Phosphorylated self-peptides alter human leukocyte class I-restricted and generate tumor-specific

Jan Petersena,1, Stephanie J. Wurzbacherb,1, Nicholas A. Williamsonb, Sri H. Ramarathinamb, Hugh H. Reida, Ashish K. N. Nairb, Anne Y. Zhaob, Roza Nastovskab, Geordie Rudgeb, Jamie Rossjohna,2, and Anthony W. Purcellb,2

aProtein Crystallography Unit, Department of Biochemistry and Molecular Biology, School of Biomedical Sciences, Monash University, Victoria 3800, Australia; and bDepartment of Biochemistry and Molecular Biology and Bio21 Molecular Science and Biotechnology Institute, University of Melbourne, Victoria 3010, Australia

Communicated by Peter Doherty, University of Melbourne, Victoria, Australia, December 22, 2008 (received for review November 30, 2008) (HLA) class I molecules present a variety Cancer immunotherapy has focused on the identification of of posttranslationally modified epitopes at the cell surface, al- tumor-associated that are expressed exclusively by though the consequences of such presentation remain largely cancer cells. These antigens fall into 3 broad classes: (i) cancer unclear. Phosphorylation plays a critical cellular role, and deregu- antigens, such as testis and other embryonic or developmental lation in phosphate metabolism is associated with disease, includ- antigens that are not normally expressed in adult tissues but are ing and tumor . We have solved the expressed in a broad range of tumors (11); (ii) neoantigens high-resolution structures of 3 HLA A2-restricted phosphopeptides generated by mutation in key regulator molecules, such as p53 associated with tumor immunity and compared them with the (12) or aberrant posttranslational modification of proteins (13); structures of their nonphosphorylated counterparts. Phosphoryla- and (iii) viral antigens associated with cancer, such as Epstein- tion of the was observed to affect the structure and Barr virus antigens (14). Many of these antigens are not ex- mobility of the bound epitope. In addition, the phosphoamino acid pressed on the surface of tumor cells, and therefore are not

