Nα-Terminal Acetylation for T Cell Recognition: Molecular Basis of MHC Class I −Restricted Nα-Acetylpeptide Presentation This information is current as of June 7, 2017.
Mingwei Sun, Jun Liu, Jianxun Qi, Boris Tefsen, Yi Shi, Jinghua Yan and George F. Gao J Immunol 2014; 192:5509-5519; Prepublished online 14 May 2014; doi: 10.4049/jimmunol.1400199 http://www.jimmunol.org/content/192/12/5509
References Subscription Permissions Email Alerts
http://www.jimmunol.org/content/suppl/2014/05/14/jimmunol.140019 9.DCSupplemental This article cites 45 articles, 16 of which you can access for free at: http://www.jimmunol.org/content/192/12/5509.full#ref-list-1 Information about subscribing to The Journal of Immunology is online at: http://jimmunol.org/subscription Submit copyright permission requests at: http://www.aai.org/About/Publications/JI/copyright.html Receive free email-alerts when new articles cite this article. Sign up at: http://jimmunol.org/alerts
The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2014 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606.
Downloaded from http://www.jimmunol.org/ by guest on June 7, 2017
Supplementary Material
The Journal of Immunology
Na-Terminal Acetylation for T Cell Recognition: Molecular Basis of MHC Class I–Restricted Na-Acetylpeptide Presentation Mingwei Sun,*,† Jun Liu,‡ Jianxun Qi,† Boris Tefsen,† Yi Shi,†,x Jinghua Yan,† and George F. Gao*,†,‡,x
P
roduced by Ag processing and proteasomal degradation of intracellular proteins, polypeptides serve as CTL epitopes presented by MHC class I molecules, which play a critical role in cellular immunity (1). Eukaryotic proteins bearing various posttranslational modifications (PTMs) can generate a group of modified Ags, which contribute to a special repertoire of MHCassociated peptides presented at the cell surface as potential targets for TCR-mediated recognition. A modified peptide may become a new Ag because of the distinguished antigenicity compared with its *School of Life Sciences, University of Science and Technology of China, Hefei 230027, China; †CAS Key Laboratory of Pathogenic Microbiology and Immunology, Institute of Microbiology, Chinese Academy of Sciences, Beijing 100101, China; ‡ National Institute for Viral Disease Control and Prevention, Chinese Center for Disease Control and Prevention, Beijing 102206, China; and xResearch Network of Immunity and Health, Beijing Institutes of Life Science, Chinese Academy of Sciences, Beijing 100101, China Received for publication January 23, 2014. Accepted for publication April 15, 2014. This work was supported by the National 973 Project of the China Ministry of Science and Technology (Grant 2010CB911902) and the National Natural Science Foundation of China (Grants 31030030 and 81373141). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. G.F.G. is a leading principal investigator of the National Natural Science Foundation of China Innovative Research Group (Grant 81021003). The atomic coordinates of the crystal structures of HLA-B*3901NAc-SL9, HLAB*3901SL9, and HLA-B*3901HL8 presented in this article have been submitted to the Protein Data Bank (http://www.pdb.org/pdb/home/home.do) under accession numbers 4O2C, 4O2E, and 4O2F. Address correspondence and reprint requests to Prof. George F. Gao, CAS Key Laboratory of Pathogenic Microbiology and Immunology, Institute of Microbiology, Chinese Academy of Sciences, Building 3, 1 Beichen West Road, Chaoyang District, Beijing 100101, China. E-mail address:
[email protected] The online version of this article contains supplemental material. Abbreviations used in this article: CD, circular dichroism; b2m, b2-microglobulin; NH2, a-amino; Nt-acetylation, Na-terminal acetylation; P1, N-terminal; Pc, C-terminal; PEG, polyethylene glycol; pMHC, peptide–MHC; PTM, posttranslational modification; Tm, midpoint transition temperature. Copyright Ó 2014 by The American Association of Immunologists, Inc. 0022-1767/14/$16.00 www.jimmunol.org/cgi/doi/10.4049/jimmunol.1400199
