Structural Insights Uncover the Specific Phosphoinositide Recognition by the PH1 Domain of Arap3

Arap3, a dual GTPase-activating protein (GAP) for the small GTPases Arf6 and RhoA, plays key roles in regulating a wide range of biological processes, including cancer cell invasion and metastasis. It is known that Arap3 is a PI3K effector that can bind directly to PI(3,4,5)P3, and the PI(3,4,5)P3-mediated plasma membrane recruitment is crucial for its function. However, the molecular mechanism of how the protein recognizes PI(3,4,5)P3 remains unclear. Here, using liposome pull-down and surface plasmon resonance (SPR) analysis, we found that the N-terminal first pleckstrin homology (PH) domain (Arap3-PH1) can interact with PI(3,4,5)P3 and, with lower affinity, with PI(4,5)P2. To understand how Arap3-PH1 and phosphoinositide (PIP) lipids interact, we solved the crystal structure of the Arap3-PH1 in the apo form and complex with diC4-PI(3,4,5)P3. We also characterized the interactions of Arap3-PH1 with diC4-PI(3,4,5)P3 and diC4-PI(4,5)P2 in solution by nuclear magnetic resonance (NMR) spectroscopy. Furthermore, we found overexpression of Arap3 could inhibit breast cancer cell invasion in vitro, and the PIPs-binding ability of the PH1 domain is essential for this function.

Arap3 is a PI3K effector protein that was originally identified from a screen for PI (3,4,5)P3-binding proteins [11]. It belongs to the ARAP family and functions as a dual GTPase-activating protein (GAP) for the small GTPases Arf6 and RhoA [12]. Arap3 structurally consists of an N-terminal sterile alpha motif (SAM) domain, two catalytic GAP domains, ArfGAP and RhoGAP, a Ras-associating (RA) domain and five pleckstrin homology (PH) domains ( Figure 1A). It is uncommon for a protein to contain 5 PH domains. Arap3 plays essential roles in diverse biological processes, including cell adhesion and migration [13][14][15], developmental angiogenesis [16], and lymphangiogenesis [17]. It is also involved in several cancers. It inhibits peritoneal dissemination of scirrhous gastric carcinoma cells [18]. It interacts directly with NEDD9, which is an established marker of invasive and metastatic cancers, and is involved in regulating mesenchymal cell invasion and metastasis of breast cancer [19]. Moreover, the expression level of ARAP3 might be a useful indicator of the metastatic likelihood of the basal-like breast tumors [20].
GTPase-activating protein (GAP) for the small GTPases Arf6 and RhoA [12]. Arap3 structurally consists of an N-terminal sterile alpha motif (SAM) domain, two catalytic GAP domains, ArfGAP and RhoGAP, a Ras-associating (RA) domain and five pleckstrin homology (PH) domains ( Figure 1A). It is uncommon for a protein to contain 5 PH domains. Arap3 plays essential roles in diverse biological processes, including cell adhesion and migration [13][14][15], developmental angiogenesis [16], and lymphangiogenesis [17]. It is also involved in several cancers. It inhibits peritoneal dissemination of scirrhous gastric carcinoma cells [18]. It interacts directly with NEDD9, which is an established marker of invasive and metastatic cancers, and is involved in regulating mesenchymal cell invasion and metastasis of breast cancer [19]. Moreover, the expression level of ARAP3 might be a useful indicator of the metastatic likelihood of the basal-like breast tumors [20]. Arap2, Q8WZ64. Alignment was performed using Clustal X and illustrated with ESPript 3.0. Strictly conserved (white letters filled with red color) and conservatively substituted (red letters with blue box) residues are denoted. The secondary structure element for human Arap3-PH1 is labeled on the top. The KXnQXR motif are marked by black dots. (C) Arap3-PH1 (20 µg) mixed with liposomes (640 µg) composed of 98% PC as the fixed component and 2% of specific phospholipids, respectively. Proteins in the absence of liposome were used as a control. After centrifugation, the pellet (P) and supernatant (S) were analyzed by SDS/PAGE and Coomassie. (D) SPR measurements of the binding affinities of the Arap3-PH1 domain for diC4-PI (3,4,5)P3 and diC4-PI (4,5)P2. The upper panel shows representative sensorgrams of diC4-PI(3,4,5)P3 (left) and diC4-PI(4,5)P2 (right) when mixed with Arap3-PH1. Data were collected by injecting increasing concentrations of diC4-PI (3,4,5)P3 and diC4-PI(4,5)P2 samples over Arap3-PH1 proteins immobilized on the surface of a CM5 biochip. The lower panel shows representative binding curves fitting for diC4-PI(3,4,5)P3 (left) and diC4-PI(4,5)P2 (right) during their interaction with Arap3-PH1. A one-site binding model was utilized to fit the curves. The experiment was carried out in triplicate. The KD value is presented as mean ± SD, n = 3.
In this study, we first expressed and purified the recombinant Arap3-PH1 domain and examined its lipid binding ability using liposome pull-down assay and surface plasmon resonance (SPR). Contrary to previous studies [23], we found that Arap3-PH1 alone is capable of binding PI (3,4,5)P3 and also PI(4,5)P2, albeit with lower affinity. To understand how Arap3-PH1 and PIP lipids interact, we solved the crystal structure of the Arap3-PH1 in its free state and in a complex with diC4-PI(3,4,5)P3, a soluble analog of PI (3,4,5)P3. Moreover, we have characterized the interactions of Arap3-PH1 with diC4-PI(3,4,5)P3 and diC4-PI(4,5)P2 by NMR. In addition, we found that a cancer-associate point mutation within the Arap3-PH1 domain (R308H) abolishes its binding to PI(3,4,5)P3 lipid, and impairs the capacity of Arap3 to inhibit breast cancer cell invasion in vitro.

