Trimeric Structure of PRL-1 Phosphatase Reveals an Active Enzyme Conformation and Regulation Mechanisms

doi:10.1016/j.jmb.2004.10.061

J. Mol. Biol. (2005) 345, 401–413

Trimeric Structure of PRL-1 Phosphatase Reveals an Active Enzyme Conformation and Regulation Mechanisms Dae Gwin Jeong1, Seung Jun Kim1,2, Jae Hoon Kim1,2, Jeong Hee Son1,2 Mi Rim Park2, Sang Myoun Lim2, Tae-Sung Yoon2 and Seong Eon Ryu1,2* 1

Center for Cellular Switch Protein Structure, Korea Research Institute of Bioscience and Biotechnology, 52 Euh-eun-dong, Yuseong-gu Daejeon 305-806, South Korea 2

Systemic Proteomics Research Center, Korea Research Institute of Bioscience and Biotechnology 52 Euh-eun-dong, Yuseong-gu Daejeon 305-806, South Korea

The PRL phosphatases, which constitute a subfamily of the protein tyrosine phosphatases (PTPs), are implicated in oncogenic and metastatic processes. ˚ Here, we report the crystal structure of human PRL-1 determined at 2.7 A resolution. The crystal structure reveals the shallow active-site pocket with highly hydrophobic character. A structural comparison with the previously determined NMR structure of PRL-3 exhibits significant differences in the active-site region. In the PRL-1 structure, a sulfate ion is bound to the active-site, providing stabilizing interactions to maintain the canonically found active conformation of PTPs, whereas the NMR structure exhibits an open conformation of the active-site. We also found that PRL-1 forms a trimer in the crystal and the trimer exists in the membrane fraction of cells, suggesting the possible biological regulation of PRL-1 activity by oligomerization. The detailed structural information on the active enzyme conformation and regulation of PRL-1 provides the structural basis for the development of potential inhibitors of PRL enzymes. q 2004 Elsevier Ltd. All rights reserved.

*Corresponding author

Keywords: PRL phosphatases; crystal structure; metastasis inhibitor; active form; oligomerization

Introduction Protein tyrosine phosphatases (PTPs) play important roles in cellular signal transduction mediating cell growth, differentiation, transcription and metabolism.1,2 The PTP family proteins have a conserved active-site motif that contains a catalytic cysteine residue. The PTP family, which consists of about 120 members, can be divided into two subgroups according to their target preference and structural characteristics. The first subgroup includes authentic PTPs that dephosphorylate only phospho-tyrosine (pTyr) and the second consists of dual specificity phosphatases that catalyze dephosphorylation of phospho-serine/ threonine (pSer/pThr) as well as pTyr. The PRL (phosphatase of regenerating liver) phosphatases are small C-terminal-prenylated PTPs that have three subtypes, PRL-1–3.3 PRL-1 Abbreviations used: PTPs, protein tyrosine phosphatases; pTyr, phospho-tyrosine; PRL, phosphatase of regenerating liver. E-mail address of the corresponding author: [email protected]

was first cloned as an immediate-early gene in rat liver following hepatectomy.4 PRL-2 and 3 were later found by sequence homology searches.5 They contain the PTP signature motif in their sequences, but otherwise they do not have homology to any other previously described PTPs. Overexpression of PRL-1 or PRL-2 in epithelial cells results in a transformed phenotype and cells transfected with the proteins form tumors in nude mice.6 PRL-3 is highly overexpressed in liver metastasis of colon cancer but neither in non-metastatic tumors nor in normal colorectal epithelium.7 In addition, PRL-3 gene amplification is found in significant fraction of metastatic lesions from different patients. Pentamidine, an inhibitor of PRL proteins markedly suppressed the growth of WM9 human melanoma tumors in nude mice.8 It was also shown that overexpression of PRL-1 and PRL-3 contributes to the acquisition of metastastic properties by promoting cell migration and invasion.9 The PRL phosphatases have a consensus Cterminal CAAX sequence (C, cysteine; A, aliphatic amino acid; X, any amino acid) for prenylation. Protein prenylation plays critical roles in targeting proteins into cellular membranes, resulting in

0022-2836/$ - see front matter q 2004 Elsevier Ltd. All rights reserved.

