- Email: [email protected]
Crystal structure of inactive form of Rab3B
Biochemical and Biophysical Research Communications 418 (2012) 841–844
Contents lists available at SciVerse ScienceDirect
Biochemical and Biophysical Research Communications journal homepage: www.elsevier.com/locate/ybbrc
Crystal structure of inactive form of Rab3B Wei Zhang a, Yang Shen b, Ronghong Jiao c, Yanli Liu a, Lingfu Deng a, Chao Qi a,⇑ a
Hubei Key Laboratory of Genetic Regulation and Integrative Biology, College of Life Science, Huazhong Normal University, Wuhan 430079, PR China Structural Genomics Consortium, University of Toronto, 101 College St., Toronto, Ontario, Canada M5G 1L7 c Department of Function Inspection, Hebei Provincial People’s Hospital, Shijiazhuang 050051, PR China b
a r t i c l e
i n f o
Article history: Received 7 January 2012 Available online 31 January 2012 Keywords: G protein Vesicular trafficking Rab3B
a b s t r a c t Rab proteins are the largest family of ras-related GTPases in eukaryotic cells. They act as directional molecular switches at membrane trafficking, including vesicle budding, cargo sorting, transport, tethering, and fusion. Here, we generated and crystallized the Rab3B:GDP complex. The structure of the complex was solved to 1.9 Å resolution and the structural base comparison with other Rab3 members provides a structural basis for the GDP/GTP switch in controlling the activity of small GTPase. The comparison of charge distribution among the members of Rab3 also indicates their different roles in vesicular trafficking. Ó 2012 Elsevier Inc. All rights reserved.
1. Introduction Rab proteins, the members of the Ras GTPase superfamily, function as molecular switches mediating vesicle budding, cargo sorting, transport, tethering and fusion [1,2]. Since the discovery of Rab proteins in 1980s, their molecular mechanisms of action as molecular switch have gotten more and more attention. Members of Rab3 subfamily (Rab3A, Rab3B, Rab3C, Rab3D), which share 77–85% identity at the amino acid level [3], are believed to play different roles in cellular processes. Different Rab proteins localized the particular subcellular compartment and involved in different cellular processes. Rab3B is essential for GnRH-regulated exocytosis downstream of cytosolic Ca2+ in gonadotrophs [4] and believed to have important roles in exocytosis activity with SNAP-25 [5]. Recent studies also demonstrated Rab3B’s important roles in shortterm synaptic plasticity and cross-presentation of dendritic cells with other Rab3 members [6,7]. Like other G proteins, the members of Rab3 cycle between active (GTP-bound) and inactive (GDP-bound) states in a highly conserved molecular mechanism [8,9]. The cycle of two states allows both spatial and temporal control of Rab3 activity. On the other hand, the activity of Rab3 is coordinated by several factors including guanine nucleotide exchange factor (GEF), GTPase activating protein (GAP), GDP dissociation inhibitor (GDI) and so on. In the inactive states, accessory factors target the Rab3–GTP complex. GTP hydrolysis is catalyzed by GAP, and then the factors returns Rab3 to their inactive states [10–12]. The GDP-bound Rab3 form stable cytosolic complexes and represent a cytoplasmic reservoir of Rab3 proteins [10]. The mechanisms of the control of activity ⇑ Corresponding author. E-mail address: [email protected] (C. Qi). 0006-291X/$ - see front matter Ó 2012 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2012.01.124
in Rab3 underlying increasing attention. Dumas have shown the structural basis of activation and GTP hydrolysis in Rab3A protein [13], while the mechanisms and the structural basis of inactive Rab3 proteins remains unclear. To shed light on further biological function investigation of Rab3B, we present the high-resolution crystal structure of Rab3B:GDP complex and provides a structural basis for the GDP/GTP switch in controlling the activity of small GTPase.