stabilized the HLA peptide complex in an epitope-specific manner directly accessible to . Thus, because of the ability of IMMUNOLOGY and was observed to exhibit discrete flexibility within the antigen- CTLs to survey intracellular protein expression, vaccines that are binding cleft. Collectively, our data suggest that phosphorylation capable of eliciting such responses represent an attractive option generates neoepitopes that represent demanding targets for T-cell for cancer immunotherapy. receptor ligation. These findings provide insights into the mode of To study the intrinsic link between the deregulated signaling phosphopeptide presentation by HLA as well as providing a plat- cascade present in many cancers and the ability of antigen form for the rational design of a generation of posttranslationally processing to alert CTLs to such molecular events, we have modified tumor vaccines. investigated the structural and biophysical properties and struc- tures of 3 HLA A2 phosphopeptide complexes derived from cell ␤ antigen presentation ͉ HLA ͉ phosphopeptide ͉ T cells ͉ division cycle (CDC) 25b, -catenin, and insulin receptor sub- X-ray crystallography strate (IRS) 2 and have compared them with the structures of their nonphosphorylated counterparts. The presentation of HLA class I-restricted phosphorylated epitopes and the impli- hosphorylation plays a critical role in cellular signaling, and cations for altered self are discussed. Pchanges in phosphate metabolism are associated with virtu- ally all disease states. The immune system has evolved to survey Results and Discussion changes in phosphorylation through the action of both innate Structures of HLA A2 Bound to Phospho- and Native-Peptide Epitopes. and adaptive effector pathways that include specific recognition To gain insight into the mode of phosphopeptide presentation, of phosphoantigens. For example, Toll-like receptor (TLR) 4 HLA A2 was expressed and refolded in the presence of 3 and TLR9 perceive various forms of phosphoantigens (1, 2). phosphopeptides. Two of these peptides, a nonamer and ␥␦ Moreover, a subset of T cells recognizes pyrophosphomo- decamer, were phosphorylated at the P4 position IRS2 noesters that are found in various microbial pathogens (3). 1097RVApSPTSGV1105, ␤-catenin 30YLDpSGIHSGA39, Natural killer (NK) cell recognition of phosphoantigens has also whereas the other nonamer was phosphorylated at the P5 revealed that phosphorylation of human leukocyte antigen position CDC25b residues 38–46 38GLLGpSPVRA46. These (HLA) Cw4-bound peptide antigens reduced inhibitory signals phosphoserine-containing peptides were chosen based on their mediated via killer Ig receptors and led to enhanced NK cell natural antigen presentation on multiple HLA A2ϩ tumor cell cytolysis (4). Phosphoantigens are also recognized by the adap- lines and their in HLA A2 transgenic mice (9). tive immune system. Recognition of phosphoantigens by anti- bodies is very well documented (5), and phosphorylated autoan- tigens are implicated in human autoimmune disorders, such as Author contributions: J.R. and A.W.P. designed research; J.P., S.J.W., N.A.W., H.H.R., primary Sjo¨gren’s syndrome and lupus (6, 7). Phosphoantigen A.K.N.N., A.Y.Z., and R.N. performed research; S.H.R. and G.R. contributed new reagents/ analytical tools; J.P. and S.J.W. analyzed data; and J.P., S.J.W., J.R., and A.W.P. wrote the surveillance by T cells has been observed in major histocom- paper. patibility complex (MHC) class I- and class II-restricted antigen The authors declare no conflict of interest. presentation (8–10). In addition, HLA A2-restricted tumor- Data deposition: The atomic coordinates have been deposited in Protein Data Bank, specific phosphopeptides are immunogenic, and cytotoxic T www.pdb.org (PDB ID codes 3FQN, 3FQR, 3FQT, 3FQU, 3FQW, and 3FQX). (CTLs) that distinguish between phosphorylated 1J.P. and S.J.W. contributed equally to this work. and native peptides can be generated in HLA A2 transgenic 2To whom correspondence may be addressed. E-mail: [email protected] mice (9). or [email protected]. The exquisite sensitivity of CTLs toward subtle changes in This article contains supporting information online at www.pnas.org/cgi/content/full/ peptides presented on the cell surface allows discrimination of 0812901106/DCSupplemental. infected cells or cells undergoing malignant transformation. © 2009 by The National Academy of Sciences of the USA

www.pnas.org͞cgi͞doi͞10.1073͞pnas.0812901106 PNAS Early Edition ͉ 1of6 Downloaded by guest on September 29, 2021 Table 1. Data collection and refinement statistics IRS2 IRS2 ␤-catenin ␤-catenin CDC25b CDC25b nonphospho phospho nonphospho phospho nonphospho phospho

Resolution, Å 23.7–1.93 24.0–1.70 23.7–1.65 24.3–1.70 24.9–1.80 30–1.80

Space group P212121 P212121 P212121 P212121 P212121 P212121 Cell dimensions, Å (a, b, c) 59.98 59.85 59.72 59.68 59.68 59.91 79.38 79.68 79.71 79.71 79.84 80.04 111.29 111.00 110.87 111.56 110.21 110.37 Total no. observations 38,009 56,258 63,777 56,745 49,135 49,810 Multiplicity 4.7 5.1 6.3 7.1 3.5 4.9 Data completeness, % 93.07 (75.9) 95.25 (73.9) 98.7 (88.8) 95.8 (76.3) 99.19 (97.8) 99.8 (100)

I/␴I 22.1 (3.7) 26.1 (3.9) 27.3 (2.5) 26.9 (4.3) 18.6 (2.8) 23.9 (3.3) Rmerge*, % 5.6 (32.8) 4.4 (31.8) 5.0 (42.9) 5.1 (30.4) 5.0 (45.0) 5.2 (46.6) † Rfactor ,% 16.85 17.10 17.92 16.85 17.53 17.04 ‡ Rfree ,% 19.63 19.61 19.95 18.86 20.34 20.18 rmsd from ideality Bond lengths, Å 0.006 0.008 0.006 0.005 0.005 0.007 Bond angles, ° 1.022 1.115 1.058 0.998 0.967 1.142 Ramachandran angles, % Favored 97.61 98.13 97.84 97.61 98.14 98.40 Allowed 2.39 1.87 2.16 2.39 1.86 1.60 Outliers — — — — — — B-factors Peptide 28.7 29.0 28.0 26.4 28.0 38.3 Protein 27.3 28.5 27.3 24.4 29.4 27.6 Water ions (Cd, Co, Mg), glycerol 36.5 41.3 40.3 38.0 40.1 38.7

