was refined with 2.8 Å resolution data in CNS [13] for additional model building and adjustment and then in Refmac for TLS refinement [14,15]. The stereochemical quality of the final model of human synbindin was checked by PROCHECK [16]. Data collection and structural refinement statistics are listed in Table 1. Results and discussion Overall structure The final model of human synbindin consists of two monomers (A and B) per asymmetric unit. Because of weak electron density, some connecting residues between secondary elements are absent in the model. The disordered residues include residues 87–90 and 124–132 in the monomer A, while residues 28 and 124–130 in the molecule B. The C-terminal 7 (the monomer A) or 8 (the monomer B) residues together with the affinity His6 tag are not visible in the electron density map. The most striking feature of the synbindin structure is that the protein contains a split longin domain (LD), assembled by LDN for N-terminal fragment (residues 2–23) and the LDC for C-terminal one (residues 103–212). A PDZ-like domain with only 75 amino acid residue is inserted between LDN and LDC in the primary sequence (Fig. 1). We termed the PDZ-like domain in synbindin as an atypical PDZ domain (APD) due to its exceptionally unique structural and sequence features compared to all known PDZ domains defined by SMART (Simple Modular Architecture Research
Table 1 Statistics of data collection, MAD phasing, and structural refinement Native data MAD data
Data collection Space group Unit cell parameters Wavelength (Å) Resolution range (Å) No. of total reflections Unique reflections Completeness (%) Rsym (%) I/r Refinement Resolution range (Å) R-factor/Rfree (%) r.m.s.d bond length (Å) r.m.s.d bond angle (°) Number of atoms Average B-factor (Å2)
P213 a = 124.391 1.5418 50.0–2.8 (2.8–2.9) 254,534 16,132 98.1 (99.8) 5.4 (44.0) 24.1 (4.26) 50.0–2.8 (2.87–2.8) 24.3/29.0 (33.8/35.4) 0.008 1.067 3205 73.7
Ramachandran plot (%) Most favored region 87.9 Additionally allowed region 11.8 Generously allowed region 0.3
Peak
Edge
Remote
P213 a = 123.685 0.9787 50.0–3.5 (3.5–3.8) 257,603 8183 100 (100) 14.3 (50.1) 21.1 (6.27)
P213 a = 123.594 0.9793 50.0–3.6 (3.60–3.68) 129,419 7537 99.9 (100) 14.8 (50.1) 16.44 (5.17)
P213 a = 123.621 0.9500 50.0–3.6 (3.60–3.68) 24,470 7537 100 (100) 11.7 (35.2) 25.1 (9.19)
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Tool, http://smart.embl.de/). Additionally, very few interactions between the LD and the APD could be observed in both monomers (Fig. 1B and Fig. 2A), suggesting that the connecting sequences between the two domains are flexible. The split longin domain The fold of the synbindin LD is highly unusual, as the first two b-strand of the domain are contributed by the very N-terminal sequences prior to the PDZ-like domain of the protein. The synbindin LD represents the first split LD structure among known longin domains. Although split by the APD, the overall fold of the synbindin LD resembles those of canonical login domains, which consists of a central five-stranded antiparallel b-sheet sandwiched by one helix (a1L) in one side and two helices (a2L and a3L) in the other side (Fig. 1B). Structural similarity search results with DALI [17] and SSM[18] show that the split LD of human synbindin share highly structural similarity to other login domains, such as those in SEDL [8], Sec22b [19], SRX of SRa [20,21], Ykt6p [22], r1 or r2 adaptin of the AP1 or AP2 complex [23,24], and MP1 [25,26], although, the sequence similarities between synbindin and any of these longin domains are low (8–18% sequence identity). The LDN consists of a b-hairpin (b1L and b2L), and the LDC consists of the other three b-strands and the three a-helices (Fig. 1B). A glycine residue (Gly13) in the b-hairpin turn is strictly conserved in synbindins (Fig. 1A). It was found that the glycine was also conserved in all known LD structures and speculated to facilitate the b-hairpin turn, except that in SEDL, where the glycine is replaced by an aspartate and the lack is compensated by adjustments in the adjacent b-strand [21]. In the LDC, a1L connects the APD and the central b-sheet. The orientation of a1L is different in the monomer A and B as well as the length by superposing the LDs of the two monomers (Fig. 2A), suggesting a1L is flexible. The flexibility of a1L results in two conformations proposed as two states: the ‘open’ state (the monomer A) and the ‘closed’ state (the monomer B). In the ‘closed’ state, a1L lies on the middle of the central b-sheet and there are two hydrophobic patches formed by about a dozen of conserved hydrophobic residues on both two sides of a1L (Fig. 1A and 2B). However, in the ‘open’ state, a1L move to one side of the b-sheet to expose most of the conserved hydrophobic residues together with aliphatic side-chain atoms of a strictly conserved lysine (Lys154) forming a large hydrophobic