2. Materials and methods 2.1. Protein expression and purification The fragment of human Rab3B (residues 18–190) encompassing the GTPase domain was subcloned into a pET-28a-MHL vector via ligase-independent cloning. The recombinant protein was over expressed in Escherichia coli BL21 (DE3) with the pRARE plasmid for codon-biased expression. Cells were grown in minimal media (Terrific Broth) at 37 °C to an optical density of approximately 3.0. Protein expression was induced with 0.5 mM isopropyl-1thio-D-galactopyranoside (IPTG) and the cell cultures were grown at 15 °C after induction. The cells were allowed to grow overnight before they were harvested and flash frozen in liquid nitrogen and stored at 80 °C. The protein was purified by affinity chromatography on Ni–NTA resin (Qiagen Mississauga, ON) and size exclusion chromatography using a Superdex-75 26/60 gel filtration column (GE Healthcare, Tyrone, PA). The purity of proteins was higher than 95% judged by SDS–PAGE. The his-tag was cleaved from Rab3B by the addition of 0.05 mg of TEV protease per milligram of GDP-bound Rab3B protein, followed by incubation in ice for 12 h. The sample was then
842
W. Zhang et al. / Biochemical and Biophysical Research Communications 418 (2012) 841–844 Table 1 Data collection and refinement statistics. Data collection Space group Cell dimensions a, b, c (Å) a, b, c (°) Wavelength (Å) Resolution (Å) Rmerge (%) I/rI Completeness (%) Redundancy Refinement Resolution (Å) No. of reflections Rwork/Rfree No. of atoms Protein Heterogen atoms Solvent atoms R.m.s. deviations Bond lengths (Å) Bond angles (°)
Fig. 1. Ribbon diagram of the structure of the Rab3B–GDP complex: The overall structure of the complex exhibits seven-stranded b, comprised of six parallel strands and one antiparallel strand, surround by six a helices. The nucleotide with the complex is highlighted in purple. The segments corresponding to the proposed switch I and switch II are highlight in dark red. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
passed through a Ni–NTA column and the flow-through was collected and concentrated to 18 mg/mL for crystallization. 2.2. Crystallization, X-ray data collection and structure determination Crystals suitable for X-ray diffraction analysis were obtained by the sitting-drop vapor diffusion method at 291 K by mixing equal volumes of the GDP-bound Rab3B protein solution (18 mg/mL) and the reservoir solution, which consists of 2 M (NH4)2SO4, 0.2 M NaAc, 0.1 M HEPES, 5% MPD (pH 7.5). Diffraction data from GDPbound Rab3B crystals were collected at wavelengths of 0.803 Å at beamline 19ID of the Advanced Photon Source (Argonne, Illinois, USA) and reduced with the HKL software suite [25]. Further experimental details are listed in Table 1. The structure was solved by the multiple wavelength anomalous diffraction [26] method using aforementioned high energy remote and peak wavelength data sets SHELX [27] software. The protein model was traced automatically with ARP/WARP [28]. Further refinement was performed with the programs COOT [29], PHENIX [30], and REFMAC [31]. The MOLPROBITY serve was used periodically for validation of the model’s geometry [32]. 3. Results and discussion 3.1. Overall structure The crystal structure of the Rab3B:GDP complex (residues 18– 190) is well defined. Rab3B has a typical GTPase fold consisting of seven-stranded b, comprised of six parallel strands and one antiparallel strand, surrounded by six a helices (Fig. 1). The overall structure is similar to the complex Rab3D–GDP and GppNHpbound Rab3A, but some structural differences are also observed in the C-terminal end and the putative switch regions of the proteins. More importantly, the putative switch regions have been previously implicated in the function of Rab proteins [14–17] and indicated a additional interactions with the nucleotide in the activ-
C121 56.97, 50.88, 73.55 90, 110.91, 90 0.803 25.0–1.90 (1.97–1.90) 6.1 (60.9) 7.6 (2.6) 99.7 (97.9) 6.7 (3.3) 25.0–1.90 15,627 21.1/25.1 1390 34 30 0.014 1.381
Values in parentheses correspond to the highest resolution shells.