*Rmerge ϭ͚͉IhklϪ͗Ihkl͉͘/͚Ihkl. † ‡ Rfactor ϭ͚hkl ͉͉Fo͉Ϫ͉Fc͉͉/͚hkl ͉Fo͉ for all data except for 5%, which was used for the Rfree calculation. Numbers in parentheses refer to statistics in the highest resolution bin.

The 3 HLA A2 epitopes were subsequently crystallized, and goes local conformational changes around the site of phosphor- their respective structures were solved and refined to a resolution ylation at P5 to avoid steric clashes with Ala 69 and Thr 73 of the of 1.8 Å or better (Table 1). In addition, to gain insight into how HLA A2 heavy chain (Fig. 2 B and C). This results in the peptide the incorporation of the phospho-moiety influenced the pHLA pushing away from the ␣1-helix toward center of the antigen- A2 structure, we determined the structures of the nonphospho- binding cleft, resulting in a shift of2ÅintheC␣ position rylated counterparts of these peptides bound to HLA A2 to a between P5 and P5-phosphoSer and also a change in the resolution of 1.93 Å or better (Table 1). With the exception of conformation of P3-Leu. Moreover, the phosphate group is the HLA A2YLDSGIHSGA, in which the central region of the observed in 2 discrete conformations (Fig. 2B), indicative of epitope demonstrated high mobility (see below), the mode of discrete mobility in this moiety [Table S1]. One conformer forms binding of the peptides was unambiguous (Fig. 1). a salt bridge with P8-Arg, and both conformers interact with the All 6 pHLA A2 structures determined were crystallized under peptide backbone through a water-mediated H-bond to P6-Pro, the same conditions, in the same space group and unit cell which appear to be the only interactions the phosphate head dimensions (Table 1). In addition, an alignment of the antigen- group makes (Fig. 2 A and B). In this instance, these pHLA binding cleft (residues 1–185) of HLA A2 indicated no signifi- complexes define a case of altered self in which the peptide cant structural rearrangement in the corresponding phospho- antigen demonstrates significantly altered conformation. These and nonphospho-structures (rmsd: CDC25b/CDC25b-phospho, data demonstrate the alteration of a self-pHLA by a posttrans- HLA A2 ϭ 0.079 Å, peptide ϭ 0.27 Å; IRS2/IRS2-phospho, lational modification. HLA A2 ϭ 0.13 Å, peptide ϭ 0.09 Å; ␤-catenin/␤-catenin- phospho, HLA A2 ϭ 0.11 Å, peptide ϭ 0.45 Å). In all cases, the IRS2. The IRS2 peptide (RVASPTSGV) also bound to HLA A2 phosphate group is prominently surface-exposed and contrib- in an extended manner with P2-Val and P9-Val as main anchor utes to increased electronegativity at the candidate T-cell re- residues (Fig. 1B). In addition, P3-Ala and P6-Thr point down- ceptor (TCR) binding site (Figs. 2 and 3). ward into the antigen-binding cleft. P1-Arg, P4-Ser, P5-Pro, and P7-Ser are solvent-exposed and potential TCR contact sites, CDC25b. The CDC25b peptide (GLLGSPVRA) bound to HLA although P4-Ser is not involved in any significant interactions. A2 in a linear and extended manner with a central bulge around The phosphorylated IRS2 peptide is accommodated within the the P4-P5 position (Fig. 1A). P2-Leu and P9-Ala are the main HLA A2 binding cleft with little change when compared with anchor residues, whereas P3-Leu also pointed down toward the the nonphosphorylated counterpart (Fig. 1E). The differences in antigen-binding cleft and contributes to peptide binding. P5-Ser the phosphorylated and nonphosphorylated complexes reside and P8-Arg interact via a water-mediated H-bond, and both predominantly in the addition of an electronegative charge and project upward, comprising potential TCR contact sites, with small changes in the conformation of Lys 66 and Arg 65 (Fig. 2 P5-Ser leaning toward the ␣1-helix, and forming van der Waals D–F). As observed for the CDC25b pHLA complex, the phos- interactions with Ala 69 of HLA A2 (Fig. 2A). There are no phate group at P4 of the IRS2 peptide was observed in 2 conformational changes in the HLA A2 heavy chain associated conformations, again reflecting flexibility in the phospho- with the accommodation of the phospho-moiety in the HLA moiety. One conformer interacts with Lys 66 on the ␣1-helix and A2GLLGpSPVRA complex (Fig. 1D). However, the peptide under- with Gln 155 on the ␣2-helix through a water-mediated H-bond

