Tetrakisphosphate: a Novel Adaptor Protein Involved in Human Cerebral Cavernous Malformation

Biochemical and Biophysical Research Communications 399 (2010) 587–592

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Biochemical and Biophysical Research Communications

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Crystal structure of human programmed cell death 10 complexed with inositol-(1,3,4,5)-tetrakisphosphate: A novel adaptor protein involved in human cerebral cavernous malformation

Jingjin Ding, Xiaoyan Wang, De-Feng Li, Yonglin Hu, Ying Zhang, Da-Cheng Wang *

National Laboratory of Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, Beijing 100101, People’s Republic of China article info abstract

Article history: Programmed cell death 10 (PDCD10) is a novel adaptor protein involved in human cerebral cavernous Received 26 July 2010 malformation, a common vascular lesion mostly occurring in the central nervous system. By interacting Available online 1 August 2010 with different signal proteins, PDCD10 could regulate various physiological processes in the cell. The crystal structure of human PDCD10 complexed with inositol-(1,3,4,5)-tetrakisphosphate has been deter- Keywords: mined at 2.3 Å resolution. The structure reveals an integrated dimer via a unique assembly that has never Adaptor protein been observed before. Each PDCD10 monomer contains two independent domains: an N-terminal Programmed cell death 10 domain with a new fold involved in the tight dimer assembly and a C-terminal four-helix bundle domain Unique dimeric assembly that closely resembles the focal adhesion targeting domain of focal adhesion kinase. An eight-residue Conformational variability Phosphatidylinositide binding ﬂexible linker connects the two domains, potentially conferring mobility onto the C-terminal domain, Binding partner recruitment resulting in the conformational variability of PDCD10. A variable basic cleft on the top of the dimer interface binds to phosphatidylinositide and regulates the intracellular localization of PDCD10. Two potential sites, respectively located on the two domains, are critical for recruiting different binding partners, such as germinal center kinase III proteins and the focal adhesion protein paxillin. Ó 2010 Elsevier Inc. All rights reserved.

Introduction strin-homology (PH) domains, bind phosphatidylinositides and determine the localization of the corresponding adaptor proteins Adaptor proteins are attracting more interest due to their essen- [3]. tial roles in governing multi protein cross-talk and signaling spec- Programmed cell death 10 (PDCD10) was initially characterized ificity of numerous signal transduction pathways that mediate as a gene whose expression was upregulated upon the induction various physiological processes in the cell [1,2]. They can recruit of apoptosis in human myeloid cell lines [4]. Later, PDCD10 was different signal proteins to a specific location and regulate the identified as the cerebral cavernous malformation 3 (CCM3) gene, interactions between these binding partners, thus driving the for- in which loss-of-function mutations could result in cerebral cavern- mation of larger signaling complexes. Adaptor proteins tend to lack ous malformation (CCM) [5]. CCM is a common vascular lesion found any intrinsic catalytic activity but instead consist of different pro- mostly in the central nervous system. Individuals with CCM may be tein–protein and protein–lipid-interaction modules. For example, under a risk of focal hemorrhage, resulting in headaches, seizures the Src-homology 2 (SH2) domains recognize specific sequences and stroke in midlife [6]. The protein encoded by the PDCD10 gene within their binding partners that contain phosphotyrosine resi- was predicted to be an adaptor protein because it lacks any known dues, and Src-homology 3 (SH3) domains bind proline-rich se- catalytic domains. As a novel adaptor protein, PDCD10 was identi- quences within specific peptide sequence contexts of their fied as a component of the larger CCM signaling complex, which binding partners. Lipid-interaction modules, such as the pleck- consists of KRIT1 (Krev1/Rap1A Interaction Trapped 1, also known as CCM1), OSM (Osmosensing Scaffold for MEKK3, also known as CCM2) and PDCD10 [7,8]. Both proliferative and apoptotic functions Abbreviations: PDCD10, programmed cell death 10; CCM, cerebral cavernous malformation; 4IP, inositol-(1,3,4,5)-tetrakisphosphate; SH2, Src-homology 2; SH3, have been identified for this adaptor protein [9,10]. Recently, Src-homology 3; PH, pleckstrin-homology; KRIT1, Krev1/Rap1A Interaction Trapped PDCD10 has been found to localize to the Golgi apparatus, forming 1; OSM, Osmosensing Scaffold for MEKK3; GCKIII, germinal center kinase III; FAT, a complex with proteins of the germinal center kinase III (GCKIII) focal adhesion targeting; MAD, multiple-wavelength anomalous dispersion. family, which are involved in signal pathways of cell orientation * Corresponding author. Address: National Laboratory of Biomacromolecules, and migration [11]. Although understanding of the physiological Institute of Biophysics, Chinese Academy of Sciences, 15 Datun Road, Chaoyang District, Beijing 100101, People’s Republic of China. Fax: +86 10 64888560. role of PDCD10 has increased, the structural basis for the function E-mail address: [email protected] (D.-C. Wang). of PDCD10 remains unknown and is of great interest.