unmodified homolog. A variety of natural peptide Ags containing modification have been observed that can be immunologically discriminated by T cells from their unmodified homologs as “altered self” (2). Thus, the significance of PTMs on epitopes and the application of modified peptides in vaccine development for immunotherapy against cancer and autoimmune diseases have been increasingly appreciated (3, 4). The molecular bases of the presentation of peptides with several PTMs by MHC class I molecules have been successfully explicated. For instance, the formyl group on an Nt-formylated peptide binds to the bottom of the peptide-binding groove of H2-M3 (5); both the glycan and the phosphate moieties of the central region of the glycopeptides (6, 7) and the phosphopeptides (8, 9), respectively, are exposed to enable TCR binding, and the deimination (citrullination) of arginine on a peptide presented by two HLAB27 subtypes induces distinct peptide conformations (10). Na-terminal acetylation (Nt-acetylation) is one of the most common PTMs, occurring on the vast majority of eukaryotic proteins. In humans, .80% of the different varieties of intracellular proteins are irreversibly Nt-acetylated by Na-acetyltransferases, often after the removal of the initiator methionine. Only a subset of the penultimate residues (Ala, Ser, Thr, Cys, and Val) or the retained initiator methionine can be acetylated at the a-amino (NH2) groups (11). A recent study found that acetylated N-terminal residues of eukaryotic proteins act as specific degradation signals (Ac-N-degrons) that are recognized by specific ubiquitin ligases (12). A subsequent systematic analysis demonstrated that Ntacetylation can also represent an early determining factor in the cellular sorting for prevention of protein targeting to the secretory pathway (13). These findings suggested that Nt-acetylation–mediated inhibition of secretion could contribute to the retention of proteins in the cytosol where they may subsequently be ubiquitinylated through the specific recognition of their Ac-N-degrons and thereby
Downloaded from http://www.jimmunol.org/ by guest on June 7, 2017
As one of the most common posttranslational modifications (PTMs) of eukaryotic proteins, Na-terminal acetylation (Ntacetylation) generates a class of Na-acetylpeptides that are known to be presented by MHC class I at the cell surface. Although such PTM plays a pivotal role in adjusting proteolysis, the molecular basis for the presentation and T cell recognition of Naacetylpeptides remains largely unknown. In this study, we determined a high-resolution crystallographic structure of HLA (HLA)-B*3901 complexed with an Na-acetylpeptide derived from natural cellular processing, also in comparison with the unmodified-peptide complex. Unlike the a-amino–free P1 residues of unmodified peptide, of which the a-amino group inserts into pocket A of the Ag-binding groove, the Na-linked acetyl of the acetylated P1-Ser protrudes out of the groove for T cell recognition. Moreover, the Nt-acetylation not only alters the conformation of the peptide but also switches the residues in the a1-helix of HLA-B*3901, which may impact the T cell engagement. The thermostability measurements of complexes between Na-acetylpeptides and a series of MHC class I molecules derived from different species reveal reduced stability. Our findings provide the insight into the mode of Na-acetylpeptide–specific presentation by classical MHC class I molecules and shed light on the potential of acetylepitope-based immune intervene and vaccine development. The Journal of Immunology, 2014, 192: 5509–5519.
Na-ACETYLPEPTIDE–HLA-B*3901 STRUCTURE AND FUNCTION
5510 Table I. Characteristics of the peptides used in this study
Tm (˚C)c Sequencea
MHC Restriction
Derived Ag
Position
IC50 (nM)b
Unmodified
Nt-acetylated
Ref.
SHVAVENAL HVAVENAL SLLMWITQC SIYRYYGL AIFQSSMTK TVSGLAWTR CYTWNQMNL VPLRPMTY MLLSVPLLL
HLA-B*3901 HLA-B*3901 HLA-A*0201 H-2Kb HLA-A*1101 HLA-A*3303 HLA-A*2402 HLA-B*3501 HLA-A*0201
RNA helicase DBX/CAP-Rf RNA helicase DBX/CAP-Rf Cancer/testis Ag Superagonist HIV reverse transcriptase CMV phosphoprotein 65 Modified Wilms’ tumor protein HIV Nef protein Calreticulin precursor
2–10 3–10 157–165 — 313–321 162–170 235–243 75–82 1–9
10 387 1015 2 13 29 193 51 12
49.2 43.9 49.4 42.3 55.5 48.7 47.8 47.1 47.5
40.1 — 36 36.7 35.8 38.4 39 37.5 32.3
17 18 19 20 21 22 23 24 25
a
Amino acid residues in bold format are the (synthetic) Nt-acetylation sites. Artificial neural network prediction of the peptide binding affinity as IC50 value in nM calculated using the NetMHC 3.4 server (http://www.cbs.dtu.dk/services/NetMHC/). Thermal denaturation midpoint (Tm) of the pMHC complexes with unmodified and Nt-acetylated peptides corresponding to Fig. 6.
