Structure determination of SH2–phosphopeptide complexes by X-ray crystallography - the example of p120RasGAP

Abstract

The p120RasGAP protein contains two Src Homology 2 (SH2) domains, each with phosphotyrosine binding activity. We describe the crystallization of the isolated and purified p120RasGAP SH2 domains with phosphopeptides derived from a binding partner protein, p190RhoGAP. Purified recombinant SH2 domain protein is mixed with synthetic phosphopeptide at a stoichiometric ratio to form the complex in vitro. Crystallization is then achieved by the hanging drop vapor diffusion method over specific reservoir solutions that yield single macromolecular co-crystals containing SH2 domain protein and phosphopeptide. This protocol yields suitable crystals for X-ray diffraction studies, and our recent X-ray crystallography studies of the two SH2 domains of p120RasGAP demonstrate that the N-terminal SH2 domain binds phosphopeptide in a canonical interaction. In contrast, the C-terminal SH2 domain binds phosphopeptide via a unique atypical binding mode. The crystallographic studies for p120RasGAP illustrate that although the three-dimensional structure of SH2 domains and the molecular details of their binding to phosphotyrosine peptides are well defined, careful structural analysis can continue to yield new molecular-level insights.

1. Introduction

The Src homology 2 (SH2) domain is one of the best studied and well characterized modular protein signaling domains [1]. The SH2 domain was first identified as a conserved non-catalytic region in Src and other nonreceptor tyrosine kinases [2] and is well represented in the human genome, with approximately 120 SH2 domains in 111 human proteins [3]. The central function of the SH2 domain is to bind phosphotyrosine residues in target proteins, thereby regulating both recruitment and enzymatic activity in tyrosine kinase signaling cascades.

The three-dimensional structure of the SH2 domain is well described through a combination of many structural determinations by both X-ray crystallography and NMR spectroscopy [4]. The SH2 domain is approximately 100 residues in length and adopts a conserved overall fold consisting of a central beta sheet flanked by two alpha helices [5,4,6]. The phosphotyrosine peptide binds in a two-pronged manner to two binding pockets on either side of the central beta sheet: the phosphotyrosine site and the specificity site [7,8]. In canonical SH2 domains, the positively charged phosphotyrosine site contains a highly conserved arginine residue in a FLVR sequence (Phe-Leu-Val-Arg) that directly binds the phosphotyrosine and contributes a majority of the binding free energy [9]. The specificity pocket binds the peptide residue at the +3 position to the phosphotyorosine (defined as the 0 position), and the characteristics of the residues lining this pocket establish the binding specificity.

Co-crystal structures of isolated SH2 domains with phosphopeptide helped establish the two-pronged binding mode and specificity determinants [4–6]. Typically, studies utilize synthetic phosphopeptides derived from target protein sequences of approximately 4–10 residues in length, beginning at residues −3 to −1 from the 0 position phosphotyrosine and extending to the +3, or as long as the +5 position. Dissociation constant (K_d) values range from 0.1 to 10 μM [10]. Thus, the achievable in vitro concentrations of recombinantly expressed and purified SH2 domain protein (0.1 mM or higher) and phosphopeptide (~1 mM or higher) are amenable to complex formation and co-crystallization. Additionally, recombinant isolated SH2 domain proteins can often be easily overexpressed in bacterial systems and purified to high quality necessary for crystallization trials.

One of the earliest identified SH2 domain-containing proteins is p120RasGAP (RASA1). p120RasGAP was the first GTPase activating protein (GAP) identified for the Ras small GTPases and the only one to contain Src homology domains, namely two SH2 domains and one SH3 domain [11–13] (Figure 1A). Additionally, p120RasGAP contains plekstrin homology (PH) and C2 domains which may aid membrane binding, and a C-terminal catalytic GAP domain (Figure 1A). p120RasGAP enhances the intrinsic GTP hydrolysis activity of Ras GTPases by two to five orders of magnitude and promotes the GDP-bound form of Ras to turn off signaling [11,14]. This is achieved by p120RasGAP binding directly to Ras-GTP and contributing a so-called ‘arginine finger’ residue to the active site to stabilize the reaction intermediate and promote catalysis. Thus, p120RasGAP is an essential factor in the Ras GTPase cycle and a key regulator of Ras signaling.