Here we present the crystal structure of PDCD10 complexed Crystallization with inositol-(1,3,4,5)-tetrakisphosphate (4IP) and discuss the unique dimeric assembly, possible conformational variability and The crystallization experiments were conducted using the phosphatidylinositide binding of this novel adaptor protein. The hanging-drop vapor-diffusion method at 293 K with 2 ll drops potential sites of PDCD10 for binding partner recruitment are also containing 1 ll protein solution and 1 ll reservoir solution equili- investigated. brated over 0.5 ml reservoir solution. Initial crystallization screen- ing was performed using Index kit (Hampton Research). After two Material and methods days, two conditions produced microcrystals: (i) 3.0 M NaCl and 100 mM Bis–Tris pH 5.5 and (ii) 200 mM ammonium sulfate, 25% Cloning, expression, and purification (w/v) PEG 3350 and 100 mM Bis–Tris pH 5.5. Well-shaped crystals were obtained in the optimized conditions by varying the concen- The truncated DNA comprising residues 8–212 of the human tration of precipitants, the pH, and the type and concentration of PDCD10 gene (GenBank: NP_009148) was amplified from the re- additives. However, the crystals diffracted poorly to 8 Å on a Riga- verse transcriptase-polymerase chain reaction products of human ku FRE diffraction system using a Rigaku R-Axis IV++ image plate hemopoietic stem cell by PCR and subcloned into a pET22b(+) detector. In an attempt to produce better crystals, we added inosi- vector (Novagen) using Nde I and Xho I restriction sites. The recom- tol-(1,3,4,5)-tetrakisphosphate (4IP, Avanti Polar Lipids) to the pro- binant protein was overexpressed as a fusion protein with a tein to a final concentration of 5 mM and reset the crystallization C-terminal six-histidine tag in Escherichia coli BL21(DE3). The cells trials. Well-diffracting crystals of both the native protein and the were grown in LB medium supplemented with 100 lg/mL ampicil- SeMet derivative were obtained under the following condition: lin at 310 K until the culture reached an OD600 of 0.6, and expres- 30% (v/v) ethylene glycol, 100 mM sodium cacodylate pH 6.0. Dia- sion was induced with 0.5 mM IPTG at 289 K overnight. The mond-shaped crystals (300 Â 200 Â 200 lm) typically appeared in harvested cells were resuspended in lysis buffer (50 mM NaH2PO4, 24 h. pH 8.0; 300 mM NaCl; 10 mM imidazole) with 0.1 mM PMSF and lysed by sonication. The lysate was clarified by centrifugation Data collecting and processing and purified on a nickel-affinity column followed by size-exclusion chromatography using a Superdex 200 column (Amersham Phar- All diffraction data were collected at the beamline 17A (BL17A) macia). The purified protein was concentrated to 40 mg/ml and at the Photon Factory (Tsukuba, Japan) using an ADSC Quantum- stored in 20 mM Tris–HCl pH 8.0, 150 mM NaCl, 0.2 mM EDTA, 270 charge-coupled device (CCD) detector. Prior to data collection, 5 mM DTT at 193 K. The selenomethionine-substituted (SeMet) PDCD10 crystals were transferred to a cryoprotectant solution con- derivative was produced by expression in E. coli methionine auxo- taining 20% (v/v) dimethyl sulfoxide, 25% (v/v) ethylene glycol, trophic strain B834(DE3). The purification procedure of the SeMet 100 mM sodium cacodylate pH 6.0 and soaked for about 2 min. derivative was the same as that of the native protein. The crystals were then flash-frozen in a nitrogen-gas stream at

Table 1 Data collection and structural reﬁnement statistics.

