Studies of the molecular mechanism of CagA mediated . · PDF file1 Studies of the molecular...

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1 Studies of the molecular mechanism of CagA mediated virulence. Nam Ky Tonthat Maria Anne Schumacher, Ph.D. A. Abstract: The bacterium Helicobacter pylori is the etiological agent of gastritis and peptic ulcer. Infection with H. pylori also poses a strong risk factor for the development of gastric adenocarcinoma 1 . In particular, H. pylori strains carrying the cytotoxin-associated antigen A (cagA) gene have been shown to be more virulent than those that do not 5 . In fact, it has been demonstrated that transgenic expression of CagA can induce gastrointestinal neoplasm in mice 6 . The cagA gene encodes a 120-145 kDa CagA protein that is delivered into host gastric epithelial cells 7, 8 . Once inside the host cell, CagA localizes to the inner surface of the plasma membrane and is phosphorylated by members of the Src family kinases 3, 9, 10 . The phosphorylation of CagA occurs on tyrosines within EPIYA motifs in the CagA C-terminal domain. Because of homologous recombination that occurs in the cagA gene, different CagA proteins are formed that differ in their EPIYA site. As a result four distinct EPIYA sites with varying sequence, called A, B, C, and D, are found. CagA proteins from regions such as Europe, North America, and Australia, contain an A and B site followed by up to three C sites and are thus termed the A-B-C-type CagA or Western CagA. H. pylori strains from regions of Japan, Korea, and China contains an A, a B and a D site and are called East-Asian CagA or A-B-D type CagA 3, 11 . Phosphorylation at the C or D site of CagA allows the SHP-2 tyrosine phosphatase of the host cell to bind. The crystal structure of SHP-2 shows that it contains two SH2 domains and a C-terminal phosphatase domain. It is thought that binding of CagA to the SH2 domains causes a conformational change leading to phosphatase activation. The activated form of SHP-2 can then dephosphorylate focal adhesion kinase (FAK), resulting in a morphological change to the cell termed the “hummingbird” phenotype 12 . Additionally, activated SHP-2 is able to positively regulate Erk signaling through Ras 13 . In summary, studies of cagA-positive and cagA-negative H. pylori strains have shown that those strains producing CagA are more virulent 5 . CagA acts as a virulence factor in gastric epithelial cells, and one such target of CagA is SHP-2. The interaction of CagA with SHP-2 can disrupt the Ras signal transduction pathways and modify cellular functions 12 . Although the downstream effects of CagA have been well documented, the details of how CagA is able to initiate signal cascades are not known. The goals of this proposal are to determine the molecular mechanisms of the CagA-SHP-2 interaction and elucidate the pathway by which CagA acts to activate Ras/Erk signaling. We hypothesize that SHP-2 is able to bind preferentially to the EPIYA-D site over the EPIYA-C site, and this leads to higher level of SHP-2 activation by East-Asian CagA. Furthermore, we also hypothesize that the CagA-SHP-2 complex can stimulate Erk signaling through the indirect regulation of Ras.

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Studies of the molecular mechanism of CagA mediated virulence. Nam Ky Tonthat

Maria Anne Schumacher, Ph.D. A. Abstract:

The bacterium Helicobacter pylori is the etiological agent of gastritis and peptic ulcer. Infection with H. pylori also poses a strong risk factor for the development of gastric adenocarcinoma1. In particular, H. pylori strains carrying the cytotoxin-associated antigen A (cagA) gene have been shown to be more virulent than those that do not5. In fact, it has been demonstrated that transgenic expression of CagA can induce gastrointestinal neoplasm in mice6. The cagA gene encodes a 120-145 kDa CagA protein that is delivered into host gastric epithelial cells7, 8. Once inside the host cell, CagA localizes to the inner surface of the plasma membrane and is phosphorylated by members of the Src family kinases3, 9, 10. The phosphorylation of CagA occurs on tyrosines within EPIYA motifs in the CagA C-terminal domain. Because of homologous recombination that occurs in the cagA gene, different CagA proteins are formed that differ in their EPIYA site. As a result four distinct EPIYA sites with varying sequence, called A, B, C, and D, are found. CagA proteins from regions such as Europe, North America, and Australia, contain an A and B site followed by up to three C sites and are thus termed the A-B-C-type CagA or Western CagA. H. pylori strains from regions of Japan, Korea, and China contains an A, a B and a D site and are called East-Asian CagA or A-B-D type CagA3, 11.

Phosphorylation at the C or D site of CagA allows the SHP-2 tyrosine phosphatase of the host cell to bind. The crystal structure of SHP-2 shows that it contains two SH2 domains and a C-terminal phosphatase domain. It is thought that binding of CagA to the SH2 domains causes a conformational change leading to phosphatase activation. The activated form of SHP-2 can then dephosphorylate focal adhesion kinase (FAK), resulting in a morphological change to the cell termed the “hummingbird” phenotype12. Additionally, activated SHP-2 is able to positively regulate Erk signaling through Ras13.

