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Phosphorylated Self-Peptides Alter Human Leukocyte Antigen Class I-Restricted Antigen Presentation and Generate Tumor-Specific Epitopes

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Phosphorylated Self-Peptides Alter Human Leukocyte Antigen Class I-Restricted Antigen Presentation and Generate Tumor-Specific Epitopes Phosphorylated self-peptides alter human leukocyte antigen class I-restricted antigen presentation and generate tumor-specific epitopes 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) Human leukocyte antigen (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 antigens 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 autoimmunity and tumor immunity. 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 epitope 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 antibodies. 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 immunogenicity 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). lymphocytes (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).
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