Data collection statistics Crystal Selenomethionine derivative Native

Space group P41212 P41212 Cell parameters (Å) a = 90.34 a = 89.83 b = 90.34 b = 89.83 c = 114.14 c = 114.20 Wavelength (Å) 0.97888 (peak) 0.97928 (edge) 0.96405 (remote) 1.0000 Resolution range (last shell) (Å) 50.0–2.70 (2.85–2.70) 50.0–2.30 (2.42–2.30) Total reﬂections 256873 128264 128390 152206 Unique reﬂections 13582 13604 13615 21428 Completeness (last shell) 100% (100%) 100% (100%) 100% (100%) 99.9% (100%) Redundancy (last shell) 18.9 (19.4) 9.4 (9.7) 9.4 (9.7) 7.1 (7.3) I/(I) (last shell) 21.7 (8.3) 14.3 (4.8) 13.8 (4.2) 14.6 (5.8)

Rsym (last shell) 9.8% (36.9%) 10.1% (42.5%) 10.3% (49.4%) 8.3% (31.7%) Reﬁnement statistics

Rwork 21.35%

Rfree 26.41% Nonhydrogen atoms Protein atoms 3193 Water molecules 99 Heteroatoms 28 Average B factors (Å2) Protein 55.9 Solvent 56.84 Ligand 76.37 r.m.s. Deviations Bond lengths (Å) 0.005 Bond angles (°) 0.676 Chirality (Å) 0.051 Planarity (Å) 0.002 Dihedral angles (°) 18.192 Ramachandran plot statistics Favored regions 97.14% Allowed regions 2.86% Outlier regions 0% J. Ding et al. / Biochemical and Biophysical Research Communications 399 (2010) 587–592 589

Fig. 1. Structural characteristics and unique dimeric assembly of PDCD10. (A) Overall structure of PDCD10 complexed with 4IP. The two monomers of PDCD10 are colored in green and yellow, respectively. The 4IP molecule is shown as a ball-and-stick model. The disordered loop in monomer B is shown as a broken line. (B) The structural architecture of the PDCD10 monomer. The N-terminal domain, the C-terminal domain, and the ﬂexible linker are labeled and colored in orange, cyan and gray, respectively. The hinge (Lys69-Lys70) in a3 is also labeled. (C) Two N-terminal domains ﬁrmly inter-clasp with each other to form a compact six-helix bundle, mediating a unique dimeric assembly.

95 K on nylon cryoloops (Hampton Research). The native data were DM [12]. Automated model building was completed using RE- collected at a wavelength of 1.0000 Å with 2.3 Å resolution. The 3- SOLVE [14,15], which provided a 50%-complete initial model. The wavelength MAD data from a SeMet derivative crystal diffracted remaining residues of the final model were manually built with up to 2.7 Å resolution. MOSFLM and SCALA from the CCP4 program Coot [16] and O [17]. The structure was refined to 2.3 Å resolution suite [12] were used to process, reduce and scale the diffraction using PHENIX [18]. Translation libration screw (TLS) refinement data. Data statistics are summarized in Table 1. was used in the final cycles of the refinement. The two domains of PDCD10 were processed separately as two TLS groups. The 4IP Structure determination and refinement molecule was identified at the last stage of refinement according to the omit-annealing electron density maps. The original coordi- The structure of PDCD10 was determined by the MAD method nates of 4IP were obtained from the HIC-Up database (http:// using SOLVE [13]. Eleven Se atoms were found per asymmetric xray.bmc.uu.se/hicup/). The quality of the final model was checked unit. Phasing extension to 2.3 Å resolution were performed using by MolProbity [19]. The statistics of the refinement and the