b c
For MHC class I, the first Nt-acetylated natural ligand was identified more than a decade ago (17). However, the mode of interaction of this acetylated peptide with class I molecules remained largely enigmatic. To understand this, we determined the crystal structures of a naturally occurring Nt-acetylated selfpeptide (NAc-SL9) and two nonmodified variants (SL9 and HL8), respectively, in complex with HLA-B*3901. Taken together with the thermostability analyses of Na-acetylpeptides complexed with a series of class I molecules of human and murine origin, we elu-
Table II. Data collection and refinement statistics HLA-B*3901NAc-SL9
HLA-B*3901SL9
HLA-B*3901HL8
P212121
P212121
P212121
50.85, 81.46, 110.96 90.00 0.97916 50.0-1.8 (1.86-1.8) 278,398 41,844 1 6.5 (5.8) 99.6 (99.2) 10.9 (44.2) 17.5 (5.3)
79.94, 103.97, 122.42 90.00 0.97924 50.0-2.0 (2.07-2.0) 469,154 66,979 2 6.8 (5.9) 97.2 (95.5) 7.2 (40.1) 24.0 (3.8)
78.37, 95.92, 122.93 90.00 0.97924 50.0-1.9 (1.97-1.9) 254,849 68,094 2 3.6 (3.2) 96.7 (97.4) 7.9 (37.0) 13.9 (2.5)
30.6-1.8 17.45 20.15
23.9-2.0 18.56 20.80
29.6-1.9 18.54 21.32
3,134 495
6,263 787
6,251 882
0.002 0.701
0.009 0.875
0.010 1.502
91.3 8.1 0.6 0
91.2 8.4 0.4 0
91.6 8.1 0.3 0
21.8 16.6 32.5
27.7 30.6 37.9
26.1 23.3 33.9
a
Data collection Space group Unit cell dimensions ˚) a, b, c (A a = b = g (˚) ˚) Wavelength (A ˚) Resolution (A Total no. of reflections No. of unique reflections No. of molecules in the asymmetric unit Multiplicity Data completenessb (%) Rmergec (%) I/s I Refinement statistics ˚) Resolution (A Rfactord (%) Rfree (%) Nonhydrogen atoms Protein Water r.m.s.d from ideality ˚) Bond lengths (A Bond angles (˚) Ramachandran plote Most favored (%) Additional allowed (%) Generously allowed (%) Disallowed (%) ˚ 2) B factors (A Peptide Protein Water molecule a
Values in parentheses refer to statistics in the outermost resolution shell. Data completeness = (no. of independent reflections)/(total theoretical number). c Rmerge = +i+hkl|Ii – , I . |/+i+hklIi, where Ii is the observed intensity and , I . is the average intensity of multiple observations of symmetryrelated reflections. d Rfactor = +hkl||Fo| – |Fc||/+hkl|Fo|, where Fo and Fc are the structure-factor amplitudes from the data and the model, respectively; Rfree is the R factor for a subset (5%) of reflections that was selected prior to refinement calculations and was not include in the refinement. e Ramachandran plots were generated by using the PROCHECK program of the CCP4i suite. b
Downloaded from http://www.jimmunol.org/ by guest on June 7, 2017
generating Nt-acetylated proteasomal digestion products (14). Hence, these Nt-acetylated polypeptides in the form of MHCassociated neoantigens stand a good chance to be recognized by T cells. This has indeed been illuminated in an Nt-acetylated MHC class II–restricted peptide derived from myelin basic protein, which stimulates murine T cells to elicit experimental autoimmune encephalomyelitis, whereas the nonacetylated form does not (15). A structural study subsequently suggested that the Nt-acetylation of this peptide is essential for MHC class II binding (16).