groove on the other side (Fig. 2C). In addition, the Ne atom of Lys154 forms a hydrogen bond with the carbonyl oxygen of another strictly conserved residue (Gly152). It causes the aliphatic side-chain atoms of Lys154 to be fixed to face the solvent side, thereby involved in the hydrophobic groove. The hydrophobic groove is the major part of the interface between the two monomers (Fig. 2A and B). Recently, the crystal structure of a subcomplex of mammalian TRAPP I containing human synbindin was determined [27]. Structural comparison shows that the synbindin structure in the subcomplex is highly similar with our structure except of a1L (Fig. 3). Compared with the synbindin structure in the subcomplex, the a1L orientation in the ‘closed’ state is more similar than that in the ‘open’ state, but the a1L length in the ‘closed’ state is shorter while that in the ‘open’ state is similar (Fig. 3). It further confirms that a1L is highly flexible, especially at its C-terminal part. Hexamerization of human synbindin in crystal Analysis of the crystal packing of human synbindin suggested a hexameric structure, formed by three equals of the monomers A and B related by the cubic threefold axis (Fig. 4A). In the hexamer structure, each of the six monomers interacts with two adjacent ones through two different interfaces, respectively, which are both
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Fig. 1. (A) Multiple-sequence alignment of synbindins. The synbindin sequences of Homo sapiens (Human, Swiss-Prot Accession No. Q9Y296), Mus musculus (Mouse, Q9ES56), Drosophila melanogaster (Fruit fly, Q9VLI9), Caenorhabditis elegans (Q20100), Arabidopsis thaliana (Q9LZ97), Schizosaccharomyces pombe (Fission yeast, O43041), and Saccharomyces cerevisiae (Baker’s yeast, Q03784) are aligned using Tcoffee [28]. The secondary structures of human synbindin, which is defined by the DSSP program [29], are indicated above the alignment diagram. Because the length of a2L in the monomer A is more than that in the monomer B, the extra part of a2L in the monomer A is colored in orange. The regions of LDN, APD, and LDC are labeled above the secondary structure and colored in purple, cyan, and red, respectively. Identical and chemically similar residues in the alignment are boxed in red and yellow, respectively. The conserved residues of the LDC, which are involved in forming the hydrophobic patches in the ‘closed’ state and the hydrophobic groove in the ‘open’ state, are indicated by cyan cycles. Red cycles and purple triangle ups denote the interfaces for interacting with Leu109 and the hydrophobic groove, respectively. The conserved h-Asp-h motif (‘h’ standing for a hydrophobic residue) of the APD is boxed with orange. (B) Stereo view of the overall structure of human synbindin. The monomer B structure is used in this representation. The LDN, APD, and LDC are in the colors corresponding to (A). The secondary structural elements are labeled in the left diagram. The disordered region (residues 124–130) is indicated by a dashed line. Figures are prepared with ESPript [30], Ribbons [31], PyMol [32]. (For interpretation of color mentioned in this figure the reader is referred to the web version of the article.)
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Fig. 2. Comparing the monomers with the closed and the ‘open’ states of human synbindin. (A) Ribbons view of the two monomers with their LDs superposed. The LD and the APD are colored in orange, respectively, in the monomer A and in blue, respectively, in the monomer B. (B,C) Surface distribution of conserved hydrophobic residues in the LD with the closed (the monomer B) and the open (the monomer A) states, respectively. Hydrophobic patches in the ‘closed’ state and the hydrophobic groove in the ‘open’ state are colored in cyan. The two major patches are circled and the groove is indicated an arrow. The disordered regions are indicated by dashed lines. (For interpretation of color mentioned in this figure the reader is referred to the web version of the article.)
located on the LDs (Fig. 4A). For clarity and convenience, the interfaces between the monomers A and B and between the monomers B and A0 (related with the monomer A through the threefold axis) are named as the interface I and II, respectively. The interface I, as
Fig. 3. Comparison between the ‘open’ state, the ‘close’ state of our structure, and the human synbindin in the subcomplex of TRAPP I. The ‘open’ state, the ‘close’ state, and the human synbindin in the subcomplex are colored in orange, blue, and green, respectively. The different orientations of a1L from the three structures are shown in the front. The residue ranges of a1L of the ‘open’ state, the ‘close’ state, and the human synbindin in the subcomplex are labled. (For interpretation of color mentioned in this figure the reader is referred to the web version of the article.)