402

Crystal Structure of PRL-1

activation of cellular signaling processes as shown in Ras and G-protein coupled receptor complexes.10,11 Prenylation of PRLs directs them to the plasma membrane and the early endosome.12 Localization of PRLs was shifted to nucleus by farnesyltransferase inhibitors or prenylation site mutations.12 More recent reports showed that PRL-1 exhibited cell cycle dependent localization.13 In non-mitotic cells, the protein is localized to endoplasmic reticulum in a farnesylation dependent manner, whereas, in mitotic cells, the protein was relocalized to the centrosomes and the spindle apparatus, indicating that PRL-1 may play an important role in cell cycle progression by modulating spindle dynamics.13 Although accumulating data on cell proliferative and oncogenic activities of PRL phosphatases suggest that inhibitors of the proteins have high potential for the development of new anticancer therapeutic agents, the exact cellular mechanisms of PRL enzymes are not well studied yet. Recently, the NMR structure of PRL-3 was reported.14 The structure exhibits a loosely folded active-site with catalytic residues displaced from their canonical positions, suggesting that the structure is most likely not the active form and the protein needs to interact with other factors for activation. To understand detailed mechanisms for the substrate recognition and activity regulation of PRL enzymes, we determined the crystal structure of human PRL-1 at ˚ resolution. Our crystal structure reveals a 2.7 A well-ordered active-site structure with catalytic residues in active conformations, providing important information for the design of specific inhibitors. In the crystal, PRL-1 was found to form trimers with the C-terminal farnesylation sites lined on one side of the trimers, indicating that trimerization may

play an important role in the transportation of the protein into membranes and functional regulation. Subsequent biochemical analyses proved the existence of trimers in solution and provide evidence for the role of trimerization in the biological function of PRL-1.

Results and Discussion Overall structure The structure of PRL-1(C104S) was determined by using the multiwavelength anomalous diffraction method (Table 1). The PRL-1 structure comprises a central five-stranded b-sheet that is surrounded by six a-helices (Figure 1(a)). An approximate molecular dimension is 45!40! ˚ . One side of the b-sheet is covered with 35 A two a-helices (a1 and a2) and the other with four a-helices (a3–a6). Although PRL-1 shares low sequence identity (!30%) with other phosphatases, the backbone structure of PRL-1 is similar to those of dual specificity phosphatases (Figure 1(b)). In a search for homologous structures by using the Dali server,15 the dual specificity phosphatases VHR,16 MKP17 and PTEN18 were identified to have Z-values ranging from 18.0 to 16.0, indicating their structural homology with PRL-1. When we aligned structures of PRL-1 and VHR, 124 Ca atoms were superimposed with a root mean ˚ (Figure 1(b)). square deviation (rmsd) of 1.63 A Although there are good alignments in the core regions, substantial differences are found in various regions including loop and secondary structural elements. The most prominent differences are the deletions of N-terminal helix a0, loop a0-b1 and

Table 1. Crystallographic data Peak (l1) A. Data collection statistics ˚) Wavelength (A 0.9792 Space group P21 ˚) Cell dimensions a, b, c (A 59.29, 84.76, 122.18 a, b, g (8) 90.00, 99.79, 90.00 ˚) Highest resolution (A 2.7 (2.85–2.70) Unique reflections/total 30,169/128,838 a 92.0 (95.7) Completeness (%) 7.9 (23.5) Rmergeb(%)a 6.9 (3.1) I/s(I)a B. Refinement ˚) Resolution range (A Number of reflections Number of atoms (protein/non-protein) Rcryst Rfree rmsd ˚) Bond lengths (A Bond angles (8) Impropers (8) Dihedrals (8) a b

Edge (l2)

Remote (l3)

0.9794

0.9716

2.7 (2.85–2.70) 30,188/128,939 92.0 (95.7) 7.7 (23.2) 6.9 (3.1)

2.7 (2.85–2.70) 31,154/135,809 95.2 (97.8) 7.8 (23.5) 7.3 (2.9) 99–2.7 31,124 7086/122 23.7 29.6 0.008 1.4 0.9 23.2

The values P in parentheses P (completeness and Rmerge) are for the highest resolution bin. Rmerge Z I jIi K hIijI= jhIij, where I is the intensity for the ith measurement of an equivalent reflection with the indices h, k and l.

403

Crystal Structure of PRL-1

Figure 1 (legend next page)

loop b3-a2 in PRL-1 (Figures 1(b) and 2). Helix a0 and loop a0-b1 play important roles in substrate recognition of VHR and other phosphatases. Deletion of the N-terminal sequences implicates a different substrate recognition mechanism of PRL-1. In addition, deletion of loop b3-a2 results in a significant difference in the active site pocket entrance (see below). Other regions that cannot be aligned between PRL-1 and VHR structures are found in loops b1-b2, b2-a1, and b4-a3 (Figure 1(b)). In the crystal, three PRL-1 molecules form a threefold symmetry-associated trimer (Figure 1(c)

and (d)). The threefold symmetry-associated trimer presents C-terminal tails in one face of the trimer (Figure 1(d)), suggesting that the face of C-terminal tails may interact with cellular membranes upon farnesylation of CAAX motif in the C termini (see below). The active sites are in the outside of the trimer (Figure 1(c) and (d)), indicating that the trimeric association should not affect the PRL-1’s enzyme activity. The asymmetric unit contains six PRL-1 molecules (two sets of the trimer) that have very similar conformations throughout the entire molecule (Figure 1(e)).