The Journal of Immunology cidated that Nt-acetylation exerts a destabilizing effect on peptide– MHC (pMHC) complex, thereby influencing TCR recognition.
Materials and Methods Peptide synthesis All peptides used in this study were synthesized and purified to .90% by ChinaPeptides (Shanghai, China). Each peptide was stored in lyophilized aliquots at 280˚C and dissolved in DMSO to a final concentration of 10– 50 mg/ml before use. A summary of the peptide characteristics is presented in Table I.
Expression, purification, crystallization and data collection
(NAc-SHVAVENAL) in our study was chosen based on its natural presentation by at least three allotypes of HLA-B39 (B*3901, B*3905, and B*3909), and its direct generation by the 20S proteasome as a major breakdown product of the corresponding parental protein (Table I) (17, 18). The structure of HLA-B*3901 bound with NAc-SL9 was solved in this study to a resolution of 1.8 ˚ (Table II). The overall structure of HLA-B*3901 resembles that A of other published class I allotypes, with a H chain containing three extracellular domains (a1, a2, and a3) for binding peptide ligands (Fig. 1A, 1B). To assess the influence of Nt-aceylation in the interaction of the acetylpeptide with MHC class I, we also solved the structures of HLA-B*3901 bound with the nonacetylated peptide SL9 (SHVAVENAL). Both the unmodified and Nt-acetylated peptide bind to HLA-B*3901 in a featured mode with a central bulge revealed by the unambiguous electron density (Fig. 2A, 2B). In both peptides, P2-His and Pc-Leu are completely buried as the primary anchor residues, whereas P3-Val also points downward toward pocket D of the Ag-binding groove and subdominantly contributes to
Downloaded from http://www.jimmunol.org/ by guest on June 7, 2017
Briefly, truncated HLA-B*3901 H chain (residues 1–274) and human b2microglobulin (b2m) were expressed respectively as inclusion bodies using BL21(DE3) strain of Escherichia coli and subsequently refolded and assembled in the presence of peptides as described previously (26, 27). After incubation at 277 K for 24–48 h, the soluble portion of the complexes were concentrated and purified by size exclusion and subsequently by anionexchange chromatography. The three pHLA-B*3901 complexes were finally concentrated to 4–8 mg/ml (20 mM Tris [pH 8] and 50 mM NaCl) and subsequently crystallized after being mixed in a 1: 1 ratio with crystallization reservoir solution (NAc-SL9: 0.1 M ammonium acetate, 0.1 M Bis-Tris [pH 6.5], and 17% [w/v] polyethylene glycol [PEG] 10,000; SL9: 0.1 M ammonium acetate, 0.1 M Bis-Tris [pH 6.5], and 20% [w/v] PEG 10,000; HL8: 0.2 M sodium formate, and 20% [w/v] PEG 3,350) by the hanging-drop vapor diffusion method at 277 K. For cryoprotection, each crystal was first soaked in reservoir solution supplemented with 17% (v/v) glycerol, and then flash-cooled and maintained at 100 K in a cryostream (28). X-ray diffraction data were collected at beamline BL17U on Shanghai Synchrotron Radiation Facility. Raw data were indexed, integrated, corrected for absorption, scaled, and merged using HKL2000 program (29).
5511
Structure determination, refinement, and analysis All crystals of pHLA-B*3901 belong to the P212121 space group. The structures were solved by molecular replacement method using MOLREP program with HLA-B*2705 (Protein Data Bank 2BST) as a search model (30), which were manually rebuilt with COOT program under the guidance of Fo-Fc and 2Fo-Fc electron density maps (31). Restrained refinements were performed with REFMAC5 program (30) and followed by isotropic atomic displacement parameter refinement and bulk solvent modeling in phenix.refine (32). During the process of model building and refinement, the peptides and water molecules were built into unambiguous electron density. The stereochemical quality of the structure was verified with PROCHECK program (33). Different from the NAc-SL9 complex, two clear solutions in both the rotation and translation functions corresponded to the two molecules in one asymmetric unit of SL9 or HL8 complex. A summary of X-ray data collection, refinement, and validation statistics are presented in Table II. Structural figures were produced with PyMOL program (DeLano Scientific) for displaying molecular three-dimensional model and electron density. Unless otherwise specified, figure generation and calculational analysis were performed based on one uniform molecule in the asymmetric units. The atomic coordinates of the crystal structures of HLA-B*3901NAc-SL9, HLA-B*3901SL9, and HLA-B*3901HL8 have been deposited in the Protein Data Bank (http://www.pdb.org/pdb/home/home.do) with accession number 4O2C (http://pdb.org/pdb/search/structidSearch.do?structureId=4O2C), 4O2E (http://pdb.org/pdb/search/structidSearch.do?structureId=4O2E), and 4O2F (http://pdb.org/pdb/search/structidSearch.do?structureId=4O2F), respectively.