Fig. 2. Conformational variability based on C-terminal domain mobility. (A) C-terminal domain mobility. The orientation of the C-terminal domain varies approximately 30° from monomer A to monomer B. (B) Surface representation of the hetero-state (AB) observed in the present structure. (C) Surface representations of the closed-state (BB) and the open-state (AA) by modeling monomer A and B into homo-states. The size of the cleft on the top of the dimer interface varies with the conformational variety of PDCD10. 590 J. Ding et al. / Biochemical and Biophysical Research Communications 399 (2010) 587–592 stereochemistry of the final model are summarized in Table 1. Fig- chromatography analysis reveals that PDCD10 is a homodimer in ures were prepared using PYMOL (http://www.pymol.org/). solution. However, a truncated constructs (residues 83–212), which include the flexible linker and the intact C-terminal domain, Results and discussion acts as a monomer in solution by size-exclusion chromatography analysis (data not shown). These data imply that the unique Overall structure dimeric assembly of PDCD10 is completely mediated by the N-terminal domain, and the dimer is the basic unit of PDCD10 The crystal structure of PDCD10 complexed with 4IP was deter- for both the structure and function. A search in the Dali database mined at 2.3 Å resolution. The asymmetric unit contains two [20] with the single N-terminal domain or the dimeric N-terminal PDCD10 monomers (residues 11–211 for monomer A, residues domains of PDCD10 did not reveal any proteins with a similar 12–86 and 95–209 for monomer B), one 4IP molecule and 99 water architecture. Thus, it could be concluded that the N-terminal do- molecules. The two monomers assemble into an integrated dimer main of PDCD10 represents a new fold, and PDCD10 is a novel via a unique assembly (Fig. 1A). Each monomer consists of seven adaptor protein with a unique dimeric assembly by the N-terminal a-helices (a1–a7), which fold into two independent domains, domain. termed the N- and C-terminal domain (Fig. 1B). The N-terminal domain is comprised of three helices (a1, a2 and a3) and is shaped Conformational variability based on C-terminal domain mobility like a hook. Directed by the loop (Asn55–Gly57) and the hinge (Lys69–Lys70), the a3 helix is oriented at a 70° angle from the The two domains of one PDCD10 monomer are very similar to a2 helix, and forms a long arm that points to the C-terminal do- their counterparts in the dimer-related monomer with r.m.s. devi- main (Fig. 1B). The C-terminal domain, comprised of a4, a5, a6 ations of 0.806 Å and 0.666 Å for N- and C-terminal domains, and a7 helices, folds into a canonical four-helix bundle (Fig. 1B). respectively. Structural superposition of the two monomers shows All four helices are closely antiparallel. They are amphiphilic with that the orientation of the C-terminal domain with respect to the the polar residues exposing their side-chains to the solvent and the N-terminal domain varies by approximately 30° from monomer non-polar residues extending their side-chains into the core of the A to monomer B (Fig. 2A). The flexible linker connecting the two bundle. An eight-residue long loop (residues 87–94), which is dis- domains should be the structural basis which induces the potential ordered in monomer B, creates a flexible linker connecting the two C-terminal domain mobility. Based on the C-terminal domain domains (Fig. 1B). This loop is like a cable and confers mobility mobility, the PDCD10 dimer could adopt variable conformations. onto the C-terminal domain. The PDCD10 structure observed in our experiment is a conforma- The integrated dimeric structure of PDCD10 is shaped like a tional hetero-state (AB) (Fig. 2B). Respectively, modeling monomer two-armed crane, in which the dimeric N- and C-terminal domains A and B into their conformational homo-states with the same di- act as the base and carrier of the crane, respectively. The flexible meric assembly of AB, we could obtain two conformational linker connecting the two domains makes this system adjustable. homo-states. One is an open-state (AA), and the other is a closed-state (BB) (Fig. 2C). Referring to the hetero-state AB, the Unique dimeric assembly by N-terminal domain two C-terminal domains are shifted down and up about 30° for homo-states AA and BB (Fig. 2C). It is reasonable to propose that One of the notable structural characteristics of the PDCD10 di- the variable dimer conformations similar to those mentioned mer is located at the N-terminus. Similar to a pair of hooks, two above may be adopted by PDCD10 in some specific physiological N-terminal domains firmly inter-clasp with each other and form conditions for achieving its functional roles. a compact six-helix bundle (Fig. 1C). A number of non-polar residues of a1, a2 and a3 helices point their hydrophobic side-chains A variable basic cleft for phosphatidylinositide binding into the center of the bundle and play an essential role in dimeriza- tion. Based on this unique dimeric assembly, an integrated PDCD10 In the final structural model, a 4IP molecule, which is the head dimer is constructed. A total of 6319 Å2 of surface area is buried in group of a biological phosphatidylinositide, was found binding in a the dimer interface, which stabilizes the dimer. Size-exclusion basic cleft. The cleft is located on the top of the dimer interface