2of6 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0812901106 Petersen et al. Downloaded by guest on September 29, 2021 A B C

D E F

G HI

Fig. 2. Interactions of the phosphorylation site in nonphosphopeptide and phosphopeptide HLA A2 complexes. Accommodation of the phosphate moi- ety by HLA A2 is accompanied by changed interactions in the complex, adding to the differential presentation of altered self. Stick representation of pep- tides and of heavy-chain side chains that interact with the phosphorylation site. Yellow indicates nonphospho-pHLA A2 complexes. Green indicates

phospho-pHLA A2 complexes. (A–C) Phosphorylation of P5-Ser in CDC25b IMMUNOLOGY leads to an altered peptide conformation attributable to steric constraints. (D–F) Phosphorylation of P4-Ser in IRS2 gives rise to numerous interactions and subtly alters the conformation of Arg 65 and Lys 66. (G–I) Phosphorylation of P4-Ser in ␤-catenin stabilizes the mobile peptide residues P3 to P6.

(15). The binding mode of the phosphorylated IRS2 peptide only partly follows this motif, however, because the salt bridge to P1-Arg is absent in the HLA A2RVApSPTSGV complex. This can be attributed to the extended binding geometry of P5-Pro and P6-Thr, which prevents the movement of P4-Ser toward P1 into the position observed by Mohammed et al. (15). Instead, P1-Arg and Lys 66 adopt side-chain conformations that place the center of the positively charged region somewhat closer to the phos- phate moiety.

Fig. 1. Peptide conformations within the antigen-binding cleft. CDC25b (A), IRS2 (B), ␤-catenin (C), CDC25b-phospho (D), IRS2-phospho (E), and ␤-catenin– phospho (F). Blue mesh indicates unbiased 2Fo-Fc maps contoured at 1␴. Yellow indicates nonphosphorylated peptides. Green indicates phosphory- lated peptides. The bound peptide is shown from a side-on view with the ␣2-helix removed for clarity.

Fig. 3. Altered surface potential for TCR recognition. Surface representation (Table S1). The second conformer interacts with the Lys 66 and of the HLA A2 with bound peptides. CDC25b (A), IRS2 (B), CDC25b-phospho Arg 65 of HLA A2. Thus, the phospho-moiety forms stabilizing (C), and IRS2-phospho (D). Gray indicates ␣-chain, with putative TCR contact contacts with the antigen-binding cleft without disrupting the residues in purple, based on the structure of the A6/HLA A2-Tax complex peptide conformation. structure (25). The arrows indicate the peptide phosphorylation sites. The negatively charged phosphate groups are located within the area of a typical A recent structural study of phosphopeptide/HLA A2 com- TCR footprint and are likely to dominate TCR discrimination. Electrostatic plexes suggested a common binding motif for the P4-phosphoSer potentials (blue, positive; red, negative) were calculated with APBS (22). The moiety of peptides, with a positively charged N-terminal residue phosphoserine residues were assumed to carry 2 negative charges.