Thermostability measurements using circular dichroism The thermostability of MHC class I complexes formed with Na-acetylpeptides and nonacetylpeptides were tested by circular dichroism (CD) spectroscopy. All complexes were refolded and purified beforehand as described above and measured at 150 mg/ml in a solution of 20 mM Tris (pH 8) and 50 mM NaCl. CD spectra at 218 nm were measured on a Chirascan spectrometer (Applied Photophysics) using a thermostatically controlled cuvette at temperature intervals of 0.1˚C at a rate of 1˚C/min between 25 and 90˚C. The denaturation curves were generated by nonlinear fitting with OriginPro 8.0 program (OriginLab).
Results Overall structures Eukaryotic proteins having an N-terminal serine are most frequently acetylated (11). The acetylserine-containing peptide NAc-SL9
FIGURE 1. Overall structure of HLA-B*3901 in complex with Naacetylpeptide. (A) Overview of HLA-B*3901NAc-SL9 structure. Cyan, a1 domain; green, a2 domain; chartreuse, a3 domain; blue, b2m; yellow, NAc-SL9, including an acetyl group (red) covalently linked to P1-Ser. (B) Vertical view of the HLA-B*3901NAc-SL9 peptide-binding surface with a1a2 platform in orthogonal model. White, a1-a2 platform; yellow, NAcSL9; black and red in ball-and-stick model, acetyl group. Pockets A–F are labeled in black letters. (C) Orientation of the NAc-SL9 and SL9 side chains relative to HLA-B*3901 and the TCR (refer to the direction sign above), viewing along the peptide from the N terminus to the C terminus. Arrows labeled with residues indicate side-chain orientation. Red “Ace” (acetyl) and blue “S1” (P1-Ser) lettering shows a significant positional swap between the acetyl and the P1-Ser side chain.
5512
Na-ACETYLPEPTIDE–HLA-B*3901 STRUCTURE AND FUNCTION
peptide binding (Fig. 1C). The surface-exposed residues in the central region of the peptide interact with the H chain via watermediated hydrogen (H)-bonds (Table III). Moreover, the side chains of P6-Glu and P7-Asn point upward and both comprise potential TCR contact sites. The third peptide of which we solved the structure in complex with HLA-B*3901 is HL8 (HVAVENAL), a natural truncated analog of NAc-SL9 isolated from the same HLAB39–bound peptide pool (18). The side chain orientations of HL8 residues are essentially identical to those of SL9 residues except for the absence of P1-Ser (Fig. 2C). Summarizing, there is no change in extended direction of the residue side chains between the NAc-SL9 and SL9/HL8 peptides except the P1 residue (Fig. 1C). Unique rotation of the Nt-acetylated P1 residue
FIGURE 2. Peptide conformations, interactions of the peptide NH2 termini within Ag-binding groove. The peptides have differently colored C-atoms (yellow, NAc-SL9; magenta, SL9; cyan, HL8) and the acetyl is colored pale cyan in all panels. (A–C) The 2Fo-Fc electron density maps of NAc-SL9 (A), SL9 (B), and HL8 (C) contoured at 1.0 s are shown as colored mesh viewed in profile with the a2 helix removed for clarity. (D–F) H-bonding interactions between the NH2 termini of peptides and the residues constituting components of pocket A. Green indicates H chain side chains. Cyan spheres indicate conserved water molecules. (G–I) Peptide-binding grooves are shown in surface representation with pocket A highlighted in green. The residues that compose pocket A are shown in lines representation and labeled. The P1 residues of three peptides (NAc-Ser in NAc-SL9, Ser in SL9, and His in HL8) are shown in stick representation.