described above, involves the hydrophobic groove of the monomer A. The region of the monomer B interacting with the groove, is comprised of the C-terminus of a2L (Leu182/B and Lys183/B, B standing for the monomer B) and the following loop connecting a2L and a3L (Asn184/B, Pro185/B, and Phe186/B) (Fig. 4B). The hydrophobic side-chains of Leu182/B and Pro185/B together with aliphatic side-chain atoms of Lys183/B are involved in the hydrophobic interaction with the groove. In addition, Lys183/B forms a salt bridge with a strictly conserved residue Asp179/B, which avoids its Ne atom to stretch into the groove. The highly conserved residue Leu109/A also contributes to the interface I by protruding from a2L and inserting into a hydrophobic pocket of the monomer B and interacting with some conserved hydrophobic residues Tyr174/B, Tyr177/B, Ala181/B, and Leu189/B (Fig. 4B). Except for a1L, the LDs of the monomer B and A0 are related by a non-crystallographic twofold axis and interact with each other through an edge-to-edge contact of their central b-sheets to form a large 10-stranded antiparallel b-sheet (Fig. 4). The association of the two central b-sheets are through four hydrogen bonds between the two ‘edge’ b-strand (b3L) and two salt bridges between Lys143/B and Glu138/A0 or Lys143/A0 and Glu138/B (Fig. 4C), which forms the first part of the interface II. The hydrophobic interaction between one hydrophobic patch (on one side of a1L) of the monomer B and three hydrophobic residues (Met114/A0 , Leu118/A0 , and Leu137/A0 ) of the monomer A0 produce the second part of the interface II (Fig. 4C). Differing from a homohexamer in crystal, human synbindin is a monomer in solution indicating by gel-filtration chromatography (data not shown). It shows that the interactions between synbindin in crystal is not enough for maintain the oligomer, suggesting that
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Fig. 4. The homohexamer structure of human synbindin and their interacting modes. (A) Ribbons representation of the homohexamer of human synbindin. The APDs of the six monomoers are omitted to emphasize the interactions between the six LDs. The homohexamer is assembled through the threefold axis, which is indicated by a red triangle up. The monomers A, B, and A0 is colored in orange, blue, and green, respectively, with other three monomers are colored in grey. Green and orange boxes denote the interfaces I (between the monomers A and B) and II (between B and A0 ), respectively. The right picture is a vertically inverted view of the left. (B) The interface I. The surface of the monomer A is colored as same as Fig. 2C and the conserved residue Leu109 is labeled and showed as sticks and balls. In the monomer B, the conserved residues interact with the hydrophobic groove and interact with Leu109 are labeled and showed as sticks and balls with purple and red color. (C) The interface II. In the left diagram, the residues (Ile134, Glu135, Met136, Leu137, and Glu138) forming b3L together with Lys143 from both of the two monomers are showed as sticks and balls. Black and purple dashed lines indicate the hydrogen bonds formed by the residues of the two b3L and the salt bridge formed by Lys143 and Glu138, respectively. In the right diagram, the residues involved in hydrophobic interactions between the two monomers are labeled and showed as sticks and balls. The view of the interface in the left picture is rotated by 90° around a horizontal axis in the right one. (For interpretation of color mentioned in this figure the reader is referred to the web version of the article.)
the homohexamer of synbindin does not exist physiologically. Nevertheless, the two interaction modes with the two different interfaces found in the LD are meaningful for biological functions of synbindin. The interaction mode through the two ‘edge’ b-strands (interface II) is consists with the similar interface found in the subcomplex structure of TRAPP I, of which the ‘edge’ b-strand of synbindin/Trs23 interacts with that of Mum2/Bet5 [27]. As to the other interaction mode (interface I), the conserved region of Bet5 corresponding the C-terminus of a2L and the followed loop in human synbindin are found to be involved in interacting with Trs33-Bet3 heterodimer [27]. These consistencies on the interaction modes suggest that synbindin could form oligomers or even huge polymer by the two interaction modes with the corresponding interfaces. Synbindin has been observed that could cluster in dendrites, which is required for the interaction between the ‘‘EFYA” tail of synbindin and syndecan-2 [9]. The clustering of synbindin could be mediated by the interaction modes of the LD. Protein data bank accession codes The atomic coordinates have been deposited in the RCSB Protein Data Bank with the accession codes 2ZMVfor synbindin crystal structure.