The PH1 Domain of Arap3 Is Sufficient to Bind PIPs and Prefers PI(3,4,5)P3
Previous experiments, based on PI(3,4,5)P3-conjugated bead binding assay using cell lysates, have shown that the PH1 domain is essential for Arap3 to bind PI(3,4,5)P3 [11], but it alone cannot bind PI(3,4,5)P3 [23]. However, sequence analyses show that Arap3-PH1 contains a KXnQXR motif in the β1-β2 region ( Figure 1B), which is identical to the canonical KXn(K/R)XR motif in PIPs binding-PH domains [27], except for the substitution of (K/R) residue by Gln (Q306) in Arap3-PH1. Such a Gln residue substitution at this position can also been found in a few PIPs-binding PH domains, such as the PH domain of P-Rex1 [28]. The sequence analysis led us to ask whether the Arap3-PH1 alone is sufficient to bind PI (3,4,5)P3 and/or other PIPs.
Together, in contrast to a previous study [23], our data clearly demonstrated that Arap3-PH1 alone is sufficient to bind PIPs and it prefers PI(3,4,5)P3 for binding, which is consistent with the reported in-cell data that full-length Arap3 is a PI(3,4,5)P3 binding protein [11].

Crystal Structures of Unliganded and diC4-PI(3,4,5)P3-Bound Arap3-PH1 Domain
To understand how Arap3-PH1 domain recognizes PI(3,4,5)P3, we determined the crystal structures of unliganded and diC4-PI(3,4,5)P3-bound Arap3-PH1 domain, which refined to 2.1 and 3.3Å resolution, respectively (Table 1). Arap3-PH1 domain adopts a canonical PH domain fold, which consists of a seven-stranded β-barrel that is capped at one end by a long C-terminal α-helix ( Figure 2A). The other end of the β-barrel is open and features three loops (β1/β2, β3/β4 and β6/β7), which are hypervariable in both length and sequence in presently known PH domain structures. The overall structures of Arap3-PH1 in free and in complex are almost identical, with an overall Ca RMSD of 0.85 Å. However, a significant structural change upon diC4-PI(3,4,5)P3 binding is observed in the region of β1/β2 loop (S298-V304), which exhibits an outward movement, with a Ca RMSD of 1.52 Å ( Figure S1).   In the complex, the diC4-PI(3,4,5)P3 binds within a highly positively charged pocket on the open end of the β-barrel, which corresponds to the canonical phosphoinositide-binding site ( Figure 2B). The 1-phosphate group of diC4-PI(3,4,5)P3 forms hydrogen bonds with the side chains of S298 in β1/β2 loop and Q306 in β2 ( Figure 2C). The 3-phosphate group interacts with the side chains of K296 in β1, R308 in β2 and K329 in β4. The 4-phosphate group forms a hydrogen bond with the side chains of Y319 in β3. It also interacts with R355 in β6/β7 loop. It is interesting to note that the 5-phosphate is orientated toward the solvent and does not make hydrogen bonds with the protein.