ity with Rab3. To facilitate the discussion, we designated the putative conformational switch regions in Rab3B that correspond with the experimentally established switch regions in Rab3A and Ras [18,10]. The two putative switch regions of Rab3B span a dozen residues, including DTFTFAFAST (single-letter amino acid code) for switch I and DTAGQERYRTITTAYY for switch II, respectively (Fig. 1). As depicted in Fig. 1, there is one more b strand and a helix in Rab3B than its counterparts in Rab3, accounting for at least part of the difference in the overall structural conformation. However, the additional b strand and a helix did not localize in the switch regions, indicating little effect in Rab3 activity but some influences on function among the Rab3 subfamily members. Consequently, the following discussion will focus on the structural differences within the switch regions and their implications in Rab3 activity. 3.2. Comparison among the Rab3 subfamily Besides Rab3B, the crystal structures of other Rab3 subfamily members had been reported as well, including Rab3A and Rab3D. To better understand the structural differences and their implication in molecular function among the Rab3 subfamily members, the multiple sequence and crystal structure alignments of these proteins (Fig. 2(A), (B1), (B2)) was shown. The comparison of amino acid sequences exhibit a highly conserved switch regions with the only one difference in amino acid in switch I, while N- and C-terminal of the proteins that are hypervariable in length and sequence (Fig. 2(A)). The recent studies have shown that these regions were required for prenylation and targeting to the specific intracellular membranes but were not essential for nucleotide binding and interaction with effect or regulatory proteins [19– 21], and suggest different cellular roles in Rab3 members. Comparison of the structures of Rab3B–GDP and Rab3A– GppNHp following least squares superposition was shown (Fig. 2(B1)), the significant differences are observed switch I and switch II regions. Rab3 act as a molecular switches, cycling between inactive (GDP-bound) and active (GTP-bound) states [22], GDP–Rab3B forms stable complex in cytoplasm. So, the structural differences in the Fig. 2(B1) indicated the different conformation of different active states in Rab3. On the other hand, the main structural difference between the Rab3B–GDP and Rab3D–GDP localized in switch I (Fig. 2(B2)). Thr46 of the Rab3B is the same position as
W. Zhang et al. / Biochemical and Biophysical Research Communications 418 (2012) 841–844
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Fig. 2. Structure-based comparison of Rab3 subfamily: (A) structure-guided sequence alignment of three Rab3 subfamily members (Rab3A, Rab3B and Rab3D). The dashed lines indicate gaps introduced to optimize alignments. The sequences corresponding to the switch I and switch II region of Rab3 family are boxed. (B) Structural comparison of Rab3 subfamily. (B1) Superimposition of Rab3B (purple; Protein Data Bank code 3DZ8) and Rab3A (green; Protein Data Bank code 3RAB). (B2) Superimposition of Rab3B (light blue) and Rab3D (gray; Protein Data Bank code 2GF9). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
Ser45 of Rab3D, the additional –CH3 of Thr46 of Rab3B perturbates the interaction with nucleotide and indicates a effect on the activity of Rab3. The high degree of sequence conservation within the switch regions of Rab3 members indicates that there are other regions to contribute the difference of structure and the specificity of the proteins. So, the comparison of the solvent accessible surface of the protein was given (Fig. 3). In the vesicle transport, the positively charged and flat surface of Rab3 proteins interact with the negatively charged vesicle membranes [23,24]. So, the significant defences with charge distribution of the members of Rab3B indicate that the predominantly negative charge distribution of Rab3B (Fig. 3) has a easy interaction with membrane-associated regulators and effectors in vesicle transport. Acknowledgments This research was supported by the Structural Genomics Consortium, a registered charity (No. 1097737) that receives funds from the Canadian Institutes for Health Research, the Canadian Foundation for Innovation, Genome Canada through the Ontario Genomics Institute, GlaxoSmithKline, Karolinska Instituted, the Knut and Alice Wallenberg Foundation, the Ontario Innovation Trust, the Ontario Ministry for Research and Innovation, Merck Co., Inc., the Novartis Research Foundation, the Swedish Agency for Innovation Systems, the Swedish Foundation for Strategic Research and the Welcome Trust. Additional support was provided by the National Science Foundation of China (No. 30670429 to J.M. and C.Q.) and Natural Science Foundation of Hubei Province of China (No. 2010CDB01206) and self-determined research funds of CCNU from the colleges’ basic research and operation of MOE (No. CCNU11A02013). Fig. 3. Comparison of the solvent accessible surface of Rab3A–GppNHp, Rab3B– GDP and Rab3D–GDP: Three orthogonal views of solvent accessible Grasp surface with charge distribution (blue for positive charge, and red for negative charge). All molecules are viewed from above and below as defined by the orientation in the figure. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
References [1] S.R. Pfeffer, Rab GTPases: specifying and deciphering organelle identity and function, Trends Cell Biol. 11 (2001) 487–491. [2] M. Zerial, H. McBride, Rab proteins as membrane organizers, Nat. Rev. Mol. Cell Biol. 2 (2001) 107–117.