Figure 1. Domain architecture of p120RasGAP and p190RhoGAP.

a) p120RasGAP (UniProt ID: P20936) contains a predicted unstructured N-terminus, N-terminal Src homology 2 (N-SH2) domain, Src homology 3 (SH3) domain, C-terminal Src homology 2 (C-SH2) domain, pleckstrin homology (PH) domain, protein kinase C conserved 2 (C2) domain, and Ras GTPase activating protein (GAP) domain. The isolated N-SH2 (residues 174–280) and C-SH2 (340–444) domains are used for co-crystallization experiments with p190RhoGAP phosphopeptides. b) p190RhoGAP (UniProt ID: Q9NRY4) contains: N-pseudoGTPase, FF, pseudoGTPase, RhoGAP domains.

Phosphotyrosine-containing binding partners for p120RasGAP SH2 domains have been identified. They include the Rho GTPase activating protein p190RhoGAP [15,16], the adaptor proteins p62Dok, p65Dok [17,18] and SH2 domain-containing adapter protein B (SHB) [19], and receptor tyrosine kinases including the platelet derived growth factor (PDGF) receptor [20,21] and the ephrin type B (EphB) receptors [22]. Several binding partners contain multiple sites of tyrosine phosphorylation, and simultaneous engagement of the two SH2 domains to the same protein target has been postulated [23].

p190RhoGAP is a dual tyrosine phosphorylated GAP protein for the Rho subfamily of GTPases and helps regulate the actin cytoskeleton. Like p120RasGAP, p190RhoGAP is a multidomain protein that, in addition to its catalytic GAP domain, contains other modular protein domains which may play a role in its recruitment and/or activation [24,25] (Figure 1B). Phosphorylation of tyrosine residues in the unstructured ‘middle domain’ (Figure 1B) by kinases such as Src and Abl has been shown to promote interaction with the SH2 domains of p120RasGAP [16,26–28]. As p120RasGAP is believed to be membrane-associated, formation of this complex leads to recruitment of p190RhoGAP to the membrane where it is in closer proximity to Rho GTPases and can regulate Rho signaling [29]. Therefore, understanding the molecular details of the interaction between p120RasGAP SH2 domains with p190RhoGAP phosphotyrosines have helped establish the structural requirements for this important signaling complex.

The three dimensional structures of the individual p120RasGAP SH2 domains have been determined, both alone and in complex with phosphotyrosine peptides derived from the phosphotyrosine sites of p190RhoGAP [30,31] (Figures 2a, 2b, 2c and 2d). These studies revealed that both domains adopt the canonical SH2 domain fold, comprised of a central beta sheet flanked by two alpha helices (Figures 2a and 2b). The phosphotyrosine and specificity sites are located on either side of the long central beta strand bD (using canonical SH2 domain secondary structure nomenclature), and together create the two-pronged binding mode for phosphotyrosine containing peptides. In detail, the crystal stuctures reveal a canonical binding mode for the N-SH2 in complex with a pTyr-1105 peptide in which the FLVR arginine, Arg-188, directly binds the phosphotyrosine, while the specificity pocket binds the preferred +3 Pro-1108 (Figure 2c and 2e). In contrast, the structure of the C-SH2 in complex with a pTyr-1087 peptide determined by X-ray crystallography revealed atypical binding interactions, in which the FLVR arginine, Arg-377, does not directly bind the phosphotyrosine, but rather is engaged with an intramolecular interaction with Asp-379 (Figure 2d and 2f). Nonetheless, the position of the phosphotyrosine binding pocket is conserved, and is instead lined with alternative residues including Arg-398 and Lys-400 to directly bind the phosphotyrosine (Figure 2f). Similar to N-SH2, the specificity pocket of C-SH2 reveals the structural basis for specificity for Pro-1090 at +3. Taken together, these structural studies reflect the importance of experimentally observing molecular level interactions between SH2 domains and their target peptides. In this paper, we detail the procedure for co-crystallization of these protein–peptide complexes by hanging drop vapor diffusion. The general procedures for co-crystallization can be applied to other SH2–phosphopeptide pairs.