NMR Characterize of Arap3-PH1 Domain Binding to PI(3,4,5)P3 and PI(4,5)P2 Head Groups in Solution
To further examine the interaction of the Arap3-PH1 domain with PIPs head groups in solution, we subsequently performed NMR experiments with diC4-PI(3,4,5)P3 and diC4-PI(4,5)P2. Assignment of the Arap3-PH1 signals was made by performing a series of triple resonance NMR experiments using 15 N, 13 C-labeled Arap3-PH1. In total, 80 backbone 1 H, 13 C, and 15 N NMR assignments out of 97 non-proline residues were obtained unambiguously ( Figure S3). Some resonance signals of the backbone amide groups could not be observed in 1 H-15 N HSQC spectra, such as the β6/β7 loop residues.

The PIPs-Binding Ability of Arap3-PH1 Domain Is Required for Arap3 to Inhibit Breast Cancer Cell Invasion In Vitro
Previous study showed that Arap3 can inhibit peritoneal dissemination of scirrhous gastric carcinoma cells [18], and it is also involved in breast cancer [19,20]. We surveyed the COSMIC cancer somatic mutation database, and found that a mutation (R308H) which occurred in Arap3 fell into the PH1 domain. Our structural study shows that R308 is a key residue that directly interacts with the head group of PI(3,4,5)P3. The mutation of this residue will interfere with the ability of Arap3-PH1 to bind PI(3,4,5)P3. We then expressed and purified the Arap3-PH1 R308H mutant, and tested its PI(3,4,5)P3-binding ability by liposome pull-down and NMR titration experiments. As expected, the introduction of the R308H mutation in the Arap3-PH1 domain completely abolishes its binding to PI(3,4,5)P3 ( Figures 4A and S4). The result is in agreement with previous observations that a double point mutation, R307A/R308A in the first PH domain, totally abolished full-length Arap3 binding to PI(3,4,5)P3 [11]. However, our crystal complex structure shows that R307 is not involved in binding PI(3,4,5)P3 and mutation of this residue may be not necessary.
Arap3 can function as a tumor metastasis suppressor and this function is dependent on its Arf and Rho-GAP activities [18,19], since the plasma membrane recruitment mediated by PI(3,4,5)P3 binding is essential for Arap3 to interact with its substrates Arf and RhoA [12]. We then test whether the PIPs binding ability of the PH1 domain is required for Arap3 to inhibit cancer cell invasion. To explore it, we performed transwell migration assay using the MDA-MB-231 human breast cancer cells. Cells were transfected with GFP-tagged fulllength wild-type Arap3, Arap3 R308H mutant or the GFP control plasmids ( Figure S5). The data show that overexpression of wild-type Arap3 can inhibit the invasion and migration of MDA-MB-231 cells compared with the control. The R308H mutation significantly reduced its inhibitory effect on cell invasion (Figure 4). These results suggest that the lipid-binding ability of PH1 domain is required for Arap3 to inhibit breast cancer cell invasion in vitro.
It should be noted that our result is in controversy with a previous study reporting that Arap3-PH1 domain alone is unable to bind PIPs [23]. The possible reason for such a discrepancy may due to the different PH1 domain boundaries we used. In the previous
It should be noted that our result is in controversy with a previous study reporting that Arap3-PH1 domain alone is unable to bind PIPs [23]. The possible reason for such a discrepancy may due to the different PH1 domain boundaries we used. In the previous study, the PH1 domain was expressed from residue L290 to R394 in Cos7 cell lysis. However, our structure data suggested that residues preceding L290, including P288 and L289, interact with the PH1 domain core and may contribute to the structural integrity and stability of the protein ( Figure S6). We, therefore, guess that the Arap3-PH1 construct starting from L290 may not fold correctly; however, this should be further studied.
In cells, the recognition of Arap3-PH1 with PIPs takes place at PM, which mediates the PM recruitment of Arap3 [11,12]. Recent studies suggested that clustering of PIP molecules as well as the lipid composition play key roles in the interactions of a PH domain to a membrane [30][31][32][33][34][35]. How Arap3-PH1 interacts with PIPs-containing membrane should be further investigated, using model membrane systems such as lipid nanodiscs [36][37][38][39]. Moreover, given that Arap3 is a large multi-domain protein, the PIPs-binding ability of its PH1 domain may be regulated by other domains or regions of the protein, which is largely unknown and should also be further investigated.