Acknowledgments This work is supported by Ministry of Science and Technology (Grant Nos. 2004CB720008 and 2006CB0D1705), 863 program (2006AA02A316), the National Natural Science Foundation of China (Grant Nos. 10490193 and 30721003) and the Chinese Academy of Sciences (KSCX2-YW-R-61). We thank Dr. Yuhui Dong and Dr. Peng Liu in Institute of High Energy Physics and Mr. Yi Han in Institute of Biophysics for diffraction data collection. References [1] M. Sacher, Y. Jiang, J. Barrowman, A. Scarpa, J. Burston, L. Zhang, D. Schieltz, J.R. Yates 3rd, H. Abeliovich, S. Ferro-Novick, TRAPP, a highly conserved novel complex on the cis-Golgi that mediates vesicle docking and fusion, EMBO J. 17 (1998) 2494–2503. [2] M. Sacher, J. Barrowman, W. Wang, J. Horecka, Y. Zhang, M. Pypaert, S. FerroNovick, TRAPP I implicated in the specificity of tethering in ER-to-Golgi transport, Mol. Cell 7 (2001) 433–442. [3] D. Kummel, J.J. Muller, Y. Roske, R. Misselwitz, K. Bussow, U. Heinemann, The structure of the TRAPP subunit TPC6 suggests a model for a TRAPP subcomplex, EMBO Rep. 6 (2005) 787–793. [4] A.P. Turnbull, D. Kummel, B. Prinz, C. Holz, J. Schultchen, C. Lang, F.H. Niesen, K.P. Hofmann, H. Delbruck, J. Behlke, E.C. Muller, E. Jarosch, T. Sommer, U. Heinemann, Structure of palmitoylated BET3: insights into TRAPP complex assembly and membrane localization, EMBO J. 24 (2005) 875–884.
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Crystal structure of human synbindin reveals two conformations of longin domain Shilong Fan a,b, Zhiyi Wei a,b, Hang Xu b, Weimin Gong a,b,* a b
School of Life Sciences, University of Science and Technology of China, Hefei, Anhui 230026, PR China National Key Laboratory of Macrobiomolecule, Center for Structural and Molecular Biology, Institute of Biophysics, Chinese Academy of Sciences, Beijing 100101, PR China
a r t i c l e
i n f o
Article history: Received 23 April 2008 Available online 6 May 2008
Keywords: Synbindin Crystal structure Longin domain
a b s t r a c t Transport protein particle (TRAPP) is a large multiprotein complex that involves in ER-to-Golgi and intraGolgi traffic. Synbindin, the human ortholog of yeast Trs23, is one component of the TRAPP complexes. In the hippocampal neurons the synbindin/syndecan complex is involved in synaptic membrane trafficking and thereby regulates the formation of dendritic spines. Here we present the three-dimensional structure of human synbindin, which contains a longin domain (LD) and an atypical PDZ domain (APD). In the crystal, synbindin forms a hexamer, in which the LD forms two different conformations and the APD is quite disordered. These conformational changes of synbindin suggest a possible interaction mode of the LD. Ó 2008 Elsevier Inc. All rights reserved.
The TRAPP complex is a large multi-subunit complex, which is found in both yeast and human [1,2]. BET3p, BET5p, Trs20p, Trs23p, Trs31p, Trs33p, and Trs85p are common subunits in TRAPP I and TRAPP II, and Trs65p, Trs120p, Trs130p are additional subunits of TRAPP II. The two forms of TRAPP act at different stages of membrane traffic [2]. TRAPP I binds endoplasmic-reticulum-derived vesicles to Golgi while TRAPP II is involved in transport within the Golgi. Both forms of the complex can exchange nucleotide on the Rab GTPase Ypt1p, which is consistent with the finding that TRAPP I and TRAPP II act in ER-Golgi and Golgi transport. Although the molecular mechanism governing TRAPP-mediated protein transport is still largely unknown, recent structural studies of various components of the TRAPP complex have provided much needed insights into the mode of assembly and possible action mechanism of the TRAPP complexes [3–8]. Synbindin, the human ortholog of yeast Trs23p, was first identified by a yeast two-hybrid screening using the syndecan-2 cytoplasmic domain as a bait. In hippocampal neurons, synbindin can associate with the synaptic membrane through direct interaction of its PDZ-like domain with the ‘‘EFYA” tail of Syndecan-2. It was
Abbreviations: PDZ, PSD-95/Dlg/ZO-1 homology; COG, conserved oligomeric Golgi; VFT, Vps fifty three; GARP, Golgi-associated retrograde protein ; HOPS, homotypic fusion and vacuole protein sorting; TRAPP, transport protein particle; COPII, coat protein complex II; SNARE, soluble NSF attachment receptor; LD, longin domain; APD, atypical PDZ-like domain; RMSD, root mean square deviation. * Corresponding author. Address: National Key Laboratory of Macrobiomolecule, Center for Structural and Molecular Biology, Institute of Biophysics, Chinese Academy of Sciences, Beijing 100101, PR China. Fax: +86 10 64888513. E-mail address: [email protected] (W. Gong). 0006-291X/$ - see front matter Ó 2008 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2008.04.143