Downloaded from http://www.jimmunol.org/ by guest on June 7, 2017
Most peptides presented on MHC class I have their terminal NH2 groups placed in pocket A (34). The free peptide NH2 group binds the class I molecules through two conserved H-bonds to Tyr7 and Tyr171, respectively, at the bottom of pocket A, essentially in a peptide sequence-independent mode (35). These interactions ensure the lowest Gibb’s free-energy for the peptide binding state, and were observed in the HLA-B*3901SL9 structure (Fig. 2E). However,
in the HLA-B*3901NAc-SL9 structure, the acetyl group added to the N terminus of P1-Ser is energetically unfavorable for burial within the pocket A, resulting in a unique rotation of the P1 dihedral angle c by ∼150˚, rotating the acetyl group into the position normally occupied by the P1 side chain. A superposition of the Ag-binding grooves reveals a considerable uplift of the P1 residue, which ˚ compared with its Ser counraises the NAc-Ser Ca-atom ∼1.4 A terpart in SL9. The methyl C-atom of the acetyl in NAc-SL9 is ˚ higher relative to the P1-Ser Og-atom in also elevated to 3.0 A SL9 (Fig. 3). Although NAc-SL9 undergoes conformational changes around the acetylation site, similar interactions to those seen in HLAB*3901SL9 are maintained at the bottom of pocket A. The rotation of the P1 residue eliminates the contacts of Tyr7 and Tyr171 with the NH2 group but the contacts are substituted by the hydroxyl group of NAc-Ser (Fig. 2D, 2E). Specifically, the acetyl interacts with a completely conserved Trp167 residue on the a2-helix of the H chain through an H-bond and indirectly through a water-mediated H-bond (Fig. 2D), both of which seem significant in stabilizing the acetyl binding. The rearrangement of the H-bonding network as well as the rotation of the P1 residue occurring in pocket A are
The Journal of Immunology
5513
Table III. Interactions between the peptides and the HLA-B*3901 residues Peptide Complex
NAc-SL9
Residue/ Moiety
Ace P1-Ser
P2-His P3-Val
Atom
O N Og O N Nd1 Nε2 N O O
P6-Glu P7-Asn P8-Ala
Oε1 Od1 O
P9-Leu
N O Oxt
SL-9
P1-Ser
N Og
P2-His
O N Nd1
P3-Val
Nε2 N O
P4-Ala P5-Val P6-Glu P7-Asn P8-Ala P9-Leu
Atoma
Trp167 Nε1 Asn63 Od1 Tyr7 Oh Tyr171 Oh Tyr159 Oh Asn63 Od1 Asn63 Od1 Ser24 Og Tyr99 Oh
(3.1) (2.9) (2.7) (2.7) (2.8) (3.2) (3.2) (3.3) (3.0)
Oxt
Van der Waals Contact Residuesb
Trp167 Nε1
Arg62, Asn63, Ile66, Thr163, Trp167 (11) Met5, Tyr7, Tyr59, Asn63, Tyr159, Trp167, Tyr171 (49)
Tyr7, Tyr9, Ser24, Glu45, Asn63, Ile66, Cys67, Tyr99, Tyr159 (76) Ile66, Arg97, Tyr99, Leu156, Tyr159 (36) Tyr9 Oh, Asn70 Nd2 Asn70 Od1, Arg97 Nh1, Arg97 Nh2 Thr69 O, Thr73 Og1 Trp147 Nε1
Lys146 Nz Trp147 Nε1 Ser77 Og Tyr84 Oh Thr143 Og1 Asn80 Nd2 Tyr84 Oh Lys146 Nz Tyr7 Oh Tyr171 Oh Arg62 Nh2 Asn63 Od1 Asn63 Nd2 Tyr159 Oh Asn63 Od1 Glu45 Oε1 Asn63 Od1 Ser24 Og Tyr99 Oh
(3.2) (3.0) (2.9) (2.8) (2.7) (2.8) (3.5) (2.8) (3.0) (2.9) (2.7) (3.0) (3.0) (2.6) (3.0) (3.4) (3.1) (3.3) (3.0)
Arg62, Ile66 (2) Val152, Gln155, Leu156 (13) Thr69, Thr73 (7) Thr73, Trp147, Ala150, Val152 (17) Thr73, Glu76, Ser77, Asn80, Lys146, Trp147 (23) Ser77, Asn80, Leu81, Tyr84, Leu95, Phe116, Tyr123, Thr143, Lys146, Trp147 (64)