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[3] F. Darchen, B. Goud, Multiple aspects of Rab protein action in the secretory pathway: focus on Rab3 and Rab6, Biochimie 82 (2000) 375–384. [4] K. Tasaka, N. Masumoto, J. Mizuki, Y. Ikebuchi, M. Ohmichi, H. Kurachi, A. Miyake, Y. Murata, Rab3B is essential for GnRH-induced gonadotrophin release from anterior pituitary cells, J. Endocrinol. 157 (1998) 267–274. [5] H. Nishioka, J. Haraoka, Significance of immunohistochemical expression of Rab3B and SNAP-25 in growth hormone-producing pituitary adenomas, Acta Neuropathol. 109 (2005) 598–602. [6] L. Zou, J. Zhou, J. Zhang, J. Li, N. Liu, L. Chai, N. Li, T. Liu, L. Li, Z. Xie, H. Liu, Y. Wan, Y. Wu, The GTPase Rab3b/3c-positive recycling vesicles are involved in cross-presentation in dendritic cells, Proc. Natl. Acad. Sci. USA 106 (2009) 15801–15806. [7] O.M. Schlüter, J. Basu, T.C. Südhof, C. Rosenmund, Rab3 superprimes synaptic vesicles for release: implications for short-term synaptic plasticity, J. Neurosci. 26 (2006) 1239–1246. [8] H R. Bourne, D.A. Sanders, F. McCormick, The GTPase superfamily: conserved structure and molecular mechanism, Nature 349 (1991) 117–127. [9] S.R. Sprang, G protein mechanisms: insights from structural analysis, Annu. Rev. Biochem. 66 (1997) 639–678. [10] M.C. Seabra, C. Wasmeier, Controlling the location and activation of Rab GTPases, Curr. Opin. Cell Biol. 16 (2004) 451–457. [11] A. Rak, O. Pylypenko, T. Durek, A. Watzke, S. Kushnir, L. Brunsveld, H. Waldmann, R.S. Goody, K. Alexandrov, Structure of Rab GDP-dissociation inhibitor in complex with prenylated YPT1 GTPase, Science 302 (2003) 646– 650. [12] S. Pfeffer, D. Aivazian, Targeting Rab GTPases to distinct membrane compartments, Nat. Rev. Mol. Cell Biol. 5 (2004) 886–896. [13] J.J. Dumas, Z. Zhu, J.L. Connolly, D.G. Lambright, Structural basis of activation and GTP hydrolysis in Rab proteins, Structure 7 (1999) 413–423. [14] P. Brennwald, P. Novick, Interactions of three domains distinguishing the Rasrelated GTP-binding proteins Ypt1 and Sec4, Nature 362 (1993) 560–563. [15] B. Dunn, T. Stearns, D. Botstein, Specificity domains distinguish the Ras-related GTPases Ypt1 and Sec4, Nature 362 (1993) 563–565. [16] J. Becker, T.J. Tan, H.H. Trepte, D. Gallwitz, Mutational analysis of the putative effector domain of the GTP-binding Ypt1 protein in yeast suggests specific regulation by a novel GAP activity, EMBO 10 (1991) 785–792. [17] A. Ignatev, S. Kravchenko, A. Rak, R.S. Goody, O. Pylypenko, A structural model of the GDP dissociation inhibitor rab membrane extraction mechanism, J. Biol. Chem. 283 (2008) 18377–18384. [18] M.V. Milburn, L. Tong, A.M. deVos, A. Brünger, Z. Yamaizumi, S. Nishimura, S.H. Kim, Molecular switch for signal transduction: structural differences between active and inactive forms of protooncogenic ras proteins, Science 247 (1990) 939–945.