further suggested that the synbindin/syndecan complex could be involved in synaptic membrane trafficking and thereby regulates the formation of dendritic spines [9]. We solved the crystal structure of synbindin at 2.8 Å resolution. Unexpectedly, we discovered that synbindin contains a longin domain split by an atypical PDZlike domain that is structurally similar to sedlin, although the two protein share very little amino acid sequence similarity. Materials and methods Protein expression and purification. The synbindin gene was amplified by PCR from a human brain library and cloned into the pET-22b vector, expressed in Escherichia coli BL21 (DE3) strain as a fusion protein containing a His6 tag at the C-terminus. Cell lysates were prepared by sonication in a buffer consisting 20 mM Tris–HCl, pH 7.5, 200 mM NaCl, and 1 mM PMSF. Synbindin was purified with Ni-affinity chromatography and further purified using a Superdex G-75 gel-filtration column (Amersham Biosciences). Selenomethionine-substituted synbindin was prepared by transforming E. coli B834 (DE3) RIL methionine auxotroph cells (Novagen) with the pET-22b vector containing the synbindin gene and growing the cells in selenomethionine-containing minimal medium. Purification of selenomethionyl synbindin was performed as described above. Crystallography. Crystals of native and selenomethionyl human synbindin were both obtained at 4 °C by the hanging-drop vapordiffusion method and drops were prepared by mixing 8 mg/ml protein with 2% MPD, 3% EG, 1% PEG 8000, and 0.1 M Hepes at pH 7.5. Data from a native crystal to 2.8 Å resolution were collected at
S. Fan et al. / Biochemical and Biophysical Research Communications 378 (2009) 338–343
100 K at a Rigaku R-AXIS IVCC imaging-plate system with a Rigaku FRE Cu rotating-anode generator. MAD (multiple-wavelength anomalous dispersion) data at three different wavelengths were collected with a selenomethionyl synbindin crystal at 100 K on beam line 3W1A of the Beijing Synchrotron Radiation Facility at Institute of High Energy Physics. All data were processed and scaled with HKL2000 [10]. Five selenium sites were located and used for phase determination at 3.5 Å by SOLVE [11] with the MAD data set, and phases were subsequently improved by density modification with RESOLVE [12]. The initial model building was performed manually with the selenium sites as references. The model was refined with 2.8 Å resolution data in CNS [13] for additional model building and adjustment and then in Refmac for TLS refinement [14,15]. The stereochemical quality of the final model of human synbindin was checked by PROCHECK [16]. Data collection and structural refinement statistics are listed in Table 1. Results and discussion Overall structure The final model of human synbindin consists of two monomers (A and B) per asymmetric unit. Because of weak electron density, some connecting residues between secondary elements are absent in the model. The disordered residues include residues 87–90 and 124–132 in the monomer A, while residues 28 and 124–130 in the molecule B. The C-terminal 7 (the monomer A) or 8 (the monomer B) residues together with the affinity His6 tag are not visible in the electron density map. The most striking feature of the synbindin structure is that the protein contains a split longin domain (LD), assembled by LDN for N-terminal fragment (residues 2–23) and the LDC for C-terminal one (residues 103–212). A PDZ-like domain with only 75 amino acid residue is inserted between LDN and LDC in the primary sequence (Fig. 1). We termed the PDZ-like domain in synbindin as an atypical PDZ domain (APD) due to its exceptionally unique structural and sequence features compared to all known PDZ domains defined by SMART (Simple Modular Architecture Research
Table 1 Statistics of data collection, MAD phasing, and structural refinement Native data MAD data
Data collection Space group Unit cell parameters Wavelength (Å) Resolution range (Å) No. of total reflections Unique reflections Completeness (%) Rsym (%) I/r Refinement Resolution range (Å) R-factor/Rfree (%) r.m.s.d bond length (Å) r.m.s.d bond angle (°) Number of atoms Average B-factor (Å2)
P213 a = 124.391 1.5418 50.0–2.8 (2.8–2.9) 254,534 16,132 98.1 (99.8) 5.4 (44.0) 24.1 (4.26) 50.0–2.8 (2.87–2.8) 24.3/29.0 (33.8/35.4) 0.008 1.067 3205 73.7
Ramachandran plot (%) Most favored region 87.9 Additionally allowed region 11.8 Generously allowed region 0.3
Peak
Edge
Remote
P213 a = 123.685 0.9787 50.0–3.5 (3.5–3.8) 257,603 8183 100 (100) 14.3 (50.1) 21.1 (6.27)
