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The crystal structure of the tumor suppressor protein pp32 (Anp32a): Structural insights into Anp32 family of proteins

Department of Biophysics and Biophysical Chemistry, Howard Hughes Medical Institute, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205-2185, USA

(RECEIVED February 3, 2007; FINAL REVISION April 4, 2007; ACCEPTED April 6, 2007)

	Abstract

TOP Abstract Introduction Results Discussion Materials and Methods Electronic supplemental material Acknowledgments References

 
The tumor suppressor protein pp32 is highly overexpressed in many cancers of the breast and prostate, and has also been implicated in the neurodegenerative disease spinocerebellar ataxias type 1 (SCA1). Pp32 is a multifunctional protein that is involved in the regulation of transcription, apoptosis, phosphorylation, and cell cycle progression, the latter through its association with the hyperphosphorylated form of the retinoblastoma tumor suppressor. We have determined the structure of an N-terminal pp32 fragment comprising a capped leucine-rich repeat (LRR) domain, which provides insight into the structural and biochemical properties of the pp32 (Anp32) family of proteins.

Keywords: crystal structure; pp32; Anp32; PHAPI; LRR

	Introduction

TOP Abstract Introduction Results Discussion Materials and Methods Electronic supplemental material Acknowledgments References

 
Pp32 (Anp32a) belongs to a family of evolutionarily conserved proteins known as the acidic nuclear phosphoprotein family (Anp32a–h). Members of the pp32 family help to modulate cellular signaling and gene expression to regulate the morphology and dynamics of the cytoskeleton, cell adhesion, neuronal development, or cerebellar morphogenesis (Matilla and Radrizzani 2005). Pp32 has been isolated several times, and has therefore been assigned a variety of names: pp32 (phosphoprotein 32) (Malek et al. 1990), Anp32a (acidic nuclear phosphoprotein 32a) (Matilla et al. 1997), PHAPI (putative HLA-associated protein I) (Vaesen et al. 1994), mapmodulin (Ulitzur et al. 1997b), Lanp (leucine-rich nuclear protein) (Matsuoka et al. 1994), and I₁ ^PP2A (inhibitor 1 protein phosphatase 2A) (Li et al. 1995, 1996).

Pp32 is expressed in normal tissues as well as in many breast, pancreas, and prostate cancers (J. Brody, pers. comm.), where it can act as a tumor suppressor (Bai et al. 2001). Pp32 has also been associated with the neurodegenerative disease spinocerebellar ataxias through binding the polyglutamine repeats of Ataxin-1 (Matilla et al. 1997). The pp32 protein contains a nuclear localization signal (NLS, import signal) at the C terminus but can also bind the nuclear export protein Crm1 through the N terminus LRR domain (Brennan et al. 2000), which allows pp32 to exist as a component of both nuclear and cytoplasmic complexes. Nuclear pp32 is found associated with the protein SET (TAF-I) in the INHAT (INHibitor of Acetyl Transferase Activity) complex (Schneider et al. 2004; Seo et al. 2002), which regulates histone modification/transcription. The endoplasmic reticulum (ER)-associated SET complex contains pp32 as one of its components. The SET complex is part of the Granzyme A-mediated apoptosis pathway, and the precise role of pp32 in this complex is unclear (Lieberman and Fan 2003). Interestingly, pp32 can promote apoptosis by accelerating caspase-9 activation after apoptosome formation (Jiang et al. 2003). Both pp32 and SET were initially isolated as potent inhibitors of protein phosphatase 2A (Li and Damuni 1998), a cell cycle regulatory phosphatase. Pp32 also helps regulate cell cycle progression by associating with the hyperphosphorylated form of the retinoblastoma tumor suppressor (Adegbola and Pasternack 2005). Pp32 is also involved in the regulation of mRNA trafficking through an association with Crm1 as part of the HuR complex (Brennan et al. 2000). Since this complex transports the mRNAs of cytokines and proto-oncogenes, it may stabilize factors involved in tumor progression and cell differentiation, such as IL-6 and vascular endothelial growth factor (Nabors et al. 1998). A structural role in cytoskeletal dynamics has also been inferred because the phosphorylated form of pp32 stimulates the localization of the Golgi apparatus in a microtubule and dynein-dependent manner via binding to the microtubule-associated proteins, MAP1, MAP2, and MAP4 (Ulitzur et al. 1997a,b; Itin et al. 1999; Opal et al. 2003).