Met5, Tyr7, Tyr59, Arg62, Asn63, Tyr159, Trp167, Tyr171 (51)
Tyr7, Tyr9, Ser24, Glu45, Asn63, Ile66, Cys67, Tyr99, Tyr159 (78)
Ile66, Arg97, Tyr99, Leu156, Tyr159 (40) Tyr9 Oh, Asn70 Od2, Asn70 Nd2 70
O Od1 O N O
Water-Mediated Bond Atom
Asn
97
Od1, Arg Nh1, Arg97 Nh2 Gln155 Oε1
Trp147 Nε1 Ser77 Og Tyr84 Oh Thr143 Og1 Lys146 Nz Asn80 Nd2 Tyr84 Oh Lys146 Nz
(3.0) (2.9) (2.7) (2.7) (3.4) (2.8) (3.5) (2.7)
Ile66 (3) Gln , Leu156 (13) 155
Thr69, Thr73 (11) Thr73, Trp147, Ala150, Val152 (18) Thr73, Glu76, Ser77, Asn80, Lys146, Trp147 (22) Ser77, Asn80, Leu81, Tyr84, Leu95, Phe116, Tyr123, Thr143, Lys146, Trp147 (59)
a
Numbers in parentheses are the length of hydrogen bonds. Numbers in parentheses are the amounts of van der Waals contacts.
b
unique features that have never been described in previous reports of MHC class I structures. Nt-acetylation alters the conformation of the peptide main chain In addition to the large rotation of the P1 residue, another sub˚ ) was observed in the central region of stantial elevation (1.2 A the peptide main chain of NAc-SL9 (Figs. 3, 4A), which shows the highest degree of solvent exposure for direct contact with the TCR. The O-atom of the acetyl interacts with the carbonyl O-atom of P2-His via another water-mediated H-bond (Figs. 2F, 4B). Thus, the acetyl group and the P2 main chain are constrained to each other, which stressfully results in deformation of the peptide main chain and an indirect lift of the P6-P7 residues.
A similar elevation of the peptide main chain also was observed when the two molecules in the same asymmetric unit of the HLAB*3901HL8 structure were compared (Fig. 4C). In both molecules, P1-His of HL8 occupies pocket B to serve as the N-terminal anchor residue without the canonical P1 interaction; the NH2 group of P1-His provides an H-bond with Tyr7 but not with Tyr171, and a water molecule bridges the NH2 group of HL8 and a Tyr159 on the a2-helix as a substitute for the carbonyl of the absent P1 residue (Fig. 2F). However, two distinct conformations of Arg62 were observed in these two molecules (Fig. 4C) (discussed below). One of them has a conformation as the Arg62 in HLA-B*3901SL9, and the other one switches its side chain to stretch over pocket A, and directly contacts the P1-His main chain by H-bonding interactions (Fig. 4D). The interactions appear to have the effect of
Downloaded from http://www.jimmunol.org/ by guest on June 7, 2017
P4-Ala P5-Val
Hydrogen Bond
5514
Na-ACETYLPEPTIDE–HLA-B*3901 STRUCTURE AND FUNCTION
FIGURE 3. Conformational comparison of the three peptides presented by HLA-B*3901. Peptide alignment by superposition of a1 helices of the HLA-B*3901 shows that the majority of conformational alterations are located in the NH2 termini and the central regions of peptide main chain. Yellow, NAc-SL9; magenta, SL9; cyan, HL8. The acetyl group is shown as ball-and-stick model and colored in pale cyan (C atoms) and red (O atom).
“pulling” the peptide up out of the binding site creating the same pattern as the water bridge in HLA-B*3901NAc-SL9 (Fig. 4B).