P213 a = 123.594 0.9793 50.0–3.6 (3.60–3.68) 129,419 7537 99.9 (100) 14.8 (50.1) 16.44 (5.17)
P213 a = 123.621 0.9500 50.0–3.6 (3.60–3.68) 24,470 7537 100 (100) 11.7 (35.2) 25.1 (9.19)
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Tool, http://smart.embl.de/). Additionally, very few interactions between the LD and the APD could be observed in both monomers (Fig. 1B and Fig. 2A), suggesting that the connecting sequences between the two domains are flexible. The split longin domain The fold of the synbindin LD is highly unusual, as the first two b-strand of the domain are contributed by the very N-terminal sequences prior to the PDZ-like domain of the protein. The synbindin LD represents the first split LD structure among known longin domains. Although split by the APD, the overall fold of the synbindin LD resembles those of canonical login domains, which consists of a central five-stranded antiparallel b-sheet sandwiched by one helix (a1L) in one side and two helices (a2L and a3L) in the other side (Fig. 1B). Structural similarity search results with DALI [17] and SSM[18] show that the split LD of human synbindin share highly structural similarity to other login domains, such as those in SEDL [8], Sec22b [19], SRX of SRa [20,21], Ykt6p [22], r1 or r2 adaptin of the AP1 or AP2 complex [23,24], and MP1 [25,26], although, the sequence similarities between synbindin and any of these longin domains are low (8–18% sequence identity). The LDN consists of a b-hairpin (b1L and b2L), and the LDC consists of the other three b-strands and the three a-helices (Fig. 1B). A glycine residue (Gly13) in the b-hairpin turn is strictly conserved in synbindins (Fig. 1A). It was found that the glycine was also conserved in all known LD structures and speculated to facilitate the b-hairpin turn, except that in SEDL, where the glycine is replaced by an aspartate and the lack is compensated by adjustments in the adjacent b-strand [21]. In the LDC, a1L connects the APD and the central b-sheet. The orientation of a1L is different in the monomer A and B as well as the length by superposing the LDs of the two monomers (Fig. 2A), suggesting a1L is flexible. The flexibility of a1L results in two conformations proposed as two states: the ‘open’ state (the monomer A) and the ‘closed’ state (the monomer B). In the ‘closed’ state, a1L lies on the middle of the central b-sheet and there are two hydrophobic patches formed by about a dozen of conserved hydrophobic residues on both two sides of a1L (Fig. 1A and 2B). However, in the ‘open’ state, a1L move to one side of the b-sheet to expose most of the conserved hydrophobic residues together with aliphatic side-chain atoms of a strictly conserved lysine (Lys154) forming a large hydrophobic groove on the other side (Fig. 2C). In addition, the Ne atom of Lys154 forms a hydrogen bond with the carbonyl oxygen of another strictly conserved residue (Gly152). It causes the aliphatic side-chain atoms of Lys154 to be fixed to face the solvent side, thereby involved in the hydrophobic groove. The hydrophobic groove is the major part of the interface between the two monomers (Fig. 2A and B). Recently, the crystal structure of a subcomplex of mammalian TRAPP I containing human synbindin was determined [27]. Structural comparison shows that the synbindin structure in the subcomplex is highly similar with our structure except of a1L (Fig. 3). Compared with the synbindin structure in the subcomplex, the a1L orientation in the ‘closed’ state is more similar than that in the ‘open’ state, but the a1L length in the ‘closed’ state is shorter while that in the ‘open’ state is similar (Fig. 3). It further confirms that a1L is highly flexible, especially at its C-terminal part. Hexamerization of human synbindin in crystal Analysis of the crystal packing of human synbindin suggested a hexameric structure, formed by three equals of the monomers A and B related by the cubic threefold axis (Fig. 4A). In the hexamer structure, each of the six monomers interacts with two adjacent ones through two different interfaces, respectively, which are both