[19] P. Chavrier, J.P. Gorvel, E. Stelzer, K. Simons, J. Gruenberg, M. Zerial, Hypervariable C-terminal domain of rab proteins acts as a targeting signal, Nature 353 (1991) 769–772. [20] O. Steele-Mortimer, M.J. Clague, L.A. Huber, P. Chavrier, J. Gruenberg, J.P. Gorvel, The N-terminal domain of a rab protein is involved in membranemembrane recognition and/or fusion, EMBO. J. 13 (1994) 34–41. [21] G.A. Gomez, J.L. Daniotti, H-Ras dynamically interacts with recycling endosomes in CHO-K1 cells: involvement of Rab5 and Rab11 in the trafficking of H-Ras to this pericentriolar endocytic compartment, J. Biol. Chem. 280 (2005) 34997–35010. [22] F. Lezoualc’h, M. Métrich, I. Hmitou, N. Duquesnes, E. Morel, Small GTPbinding proteins and their regulators in cardiac hypertrophy, J. Mol. Cell Cardiol. 44 (2008) 623–632. [23] J.S. Schonn, J.R. van Weering, R. Mohrmann, O.M. Schlüter, T.C. Südhof, H. de Wit, M. Verhage, J.B. Srensen, Rab3 proteins involved in vesicle biogenesis and priming in embryonic mouse chromaffin cells, Traffic 11 (2010) 1415–1428. [24] N.J. Pavlos, M. Grnborg, D. Riedel, J.J. Chua, J. Boyken, T.H. Kloepper, H. Urlaub, S.O. Rizzoli, R. Jahn, Quantitative analysis of synaptic vesicle Rabs uncovers distinct yet overlapping roles for Rab3a and Rab27b in Ca2+-triggered exocytosis, J. Neurosci. 30 (2010) 13441–13453. [25] Z. Otwinowski, W. Minor, Processing of the X-ray diffraction data collected in oscillation mode, Methods Enzymol. 276 (1997) 307–326. [26] J.M. Guss, E.A. Merritt, R.P. Phizackerley, B. Hedman, M. Murata, K.O. Hodgson, H.C. Freeman, Phase determination by multiple-wavelength X-ray diffraction: crystal structure of a basic ‘‘blue’’ copper protein from cucumbers, Science 4867 (1988) 806–811. [27] T.R. Schneider, G.M. Sheldrick, Substructure solution with SHELXD, Acta Crystallogr. D. Biol. Crystallogr. 58 (2002) 1772–1779. [28] A. Perrakis, M. Harkiolaki, K.S. Wilson, Lamzin VS ARP/wARP and molecular replacement, Crystallogr. D Biol. Crystallogr. 57 (2001) 1445–1450. [29] P. Emsley, B. Lohkamp, W.G. Scott, Cowtan KFeatures and development of Coot, Acta Crystallogr. D Biol. Crystallogr. 66 (2010) 486–501. [30] P.D. Adams, R.W. Grosse-Kunstleve, L.W. Hung, T.R. Ioerger, A.J. McCoy, N.W. Moriarty, R.J. Read, J.C. Sacchettini, N.K. Sauter, Terwilliger TC PHENIX: building new software for automated crystallographic structure determination, Acta Crystallogr. Biol. Crystallogr. 58 (2002) 1948–1954. [31] G.N. Murshudov, A.A. Vagin, E.J. Dodson, Refinement of macromolecular structures by the maximum-likelihood method, Acta Crystallogr. D Biol. Crystallogr. 53 (1997) 240–255. [32] I.W. Davis, A. Leaver-Fay, V.B. Chen, J.N. Block, G.J. Kapral, X. Wang, L.W. Murray, W.B. Arendall 3rd, J. Snoeyink, J.S. Richardson, D.C. Richardson, MolProbity: all-atom contacts and structure validation for proteins and nucleic acids, Nucleic Acids Res. 35 (2007) 375–383.