Plasmid Constructions
The DNA fragments encoding Arap3-PH1 domain (residues 284-385) were cloned into a pET22b (Novagen, Darmstadt, Germany) vector with a C-terminal 6 × His-tag. The expression plasmid encoding the full-length Arap3 with GFP-tagged was constructed as previously described [40]. Mutants were generated by PCR mediated site-directed mutagenesis. All the constructs were verified by DNA sequencing.

Protein Expression and Purification
All the Arap3-PH1 domain constructs with 6×His-tag were transformed into Escherichia coli BL21 (DE3) cells (Novagen). Cells were cultured in LB medium at 37 • C up to an OD 600 of 0.8-1.0, and then induced with 0.5 mM IPTG at 16 • C for 24 h. For NMR study, the isotopically labeled Arap3-PH1 proteins were expressed in cells grown in M9 medium containing 15 N-NH 4 Cl (0.5 g/L) and/or 13 C-glucose (4 g/L) as the sole nitrogen source and/or carbon source. The 15 N-NH 4 Cl and 13 C-glucose were purchased from Cambridge Isotope Laboratories, Inc. All His-tagged proteins were purified by nickel-nitrilotriacetic acid affinity chromatography after lysing cells by sonication in 20 mM Tris/HCl (pH 8.0), 0.5 M NaCl. The Arap3-PH1 proteins were eluted in a 20 mM Tris/HCl (pH 8.0), 0.5 M NaCl with 500 mM imidazole. All proteins were further purified using a Superdex 75 column (GE Healthcare, Piscataway, NJ, USA) in a buffer of 20 mM Tris/HCl (pH 7.5), 200 mM NaCl, 0.5 mM EDTA, 5 mM β-ME. The purity of proteins was confirmed by SDS-PAGE. Protein concentrations were estimated with absorbance spectroscopy, using the molar absorption coefficient.

Liposome Preparation and Liposome Pull-Down Assay
PC (phosphocholine), PI (phosphatidylinositol), PI(4,5)P2 (Phosphatidylinositol 4,5-Bisphosphate) and PI(3,4,5)P3 (Phosphatidylinositol 3,4,5-Trisphosphate) were purchased from Avanti Polar Lipids, Inc. The liposomes containing 2% PI/PI(4,5)P2/PI(3,4,5)P3, 98% PC and 1% PI (3,4,5)P3, 99% PC were prepared for the binding assay. All of the lipids used were dissolved in chloroform and mixed at the molar ratios as described above in glass tubes. The chloroform was dried using nitrogen gas to form a thin film and then further dried under vacuum over 6 h. The dry thin film was hydrated at room temperature in a buffer containing 20 mM Tris/HCl (pH 7.5), and 100 mM NaCl. The suspension was subjected to freeze-thaw cycles in liquid nitrogen and a room temperature water bath for nine cycles, and then sized by an extruder (Avanti Polar Lipids, Birmingham, AL, USA) using a 100 nm polycarbonate filter [41].
Before the experiments, the Arap3-PH1 domain proteins and mutants were dissolved in the same buffer [20 mM Tris/HCl (pH 7.5), 100 mM NaCl] and ultracentrifuged at 70,000× g/min for 30 min to remove the precipitate. For the liposome pull-down assay, 20 µg proteins were incubated with of 640 µg liposomes at 4 • C for 30 min, with a total volume of 100 µL. Then, the liposomes were pelleted by centrifugation at 70,000× g/min in a Beckman Optima MAX-XP ultracentrifuge for 30 min at 4 • C. The supernatant was removed to determine the free proteins, and the pellets were washed twice with the buffer and then dissolved in 100 µL of SDS/PAGE loading buffer. The supernatant and pellet fractions were subjected to SDS/PAGE and stained by Coomassie Blue.