Petersen et al. PNAS Early Edition ͉ 3of6 Downloaded by guest on September 29, 2021 ␤-Catenin. The native form of this peptide (YLDSGIHSGA) peptide dephosphorylation were assessed by mass spectrometric demonstrates marked flexibility in complex with HLA A2, with analysis of phosphatase-treated samples at different time points. no electron density observed for positions 5 and 6 (Fig. 1C). In Consistent with the structural studies that showed the phosphate addition, P3-Asp, P4-Ser, and P7-His exhibit flexibility. The participated in a number of interactions with HLA A2 (Table mobility of the central region of the ␤-catenin peptide is not S1), substantial protection from phosphatase activity was ob- attributable to poor crystallographic data, because the electron served when the phosphopeptides were bound to HLA A2 density surrounding this region is excellent and, moreover, the compared with the peptide in free solution (Fig. 4 C, F, and I). structure is at very high resolution (1.65 Å) and well refined [Rfree ϭ 19.95%]. There is also a degree of mobility of the Conclusions residues in the floor of the antigen-binding cleft (e.g., Tyr 99, Arg Our structures of HLA A2 complexed to both native and 97) as a result of the peptide flexibility (data not shown). phosphorylated versions of peptide epitopes provide a unique Although the peptide is highly flexible, the primary anchor opportunity to visualize the impact of phosphorylation on the (P2-Leu and P10-Ala) residues are well defined. bound conformation of the peptide ligand and HLA A2. In The mobility of the nonphosphorylated pHLA is markedly particular, conformational adjustments to some HLA A2 resi- reduced in the phosphorylated pHLA structure, and the entire dues, peptide conformation, and peptide mobility were observed epitope is clearly visible (Fig. 1F). Consistent with the other 2 in an epitope-dependent manner (Fig. 1). This distinguishes our phosphorylated complexes, the phosphate moiety is observed in study from earlier studies (15) in which only the phosphorylated 2 different conformers, which indicates a general theme of a version of the phosphopeptide epitopes was studied. Moreover, mobile phosphate group (Fig. 2 G–I). In the HLA our study focuses on epitopes that are phosphorylated at the P4 A2YLDpSGIHSGA complex, P2-Leu and P10-Ala represent the as well as P5 positions. Incorporation of the phosphate moiety primary anchor residues, with P3-Asp and P6-Ile also projecting dramatically alters the electrostatic footprint of the pHLA downward toward the antigen-binding cleft. The stabilization of complex, which, coupled to the mobility of the phosphate head the flexible nature of this peptide by phosphorylation can be group, is predicted to have a profound influence on T-cell attributed to stabilizing intrapeptide interactions as well as to recognition. interactions with Arg 65 and Lys 66 of HLA A2. In one For the IRS2 epitope, phosphorylation enhanced HLA A2 conformer, the phosphate group interacts with P7-His and Arg binding and thermostability, although phosphorylation- 65 and it appears that the P7-His is ‘‘pulled in’’ toward the dependent stabilization of the pHLA A2 was not a general phospho-moiety compared with the nonphosphorylated com- theme. In the ␤-catenin epitope, phosphorylation ordered the plex. There is also a water-mediated H-bond between the conformation of the peptide via the introduction of electrostatic phosphate group and P1-Tyr (Fig. 2 G and H). constraints. In all cases, binding to HLA A2 shielded the Overall, the 6 structures have revealed a clear alteration of self phosphoserine residue from dephosphorylation by phospha- when phosphorylated peptides are captured and presented to T tases, suggesting that once formed, the phosphopeptide com- cells—not merely via the incorporation of the phosphate moiety plexes are stable. Thus, not only are phosphopeptides trans- that alters the biophysical characteristics of the ligand but in ported into the endoplasmic reticulum actively (10), but