404

Crystal Structure of PRL-1

Figure 1. Overall structure of PRL-1. (a) Ribbon diagram: the PRL-1 structure is presented as a ribbon diagram. Secondary structural elements (helices, purple; strands, blue; loops, yellow) are labeled on the drawing. The catalytic Cys104 (mutated to serine for crystallization) and the bound sulfate ion are shown as a ball-and-stick representation. The boundaries of secondary structural elements are a1 (30–39), a2 (56–60), a3 (78–94), a4 (110–121), a5 (126–134), a6 (143–150), b1 (10–14), b2 (17–21), b3 (42–47), b4 (64–67), and b5 (99–103), respectively. (b) Ca trace: the PRL-1 structure (thick line) is superimposed with the VHR structure (thin line). Among 160 residues in the PRL-1 structure, 124 residues can be ˚ . The regions of PRL-1 that cannot be aligned are colored green and the structural aligned with an rms deviation of 1.63 A elements of VHR that are missing in PRL-1 are colored red. The viewpoint of the stereodiagram is the same as (a). (c) and (d) Trimeric structure: the structure of PRL-1 is presented as ribbon diagrams. The catalytic cysteine residues and the sulfate ions bound to the active sites in the trimer are indicated as a ball-and-stick representation. In (c), the view is along the non-crystallographic threefold axis. In (d), the side view is presented, where the C-terminal tails point towards the same direction (up) that is opposite to the location of active sites (down). (e) Superposition of protomers in the asymmetric unit: Ca traces of six protomers in the asymmetric unit were superposed in the stereo figure. Six protomers (A–F) are colored green, yellow, cyan, red, magenta and gray, respectively. The superposition was performed by using the program O.34

˚ The rmsd’s between six molecules are 0.96 A ˚ for all NCS-related atoms (132 of and 0.55 A total 160 residues) and the active-site atoms ˚ of Cys104), respectively. Among the (within 10 A six molecules, models for four molecules (Mol A, B, C and E) are of high quality and we chose

one of the four good quality molecules (Mol A) for the description of the PRL-1 structure. Active-site The active-site of PRL-1 consists of the PTP

Figure 2. Structure-based sequence alignment. The PRL-1 sequence is aligned with those of PRL-3, KAP and VHR. Sequence alignment with KAP or VHR is based on the structural superposition with PRL-1, where the aligned sequences are colored yellow and orange, respectively. The alignment between PRL-1 and PRL-3 is based on a sequence comparison. Secondary structural elements of PRL-1 are indicated above the aligned sequences. The triad residues participating in the catalytic function are also indicated above the sequences as inverted triangles. Non-conserved residues between the PRL-1 and PRL-3 sequences as defined by the program ALSCRIPT37 are colored red on the PRL-3 sequence.

406

Crystal Structure of PRL-1

Figure 3. Active-site. (a) Electron density map: the 2FoKFc electron density map around the PRL-1 active-site is presented in stereo as superposed with the refined structure. The electron density map is contoured at 1s level. (b) Comparison with VHR: active sites of PRL-1 (cyan) and VHR (magenta) were superposed and presented as worm models. PRL-1 residues (gold) are labeled as black and VHR residues (gray) are labeled as red. (c) Comparison with PRL-3 active-site of PRL-1 (cyan) and PRL-3 (gray) were superposed and presented as worm models. PRL-1 residues (gold) are labeled as black and PRL-3 residues (green) are labeled as red.

signature motif (the P-loop: His103-Cys104-(X)5Arg110, X is any amino acid) containing the catalytic Cys104. The canonical WPD loop of the PTP family is changed to WFPDD (Trp68-Phe69Pro70-Asp71-Asp72) in PRL-1 (Figure 2). Upon

substrate-binding, the WPD loop adopts a closed conformation and covers the active-site like a “flap”.2 In the PRL-1 structure, a sulfate ion is bound to the enzyme active-site coordinating atoms of main-chain amides in the P-loop (Figure 3(a)).