In summary, studies of cagA-positive and cagA-negative H. pylori strains have shown that those strains producing CagA are more virulent5. CagA acts as a virulence factor in gastric epithelial cells, and one such target of CagA is SHP-2. The interaction of CagA with SHP-2 can disrupt the Ras signal transduction pathways and modify cellular functions12. Although the downstream effects of CagA have been well documented, the details of how CagA is able to initiate signal cascades are not known. The goals of this proposal are to determine the molecular mechanisms of the CagA-SHP-2 interaction and elucidate the pathway by which CagA acts to activate Ras/Erk signaling. We hypothesize that SHP-2 is able to bind preferentially to the EPIYA-D site over the EPIYA-C site, and this leads to higher level of SHP-2 activation by East-Asian CagA. Furthermore, we also hypothesize that the CagA-SHP-2 complex can stimulate Erk signaling through the indirect regulation of Ras.

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B. Specific Aims: Specific Aim 1: Characterize the CagA-SHP-2 interaction. The major interaction partner of CagA in the cell is SHP-2. Through this interaction, CagA is able to activate SHP-2 which in turn activates the Ras/Erk signaling pathway12. Aberrant activation of the Ras/Erk signaling can promote the secretion of metalloprotease proteins, which disrupts the gastric stroma. Also, the activation of Ras can also lead to the up-regulation of growth promoting and antiapoptotic genes. Additionally, the level of pathogenicity of H. pylori is dependent on the variant of the CagA virulence factor. East-Asian CagA with the EPIYA-D site is more virulent than Western CagA with the EPIYA-C site3. Therefore the interaction between CagA and SHP-2 plays an integral part in mediating H. pylori virulence. However, details regarding the CagA-SHP-2 complex and the difference between the EPIYA-C and D sites are still unknown. We hypothesize that SHP-2 is able to bind preferentially to the EPIYA-D site over the EPIYA-C site, and this leads to higher level of SHP-2 activation by East-Asian CagA. To fulfill this aim, we will use techniques such as analytical ultracentrifugation and calorimetry to determine the stoichiometry and binding affinities of SHP-2 to variants of CagA. In addition, we will also investigate the ability of CagA to activate the phosphatase activity of SHP-2 in vitro. Specific Aim 2: Solve the crystal structures of full length SHP-2 in complex with CagA containing the EPIYA-C and EPIYA-D site. The presence and number of occurrence of the EPIYA-C and D site determines the level of virulence in H. pylori infection4, 12. However, the detailed molecular mechanism of how CagA binds to SHP-2 and activates its phosphatase function is still unknown. We propose to crystallize and determine the three-dimensional structures of SHP-2 in complex with the EPIYA-C and EPIYA-D type CagA. These structures will reveal the molecular details of the SHP-2-EPIYA interaction and how they differ in the SHP-2-EPIYA-C complex versus the SHP-2-EPIYA-D complex. Critically, the structures will also reveal how interactions between CagA EPIYA sites and the SHP-2 SH2 domains affect activation of the C-terminal phosphatase domain. In addition, the structures will show how EPIYA-D is able to more efficiently activate SHP-2. To crystallize these complexes, we will use the hanging drop vapor diffusion method and to solve the crystal structures, we will use X-ray crystallography. Specific Aim 3: Determine the pathway CagA utilizes to activate Ras/Erk signaling. H. pylori protein CagA is delivered into host gastric epithelial cells7, 8, and is phosphorylated by members of the Src family kinases (SFKs)3, 9, 10. This phosphorylated form interacts with and activates SHP-212. Activated SHP-2 then activates Erk signaling through the Ras pathway13, but how SHP-2 mediates Ras/Erk activation is still unclear. We hypothesize that CagA-SHP-2 is able to activate the Erk signaling pathway through the indirect regulation of Ras. This specific aim will establish the interaction partner(s) that allows SHP-2 to activate Ras through the following sub-aims:

1) Identify a potential interaction partner of CagA-SHP-2 (specifically we anticipate to find Gab1/Src/Spry).

2) Establish that the identified protein is an interaction partner of CagA-activated SHP-2. 3) Determine that the identified protein allows CagA-activated SHP-2 to mediate the

downstream signal to activate Ras/Erk signaling. The experimental approach for this aim will use co-IP, Western blot, and RNAi knockdown experiments.

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Figure 1. Diversity in the tyrosine phosphorylation sites of CagA. Tyrosine phosphorylation of cytotoxin-associated antigen A (CagA) by Src kinase occurs at the EPIYA motif. There are four different EPIYA sites, called EPIYA-A, -B, -C and -D, based on the sequence surrounding the EPIYA motif. Western strains of Helicobacter pylori express a form of CagA that contains the EPIYA-A and B sites, followed by 1–3 repeats of the C site (red boxes). East-Asian strains of H. pylori express a form of CagA in which the EPIYA-C site is replaced with the EPIYA-D site (yellow box). Taken from Hatakeyama, Nat. Rev. Cancer. 20043.

C. Background and Significance: CagA, virulence factor of Helicobacter pylori.

Helicobacter pylori are gram-negative bacterium that can inhabit various areas of the duodenum and the stomach. H. pylori can cause a number of gastric diseases, and more recently, epidemiological studies has determined that H. pylori infected population has a much greater risk of developing gastric adenocarcinoma in comparison to uninfected population1. Interestingly, epidemiological studies have clustered H. pylori species into two groups based on the presence of the cagA gene: cagA-positive and cagA-negative strains. The cagA gene codes for a 120-145 kDa protein, which greatly affect the virulence of H. pylori 3.