The pp32 family (Anp32a–h) belongs to the superfamily of proteins containing leucine-rich repeats (LRRs). The LRR is a short motif of 20–29 residues in length that is present in tandem arrays in a variety of cytoplasmic, membrane, and extracellular proteins (Kobe and Kajava 2001). The 28-kDa pp32 protein contains three capped LRR motifs at the N terminus and a highly acidic C terminus containing 70% aspartic and glutamic acid residues (Chen et al. 1996). Similar acidic stretches are found in the nucleosome assembly proteins, nucleomorphin, nucleoplasmin, p62, and SET, and in the HMG box proteins, where the acidic domain is likely to be involved in chromatin binding (Matilla and Radrizzani 2005). Acidic stretches are also found in proteins such as tubulin, which are responsible for microtubule association (Matilla and Radrizzani 2005).

The LRR domain is of particular importance, as it is the minimal fragment of pp32 necessary to bind to a hyperphosphorylated form of the retinoblastoma protein (Rb) (Adegbola and Pasternack 2005). In cancers where p16 is inactivated, such as pancreatic cancer, the cyclin D:CD4/6 complex is unregulated and hyperphosphorylated Rb is predominant (Rozenblum et al. 1997). In these cases, hyperphosphorylated Rb both sequesters the pro-apoptotic activity of pp32 and is unable to correctly regulate E2F mediated gene transcription, allowing proliferation of tumor cells (Weinberg 1995; Adegbola and Pasternack 2005). The pp32:Rb protein complex therefore represents a potential target for the design of novel peptides or small molecules that could be used in the treatment of pancreatic cancer, for which there are currently limited treatments available.

	Results

TOP Abstract Introduction Results Discussion Materials and Methods Electronic supplemental material Acknowledgments References

 
We determined the crystal structure of the pp32 N-terminal LRR domain from two crystal forms at 2.4 Å and 2.7 Å resolution, respectively (see Materials and Methods). Data collection and refinement statistics are given in Table 1. The C2 crystal form contains six molecules and the P6₃ crystal form contains four molecules in the asymmetric unit. The structures were refined with tight noncrystallographic symmetry restraints because of the low data:parameter ratio (see Materials and Methods). The C positions of monomers from the C2 and P6₃ superimpose with an RMSD of 0.18 Å, indicating that the structure of the LRR domain is virtually identical in both crystal forms. Unlike the structures of decorin (Scott et al. 2004), biglycan (Scott et al. 2006), and slit (Howitt et al. 2004) which dimerize through the LRR concave surface, the noncrystallographic symmetry in our pp32 crystal forms has no biological significance. The pp32 fragment used in this study is both monomeric by size-exclusion chromatography (see Materials and Methods), and lacks the C-terminal residues which are essential for chemical cross-linking of pp32 dimers and trimers (Ulitzur et al. 1997b). For clarity, further structural analysis is of a monomeric LRR domain from the high-resolution C2 crystal form.

View larger version (55K): [in this window] [in a new window]   Figure 1. (A) Stereo ribbon representation of the structure of pp32CT illustrating the canonical curved structure of the LRR domain. The ribbon is colored according to secondary structure, with -strands colored blue, -helices colored red, and coil colored yellow. (B) A ribbon representation face on to the -strands which line the concave surface, the ribbons are colored: red (N-terminal cap residues 1–40), orange LRR1 (residues 42–62), yellow LRR2 (residues 63–86), green LRR3 (residues 87–111), and blue (C-terminal cap residues 112–149). Illustrated as sticks are residues K68, E70, H92, N94, S117, and D119, which surround a shallow cleft on the concave surface of the LRR. (C) A surface representation created with GRASP (Baker et al. 2001) and colored according to surface curvature potential to highlight the shallow cleft on the concave surface of the LRR domain.

View larger version (32K): [in this window] [in a new window]   Figure 2. Sequence alignment of the individual LRRs from pp32. The central repeats belong to the SDS22-like class of LRRs.

View larger version (48K): [in this window] [in a new window]   Figure 3. (A) Ribbon representation to illustrate the structural homology between pp32 (green), U2A' spliceosomal protein (blue), and Tap mRNA export factor (magenta). (B) as in A, rotated 90°. Conserved and structurally important residues capping the LRR that are part of the YRxxxxxPxxxLD motif are illustrated as sticks.