407

Crystal Structure of PRL-1

The active-site bound sulfate ion in PTPs is known to mimic the substrate phosphate group.19 The negatively charged oxygen atoms of the sulfate ion make strong interactions with the P-loop amides having partial positive charges. The interaction distances between the amides of Val105-Arg110 ˚. and one of the sulfate oxygen atoms are 2.7–3.1 A The sulfate ion also interacts with side-chain atoms of Arg110 that in turn form charge interactions with Asp72 stabilizing the WPD loop in a closed conformation (Figure 3(b)). The aspartic acid residue of the WPD loop acts as a general acid during the enzyme reaction.2 A structural superposition between the active sites of PRL-1 and VHR (Figures 2 and 3(b)) reveals that the second aspartic acid (Asp72) of the WFPDD loop is aligned to the general acid residue of VHR, indicating that Asp72 is likely to function as the general acid in PRL-1. Our structural observation is consistent with the previous report where the D72A mutation resulted in a significant activity loss, whereas the D71A mutation yielded no effect on activity.13 In the wild-type PTPs, the active-site sulfide group interacts with main-chain amides in the P-loop, stabilizing the active-site conformation.2 The cysteine-to-serine substitution in the activesite was reported to disturb the P-loop conformation of apo-PTP1B due to the loss of interactions between the nucleophilic Sg atom with main-chain amides of the P-loop.20 However, inhibitors or sulfate ion bound to the active-site provide further interactions in the active-site and stabilize the active P-loop conformation even in the mutant PTPs lacking the active-site cysteine.19,21 The PRL-1 structure contains the active-site mutation C104S lacking the nucleophilic Sg atom. Despite the mutation, the P-loop of PRL-1 is stabilized by the active-site bound sulfate ion. The P-loop structure exhibits an active conformation with main-chain amides in the loop (Val105-Arg110) pointing to the Og atom of Ser104 with interaction distances of ˚ (Figure 3(a)). The interactions are similar 2.8–4.5 A to those found in the wild-type PTPs with active conformations.2 The PRL-1 structure reveals an extra cysteine near the catalytic Cys104. The extra cysteine (Cys49) is ˚ apart from Cys104 and there are no intervenw5 A ing structures (Figure 3(a)), indicating that the two cysteine residues would readily form a disulfide bond without large conformation changes. To verify the potential for disulfide bond formation between

the two cysteine residues, we performed matrixassisted laser desportion ionization-A of flight (MALDI-TOF) mass spectrometry analyses of the reduced and oxidized PRL-1 (Table 2). For the reliable mass estimations, we subjected the proteins to iodoacetamide (IA) alkylation and trypsin digestion before the mass analyses. The reduced PRL-1 yields peaks at m/zZ1528.7 and 1884.8 corresponding to the peptides containing IA-modified Cys49 and Cys104, respectively. The Cys104 peptide includes two more IA-modifications in Cys98 and Cys99. The oxidized PRL-1 yields a new peak at m/zZ3299.6 indicating a disulfide bond formation between Cys104 and Cys49. In comparison, the PRL-1(C104S) mutant does not yield a peak corresponding to the disulfide-bonded peptide. The reversible disulfide bond formation between Cys104 and Cys49, which is consistent with the NMR studies of PRL-3,14 suggests that PRL-1 is likely regulated by cellular redox status. Although the catalytically important residues in PRL-1 (Cys104, Arg110 and Asp72) are well aligned with those in VHR (Figure 3(b)), sequence conservation of residues near the critical residues is quite low (Figure 2), which results in various differences in the pocket surface and side-chain interactions (Figure 3(b)). Most prominently, the variable residues in the P-loop of PRL-1 (Val105-Ala106Gly107-Leu108-Gly109) have more hydrophobic character than those in VHR (Arg125-Glu126Gly127-Tyr128-Gly129). In VHR, the hydrophilic side-chains of Arg125 and Tyr128 point outside of the active-site pocket and form hydrogen bonds with His70 and Ser24, respectively, whereas the corresponding residues Val105 and Ala106 of PRL-1 point inside of the active-site pocket, resulting in a more hydrophobic pocket surface in PRL-1 (Figure 3(b)). In VHR, the protruding side-chain of Met69 in loop b3-a2 forms one side of the active-site pocket, whereas in PRL-1, the corresponding loop is truncated, resulting in a different pocket shape (Figure 3(b)). Due to the truncation of loop b3-a2 and other loops near the pocket, the active-site pocket of PRL-1 exhibits a wider pocket entrance compared to VHL. Whereas the pocket width of ˚ (PTP1B: other phosphatases is less than 6 A ˚ , VHR: 5.0 A ˚ ), that of PRL-1 is as large as 8 A ˚. 5.8 A The highly hydrophobic pocket interior and the large pocket entrance of PRL-1 may be exploited in designing specific drugs towards PRL phosphatases.