Although the function of the N-terminal region of CagA is still unknown, it is required for the transport of CagA into a cell14. The carboxyl-terminal region is variable, and it is within this region that the phosphorylation of CagA occurs. Specifically, phosphorylation occurs on the tyrosine residue within the EPIYA sites, which are present at different frequencies and with varying sequences. Because of homologous recombination events that occurred in the cagA gene, different CagA proteins were formed that differ in their EPIYA site. The results are four distinct EPIYA sites, called A, B, C, and D. CagA proteins from regions such as Europe, North America, and Australia, contain an A and a B site followed by up to three C sites and are thus termed the A-B-C-type CagA or Western CagA. H. pylori strains from regions of Japan, Korea, and China contain an A, a B and a D site and are called East-Asian CagA or A-B-D type CagA 3,

11. There are four variants of CagA based on the occurrence of the EPIYA sites: A-B-C, A-B-C-C, A-B-C-C-C (Western CagA), and A-B-D (East-Asian CagA) (Figure 1).

CagA is able to form a higher ordered complex, and this process is mediated by a CagA multimerization motif (CM motif). The CM motif occurs once and is C-terminal to the last EPIYA site; uniquely, the EPIYA-C site incorporates an additional CM motif (Figure 2). Thus in East-Asian CagA, there is only one CM site, while in Western CagA, there can be up to four. This suggests that CagA variants with EPIYA-C sites are able to multimerize more efficiently,

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Figure 2. Structural composition of the EPIYA sites of Western and East-Asian CagA proteins. The N-terminal region of the EPIYA-C segment contains the CagA multimerization (CM) motif (hash box). In addition, both the Western and East-Asian CagA proteins possess an imperfect EPIYA-C-like sequence, which constitutes the CM motif but lacks the EPIYA motif, located immediately downstream of the last repeat of the EPIYA-C or EPIYA-D segment. As a result, a prevalent form of Western CagA (A-B-C-type) carries two CM motifs, whereas a representative form of East-Asian CagA carries a single CM motif. Taken from Ren et al., JBC. 2006 4.

which could translate to higher level of SHP-2 activation. However, the East-Asian CagA, with the EPIYA-D site, has been shown to have a relatively stronger SHP-2 binding affinity than Western CagA, which contains up to three EPIYA-C sites3.

SHP-2, Src homology-2 phosphatase.

SHP-2 is composed of an N-terminal and C-terminal SH2 domain (N-SH2 and C-SH2 respectively), and one phosphatase (PTP) domain (Figure 3A). The crystal structure of SHP-2 revealed a “closed” domain arrangement2. The N-SH2 domain binds to the PTP domain and has extensive interaction with its catalytic domain. In particular, the residues D61 and Y62 of the D’E loop are deeply inserted into the catalytic cleft. These interactions directly block the phosphatase active site (Figure 3B). Thus, the N-SH2 domain acts as an internal switch to activate or inactivate SHP-2. The C-SH2 domain, on the other hand, does not have extensive contact with the PTP domain. The PTP domain is activated when the SH2 domains bind to phospho-peptides. From the structure, we can observe that the phospho-peptide binding pocket of both SH2 domains faces away from the PTP domain and are fully solvent exposed. Hof et al. suggests that once the N-SH2 domain binds to its substrate, a conformational change will occur and the active site of the PTP domain will be exposed. Additionally, although the structure shows a close arrangement of domains, the inter-domain contacts are mediated by mainly polar and water mediated interactions. This is consistent with the notion that once SHP-2 is activated, each domain will be solvent exposed.

Lee et al. have solved the structures of the N-SH2 domain alone and in complex with multiple phospho-peptides15. These structures revealed how the N-SH2 domain is able to recognize phospho-peptides in a sequence dependent manner. The SH2 domain consists of a central core of antiparallel β-sheet, with two flanking α-helices. The peptide binds in an extended conformation perpendicular to the β-sheets. An electro-positive cavity allows for the phospho-tyrosine to bind favorably. The residues N-terminal to the pY of the peptides are not observed to make significant interactions with the SH2 domain. However, consistent with in vitro peptide screens, the five residues C-terminal to the pY do play a significant role in binding. The residues in the position pY +1 through +5 bind to a shallow channel lined with electro-neutral residues. This channel selects for peptides with hydrophobic residues in positions +1,

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+3, and +5. Additionally, the BG loop contains two lysines (Lys BG5 and Lys BG7) and can partially fold over the peptide to interact with the residue in the +4 position15 (Figure 4).

SHP-2 activation is thought to be due to structural changes that occur when its N-SH2 domains bind to a phospho-peptide. However, the structures of the N-SH2 domain of SHP-2 in complex with a high-affinity peptide do not show a significant structural difference when compared with the apo structure. Therefore, the activation of SHP-2 may involve changes in the orientation of the SH2 domains relative to each other and relatively small internal changes in the domain itself.