Figure 2. Ribbon diagram of crystal structures of p120RasGAP SH2 domains.

a) N-SH2 apo crystal structure (PDB ID: 6PXB [31]). b) Crystal structure of apo C-SH2 domain (PDB ID: 6WAX [30]). c) Co-crystal structure of N-SH2 bound to p190RhoGAP pY1105-containing phosphopeptide (PDB ID: 6PXC [31]). d) Co-crystal structure of C-SH2 bound to p190RhoGAP pY1087-containing phosphopeptide (PDB ID: 6WAY [30]). In all panels, secondary structure elements are labeled. In b) and d), the phosphopeptides are drawn as all atom cylinders and transparent surfaces in yellow, and the amino- (‘N’) and carboxy-termini (‘C’) of the peptides are labeled. The positions of the phosphotyrosine and +3 residues are labeled and shown with arrows. e) The FLVR arginine Arg-207 of N-SH2 bound directly to pY-1105. f) FLVR-unique binding of pY-1087 to C-SH2. The FLVR arginine R377 is bound to D379; the phosphotyrosine is bound by R398 and K400 in the phosphotyrosine binding site. All Illustrations are rendered in CCP4mg [34].

2. Materials

2.1. SH2 domain proteins

1. Purified recombinant p120RasGAP N-SH2 domain and C-SH2 domain proteins.

2. Protein Storage buffer: 20 mM Tris HCl pH 8.0, 150 mM NaCl.

3. Centrifugal filters with nominal molecular weight limit (NMWL) 3 kilodalton. We use Amicon Ultra-4 Centrifugal Filters (Sigma Millipore).

4. Refrigerated centrifuge that fits 15 ml conical tube equivalent.

5. Spectrophotometer to measure protein concentration. We use Nanodrop (Thermofisher).

6. 18% SDS PAGE gel, Tris Glycine running buffer.

7. Coomassie Blue staining solution: 1 g Coomassie brilliant blue, 40% ethanol, 10% acetic acid.

8. Refrigerated microcentrifuge.

9. Microcentrifuge tubes, 1.5 – 1.7 ml capacity.

2.2. Phosphopeptide

1. Lyophilized phosphopeptides, commercially synthesized and HPLC purified to greater than 98%. The peptides are modified at the amino and carboxy termini with acetyl and amide groups, respectively, to neutralize charge and improve stability. In this example we use pTyr-1105: EEENI(pY)SVPHDST (p190RhoGAP residues 1100–1112, UniProt Q9NRY4); and pTyr-1087: DpYAEPMD (p190RhoGAP residues 1086–1092, UniProt Q9NRY4).

2. Peptide Reconstitution buffer: 10 mM Tris pH 7.4.

2.3. Crystallization

1. Crystallization plate with 18 mm well diameter. We use VDXm^™ Crystallization Plate with sealant

2. Plastic coverslips, 18 mm square or circle.

3. Tweezers.

4. 70% ethanol and lint-free labwipe or lens paper.

5. Reservoir stock solutions for apo N-SH2 crystals.

   • 5.1
     50% (w/v) polyethylene glycol (PEG) 10,000
   • 5.2
     1 M ammonium acetate
   • 5.3
     1 M Tris pH 8.0, pH adjusted with HCl
6. Reservoir stock solutions for N-SH2–pY-1105 co-crystals.

   • 6.1
     1 M Bis-Tris pH 6.5, pH adjusted with HCl
   • 6.2
     100% Polyethylene glycol monomethyl ether 550 (PEG MME 550)
7. Reservoir stock solutions for apo C-SH2 crystals.

   • 7.1
     4 M ammonium sulfate
   • 7.2
     1 M Bis-Tris pH 6.5, pH adjusted with HCl
8. Reservoir stock solutions for C-SH2–pY-1087 co-crystals.

   • 8.1
     1.6 M sodium citrate tribasic dihydrate
   • 8.2
     1 M Tris pH 8.5, pH adjusted with HCl

9. Stereo light microscope for viewing crystallization drops.

3. Methods

3.1. Prepare purified SH2 domain protein for crystallization.

1. Purify recombinant SH2 domain protein using optimal protocols for your SH2 domain; for example, purification procedures for the SH2 domains of p120RasGAP are established [30,31]. See Note 1.

2. Buffer exchange approximately 2 mg protein into Protein Storage buffer in the centrifugal filter device. See Notes 2 and 3.

3. Using a pipet, transfer 2 mg of purified protein to the filter device. Bring final volume up to 4 ml with Protein Storage buffer.

4. Centrifuge in centrifugal filter device at 4°C 7,500 × g maximum speed in 10 minute intervals until the protein sample volume is less than 0.5 ml.