Table 2. MALDI-TOF mass spectrometry Peptide

Sequences

Modified

Expected

Reduced PRL-1

48–60 94–110

VCEATYDTTLVEK EEPGCCIAVHCVAGLGR EEPGCCIAVHSVAGLGR

IA 3IA

1528.7 1884.8

1528.7 1884.8

2IA C49–C104 C 2IA

1811.8 3299.5

94–110(C104S) C49–C104

Oxidized PRL-1

3299.6

Reduced PRL-1(C104S)

Oxidized PRL-1(C104S)

1528.5

1528.4

1811.9

1811.9

408

Crystal Structure of PRL-1

Figure 4. Surface characteristics. (a) and (b) Sequence similarity between PRL-1 and PRL-3 is mapped onto the PRL-1 surface. The 100% identical, similar and different residue properties, as defined by the program ALSCRIPT37 are colored white, green and cyan, respectively. The active-site surface and the opposite surface are presented in (a) and (b), respectively. (c) and (d) Electrostatic potential surfaces of PRL-1 (c) and VHR (d) are presented. Positive and negative potentials are colored blue and red, respectively. The point of view is the same as (a). Residues near the active-site are labeled.

Structural comparison with PRL-3 A sequence alignment (Figure 2) indicates that PRL-3 is likely to have very similar structure to PRL-1. There are only 14 non-homologous residues in the total 173 residues. Others are either identical (135 residues, 78%) or highly homologous (24 residues, 14%). More importantly, identical residues are concentrated in the molecular surface including the enzyme active-site (Figure 4(a) and (b)). Thus, the active-site structure of PRL-3 is likely to be highly similar to PRL-1. Despite the expected structural similarity

between PRL-1 and PRL-3, the recently reported NMR structure of PRL-314 exhibits large differences in the active-site region as compared to the activesite of PRL-1 (Figure 3(c)). Both the P-loop and the WPD loop cannot be aligned between the two structures. A structural comparison of the P-loop between the two structures (PRL-1 and PRL-3) indicates that the loop of PRL-3 is shifted towards strands b1 and b2 making the active-site pocket flatter than that of PRL-1. The catalytic Cys104 of PRL-3 also is displaced from the position of the corresponding residue of PRL-1 and other PTPs with active conformation. Moreover, all main-chain

409

Crystal Structure of PRL-1

amide nitrogen atoms in the P-loop of PRL-3 do not point towards the catalytic cysteine, indicating that the NMR structure may not represent the stabilized active conformation. The structural differences in the WPD loop are more pronounced. Structural comparison shows that the general acid residue Asp72 in the WPD loop ˚ away from the corresponding of PRL-3 is 11.3 A residue of PRL-1 (Figure 3(c)). The displacement in the WPD loop conformations may result from the flexible nature of the loop. In the PRL-3 structure, the WPD loop exhibited an unusual conformation and high mobility.14 In the PRL-1 structure, the flexible loop conformation is stabilized by the substrate-mimicking sulfate ion bound to the activesite of the enzyme. Another contributing factor for the more flexibility of the WPD loop in PRL-3 may be the three consecutive proline residues and one glycine following the loop, whereas there are only two consecutive proline residues following the corresponding loop in PRL-1 (Figure 2). Substrate specificity In the recognition of PTP substrates, various surface properties near the active-site play important roles.22–24 PTP1B and VHR have the second phosphate-binding sites that bind the bisphosphorylated peptides of insulin receptor kinase22 and MAP kinase peptide,23 respectively. The second phosphate-binding sites, which are different depending on PTPs,22,23 provides an extra-binding site for the bidentate inhibitors.2 The interaction of map kinase phosphatase MKP3 with its substrate ERK2 involves multiple regions of MKP3 including the kinase interaction motif (KIM) and two other ERK-binding motifs.24 In the PRL-1 structure, there is a positively charged patch near the active-site that may play as a second phosphate-binding site (Figure 4(c)). The patch consists of Arg134, Arg137 and Arg138. Among these charged residues, Arg138 is unique in PRL-1. Arg134 could be aligned with Arg155 of VHR (Figure 2), but Arg155 of VHR is buried by the loop 22–26 of VHR that is missing in PRL-1. These differences should contribute to the substrate specificity of PRL-1. The PRL-1 structure also exhibits a characteristic cluster of negatively charged residues consisting of Glu50, Asp71 and Asp72 (Figure 4(c)). In addition, the overall electrostatic surface of PRL-1 is much more hydrophobic compared with VHL (Figure 4(c) and (d)). Such overall surface differences should play an important role in recognizing tertiary structure of target proteins. In the substrate recognition of PTPs, loop a0-b1 plays an important role.23,25 In VHR and kinaseassociated phosphatase (KAP), the substratebinding utilizes a groove between loop a0-b1 and the active-site loop that binds peptides with an extended loop conformation. In PRL-1, helix a0 is deleted and loop a0-b1 is not present, indicating that substrate-binding interactions of PRL-1 should