Figure 3. SHP-2 tyrosine phosphatase. A) SHP-2 is composed of an N- and C-terminal SH2 domain and one phosphatase (PTP) domain. B) Crystal structure of SHP-2 in its inactive state. N-SH2 (yellow), C- SH2 (green), and PTP (blue). Residues D61 and Y62 of the D’E loop (of the N-SH2 domain) are inserted deep into cleft of the PTP catalytic site and inhibit the phosphatase activity. The phospho-peptide binding site of the N and C-SH2 domains faces away from the PTP domain and are solvent exposed2.

A

B

Figure 4. The molecular surface of the SH2 domain of SHP-2 in complex with a phospho-peptide. The electrostatic surface is shown with the positive surface in blue, the negative surface in red, and neutral in white. The pY binds into an electro-positive cavity, and the residues in positions pY +1 through +5 bind in a shallow channel. The residues N-terminal to pY are not seen to make specific contacts with the SH2 domain. The electro-neutral channel selects for peptides with hydrophobic residues (white) at positions +1, +3, and +5 (position +5 can also be aromatic). A residue in the +4 position could potentially interact with Lys BG5 and Lys BG7 in the BG loop.

pY +1 V +2 N

+3 I

+4 E

+5 F

-1 E

-2 G BG Loop

K BG7 K BG5

D61

Y62

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CagA regulates SHP-2 activity. CagA is delivered from H. pylori into gastric epithelial cells and localizes to the inner

plasma membrane where it is tyrosine phosphorylated at the EPIYA sites by SFKs (Src Family Kinases). Specifically, phosphorylation at the EPIYA-C or D site allows CagA to bind and activate the phosphatase activity of SHP-2. This interaction has been shown to lead to the hummingbird phenotype12, which is characterized by elongation and spreading of the cells16. The phenotype is attributed to the dephosphorylation and inhibition of focal adhesion kinase (FAK), which is a direct substrate of CagA-activated SHP-217. The CagA-SHP-2 complex can also positively regulate Erk signaling through Ras. The activation of Ras/Erk signaling causes the induction of cyclin D1 (which promotes G1-S progression), secretion of matrix metalloprotease-1 (which disrupts the stroma), and the up-regulation of NFκB (which is involved in the production of cytokines) (Figure 5). Although, the exact link between CagA and the development of gastric adenocarcinoma is still unclear, the Hatakeyama group recently showed that the transgenic expression of CagA in mice induced gastrointestinal neoplasms, while a phosphorylation resistant mutant of CagA did not6. Given that CagA tyrosine phosphorylation is essential for the activation of SHP-2 (a bona fide oncoprotein), this report further supports the importance of the CagA-SHP-2 complex’s ability to modulate the Ras/Erk pathway.

Significance:

Infection with cagA positive H. pylori is associated with the development of gastric adenocarcinoma. The protein CagA is directly delivered into gastric epithelial cells, where it is able to activate SHP-2 and disrupt the Ras/Erk signaling pathway in a phosphorylation dependent manner. Although the downstream effects of Ras/Erk signaling have been well documented, how CagA is able to initiate signal cascades is not known. Specifically, the details of how SHP-2 is activated by CagA and the identity of its interaction partners which are necessary for the deregulation of the Ras/Erk signaling pathway needs to be determined. The goals of this proposal are to determine the molecular mechanisms of CagA-SHP-2 interaction, SHP-2 activation by CagA, and the CagA-SHP-2 interaction partner that mediates Ras activation. By fulfilling these goals, we will understand how CagA mediates the virulence of H. pylori. Furthermore, the data yielded from these studies can be directly applied to the development of novel therapeutics to combat cagA positive H. pylori infection.

Figure 5. Helicobacter pylori CagA is delivered into the cell, and activates SHP-2. The CagA-SHP-2 complex is able to activate Ras through an unidentified pathway. Ras then activate the MAPK pathway and results in the up-regulation of growth promoting, antiapoptotic, and metalloprotease genes.

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D. Research Design and Methods: Specific Aim 1: Characterize the CagA-SHP-2 interaction. 1.1 Determine the stoichiometry of the CagA-SHP-2 complex.

SHP-2 has two SH2 domains, and each domain is capable of binding to a separate phospho-tyrosine site2. CagA variants, however, have from one to three possible SH2 binding sites. To marry these ideas, it has been suggested that CagA may form an oligomer to allow SHP-2 to bind. Indeed, Ren et al. discovered a motif that mediates CagA multimerization4. The notion that CagA multimerizes to maximize the number of SHP-2 binding sites is supported by the observation that a higher level of cellular response elicited by CagA is correlated with an increase in the number SHP-2 binding sites on CagA. However, it is not clear if CagA binds to SHP-2 as a multimer. An alternative is that CagA only needs to bind to the N-SH2 domain to activate the phosphatase domain of SHP-2, and this leaves the C-SH2 domain free to bind to additional interaction partner. We propose to determine the stoichiometry of the CagA-SHP-2 complex to determine if CagA binds to SHP-2 as a multimer and what type of multimer. Experimental design and anticipated results:

We will determine the stoichiometry of the following complexes: 1) The wild-type CagA variants. 2) The wild-type CagA variants with SHP-2. 3) To determine how the CagA mutimerization site affects the formation of CagA multimer

in solution, we will generate deletion mutants without these sites as done previously by Ren et al.4.