on changes in peptide conformation and HLA A2 residues known assembly with HLA, they appear to be protected from phospha- to influence TCR engagement (16). The phosphate moiety was tase activity, preserving the epitope for scrutiny by T cells at the observed in multiple conformations, suggesting that this moiety cell surface. This is consistent with the ability of these peptides can adopt discrete conformations, thereby potentially represent- to generate phosphopeptide-specific CTLs (9) and the detection ing a ‘‘moving target’’ for TCR engagement. In each case, the of both tumor-specific and self-phosphopeptides in the HLA A2 phosphate sits centrally in the antigen-binding cleft as a prom- immunopeptidome (9). This also suggests that phosphopeptides inent electronegative target for T-cell ligation (Fig. 3). Also will be present in the thymus during T-cell ontogeny, shaping the shown in Fig. 3 is the putative TCR docking site on the surface T-cell repertoire and selecting phosphopeptide-specific T cells. of the pHLA complex, which indicates that the phosphate would Thus, phosphopeptides represent a class of potential vaccine be highly accessible for interaction with CDR regions of the candidates. Their presence on the surface of antigen-presenting bound TCR. cells not only directly reflects the altered signaling events occurring in the transformed cell but generates an altered Influence of Phosphate Group on Stability and Binding to HLA A2. To self-pHLA landscape providing the cytotoxic T-cell effector arm determine if the interactions between the phosphate group and of the immune system an opportunity to remove malignant cells HLA A2 residues have an impact on binding and thermostability from the host. Clearly, other disease states that have an impact of the pHLA A2 complexes, thermal denaturation curves and on phosphorylation and related signaling events will also poten- HLA A2 binding studies were undertaken. Good correlation tially yield distinctive pHLA landscapes that can be discerned by between thermal melt curves incorporating CD measurements surveying T lymphocytes. of complex structure and competitive binding assays were ob- served for all 3 peptide sets (Fig. 4). The ␤-catenin and CDC25b Materials and Methods complex stability and HLA A2 binding were essentially unaf- Peptides. All peptides were synthesized and purified to Ͼ85% at the Bio21 fected by the phosphorylation of P4-Ser and P5-Ser of the Peptide synthesis facility. Peptide stocks were prepared and dissolved in DMSO respective peptides. In contrast, the phosphorylation of P4-Ser to a final concentration of 10–100 mg/mL. in the IRS2 peptide resulted in enhanced complex thermostability (increase of Ϸ6°CinTm) and improved HLA A2 binding (6.3-fold Expression, Purification, Crystallization, and Structure Determination. Trun- increase in IC50). Thus, phosphorylation can increase the stability cated HLA A*0201 class I heavy chain, encompassing residues 1–2745, was of the HLA A2 complex in an epitope-dependent manner. expressed as inclusion bodies using the BL21 strain of Escherichia coli as described previously (17). Crystals of all pHLA complexes were grown by the hanging drop vapor HLA A2 Protects the Phosphate Moiety from Phosphatases. One issue diffusion method at 20 °C using similar precipitant solutions with 2–4 mM that surrounds the presence of phosphoserine within the HLA MgCl , 2–4 mM CdCl , 0.1 M Hepes (pH 7.4), 100–200 mM NaCl, and 12–13% A2 binding cleft is the general labile nature of the phosphate 2 2 PEG3350 (vol/vol). For the IRS2 nonphospho-complex, CoCl2 was used instead modification. We therefore undertook a series of experiments to of MgCl2. Streak-seeding was required to nucleate crystals of both CDC determine if the HLA A2 antigen-binding cleft afforded pro- complexes. Crystals appeared after 12–48 h and grew to maximal size in 7–14 tection to the peptide from phosphatase activity. The kinetics of days. Crystals were flash-cooled to 100 K before data collection using 20%