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Fig. 1. (A) Multiple-sequence alignment of synbindins. The synbindin sequences of Homo sapiens (Human, Swiss-Prot Accession No. Q9Y296), Mus musculus (Mouse, Q9ES56), Drosophila melanogaster (Fruit fly, Q9VLI9), Caenorhabditis elegans (Q20100), Arabidopsis thaliana (Q9LZ97), Schizosaccharomyces pombe (Fission yeast, O43041), and Saccharomyces cerevisiae (Baker’s yeast, Q03784) are aligned using Tcoffee [28]. The secondary structures of human synbindin, which is defined by the DSSP program [29], are indicated above the alignment diagram. Because the length of a2L in the monomer A is more than that in the monomer B, the extra part of a2L in the monomer A is colored in orange. The regions of LDN, APD, and LDC are labeled above the secondary structure and colored in purple, cyan, and red, respectively. Identical and chemically similar residues in the alignment are boxed in red and yellow, respectively. The conserved residues of the LDC, which are involved in forming the hydrophobic patches in the ‘closed’ state and the hydrophobic groove in the ‘open’ state, are indicated by cyan cycles. Red cycles and purple triangle ups denote the interfaces for interacting with Leu109 and the hydrophobic groove, respectively. The conserved h-Asp-h motif (‘h’ standing for a hydrophobic residue) of the APD is boxed with orange. (B) Stereo view of the overall structure of human synbindin. The monomer B structure is used in this representation. The LDN, APD, and LDC are in the colors corresponding to (A). The secondary structural elements are labeled in the left diagram. The disordered region (residues 124–130) is indicated by a dashed line. Figures are prepared with ESPript [30], Ribbons [31], PyMol [32]. (For interpretation of color mentioned in this figure the reader is referred to the web version of the article.)
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Fig. 2. Comparing the monomers with the closed and the ‘open’ states of human synbindin. (A) Ribbons view of the two monomers with their LDs superposed. The LD and the APD are colored in orange, respectively, in the monomer A and in blue, respectively, in the monomer B. (B,C) Surface distribution of conserved hydrophobic residues in the LD with the closed (the monomer B) and the open (the monomer A) states, respectively. Hydrophobic patches in the ‘closed’ state and the hydrophobic groove in the ‘open’ state are colored in cyan. The two major patches are circled and the groove is indicated an arrow. The disordered regions are indicated by dashed lines. (For interpretation of color mentioned in this figure the reader is referred to the web version of the article.)
located on the LDs (Fig. 4A). For clarity and convenience, the interfaces between the monomers A and B and between the monomers B and A0 (related with the monomer A through the threefold axis) are named as the interface I and II, respectively. The interface I, as
Fig. 3. Comparison between the ‘open’ state, the ‘close’ state of our structure, and the human synbindin in the subcomplex of TRAPP I. The ‘open’ state, the ‘close’ state, and the human synbindin in the subcomplex are colored in orange, blue, and green, respectively. The different orientations of a1L from the three structures are shown in the front. The residue ranges of a1L of the ‘open’ state, the ‘close’ state, and the human synbindin in the subcomplex are labled. (For interpretation of color mentioned in this figure the reader is referred to the web version of the article.)
described above, involves the hydrophobic groove of the monomer A. The region of the monomer B interacting with the groove, is comprised of the C-terminus of a2L (Leu182/B and Lys183/B, B standing for the monomer B) and the following loop connecting a2L and a3L (Asn184/B, Pro185/B, and Phe186/B) (Fig. 4B). The hydrophobic side-chains of Leu182/B and Pro185/B together with aliphatic side-chain atoms of Lys183/B are involved in the hydrophobic interaction with the groove. In addition, Lys183/B forms a salt bridge with a strictly conserved residue Asp179/B, which avoids its Ne atom to stretch into the groove. The highly conserved residue Leu109/A also contributes to the interface I by protruding from a2L and inserting into a hydrophobic pocket of the monomer B and interacting with some conserved hydrophobic residues Tyr174/B, Tyr177/B, Ala181/B, and Leu189/B (Fig. 4B). Except for a1L, the LDs of the monomer B and A0 are related by a non-crystallographic twofold axis and interact with each other through an edge-to-edge contact of their central b-sheets to form a large 10-stranded antiparallel b-sheet (Fig. 4). The association of the two central b-sheets are through four hydrogen bonds between the two ‘edge’ b-strand (b3L) and two salt bridges between Lys143/B and Glu138/A0 or Lys143/A0 and Glu138/B (Fig. 4C), which forms the first part of the interface II. The hydrophobic interaction between one hydrophobic patch (on one side of a1L) of the monomer B and three hydrophobic residues (Met114/A0 , Leu118/A0 , and Leu137/A0 ) of the monomer A0 produce the second part of the interface II (Fig. 4C). Differing from a homohexamer in crystal, human synbindin is a monomer in solution indicating by gel-filtration chromatography (data not shown). It shows that the interactions between synbindin in crystal is not enough for maintain the oligomer, suggesting that