4) The CagA multimerization site deletion mutant (from #3) with SHP-2. 5) Generate double mutant R32A and H53A to disable the ability of the N-SH2 domain of

SHP-2 to bind to a phospho-peptide. Determine whether CagA is able to bind to the mutant N-SH2 disabled SHP-2. Perform the same studies with the C-SH2 domain. The double mutant R318A and H169A will equivalently disable the C-SH2 domain. Each CagA variant and SHP-2 protein will be expressed in Escherichia coli and purified to

homogeneity as done previously 2, 10. CagA will be phosphorylated in vitro by using commercially available Src kinases 10, 18. We will then incubate the phosphorylated CagA and SHP-2 proteins, to allow the complex to form. The complex will then be analyzed by analytical ultracentrifugation. This will allow us to determine the molar mass of the complex and consequently the stoichiometry.

We anticipate that CagA will form homodimers and binds to SHP-2 in this form. Thus the number of C or D sites per CagA determines the number of SHP-2 proteins that are recruited (Figure 6). Additionally, we anticipate that the CM motif will play a critical part in recruiting SHP-2. This will be especially interesting in the case of Western CagA because of the additional CM site embed in each C site.

Figure 6. Anticipated stoichiometry of SHP-2 in complex with CagA variants.

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Alternatives and potential pitfalls:

We will perform the analytical ultracentrifugation experiments with varying concentrations of protein to thoroughly analyze the association behavior of the complex. Performing the experiment with various protein concentrations will help to ensure that our analyses are accurate. Furthermore, we will also perform these studies in different buffers and salt concentrations to offset the primary charge effects that may occur and to analyze the solution conditions, which allow the complex to form.

Some alternatives to the analytical ultracentrifugation methodology to determine the stoichiometry include: 1) Native Polyacrylamide Gel Electrophoresis (Native PAGE), 2) Dynamic Light Scattering (DLS), and 3) Static Light Scattering (SLS).

1) Native PAGE will provide information on the relative size of the complex and from that we can determine the stoichiometry directly.

2) In a DLS experiment, the light scattered by the macromolecules in solution are measured, and the hydrodynamic radius of the macromolecules can be calculated. This hydrodynamic radius can be correlated with a mass and thus will allow us to determine the stoichiometry.

3) In STS, the intensity of the light scattered by the macromolecules in solution is measured as a function of angle, and this information can yield the molar mass and radius of gyration, which can be extrapolated to determine the stoichiometry of a complex.

1.2 Determine the binding affinities of SHP-2 to CagA.

The CagA variant with the EPIYA-D site conveys greater virulence than those variants containing up to three EPIYA-C sites, and the level of virulence is determined by the interaction between CagA and SHP-23. It is possible that SHP-2 prefers the D-type to the C-type binding site. By measuring and comparing the binding affinities between SHP-2 and CagA variants, we will be able to understand an integral component of this interaction. Experimental design and anticipated results: CagA and SHP-2 sample will be prepared as described in Aim 1.1. We will utilize Isothermal Titration Calorimetry (ITC) to determine the binding constants for each complex. We will determine the binding affinities of the following samples to SHP-2:

1) Individual EPIYA sites (A, B, C, and D sites). 2) Wild-type CagA variants. 3) The CagA multimerization site deletion mutants (same as those in Aim 1.1).

Additionally, the structure of the N-SH2 domain of SHP-2 showed it is able to bind six contiguous residues of a phospho-peptide. These residues are the pY and the five residues directly C-terminal to it. In the case of the C and D site, the only residue, within the predicted binding site, that differs is in the pY +5 position. In the D site, a Phe is present, while the C site contains an Asp. We would like to investigate whether this residue can affect the binding of CagA to SHP-2, and consequential be the key difference between the C and D site. Therefore, in addition to the above experiments, we will also generate a point mutation at the pY +5 position of Western CagA from an Asp to a Phe and determine its binding affinity to SHP-2. We will also generate the pY +5 Phe to Asp mutation for East-Asian CagA and carryout the same analyses.

Regarding our proposed methodology, ITC experiments will measure the heat of a reaction. In our proposed binding experiment. We will titrate CagA into a sample cell containing a constant amount of SHP-2. As we titrate more and more CagA into the sample cell, more heat will be given off until equilibrium is reached. The measurements of heat can then be fitted to a curve and thermodynamic parameters can be determined. Consequentially, these parameters will allow us to calculate binding affinities. Alternatives and potential pitfalls:

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Although ITC is a very sensitive technique, the level of heat required to delineate the signal over the noise is dependent on sample concentrations. The ability of a sample to be concentrated is dependent on the buffer and the presence of counter-ions. Therefore we will explore the different physiologically relevant buffer conditions, which will allow us to concentrate the proteins to a level suitable for the studies proposed. An alternative method to determine the binding constants is fluorescence anisotropy (FA). FA assays utilizes fluorescence to measure the amount of Brownian motion (proportional to anisotropy) of a macromolecule. We can titrate CagA into a sample cell with a constant amount of SHP-2, and as we add more CagA, the anisotropic value will increase until equilibrium is reached. A binding curve can be generated by plotting the protein concentration versus the anisotropic value. Aim 1.3 Determine the ability of CagA variants to activate SHP-2. Western CagA contains up to three C-type SHP-2 binding site, yet it is less virulent than the East-Asian CagA, which contains only one D-type site. It has been suggested that this is due to the ability of the D-type site to bind more strongly to SHP-2. This sub-aim will determine the level of phosphatase activity of CagA-activated SHP-2.