5. Remove the filtrate from the bottom tube and set aside.

6. Add up to 4 ml Protein Storage buffer to the protein solution in the sample reservoir. Resuspend solution carefully.

7. Repeat steps 2–4 two additional times, for a total of three buffer exchanges.

8. Centrifuge in centrifugal filter device until volume reaches ~ 100 μl.

9. Using a micropipettor, transfer volume from filter device to fresh microcentrifuge tube.

10. Determine protein concentration: measure Absorbance at 280 nm (A₂₈₀) on the spectrophotometer. Zero the instrument using Protein Storage buffer.

11. From A₂₈₀ values, calculate the concentration using the extinction coefficient and the molecular weight values from Table 1. 2 mg of protein in 100 μl should be approximately 20 mg/ml, or 1.6 mM.

12. Aliquot protein to 20–50 μl in microcentrifuge tubes. Flash freeze in liquid nitrogen and store at −80°C. See Note 4.

13. Assess protein purity by SDS-PAGE chromatography and Coomassie staining. Purity should be > 98%.

Table 1.

p120RasGAP SH2 domain parameters.

	Molecular weight (Da)	Extinction coefficient (M−1 cm−1) at 280 nm	Residue #s	Length (# amino acids)
N-SH2	12251	15930	174–280	109
C-SH2	12453	17420	340–444	107

3.2. Preparation of Phosphopeptides.

1. Reconstitute lyophilized phosphopeptides to maximum solubility in Peptide Reconstitution buffer in a microcentrifuge for final concentrations as shown in Table 2. For pY-1087 peptide, we add 500 μl of Peptide Reconstitution buffer to 5 mg lyophilized powder, to obtain 10 mg/ml (9.7 mM). For pY-1105 phosphopeptide, we add 1000 μl of peptide Reconstitution buffer to 5 mg lyophilized powder, to obtain 5 mg/ml (3.0 mM). See Notes 5, 6 and 7.

2. Centrifuge solution for 15 minutes at 4°C at 20,000 RCF to pellet any remaining particulates or aggregates. Aliquot supernatant to microcentrifuge tubes in 50 μl. Flash freeze in liquid nitrogen. Store at −80°C.

Table 2.

p190RhoGAP phosphopeptide parameters.

Peptide	Residue #s	Sequence	Length (# amino acids)	Molecular weight (g/mol)	Maximum Solubility (mg/ml)	Final stock concentration (mM)
pY-1087	1086 – 1093	D(pY)AEPMDA	8	1032	10	9.7
pY-1105	1100 – 1112	EEENI(pY)SVPHDST	13	1641	5	3.0

3.3. Formation of complex between SH2 domain and phosphopeptide.

1. Calculate the volume of protein and phosphopeptide required to achieve a 1:1 molar ratio in solution. For example: 85 μl of 1.6 mM SH2 domain protein plus 15 μl of 9.7 mM phosphopeptide equals 1.4 mM each component in 100 μl. See Notes 8 and 9.

2. Mix the components on ice. Incubate for 1 hour.

3. Centrifuge the sample to pellet particulates and/or aggregates prior to setting crystallization drops.

3.4. Crystallization using hanging drop vapor diffusion method.

1. Calculate a series of reservoir solutions according to the crystallization conditions specified for each sample type in Table 3 (example in Table 4). 500 μl reservoir solution is needed per single well of the 18 mm diameter crystallization plate. See Notes 10, 11 and 12.

2. Mix reservoir components thoroughly in separate tubes.

3. Pipet 500 μl of reservoir solution to a single well of a pre-greased 24-well crystallization plate (for example, VDX plate).

4. Prepare a single coverslip by cleaning with 70% ethanol and drying with lint-free labwipe or lens paper. See Note 13.

5. Pipet 1 μl of protein/peptide mixture onto the center of a coverslip.

6. Pipet 1 μl of reservoir solution from the well and add to the protein/peptide drop on the coverslip. See Notes 14, 15, 16.

7. Handle the coverslip by its edges using tweezers or your fingers and carefully invert, so that the drop is hanging.

8. Center coverslip over greased reservoir, place down gently, then press along the edge of the coverslip to seal.

9. Repeat steps 3–8 with additional reservoir positions.

10. Incubate crystallization plate at ambient (20°C) temperature for several days. Monitor crystal growth by viewing under stereo light microscope daily. See Note 17.

11. These crystals are suitable for X-ray diffraction data collection.

Table 3.

Crystallization conditions.