Fig. 3. Phosphatidylinositide binding. (A) A basic cleft on the top of the dimer interface contains a 4IP molecule. The 4IP molecule is represented as a sphere model. (B) Binding of the 4IP molecule. The residues interact with the 4IP molecule through an extended hydrogen-bond network. The 4IP molecule is shown as a ball-and-stick model. The hydrogen bonds are shown as broken lines. J. Ding et al. / Biochemical and Biophysical Research Communications 399 (2010) 587–592 591 between the two C-terminal domains (Fig. 3A). The binding pocket is mainly formed by four lysine residues (Lys70 and Lys186 of both monomers). The 4IP molecule interacts with the PDCD10 dimer through an extended hydrogen-bond network (Fig. 3B). The 1- phosphate group of inositol is exposed to the solvent, thus leaving room for the lipid chain when PDCD10 interacts with biological phosphatidylinositide (Fig. 3). This observation indicates that the basic cleft is a significant site for binding to critical biological li- gands, such as phosphatidylinositides. Interestingly, the size of this basic cleft varies (8.93–12.26 Å) based on the conformational variability of PDCD10 (Fig. 2B and C). It is reasonable to propose that the phosphatidylinositide binding affinity could vary along with the conformational variability of PDCD10. In fact, the rather diffuse electron density of the 4IP molecule in the final model indicates that the 4IP molecule is partially occupied and that PDCD10 does not bind 4IP with high affinity under the conformational hetero- state. Previous biochemical research has revealed that PDCD10 can bind phosphatidylinositides, which helps localize and stabilize its interaction with membrane-associated proteins [7]. Here we present the possible structural basis of phosphatidylinositide binding in PDCD10. The potential conformational variability of PDCD10 based on mobility of the C-terminal domain also provides the dy- namic structural basis for regulation of the phosphatidylinositide binding, which in turn, mediates the intracellular localization and functional role of PDCD10.

Potential sites for binding partner recruitment

As a typical adaptor protein, PDCD10 contains a few potential sites for recruiting different binding partners. Checking the molecular surface of the dimeric N-terminal domains, we found two qua- si 2-fold symmetric hydrophobic patches (termed Site I) (Fig. 4A and B). Site I is formed by three methionine residues (Met17 and Met20 of one monomer and Met83 of the other) and two tyrosine residues (Tyr23 and Tyr27). It stacks with its counterpart in the crystallographic symmetric molecule through broad hydrophobic contacts, which induce compact packing in the 4-fold direction (Fig. 4B). This hydrophobic patch has a large surface area suitable for potential intermolecular interactions. It has been reported that PDCD10 could interact with GCKIII kinases and stabilize them to promote Golgi assembly and cell orientation [11]. The binding site of PDCD10 for GCKIII kinases has been localized on the N-terminal domain [21]. Here the Site I of PDCD10 presents a potential site for GCKIII kinase binding and will be investigated in our future research. The four-helix bundle fold represented by the C-terminal domain of PDCD10 is also found in some cell adhesion proteins. A Dali database search [20] reveals that the closest structural homologs of the C-terminal domain of PDCD10 are the focal adhesion targeting Fig. 4. Potential sites for recruiting different binding partners. (A) Electrostatic potential analysis of PDCD10. Two hydrophobic patches (Site I and Site II), (FAT) domains of focal adhesion kinases such as FAK and Pyk2 respectively located on the N- and C-terminal domain, for potential protein– (1K40 for FAK, Z-score = 14.8, r.m.s. deviation = 2.1 Å; 3GM2 for protein interactions are boxed. (B) Homo-interactions between two Site I induce a Pyk2, Z-score = 15.6, r.m.s. deviation = 1.8 Å). The FAT domains of compact packing in the 4-fold direction. The PDCD10 dimer and 4-fold-related FAK and Pyk2 mediate focal adhesion targeting of these two pro- symmetric dimer are colored in pink and cyan, respectively. The residues forming teins by interacting with the LD motifs of the focal adhesion pro- Site I are labeled and shown as stick models. (C) Superimposing the C-terminal domain of PDCD10 with the FAT domains of focal adhesion kinases indicates the tein paxillin. Each FAT domain contains two hydrophobic patches conservation of Site II. The hydrophobic residues forming Site II and the basic that bind to different LD motifs on the two opposite faces of the residues surrounding Site II are labeled and shown as stick models. four-helix bundle. Focal adhesion kinases are recruited to focal adhesion due to their interaction with paxillin, leading to activa- patch on the a4a7 face is occupied by the C-terminal part of a3 tion of their kinase activities and transduction of adhesion signals and a flexible linker that connects the two domains. This observa- [22–24]. Structural superposition of the C-terminal domain of tion implies that Site II could potentially mediate paxillin binding PDCD10 with the FAT domains of focal adhesion kinases suggests in PDCD10 and could implicate PDCD10 in the cell adhesion. that the hydrophobic patch on the a5a6 face for paxillin binding is conserved (termed Site II) and is composed of Ile131, Ala135, Addendum Ile138, Leu142, Val168, Ser171, Ser175 and Leu178 (Fig. 4A and C). It is surrounded by four conserved basic residues (Lys132, During the preparation of this manuscript, another group Lys138, Lys165 and Lys179) (Fig. 4C). 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