Experimental design and anticipated results:

CagA and SHP-2 samples will be prepared as described in Aim 1.1. A SHP-2 phosphatase assay will be done with the follow CagA proteins:

1) Wild-type CagA variants. 2) CagA multimerization site deletion mutants (same as those generated in Aim 1.1). 3) pY +5 Asp to Phe point mutation of Western CagA. 4) pY +5 Phe to Asp point mutation of East-Asian CagA.

We anticipate that the level of activity of the East-Asian CagA will be higher than that of the Western CagA. Additionally, the deletion of the CM sites should inhibit the ability of CagA to activate SHP-2. We expect that these results will correlate with the binding and stoichiometry studies done in the previous sub-aims.

There are commercially available phosphatase assays designed for SHP-2. The CagA-SHP-2 complex can be reconstituted in vitro and tyrosine phospho-peptides will be used as the substrate. The free phosphate in solution can be measured and indirectly provide the rate of the reaction. Alternatives and potential pitfalls:

Phosphatase assays utilize a dye-binding assay to determine the amount of free phosphate. These dye-binding assays can be sensitive to the buffer that is used. Therefore, we may potentially have to optimize our buffer conditions to match the requirement of the assay whilst still staying in physiologically relevant conditions. Alternatives include the usage of phospho-specific antibodies or thin layer chromatography to indirectly detect the phosphatase activity. Specific Aim 2: Solve the crystal structures of full length SHP-2 in complex with CagA containing the EPIYA-C and EPIYA-D motif. 2.1 Crystallization of the CagA-SHP-2 complex. Experimental design and anticipated outcome:

Regarding sample preparations, CagA and SHP-2 have both been successfully over-expressed and purified in previous work2, 10; therefore, we do not anticipate any major difficulty. We will then perform de novo crystallization experiments using the hanging-drop vapor diffusion method. The conditions used in these crystallization experiments will employ an incomplete factorial or sparse matrix approach and crystallization screens will test the effects of wide ranges of pH, ionic strength, counter-ions, temperature and a variety of precipitating

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agents, which include polyethylene glycol, ammonium sulfate, sodium-potassium phosphate and 2-methyl-2, 4-pentanediol 19. Initial crystals yielded from these efforts will be optimized by: increasing the crystallization drop volume or the macromolecule concentration, varying the pH and temperature of the crystallization condition, and by performing micro- and macro-crystal seeding.

Due to the nondeterministic nature of crystallography, we will attempt to crystallize the CagA-SHP-2 complex with multiple constructs:

1) Full length A-B-C, A-B-C-C, A-B-C-C-C, A-B-D type CagA. 2) The N-terminal region of CagA does not seem to play a role in the activation of SHP-

2. The EPIYA sites, in the C-terminal region, play the most critical role in the activation of SHP-2. We will utilize the portion of CagA, which encodes the C, C-C, C-C-C, and D sites with and without the CM site.

3) We will attempt to identify stable domains of CagA through limited proteolysis. We will then utilize the domain, which contains the EPIYA sites for crystallization.

4) We will also attempt to crystallize the complex with peptides of the predicted binding site, pY and residues +1 through +5.

5) The structure of SHP-2 has been solved and we can utilize this knowledge to determine the N- and C-terminal portions of the protein that are highly flexible. We can design a construct that does not contain these flexible regions, and utilize it in our crystallization efforts.

6) We can perform limited proteolysis experiments with the CagA-SHP-2 complex to identify the portion of CagA and SHP-2 that are highly flexible. However, the regions to be used in crystallization attempts must still be relevant to our aim to observe how CagA is able to activate the PTP domain through its interaction with the SH2 domains.

7) We will also investigate whether the Drosophila and Caenorhabditis elegans SHP-2 homolog, Csw and Ptp-2 respectively, can be substituted for SHP-2. We will first have to assess if they are able to bind and be activated by CagA. These homologous proteins may be more amendable to crystallization efforts.

Information regarding the stoichiometry and binding affinity from Aim 1 will be used to determine the ratio of CagA to SHP-2 to use in the crystallization efforts. Additionally, we will also explore other methods of crystallization such as: sitting and sandwich drop vapor diffusion, and dialysis crystallization.