be different from other PTPs. In addition to the absence of helix a0, loop a0-b1, the active-site surface of PRL-1 lacks several other protruding loops, resulting in an almost flat active-site surface. This unusual surface property is consistent with that the only known PRL-1 target protein ATF7 is a helical coiled-coil protein,26 in comparison to other PTP substrates with extended loop conformations. Binding of helices would require a flat surface, whereas surface grooves are necessary to fit extended loops. The PRL-1’s flat surface may also be used to interact with the helical protein ab-tubulins27 during the modulation of spindle dynamics.13 Oligomeric state and C-terminal prenylation PRL-1 forms trimers in the crystal (Figure 1(c) ˚2 and (d)). The trimeric association buries 2027 A (25.8% of the total monomeric surface) in each monomer. The trimeric interface is made of numerous hydrogen bonds and van-der-Waals interactions. Residues of loop a5-a6 (Asp128, Gln131, Arg134, Gln135 and Arg138) from the first PRL-1 molecule interact with residues of strand b1 (Glu11 and Thr13), loop a1-b3 (Lys39 and Tyr40), loop a3-b5 (Pro96 and Gly97) from the second molecule. In the trimeric core resides a hydrophobic patch comprising Tyr14 and Phe132 whose trimeric interactions stabilize the core region. The extensive trimeric interactions and the direction of C-terminal tails towards one face of the trimer (Figure 1(d)) add to suggest that the trimer found in the crystals may have a role in the physiological function of PRL-1. To characterize the potential for PRL-1 trimerization in cells, we first analyzed the trimerization with the C-terminal-truncated PRL-1 (residues 4–163) that was used in the crystallization. In the dynamic light-scattering experiments, the truncated PRL-1 protein appears to be monomers in 1 mg/ml concentration, whereas the estimated mass of the protein in 7 mg/ml concentration is between those of dimers and trimers, indicating that the protein exists as a mixture of different oligomers in high concentration (Table 3). The gel filtration and analytical ultracentrifugation experiments with diluted proteins did not show a clear indication for oligomeric association. The mixture of different oligomeric status of PRL-1 in high concentration is reminiscent of the heterogeneous oligomerization of receptor protein-tyrosine phosphatase-a depending on protein concentration.28 In contrast to the weak indication for oligomerization in the C-terminal-truncated PRL-1 protein, the in vivo farnesylated PRL-1 exhibited clear evidence for the trimerization (Figure 5). We Table 3. Dynamic light-scattering Protein concentration (mg/ml) 1 7

Hydrodynamic radius (nm)

Estimated mass (kDa)

2.2 2.8

21.4 45.7

410

Crystal Structure of PRL-1

Figure 5. Subcellular fractionation and crosslinking. (a) Fractionated samples of the full-length (1– 173) and the C-terminal-truncated (1–163) PRL-1 proteins were analyzed by SDS-PAGE. Lanes 1–3 represent the total cell lysate, S100 and P100 fractions, respectively. The Flag-tagged PRL-1 proteins were detected by anti-Flag monoclonal antibody. (b) The P100 membrane fractions of the wildtype and mutant (T13F and Q131A) PRL-1 proteins were subjected to glutaraldehyde crosslinking. Lanes 1–4 for each protein represent 0, 0.02, 0.04 and 0.05% glutaraldehyde, respectively. The PRL-1 proteins were detected as in (a). Arrow indicates the position of the PRL-1 trimer.

expressed different PRL-1 proteins in HEK293 cells and isolated the membrane fractions to analyze the relationship between PRL-1’s membrane localization and trimerization. The tested PRL-1 constructs included the full-length protein containing the C-terminal farnesylation site (residues 1–173) and the protein lacking the farnesylation site (residues 1–163). The full-length protein, which was presumably farnesylated in cells, was found predominantly in the membrane fraction, whereas the PRL-1 lacking farnesylation site was not detected in the membrane fraction. The cross-linking of the membrane-localized PRL-1 showed clear trimerization of PRL-1. In the PRL-1 proteins carrying mutations at the trimeric interface (T13F and Q131A), the trimerization was significantly reduced. These results indicate that PRL-1 forms a trimer in the membrane with the conformation observed in the crystal. The membrane localization of PRL-1 is important in the PRL-1’s mitotic regulation activity.13 The trimerization of the membrane-associated PRL-1 indicates that the trimerization may have a role in the physiological function of PRL-1. The protein farnesylation alone is often insufficient for the translocation of the protein to the plasma membrane.11 In the translocation of the heterotrimeric G-protein, in addition to the prenylation of one subunit, oligomerization with other subunits is essential for the membrane localization.11 The