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Fig. 4. The homohexamer structure of human synbindin and their interacting modes. (A) Ribbons representation of the homohexamer of human synbindin. The APDs of the six monomoers are omitted to emphasize the interactions between the six LDs. The homohexamer is assembled through the threefold axis, which is indicated by a red triangle up. The monomers A, B, and A0 is colored in orange, blue, and green, respectively, with other three monomers are colored in grey. Green and orange boxes denote the interfaces I (between the monomers A and B) and II (between B and A0 ), respectively. The right picture is a vertically inverted view of the left. (B) The interface I. The surface of the monomer A is colored as same as Fig. 2C and the conserved residue Leu109 is labeled and showed as sticks and balls. In the monomer B, the conserved residues interact with the hydrophobic groove and interact with Leu109 are labeled and showed as sticks and balls with purple and red color. (C) The interface II. In the left diagram, the residues (Ile134, Glu135, Met136, Leu137, and Glu138) forming b3L together with Lys143 from both of the two monomers are showed as sticks and balls. Black and purple dashed lines indicate the hydrogen bonds formed by the residues of the two b3L and the salt bridge formed by Lys143 and Glu138, respectively. In the right diagram, the residues involved in hydrophobic interactions between the two monomers are labeled and showed as sticks and balls. The view of the interface in the left picture is rotated by 90° around a horizontal axis in the right one. (For interpretation of color mentioned in this figure the reader is referred to the web version of the article.)
the homohexamer of synbindin does not exist physiologically. Nevertheless, the two interaction modes with the two different interfaces found in the LD are meaningful for biological functions of synbindin. The interaction mode through the two ‘edge’ b-strands (interface II) is consists with the similar interface found in the subcomplex structure of TRAPP I, of which the ‘edge’ b-strand of synbindin/Trs23 interacts with that of Mum2/Bet5 [27]. As to the other interaction mode (interface I), the conserved region of Bet5 corresponding the C-terminus of a2L and the followed loop in human synbindin are found to be involved in interacting with Trs33-Bet3 heterodimer [27]. These consistencies on the interaction modes suggest that synbindin could form oligomers or even huge polymer by the two interaction modes with the corresponding interfaces. Synbindin has been observed that could cluster in dendrites, which is required for the interaction between the ‘‘EFYA” tail of synbindin and syndecan-2 [9]. The clustering of synbindin could be mediated by the interaction modes of the LD. Protein data bank accession codes The atomic coordinates have been deposited in the RCSB Protein Data Bank with the accession codes 2ZMVfor synbindin crystal structure.
Acknowledgments This work is supported by Ministry of Science and Technology (Grant Nos. 2004CB720008 and 2006CB0D1705), 863 program (2006AA02A316), the National Natural Science Foundation of China (Grant Nos. 10490193 and 30721003) and the Chinese Academy of Sciences (KSCX2-YW-R-61). We thank Dr. Yuhui Dong and Dr. Peng Liu in Institute of High Energy Physics and Mr. Yi Han in Institute of Biophysics for diffraction data collection. References [1] M. Sacher, Y. Jiang, J. Barrowman, A. Scarpa, J. Burston, L. Zhang, D. Schieltz, J.R. Yates 3rd, H. Abeliovich, S. Ferro-Novick, TRAPP, a highly conserved novel complex on the cis-Golgi that mediates vesicle docking and fusion, EMBO J. 17 (1998) 2494–2503. [2] M. Sacher, J. Barrowman, W. Wang, J. Horecka, Y. Zhang, M. Pypaert, S. FerroNovick, TRAPP I implicated in the specificity of tethering in ER-to-Golgi transport, Mol. Cell 7 (2001) 433–442. [3] D. Kummel, J.J. Muller, Y. Roske, R. Misselwitz, K. Bussow, U. Heinemann, The structure of the TRAPP subunit TPC6 suggests a model for a TRAPP subcomplex, EMBO Rep. 6 (2005) 787–793. [4] A.P. Turnbull, D. Kummel, B. Prinz, C. Holz, J. Schultchen, C. Lang, F.H. Niesen, K.P. Hofmann, H. Delbruck, J. Behlke, E.C. Muller, E. Jarosch, T. Sommer, U. Heinemann, Structure of palmitoylated BET3: insights into TRAPP complex assembly and membrane localization, EMBO J. 24 (2005) 875–884.
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