Alternatives and potential pitfalls: There are many factors that can inhibit the crystallization process, and the strategies described above are meant to increase the chances for successful crystallization. Fundamentally, crystallization is the orderly aggregation of the macromolecule. If the sample is not structurally homogeneous, crystallization is less likely to occur. The presence of flexible linkers between domains can adversely affect the structural homogeneity the macromolecule in solution. To address this issue, we proposed to do limited proteolysis analysis and identify the regions/domains, which are likely to be labile and are susceptible to cleavage. We can then generate truncation mutants or site mutants to trap the macromolecule in a certain conformation. Additionally, we can also use the available knowledge to truncate domain/regions of the protein that does not play an active role in this specific interaction. For example, the N-terminal domain of CagA may not be necessary for the interaction of CagA and SHP-2, and we can truncate this portion of CagA to eliminate the potential complication it can pose. The SHP-2 homologs, Csw and Ptp-2, can also provide viable substitution of its human counterpart. In the case of Csw, the Drosophila homolog, the loop that connects the PTP domain and the C-SH2 domain is shorter than the respective loop in SHP-2. This information allows us to either shorten the loop in SHP-2 or use Csw instead of SHP-2 in the crystallization effort. 2.2 Data collection, structure determination, and model building.

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X-ray intensity data will be collected in-house using an R-AXIS IV imaging plate at 100K and x-rays generated by a Rigaku RU-300HB x-ray generator fitted with osmic mirrors. The Stanford Synchrotron Radiation Laboratory (SSRL) or Advanced Light Source at Berkeley (ALS) synchrotron sources will be used to collect the best quality and highest resolution x-ray intensity data. Intensity data will be processed with MOSFLM20 as implemented in CCP421. The structure of SHP-2 has been solved, and it may allow us to solve the structure of the complex with molecular replacement methods. However, since there might be major structural changes of SHP-2, molecular replacement may not provide a reasonable solution to the phase problem. We will then perform de novo structure determination by the multiwavelength anomalous dispersion method, using selenomethionine-substituted protein. Initial electron density maps will be displayed and traced using the software package O22. Model refinement will be done using CNS23 and model rebuilding will be done in O. Structural and stereochemical analysis will be done using CNS, O, PyMOL24, and PROCHECK25. Specific Aim 3: Determine the pathway CagA utilizes to activate Ras/Erk signaling. 3.1 Identify potential interaction partner(s) of CagA-SHP-2. Experimental design and anticipated results:

We shall transfect the tagged CagA expression vector into AGS human gastric epithelial cells or monkey COS-7 cells as described previously12. The vector utilizes an inducible Tet-On system for expression. The cells will be stimulated for 48 hrs and then harvested for co-IP experiments. We will immuno-precipitate with both CagA and SHP-2, and immunoblot for our target protein. Based on current data, it is possible that CagA-SHP-2 is able to activate Ras signaling through: A) Gab1, B) Src, or C) Spry. A) CagA-SHP-2 activates Ras signaling through the regulation of Gab1. Ras has a slow intrinsic GTPase activity and in its GTP bound form, Ras is active and is able to mediate a variety of cellular processes26. However, RasGap (Ras GTPase-Activating Protein) can stimulate the intrinsic GTP-hydrolysis activity and convert the active Ras-GTP form to the inactive Ras-GDP form. Gab1 is able to recruit RasGap to the inner membrane, allowing it to inhibit Ras. However, SHP-2 can dephosphorylate Y317 of Gab1 to disrupt its ability to bind to RasGap and thus inhibits its ability to recruit RasGap to the inner membrane27. This suggests that CagA-activated SHP-2 may also be able to activate Ras signaling by inhibiting Gab1 recruitment of RasGap (Figure 7).

B) CagA-SHP-2 activates Ras signaling through the regulation of Src.

Src is able to regulate Ras/Erk signaling28, and Src can be regulated through phosphorylation. Src has a tyrosine phosphorylation site at position 527, and when Y527 is

Figure 8. CagA-SHP-2 activates Ras signaling by dephosphorylating the inhibitory pY527 of Src.

Figure 7. CagA-SHP-2 activates Ras signaling by disrupting Gab1 recruitment of RasGap.

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phosphorylated, Src activity is inhibited29. It is therefore possible that CagA-activated SHP-2 is positively modulating Ras/Erk signaling by dephosphorylating the inhibitory tyrosine phosphorylation of Src (Figure 8).

C) CagA-SHP-2 activates Ras signaling through regulation of Sprouty (Spry) proteins.

Sprouty (Spry) proteins can inhibit Ras signaling by binding to Grb2-Sos and sequestering it. The Grb2-Sos complex function as a Ras GEF (Guanine-nucleotide-exchange factor) and is able to promote Ras activity. SHP-2 is able to dephosphorylate Y53 and Y89 of Spry and suppresses its inhibition of Ras30, 31. This suggests that CagA-activated SHP-2 can act as an inhibitor of Spry by dephosphorylating Y53 and Y89, allowing Grb2-Sos to activate Ras/Erk signaling (Figure 9).