The pp32 gene knockout shows no phenotype (Opal et al. 2004), indicating some functional overlap between the pp32 (Anp32) family members. We therefore analyzed the conservation of residues within the pp32 (Anp32) protein family to identify residues that could form a common binding interface. There are many sequences of pp32 (Anp32) proteins in the database from multiple species, a sequence alignment of the human sequences is shown in Figure 4A. The sequence conservation of the human pp32 (Anp32) family has been mapped onto the surface of the pp32 LRR domain structure (Fig. 4 B,C) and clearly indicates that LRR3 and the C-Cap are the most conserved regions within the domain. We note a shallow cleft in the concave surface (Fig. 1C), which is formed by the surface residues K68 and E70 in 3 (LRR2), H92 and N94 in 4 (LRR3), and S117 and D119 in 5 (C-Cap) (Fig. 1B). These residues lining the cleft are highly conserved among the members of the pp32 (Anp32) family (residues highlighted with an asterix in Fig. 4A). In pp32r1 (Anp32c), which is overexpressed in prostate cancer (Kadkol et al. 2001), there is a deletion of four residues in LRR2 and two residue changes (E70R and N94Y) in the residues surrounding the cleft (Fig. 4A). Pp32r1 (Anp32c) does not associate with hyperphosphorylated retinoblastoma (Adegbola and Pasternack 2005), consistant with the possible involvement of these cleft residues in this interaction. Additional mutational analysis of residues on the concave surface of the pp32 LRR will be necessary to establish whether the cleft region mediates interactions with retinoblastoma and other protein ligands, or whether other residues on the pp32 surface are required.

View larger version (63K): [in this window] [in a new window]   Figure 4. (A) Sequence alignment of human pp32 (Anp32) family homologs a–h. Identical residues from all sequences are boxed black and similar residues are open boxed. The consensus sequence is shown below, and the secondary structure taken from the pp32 (Anp32a) crystal structure is shown above. (*) Denotes the position of the residues K68, E70, H92, N94, S117, and D119 that surround a surface cleft in the pp32 crystal structure. The position of the CKII phosphorylation sites S158 and S204 are indicated with # and the nuclear localization signal KRKR indicated with ++++. (B) Sequence variability of the pp32 (Anp32) family with a view of the concave surface of the pp32 LRR domain. The surface is colored according to sequence variability (derived from the alignment in A) with blue being the most conserved through the spectrum to red being the least conserved. (C) As in B, rotated by 180° viewing the convex surface.

	Discussion

TOP Abstract Introduction Results Discussion Materials and Methods Electronic supplemental material Acknowledgments References

The structure provides insights into the molecular architecture of potential protein interactions mediated by the pp32 LRR domain, such as the interaction with the hyperphosphorylated form of Rb, which is predominant in p16 inactivated pancreatic tumors. The structure also provides the basis to identify possible small molecule binding partners by virtual screening that could be used to disrupt this interaction and be developed as therapeutics. We can also selectively probe the interactions made by pp32 (Anp32a) and other family members through rational site-directed mutagenesis to provide further insight into the structure and function of the pp32 (Anp32) family.

	Materials and Methods

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Protein expression and purification
The pp32 (Anp32a) gene fragment pp32CT comprising residues 1–149 was amplified by PCR from a full-length clone (RZPD). The fragment was cloned into the pProEX HTb expression vector in frame with an N-terminal His₆ tobacco etch virus (TEV) cleavable tag. Cultures of Escherichia coli Rosetta 2 (DE3) (Novagen) containing the pp32 expression construct were grown in LB at 37°C, induced at mid-log phase at 28°C with 0.5 mM IPTG, and harvested after 3–4 h. Cells were lysed using a microfluidizer in Buffer A (25 mM Tris, 500 mM NaCl, 2 mM 2-mercaptoethanol, 10 mM Imidazole pH 8.0) supplemented with DnaseI (Roche) 2 mM MgCl₂, 1 mM PMSF, and EDTA free protease inhibitor cocktail (Roche). Lysed cells were then centrifuged at 20,000g before loading onto a 5-mL Hi-Trap chelating column equilibrated in buffer A. After washing with 10 column volumes of buffer A protein was eluted in buffer B (25 mM Tris, 500 mM NaCl, 2 mM 2-mercaptoethanol, 250 mM Imidazole pH 8.0). Protein was dialyzed into TEV cleavage buffer (25 mM Tris, 150 mM NaCl, 2 mM 2-mercaptoethanol, pH 8.0) and the His₆ tag removed with His₆-TEV protease for 3–4 h at room temperature. Subsequent repurification on a Hi-Trap chelating column removed contaminants and TEV protease. Size-exclusion chromatography, using a Superdex 200 16/60 (Pharmacia) was then used as the final purification step, where the fragment elutes as a monomer compared to standards. Fractions containing pure pp32CT were then pooled and dialyzed into crystallization buffer (25 mM Tris, 150 mM NaCl, 2 mM 2-mercaptoethanol, pH 8.0) before concentrating to 20–40 mg/mL.