4of6 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0812901106 Petersen et al. Downloaded by guest on September 29, 2021 IMMUNOLOGY

Fig. 4. Stabilization experiments with the peptides GLLGpSPVRA, RVApSPTSGV, and YLDpSGIHSGA and their native forms. (A, D, and G) Thermal denaturation of the phosphopeptide- and nonphosphopeptide-MHC complexes using CD spectroscopy. (B, E, and H) Competition-based peptide binding assay with the phosphopeptides and nonphosphopeptides. (C, F, and I) Dephosphorylation of the single phosphopeptides and the phosphopeptide-MHC complexes by alkaline phosphatase. Nonphosphorylated peptide/complexes are shown by gray lines and phosphopeptide/complexes are shown by black lines.

glycerol (vol/vol). X-ray diffraction experiments were performed using a Rik- CD. CD spectra were measured on a Jasco 815 spectropolarimeter using a agu RU-3HBR rotating anode generator with helium-purged OSMIC focusing thermostatically controlled cuvette at temperatures between 30 and 90 °C. ϩϩ mirrors coupled to an R-AXIS IV detector (Rikagu). All crystals belong to Far-UV spectra were collected and analyzed as described (17). space group P212121, with very similar unit cell dimensions. For a full summary of the data collection statistics, refer to Table 1. Competitive HLA A2 Binding Assay. Cells were treated with ice-cold citric acid The structures were solved by molecular replacement using the program for 90 s before the binding assay to remove HLA-bound peptides. The com- Phaser (18). A modified protomer of a previously solved HLA A2 structure with petition-based peptide binding assay was performed according to van der the peptide residues removed was used as the search probe. Refinement was Burg et al. (23). Briefly, 25 ␮L of competitor peptide (different end concen- monitored by the R value (5% of the data), using the same set of reflections free trations) was mixed with 25 ␮L of fluorescence-labeled reference peptide for all data sets. Rigid body refinement and restrained refinement were [GILGK(FITC)VFTL, end concentration ϭ 150 ng/mL] in a 96-well V-bottom performed using the program Refmac (19). This was followed by simulated plate. One hundred microliters of mild acid-treated JY cells (5 ϫ 104/well) was annealing and individual B-factor and total least squares (TLS) refinement in added to the wells and incubated at 4 °C for 24 h. Cells were washed with PBS Phenix (20). Model building was performed using the program Coot (21). ␮ Water molecules and peptides were built into unambiguous electron density containing 1% BSA; 10 L of propidium iodide (1 mg/ml solution) was added, during the refinement process. Cd and Co ions were modeled into strong and the mean fluorescence (MF) was then measured by FACScan (Becton spherical peaks (␴ Ͼ 8) of the 2Fo-Fc maps, and their occupancies were Dickinson). Percentage inhibition of fluorescent peptide binding was calcu- adjusted manually to fit the maps. Figures were generated with Pymol lated using the following formula: (DeLano Scientific LLC) and APBS (22). For the calculation of the unbiased ͑ Ϫ ͑ Ϫ ͒ 2Fo-Fc maps shown in Fig. 1, a single round of simulated annealing and B 1 MFreferenceϩcompetitor peptide MFno reference peptide / factor- and TLS refinement was performed with the peptides removed from ͑ Ϫ ͒͒ ϫ the models. MFreference peptide MFno reference peptide 100%

Petersen et al. PNAS Early Edition ͉ 5of6 Downloaded by guest on September 29, 2021 Phosphatase Treatment. The kinetics of peptide dephosphorylation were relative abundance of phosphorylated peptide was determined by MALDI- measured by MALDI-TOF mass spectrometry following alkaline phosphatase TOF mass spectrometry using an Applied Biosystems Pulsar i Q-TOF mass treatment of the free phosphopeptide and phosphopeptide-HLA A2 com- spectrometer as described previously (24). plexes in solution. Five micrograms of phosphopeptide or phosphopeptide- ␮ HLA A2 complex was incubated with 5 L of alkaline phosphatase (0.4 mg/mL ACKNOWLEDGMENTS. A.W.P. is a National Health and Medical Research for the phosphopeptide derived from IRS2, 0.02 mg/mL for the phosphopep- Council of Australia (NH&MRC) Senior Research Fellow, and J.R. is an Austra- tide derived from ␤-catenin, and 0.067 mg/mL for the phosphopeptide derived lian Research Council Federation Fellow. This work was supported by from CDC25b) for 5 to 30 min. The same peptide-optimized concentrations of NH&MRC Project Grants 491117 and 508927 and National Institutes of Health phosphatase were used for the phosphopeptide-HLA A2 complexes. The Grant GM057428-06.

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