Contents lists available at SciVerse ScienceDirect
Biochemical and Biophysical Research Communications journal homepage: www.elsevier.com/locate/ybbrc
Crystal structure of inactive form of Rab3B Wei Zhang a, Yang Shen b, Ronghong Jiao c, Yanli Liu a, Lingfu Deng a, Chao Qi a,⇑ a
Hubei Key Laboratory of Genetic Regulation and Integrative Biology, College of Life Science, Huazhong Normal University, Wuhan 430079, PR China Structural Genomics Consortium, University of Toronto, 101 College St., Toronto, Ontario, Canada M5G 1L7 c Department of Function Inspection, Hebei Provincial People’s Hospital, Shijiazhuang 050051, PR China b
a r t i c l e
i n f o
Article history: Received 7 January 2012 Available online 31 January 2012 Keywords: G protein Vesicular trafficking Rab3B
a b s t r a c t Rab proteins are the largest family of ras-related GTPases in eukaryotic cells. They act as directional molecular switches at membrane trafficking, including vesicle budding, cargo sorting, transport, tethering, and fusion. Here, we generated and crystallized the Rab3B:GDP complex. The structure of the complex was solved to 1.9 Å resolution and the structural base comparison with other Rab3 members provides a structural basis for the GDP/GTP switch in controlling the activity of small GTPase. The comparison of charge distribution among the members of Rab3 also indicates their different roles in vesicular trafficking. Ó 2012 Elsevier Inc. All rights reserved.
1. Introduction Rab proteins, the members of the Ras GTPase superfamily, function as molecular switches mediating vesicle budding, cargo sorting, transport, tethering and fusion [1,2]. Since the discovery of Rab proteins in 1980s, their molecular mechanisms of action as molecular switch have gotten more and more attention. Members of Rab3 subfamily (Rab3A, Rab3B, Rab3C, Rab3D), which share 77–85% identity at the amino acid level [3], are believed to play different roles in cellular processes. Different Rab proteins localized the particular subcellular compartment and involved in different cellular processes. Rab3B is essential for GnRH-regulated exocytosis downstream of cytosolic Ca2+ in gonadotrophs [4] and believed to have important roles in exocytosis activity with SNAP-25 [5]. Recent studies also demonstrated Rab3B’s important roles in shortterm synaptic plasticity and cross-presentation of dendritic cells with other Rab3 members [6,7]. Like other G proteins, the members of Rab3 cycle between active (GTP-bound) and inactive (GDP-bound) states in a highly conserved molecular mechanism [8,9]. The cycle of two states allows both spatial and temporal control of Rab3 activity. On the other hand, the activity of Rab3 is coordinated by several factors including guanine nucleotide exchange factor (GEF), GTPase activating protein (GAP), GDP dissociation inhibitor (GDI) and so on. In the inactive states, accessory factors target the Rab3–GTP complex. GTP hydrolysis is catalyzed by GAP, and then the factors returns Rab3 to their inactive states [10–12]. The GDP-bound Rab3 form stable cytosolic complexes and represent a cytoplasmic reservoir of Rab3 proteins [10]. The mechanisms of the control of activity ⇑ Corresponding author. E-mail address: [email protected] (C. Qi). 0006-291X/$ - see front matter Ó 2012 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2012.01.124
in Rab3 underlying increasing attention. Dumas have shown the structural basis of activation and GTP hydrolysis in Rab3A protein [13], while the mechanisms and the structural basis of inactive Rab3 proteins remains unclear. To shed light on further biological function investigation of Rab3B, we present the high-resolution crystal structure of Rab3B:GDP complex and provides a structural basis for the GDP/GTP switch in controlling the activity of small GTPase.