Crystallization and data collection
Two plate-like crystal forms of pp32CT were grown at 22°C by vapor diffusion using the hanging-drop method. Crystal form I grew from 1.6–2.0 M Ammonium sulphate, 5% PEG 3350, and 0.1 M sodium citrate buffer, pH 5.5 at a protein concentration of 20–30 mg/mL. These crystals of pp32CT belong to the monoclinic space group C2 with unit cell parameters a = 106.5, b = 183.2, c = 67.47 Å, = 104.4 and six molecules per asymmetric unit.

Crystal form II grew in 15%–25% PEG3350, 0.2 M ammonium citrate, and 0.1 M sodium citrate buffer, pH 5.0–5.5, at a protein concentration of 25–40 mg/mL. These crystals of pp32CT belong to the hexagonal space group P6₃ with unit cell parameters a = b = 105.9, c = 132.4 Å, and four molecules per asymmetric unit.

For both crystal forms, single crystals of pp32CT were transferred briefly to cryoprotectant solutions consisting of mother liquor supplemented with increasing concentrations of glycerol (up to 25%) before being frozen directly with liquid nitrogen. Data were collected at 100K at BioCars beamline 14-BM-C (APS). The data were processed, scaled, and reduced using HKL2000 (Table 1).

Structure determination and refinement
A homology model of the N-terminal LRR domain of pp32 was created using Modeller 8.01 and the coordinates of the spiceosomal protein U2A' protein (Price et al. 1998) (accession 1A9NA), which was used to solve the structure by molecular replacement with MOLREP (Vagin and Teplyakov 2000). Initially, data from crystal form II were processed in space group P6₃22 with two molecules in the asymmetric unit; however, this structure would not refine satisfactorily beyond an R _free of 39%. The correct space group was subsequently identified as P6₃ with four molecules in the asymmetric unit, in which the NCS molecules mimic pseudo P6₃22 symmetry. Crystal form I was then solved by molecular replacement with MOLREP (Vagin and Teplyakov 2000) using the coordinates of a monomer from the partially refined P6₃ structure.

Both structures were refined using REFMAC5 (Murshudov et al. 1997) and model building carried out with the program Coot (Emsley and Cowtan 2004). The progress of the refinement was monitored by the agreement between calculated and observed structure factors for the excluded reflections (R _free). Due to limited observations tight NCS restraints were used throughout the refinement process. Final cycles of refinement were carried out using TLS refinement as implemented in REFMAC5 (Winn et al. 2003); with the individual NCS-related molecules as TLS groups, final refinement statistics are shown in Table 1. Coordinates and structure factors have been deposited in the RCSB Protein Data Bank, for both crystal forms (accession 2je0 and 2je1, respectively).

Structural analysis and graphics
Sequences from human Anp32a–h were aligned using ClustalW (Thompson et al. 1994) and plotted using ESPript (Fig. 4A; Gouet et al. 1999). The Shannon entropy was calculated for each residue position for the ClustalW alignment, with H2PDB (http://bio.dfci.harvard.edu/Tools/entropy2pdb.html) and the calculated values transferred to the B-factor column of the PDB file and plotted on the surface (Fig. 3). Surface curvature was calculated with GRASP (Baker et al. 2001) and figures prepared with MolScript (Esnouf 1997) and Raster3D (Fig. 1C; Merritt and Murphy 1994). All other molecular images were generated using PyMol (http://pymol.sourceforge.net/).

	Electronic supplemental material

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Electrostatic-potential maps were calculated with APBS (Baker et al. 2001) (Suppl. Fig. 1).

	Footnotes

 
Supplemental material: see www.proteinscience.org

Reprint requests to: Cynthia Wolberger, Department of Biophysics and Biophysical Chemistry, Howard Hughes Medical Institute, Johns Hopkins University School of Medicine, 725 N. Wolfe Street, Baltimore, MD 21205-2185, USA; e-mail: cwolberg{at}jhmi.edu; fax: (410) 614-8648.

Article published online ahead of print. Article and publication date are at http://www.proteinscience.org/cgi/doi/10.1110/ps.072803507.

	Acknowledgments

TOP Abstract Introduction Results Discussion Materials and Methods Electronic supplemental material Acknowledgments References

 
We thank Jonathan Brody (Thomas Jefferson University Hospital) for critical reading of this manuscript, Michael Love (Johns Hopkins University) for computational support, Robbert Henning, Michael Bolbat, and the staff at BioCARS beamline 14-BM-C (APS, Chicago) for assistance during data collection. The U.S. Department of Energy, Basic Energy Sciences, Office of Science, under Contract No. W-31-109-Eng-38, supports use of the Advanced Photon Source. Use of the BioCARS Sector 14 is supported by the National Institutes of Health, National Center for Research Resources, under grant number RR07707.

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