SH2 domain	Phosphopeptide	Precipitant		Additive	Buffer (0.1 M)
		Precipitant	Concentration Range*		reagent	pH**
N-SH2	-	PEG 10,000	18 – 23 % (w/v)	0.2 M ammonium acetate	Tris	8.0
C-SH2	-	Ammonium sulfate	1.9 – 2.4 M	-	Bis-Tris	6.5
N-SH2	pY-1105	Sodium malonate	1.4 – 1.9 M	2% PEG MME 550	Bis-Tris	6.5
C-SH2	pY-1087	Sodium citrate tribasic	0.8 – 1.3 M	-	Tris	8.5

^*Vary this reagent stepwise. For example, 18 – 19 – 20 – 21 – 22 – 23 % PEG 10,000, or 1.9 – 2.0 – 2.1 – 2.2 – 2.3 – 2.4 – M Ammonium sulfate.

^**Tris and Bis-Tris buffers are pH adjusted with HCl.

Table 4.

Example optimization screen for crystallization of N-SH2 with pY-1105 peptide.

1	2	3	4	5	6
1.4 M Sodium malonate 2 % PEG MME 550 0.1 M Bis-Tris pH 6.5	1.5 M Sodium malonate 2 % PEG MME 550 0.1 M Bis-Tris pH 6.5	1.6 M Sodium malonate 2 % PEG MME 550 0.1 M Bis-Tris pH 6.5	1.7 M Sodium malonate 2 % PEG MME 550 0.1 M Bis-Tris pH 6.5	1.8 M Sodium malonate 2 % PEG MME 550 0.1 M Bis-Tris pH 6.5	1.9 M Sodium malonate 2 % PEG MME 550 0.1 M Bis-Tris pH 6.5

4. Notes

1. p120RasGAP SH2 domain expression constructs include substitutions at codons for native cysteine residues to serine, to prevent disulfide-mediated dimerization or heteromerization of purified proteins in solution which can introduce heterogeneity and interfere with crystallization. These substitution sites are: C236S and C261S in N-SH2, and C372S and C402S in C-SH2.

2. Amicon-4 centrifuge filters with nominal molecular weight limit (NMWL) of 3 kDa are appropriate for SH2 domain protein which has a molecular weight of approximately 10–12 kDa.

3. Alternative to using a centrifugal filter device, buffer exchange may be performed by size exclusion chromatography or dialysis.

4. Avoid repeated freeze-thaw cycles of protein and peptide.

5. Phosphopeptides have variable solubility in aqueous buffers, and solubility may be limited. Solubility testing in various reconstitution solutions may be offered by the commercial peptide vendor. Phosphopeptide reconstitution may be achieved in aqueous solutions including water or Peptide Reconstitution buffer. Alternatively, the peptide may be initially dissolved in a small amount of DMSO, ammonium hydroxide or acetic acid, followed by dilution in water or Peptide Reconstitution buffer.

6. Commercially synthesized peptides may be provided in aliquots of lyophilized powder in pre-measured mass (e.g. 5 mg per vial) to simplify the reconstitution process.

7. Phosphopeptide concentration can be verified by measuring A₂₈₀ on the spectrophotometer blanked with Peptide Reconstitution buffer. Use the extinction coefficient difference for phosphotyrosine versus tyrosine as reported in [32]. Alternatively, phosphopeptide concentration can be quantified by amino acid analysis [33].

8. The protein:peptide molar raio can be varied to account for errors in protein or phosphopeptide concentration determination and/or systematic errors in pipetting. For example, protein:peptide ratios of 1:1.5 or 1:0.8 maybe be trialed.

9. A phosphopeptide titration into SH2 domain may be monitored by native PAGE electrophoresis, isothermal titration calorimetry, etc., to experimentally establish the condition under which a stoichiometric complex is formed.

10. All solutions are prepared with deionized or doubly-distilled water and sterile filtered through a 0.22 μm membrane. Store stock solutions at 4°C.

11. Stock crystallization solutions can be made from powder, or can be purchased as pre-made formulations directly from multiple suppliers.

12. Crystallization conditions are identified by screening using commercially available crystallization kits. After initial hits are identified, the conditions are further optimized by testing the effects of altering the reservoir solution by changing precipitant concentration, buffer pH values, etc. Further optimization can include changing protein concentration and adjusting the SH2:phosphopeptide molar ratio.

13. Plastic or siliconized glass coverslips can be used.

14. Be careful not to introduce bubbles into crystallization drop when pipetting protein:peptide or reservoir solutions.

15. Mixing of drop is not necessary.

16. Total drop volume can vary from 1 μl to 10 μl.

17. Crystals should appear within the first four days, but may take longer to grow to full size, up to two weeks.