2. Materials and methods 2.1. Protein expression and purification The fragment of human Rab3B (residues 18–190) encompassing the GTPase domain was subcloned into a pET-28a-MHL vector via ligase-independent cloning. The recombinant protein was over expressed in Escherichia coli BL21 (DE3) with the pRARE plasmid for codon-biased expression. Cells were grown in minimal media (Terrific Broth) at 37 °C to an optical density of approximately 3.0. Protein expression was induced with 0.5 mM isopropyl-1thio-D-galactopyranoside (IPTG) and the cell cultures were grown at 15 °C after induction. The cells were allowed to grow overnight before they were harvested and flash frozen in liquid nitrogen and stored at 80 °C. The protein was purified by affinity chromatography on Ni–NTA resin (Qiagen Mississauga, ON) and size exclusion chromatography using a Superdex-75 26/60 gel filtration column (GE Healthcare, Tyrone, PA). The purity of proteins was higher than 95% judged by SDS–PAGE. The his-tag was cleaved from Rab3B by the addition of 0.05 mg of TEV protease per milligram of GDP-bound Rab3B protein, followed by incubation in ice for 12 h. The sample was then
842
W. Zhang et al. / Biochemical and Biophysical Research Communications 418 (2012) 841–844 Table 1 Data collection and refinement statistics. Data collection Space group Cell dimensions a, b, c (Å) a, b, c (°) Wavelength (Å) Resolution (Å) Rmerge (%) I/rI Completeness (%) Redundancy Refinement Resolution (Å) No. of reflections Rwork/Rfree No. of atoms Protein Heterogen atoms Solvent atoms R.m.s. deviations Bond lengths (Å) Bond angles (°)
Fig. 1. Ribbon diagram of the structure of the Rab3B–GDP complex: The overall structure of the complex exhibits seven-stranded b, comprised of six parallel strands and one antiparallel strand, surround by six a helices. The nucleotide with the complex is highlighted in purple. The segments corresponding to the proposed switch I and switch II are highlight in dark red. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
passed through a Ni–NTA column and the flow-through was collected and concentrated to 18 mg/mL for crystallization. 2.2. Crystallization, X-ray data collection and structure determination Crystals suitable for X-ray diffraction analysis were obtained by the sitting-drop vapor diffusion method at 291 K by mixing equal volumes of the GDP-bound Rab3B protein solution (18 mg/mL) and the reservoir solution, which consists of 2 M (NH4)2SO4, 0.2 M NaAc, 0.1 M HEPES, 5% MPD (pH 7.5). Diffraction data from GDPbound Rab3B crystals were collected at wavelengths of 0.803 Å at beamline 19ID of the Advanced Photon Source (Argonne, Illinois, USA) and reduced with the HKL software suite [25]. Further experimental details are listed in Table 1. The structure was solved by the multiple wavelength anomalous diffraction [26] method using aforementioned high energy remote and peak wavelength data sets SHELX [27] software. The protein model was traced automatically with ARP/WARP [28]. Further refinement was performed with the programs COOT [29], PHENIX [30], and REFMAC [31]. The MOLPROBITY serve was used periodically for validation of the model’s geometry [32]. 3. Results and discussion 3.1. Overall structure The crystal structure of the Rab3B:GDP complex (residues 18– 190) is well defined. Rab3B has a typical GTPase fold consisting of seven-stranded b, comprised of six parallel strands and one antiparallel strand, surrounded by six a helices (Fig. 1). The overall structure is similar to the complex Rab3D–GDP and GppNHpbound Rab3A, but some structural differences are also observed in the C-terminal end and the putative switch regions of the proteins. More importantly, the putative switch regions have been previously implicated in the function of Rab proteins [14–17] and indicated a additional interactions with the nucleotide in the activ-
C121 56.97, 50.88, 73.55 90, 110.91, 90 0.803 25.0–1.90 (1.97–1.90) 6.1 (60.9) 7.6 (2.6) 99.7 (97.9) 6.7 (3.3) 25.0–1.90 15,627 21.1/25.1 1390 34 30 0.014 1.381
Values in parentheses correspond to the highest resolution shells.