three-farnesyl groups in the trimeric PRL-1 may provide strong adhering forces to the membrane, stabilizing the membrane-association. In PRL-1, the trimerization and membrane localization appear to cooperate with each other by providing strengthened adhering forces to the membrane for the membrane localization and by increasing local concentration of monomers in the membrane for the trimerization, respectively. Whereas the multimerization-mediated functional regulation is often found in protein kinases (PKs), there are only a few examples of such regulation in PTPs. Moreover, the previously known oligomerization of PTPs is restricted to ones inhibiting enzyme activity.28,29 Thus, the presumptive positive regulation of mitotic process by the PRL-1 trimerization is unique and indicates that the activity of PTPs could be positively regulated by multimerization as observed in PKs. The involvement of trimerization in the PRL-1’s function also indicates that the trimeric interface of PRL-1 may present a new target for designing inhibitors against PRL proteins.

Materials and Methods Expression, purification and crystallization PRL-1 was cloned from human fetal brain cDNA and subcloned into pET28a. PRL-1(C104S) (residues 4–163)

411

Crystal Structure of PRL-1

lacking the C-terminal farnesylation site was expressed in Escherichia coli BL21(DE3) and B834(DE3) cells for the native and selenomethionine-labeled proteins, respectively. The C104S mutation was necessary to obtain diffraction-quality crystals. Cells were grown at 18 8C after induction with 0.1 mM IPTG. Cell pellets were resuspended in a lysis buffer containing 50 mM Tris–HCl (pH 7.5), 200 mM NaCl, 5% (v/v) glycerol, 0.04% (v/v) b-mercaptoethanol and 1 mM PMSF. After cell lysis by sonication, the His-tagged protein was purified by nickelaffinity chromatography. The His-tag was removed by thrombin digestion and the PRL-1 protein was further purified by ion exchange chromatography. Crystallization was performed at 18 8C with commercial screening solutions by using the vapor diffusion method. Crystallization drops contained 1.7 ml each of the protein and reservoir solutions. Very thin plate crystals were obtained within a week in a drop equilibrated against a reservoir containing 25% (w/v) PEG4000, 0.1 M sodium acetate (pH 4.6) and 0.2 M ammonium sulfate. Subsequent optimization in crystallization conditions resulted in the best crystals that were obtained by using a reservoir containing 15% PEG4000, 0.1 M sodium acetate (pH 4.6), 0.2 M magnesium sulfate, 7% glycerol and 10 mM DTT. The crystals belonged to the monoclinic ˚, space group P21 with unit-cell parameters of aZ59.29 A ˚ , cZ122.18 A ˚ and bZ99.798. bZ83.76 A

Structure solution, model building and refinement A crystal grown from the selenomethionyl-derivatized protein was used for the multiple anomalous dispersion (MAD)30 data collection at the Pohang Accelerator Laboratory beamline 6B. Data collected at three wavelengths, peak (l1), edge (l2) and remote (l3) were processed and scaled with the programs MOSFLM31 and SCALA,31 respectively. The asymmetric unit contained six monomers of the PRL-1 protein. Twelve out of expected 18 selenium sites in the asymmetric unit were located by the program SOLVE.32 The programs SHARP33 and DM31 were used for the heavy-atom parameter refinement and the phase improvement by solvent flattening, respectively. The resulting experimental map was of sufficient quality for building the majority of the protein molecules. The model was built with the program O34 and refined with the program CNS35 in the resolution range of ˚ . The randomly selected 5% of the data were set 99–2.7 A aside for the Rfree calculation. Data collected at the remote wavelength (l3) were used in the refinement. The refinement included an overall anisotropic B-factor refinement and bulk solvent correction. During the refinement, it was possible to apply tight NCS restraints ˚ 2) for most regions (residues 10–13, 31–49 (100 kJ/mol/A and 55–153) of the molecule without compromising the quality of electron density maps. The N- and C-terminal regions and the b2-a1 and b3-a2 loop regions displayed deviations from the NCS and atoms in those regions were excluded from the restraints. The Rcryst and Rfree of the final model are 23.7% and 29.6%, respectively (Table 1). The stereochemical analyses using the program PROCHECK36 indicate that 87.5% of the refined residues are located in the most favored region and none has disallowed conformations. The final model contains residues 8–156 in molecules A, B, C and E; residues 8–25, 28–50 and 54–156 in molecule D; residues 8–25 and 28–156 in molecule F; and six sulfate ions and 92 water molecules.