Surface Plasmon Resonance (SPR)
SPR measurements were conducted in a buffer containing 20 mM HEPES/NaOH (pH 6.8), 100 mM NaCl using a Biacore T200 Instrument (GE Healthcare). A total of 10 µg/mL Arap3-PH1 domain proteins were captured onto the surface of sensor chip CM5 (GE Healthcare) until the response unit (RU) value increased to ∼2500. DiC4-PI(3,4,5)P3 (Echelon, Salt Lake City, UT, USA) were diluted from 75 µM to 1.172 µM as 1:2 dilution series and diC4-PI(4,5)P2 (Echelon, USA) were diluted from 600 µM to 4.688 µM. They were consecutively injected onto the captured Arap3-PH1 proteins. Washing of the flow system using the 20 mM HEPES/NaOH (pH 6.8), 100 mM NaCl for 180 s was then performed following each run. Binding data were analyzed and fitted using a one-site binding model.

Crystallization and Structure
Arap3-PH1 was concentrated to~9 mg/mL in the buffer of 10 mM Tris/HCl (pH 7.2), 100 mM NaCl, 0.5 mM EDTA and the diC4-PI (3,4,5)P3 was dissolved in the same buffer. Crystals of apo-form Arap3-PH1 domain were crystallized using the sitting drop vapor diffusion method with a one-to-one admixture of the protein and a well solution consisting of 0.05 M calcium chloride dihydrate, 0.1 M BIS-TRIS (pH 6.5), 30% v/v polyethylene glycol monomethyl ether 550. Arap3-PH1 was mixed with diC4-PI(3,4,5)P3 at a 1:1.2 molar ratio and crystallized using sitting drop vapor diffusion method at 18 • C by mixing the complex with equilibration solution (0.1 M ammonium sulfate, 0.1 M sodium acetate trihydrate (pH 4.6), 30% v/v polyethylene glycol 400) in a one-to-one ratio. All the crystals were transferred to cryoprotectant (dehydration treatment in reservoir solutions containing 15% glycerol) and flash-cooled with liquid nitrogen.

Data Collection and Structure Determination
X-ray diffraction data were collected at 100 K in a liquid nitrogen stream using beamline BL18U at the Shanghai Synchrotron Radiation Facility (SSRF). The data were processed with HKL2000 [42] and programs in the CCP4 suite [43]. The structure of the Arap3-PH1 domain (PDB code: 7YIR) and Arap3-PH1 complexed with diC4-PI(3,4,5)P3 (PDB code: 7YIS) were solved by molecular replacement with co-ordinates from PDB entry 1UPR/1FAO and the program PHASER [44]. All the structural models were subsequently refined by programs REFMAC5 [45], PHENIX [46], and COOT [47]. Crystallographic parameters are listed in Table 1. All structure figures were prepared with PyMOL. The transition state complex interface was calculated in PDBePISA.

Western Blotting and Antibodies
For immunoblotting analyses, MDA-MB-231 cells were lysed in RIPA lysis buffer supplemented with protease and phosphatase inhibitors (Roche Applied Science, Manheim, Germany). Immunoblotting was performed as described previously [40]. Primary antibodies used were anti-GFP tag mAb (Cell Signaling, Danvers, MA, USA), anti-β actin mAb (Cell Signaling, Danvers, MA, USA).

Matrigel Invasion
Before the experiments, MDA-MB-231 cells were transfected with Arap3 WT , Arap3 R308H and empty control, respectively. The expression levels of proteins were verified by immunoblotting. Matrigel invasion assay was performed with Transwell membrane filter inserts (8 mm pore size, Corning costar) by using EGF (10 ng/mL, Sigma-Aldrich, Saint-Louis, MO, USA), as described [49]. Forty-eight hours after transfection, 1 × 10 5 cells were seeded on the upper wells. After incubation for 12 h, cells were fixed in 4% paraformaldehyde, and the number of cells that migrated out to the lower surface of the membranes was scored by staining with 1% crystal violet. Data were collected from three independent experiments.

Conclusions
In the present work, we have clarified the phosphoinositide recognition mechanism of Arap3 by biochemical and structural methods. Our data showed that the Arap3-PH1 domain is capable of binding PI(3,4,5)P3 and also PI(4,5)P2, albeit with lower affinity. Crystal structure and NMR titration analysis revealed the structural basis for the specific phosphoinositide recognition by Arap3-PH1. Furthermore, cell-based function analysis using a cancer-associated mutant R308H demonstrated that the lipid-binding ability of the PH1 domain is required for Arap3 to inhibit cancer cell invasion in vitro.