ity with Rab3. To facilitate the discussion, we designated the putative conformational switch regions in Rab3B that correspond with the experimentally established switch regions in Rab3A and Ras [18,10]. The two putative switch regions of Rab3B span a dozen residues, including DTFTFAFAST (single-letter amino acid code) for switch I and DTAGQERYRTITTAYY for switch II, respectively (Fig. 1). As depicted in Fig. 1, there is one more b strand and a helix in Rab3B than its counterparts in Rab3, accounting for at least part of the difference in the overall structural conformation. However, the additional b strand and a helix did not localize in the switch regions, indicating little effect in Rab3 activity but some influences on function among the Rab3 subfamily members. Consequently, the following discussion will focus on the structural differences within the switch regions and their implications in Rab3 activity. 3.2. Comparison among the Rab3 subfamily Besides Rab3B, the crystal structures of other Rab3 subfamily members had been reported as well, including Rab3A and Rab3D. To better understand the structural differences and their implication in molecular function among the Rab3 subfamily members, the multiple sequence and crystal structure alignments of these proteins (Fig. 2(A), (B1), (B2)) was shown. The comparison of amino acid sequences exhibit a highly conserved switch regions with the only one difference in amino acid in switch I, while N- and C-terminal of the proteins that are hypervariable in length and sequence (Fig. 2(A)). The recent studies have shown that these regions were required for prenylation and targeting to the specific intracellular membranes but were not essential for nucleotide binding and interaction with effect or regulatory proteins [19– 21], and suggest different cellular roles in Rab3 members. Comparison of the structures of Rab3B–GDP and Rab3A– GppNHp following least squares superposition was shown (Fig. 2(B1)), the significant differences are observed switch I and switch II regions. Rab3 act as a molecular switches, cycling between inactive (GDP-bound) and active (GTP-bound) states [22], GDP–Rab3B forms stable complex in cytoplasm. So, the structural differences in the Fig. 2(B1) indicated the different conformation of different active states in Rab3. On the other hand, the main structural difference between the Rab3B–GDP and Rab3D–GDP localized in switch I (Fig. 2(B2)). Thr46 of the Rab3B is the same position as
W. Zhang et al. / Biochemical and Biophysical Research Communications 418 (2012) 841–844
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Fig. 2. Structure-based comparison of Rab3 subfamily: (A) structure-guided sequence alignment of three Rab3 subfamily members (Rab3A, Rab3B and Rab3D). The dashed lines indicate gaps introduced to optimize alignments. The sequences corresponding to the switch I and switch II region of Rab3 family are boxed. (B) Structural comparison of Rab3 subfamily. (B1) Superimposition of Rab3B (purple; Protein Data Bank code 3DZ8) and Rab3A (green; Protein Data Bank code 3RAB). (B2) Superimposition of Rab3B (light blue) and Rab3D (gray; Protein Data Bank code 2GF9). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
Ser45 of Rab3D, the additional –CH3 of Thr46 of Rab3B perturbates the interaction with nucleotide and indicates a effect on the activity of Rab3. The high degree of sequence conservation within the switch regions of Rab3 members indicates that there are other regions to contribute the difference of structure and the specificity of the proteins. So, the comparison of the solvent accessible surface of the protein was given (Fig. 3). In the vesicle transport, the positively charged and flat surface of Rab3 proteins interact with the negatively charged vesicle membranes [23,24]. So, the significant defences with charge distribution of the members of Rab3B indicate that the predominantly negative charge distribution of Rab3B (Fig. 3) has a easy interaction with membrane-associated regulators and effectors in vesicle transport. Acknowledgments This research was supported by the Structural Genomics Consortium, a registered charity (No. 1097737) that receives funds from the Canadian Institutes for Health Research, the Canadian Foundation for Innovation, Genome Canada through the Ontario Genomics Institute, GlaxoSmithKline, Karolinska Instituted, the Knut and Alice Wallenberg Foundation, the Ontario Innovation Trust, the Ontario Ministry for Research and Innovation, Merck Co., Inc., the Novartis Research Foundation, the Swedish Agency for Innovation Systems, the Swedish Foundation for Strategic Research and the Welcome Trust. Additional support was provided by the National Science Foundation of China (No. 30670429 to J.M. and C.Q.) and Natural Science Foundation of Hubei Province of China (No. 2010CDB01206) and self-determined research funds of CCNU from the colleges’ basic research and operation of MOE (No. CCNU11A02013). Fig. 3. Comparison of the solvent accessible surface of Rab3A–GppNHp, Rab3B– GDP and Rab3D–GDP: Three orthogonal views of solvent accessible Grasp surface with charge distribution (blue for positive charge, and red for negative charge). All molecules are viewed from above and below as defined by the orientation in the figure. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
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