Mass spectrometry Purified PRL-1(4–164) proteins (wild-type and C104S mutant) were treated with 10 mM b-mercaptoethanol for 30 minutes and dialyzed overnight against 20 mM HepesNaOH (pH 7.0), 0.15 M NaCl and 2 mM b-mercaptoethanol. The dialyzed protein was diluted by tenfold in the same buffer lacking b-mercaptoethanol. The resulting protein (200 ml, 0.1 mg/ml) was treated with 20 mM DTT (ten minutes) or 500 mM H2O2 (five minutes) for reduced or oxidized protein samples, respectively. The H2O2 oxidation was terminated by addition of 1/200 volume of 1 mg/ml catalase. The proteins were alkylated by 20 mM iodoacetamide for two hours in an anaerobic chamber (Coy Laboratories) and digested with 1/100 volume of 0.5 mg/ml trypsin for 16 hours at 37 8C. The trypsin digestion was terminated with 1/100 volume of 10% (v/v) TFA and resulting peptides were analyzed by an Ettan MALDI-ToF Pro (Amersham Biosciences) mass spectrometer. Dynamic light-scattering Purified PRL-1(4–163) in 20 mM Tris–HCl (pH 8.0) and 50 mM NaCl was subjected to the dynamic light-scattering analyses using a DynaPro equipment (Protein Solutions). Before the measurements, the samples were filtered through 0.1 mm filters (Amicon). More than ten measurements were made at 4 8C with an acquisition time of 25 seconds for each measurement. The data were processed by the Dynapro data processing software (version 4.0) to estimate molecular mass. Subcellular fractionation and chemical crosslinking The full-length (residues 1–173) and C-terminal truncated (residues 1–163) PRL-1 sequences were inserted into the NheI/BamHI sites of the mammalian expression plasmid pcDNA3.1/Zeo(C) (Invitrogen) with a N-terminal FLAG tag sequence. The trimeric interface-disrupting mutants (T13F and Q131A) were constructed by using the QuickChange site-directed mutagenesis kit (Stratagene) and confirmed by DNA sequencing. HEK293 cells were grown in Dulbecco’s modified Eagle’s medium (Life Technologies, Inc) supplemented with 10% fetal bovine serum and antibiotics. The mammalian expression vector was introduced into HEK293 cells by the LipofectAMINE method (Life Technologies Inc). Transiently transfected cells were washed with an ice-cold buffer A containing 25 mM Hepes-NaOH (pH 7.0), 250 mM sucrose, 5 mM EDTA, 2 mM DTT, 1 mM PMSF and protease inhibitor cocktail, and then were centrifuged for five minutes in a microcentrifuge. The cell pellets were resuspended in 200 ml of buffer A and disrupted by 35 strokes with a tightfitting homogenizer. The unbroken cells were removed by centrifugation at 20,000g for ten seconds. The supernatant was centrifuged at 100,000g at 4 8C for one hour. The resulting supernatant (S100) and particulate fraction (P100, membrane fraction) were used for crosslinking experiments. Crosslinking of particulate fraction (P100) was performed with glutaraldehyde reaction. The particulate fraction (P100) in buffer A was incubated with 0.02, 0.04, or 0.05% (v/v) glutaraldehyde at 25 8C for 30 minutes. The crosslinking reaction was terminated by addition of 1.0 M Tris–HCl (pH 7.5) and the reaction mixture was analyzed by SDS-PAGE. The protein was detected by immunoblotting with anti-FLAG M1 monoclonal antibody (Sigma-Aldrich). Protein bands were visualized

412

Crystal Structure of PRL-1

after incubation with a horseradish peroxidase-conjugated anti-mouse secondary antibody by a chemiluminescence detection system (PIERCE). Protein Data Bank accession numbers Coordinates have been deposited with the Protein Data Bank (ID: 1XM2).

Acknowledgements We thank Dr H. S. Lee and the staffs of the Pohang Accelerator Laboratory beamline 6B for help with data collection. This study was supported by the national creative research initiatives program, the Biochallenger program (MOST), and the KRIBB molecular target program.

13.

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15. 16. 17.

18.

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Crystal Structure of PRL-1

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Edited by R. Huber (Received 5 May 2004; received in revised form 14 October 2004; accepted 21 October 2004)