Alternative and potential pitfalls:

The major pitfall of the proposed experimental design lies in the co-IP experiments. It is not guaranteed that an interaction partner will be identified with such a method. The conditions in which the binding event may occur is unknown and parameters such as salt concentration, pH, detergents, and the presence of particular ions will play a role in the binding event. An alternative to co-IP experiments to identify interaction partner(s) is to perform a pull-down experiment and identify binding partners of CagA-SHP-2 with mass spectrometry. An additionally alternative is the retrovirus-based protein-fragment complementation assay (RePCA). This assay uses a bait and prey system to identify interaction partners. The bait is fused with a fragment of green/red fluorescence protein and the prey is fused with the complimentary fragment. If the bait and prey interact, the fluorescence protein is reconstituted and a fluorescent signal can be detected32. 3.2 Establish that the identified protein is an interaction partner of CagA-activated SHP-2. We will verify that the identified proteins from Aim 3.1 are interaction partners of the CagA-SHP-2 complex. As stated in Aim 3.1, we anticipate that Gab1/Src/Spry will be among those proteins identified. Therefore, we shall refer to these three potential targets as our proteins of interest in the following sub-aims. Experimental design and anticipated results:

We shall transfect the CagA expression vector into AGS human gastric epithelial cells or monkey COS-7 cells as described previously in Aim 3.1. We will then investigating the: A) Gab1/Src/Spry’s physical interaction with CagA-SHP-2, B) tyrosine phosphorylation state of Gab1/Src/Spry, and C) Raf and Erk phosphorylation. In response to CagA expression, we expect that CagA-SHP-2 will interact with Gab1/Src/Spry, and form a complex, which can be detected with co-IP and Western blot experiments (Figure 10A). This interaction will lead to the dephosphorylation of Gab1/Src/Spry (Figure 10B). Subsequent prolonged activation of Ras will

Figure 9. CagA-SHP-2 activates Ras signaling by dephosphorylating residues Y53 and Y89 of Sprouty. These tyrosines phosphorylation allows Sprouty to bind to Grb2-Sos and inhibit its activation of Ras. Therefore CagA-SHP-2 suppresses Sprouty inhibition of Ras.

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then lead to the increase in Raf and Erk phosphorylation, which can be observed by Western blot analysis using commercially available phospho-specific antibodies (Figure 10C). Alternatives and potential pitfalls:

The inherent pitfall in detecting protein interactions with co-IP was addressed in Aim 3.1. An alternative method to detect protein-protein interactions is use the GFP-fragment reassembly technique. It is possible to express GFP in two fragments, and when these fragments are reconstituted, they will function like the wild-type protein. We can fuse one GFP-fragment to one protein and the second GFP-fragment to our target protein and look for reassembly of GFP. This will allow us to determine if two proteins are interacting.

3.3 Determine that the identified protein allows CagA-activated SHP-2 to mediate the downstream signal to activate Ras/Erk signaling. In the previous sub-aims, the interaction between the CagA-SHP-2 complex and Gab1/Src/Spry was determined. In this sub-aim, we will establish that the protein is necessary for the activation of Ras/Erk signaling. Experimental design and anticipated results:

We will establish a stably transfected knockdown of Gab1/Src/Spry by way of shRNA in AGS human gastric epithelial or monkey COS-7 cells. We will then co-transfect CagA and Gab1/Src/Spry expression vectors into the newly generated cell line. Utilizing this new cell line, we will investigate if CagA is able to mediate Erk phosphorylation in the presence and absence of Gab1/Src/Spry. In this newly create cell line, Gab1/Src/Spry is not present to act as an inhibitor of Ras activity, thus Raf and Erk phosphorylation should be higher than in normal AGS cells. When CagA is expressed, the level of Raf/Erk phosphorylation should not change

Figure 10. Anticipated Results. A) Results of Co-IP and western blot experiments will show that CagA interacts with both SHP-2 and Gab1/Src/Spry simultaneously. B) CagA-activated SHP-2 is able to dephosphorylate Gab1/Src/Spry. C) The expression of CagA will lead to Raf and Erk phosphorylation.

A B

C

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since there is not any Gab1/Src/Spry in the cell for CagA-SHP-2 to act upon. But when Gab1/Src/Spry is expressed, the level of Raf/Erk phosphorylation should decrease. This effect, however, will reverse when CagA is concomitantly expressed. Also by knocking down the expression of CagA or Gab1/Src/Spry, we can ensure that the effects observed are not mediated by an unrelated pathway (Figure 11).

Alternative and potential pitfalls: There are potential pitfalls regarding the use of RNAi. We will perform the necessary

controls to ensure that the knockdown is specific and efficient. The controls will include: scrambled (negative) siRNA control, positive siRNA control, multiple siRNAs to a single target, and we will monitor the both target mRNA and protein levels. Additionally, rescue experiments can also be done to ensure the effects we observe is not due to the contribution of an independent pathway.

An alternative to the methodology above includes the use of dominant positive and dominant negative forms of Gab1/Src/Spry. In these experiments, we can induce expression of CagA in AGS cells, it is anticipated that Ras activity will increase and that can be measured by Raf or Erk phosphorylation. If we then express the dominant positive form of Gab1/Src/Spry, the level of Raf/Erk phosphorylation should fall. Complimentarily, since Gab1/Src/Spry function downstream of SHP-2, its dominant negative form will also increase the level of Raf/Erk phosphorylation independent of CagA-SHP-2.

Figure 11. Anticipated results.

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