Comparison of the two HLA-B*3901 molecules in complex with SL9 and NAc-SL9 reveals that dramatic conformational differences in the H chain mainly focus on three residues in the a1-helix [i.e., Glu58, Gln65, and especially Arg62 (Fig. 5A, Supplemental Fig. 1)]. For HLA-B*3901SL9, the Arg62 side chain is captured and tethered down toward the peptide N terminus by an H-bond from the P1-Ser Og-atom. For NAc-SL9, in addition to the steric hindrance, the hydrophilic Arg62 guanidine group is not compatible with the hydrophobic environment created by the methyl moiety of the acetyl. Therefore, this Arg62 has to undergo a local reorientation so that its Cg-atom faces toward the methyl, thereby forming a hydrophobic interaction. The charged Arg62 guanidine group switches away and pulls the Gln65 over via an H-bond. Meanwhile, the flexible Glu58 side chain rotates to interact with its own carbonyl O-atom (Supplemental Fig. 1).
FIGURE 4. Two distinct conformations of peptides presented by HLAB*3901. (A and C) Elevated residues in the central region of peptides revealed by superposition of the a1 helices. (A) Peptide alignment of NAcSL9 (yellow) and SL9 (magenta). (B) Water-mediated H-bonds bridge the acetyl and the H2 main chain. Red frames in (A) and (B) correspond to the same area. (C) Peptide alignment of the two molecules in the same asymmetric unit of HLA-B*3901HL8. The peptides with H1 residues shown in stick and the Arg62s in their corresponding molecules were colored in cyan and green, respectively. (D) Arg62 interacts with the H2 main chain via two H-bonds. Blue frames in (C) and (D) correspond to the same area.
For ease of analysis, we have divided the conformational modes of Arg62 into different intramolecular locations. The position of the Arg62 in the HLA-B*3901SL9 is defined as position 1, where it is often found binding with TCRs (36), while that in the HLAB*3901NAc-SL9 is defined as position 2 (Fig. 5B). This conformational alteration of Arg62 also has been observed by others in two HLA-B8 structures (Fig. 5B) (37), one of which bound to a peptide with P1-Gly and its Arg62 is at position 1, whereas in the other, the P1-Phe sterically forces the Arg62 away to position 2 where it can engage and trigger various TCRs (38). Taking these previous findings into consideration, we hypothesized that in our structure, the acetyl moiety would provide a similar stereochemical effect as the phenyl group of Phe and that the Arg62 at position 2 might contact with an incoming TCR. Specially in one of the two molecules in the asymmetric unit of the HLA-B*3901HL8, Arg62 is at position 1, whereas in the other, Arg62 extends its side chain to position 3, toward the a2-helix covering the pocket A (Fig. 5B). These alternative conformations reveal the flexibility of the Arg62 in the absence of P1 residue. Thus, interestingly, the Arg62 with its mobile conformational modes in HLA-B*3901 structures can be seen as a structural knife-switch (Fig. 5C). The aliphatic moiety of Arg62 is switched “on” to position 1 for energetic contribution to peptide binding or “off” to position 2 for TCR engagement, respectively. Also, position 3 is in an intermediate state. It is tempting to speculate that these diverse modes might elicit qualitatively different TCR repertoires. Differential binding stability of Nt-acetylated and nonacetylated peptides To determine how Nt-acetylation affects the peptide-binding to MHC class I molecules, the thermostability of the three solved pHLA-B*3901 complexes was evaluated. In accordance with previous studies (17), the acetyl in NAc-SL9 exerted a negative influence on Ag-binding and thermodynamic stability of the pHLAB39 complex relative to SL9, DTm = 29.1˚C (Fig. 6A, Table I). Moreover, an excellent correlation was observed between the
Downloaded from http://www.jimmunol.org/ by guest on June 7, 2017
A “knife-switch” of Arg62
FIGURE 5. Residue switching around the acetylation site shown by superposition of the a1 helices. (A) Position of Glu58, Arg62, and Gln65 in the HLA-B*3901 bound to NAc-SL9 (orange) and SL9 (lemon). (B) Superposition of the Arg62 of different HLA-B molecules. Red dashed border frames a same position of the area with (A) but in a slightly different angle. Numbers 1–3 indicate three distinct conformational modes of the Arg62 in various HLA-B molecules. Orange, HLA-B*3901NAc-SL9; lemon, HLAB*3901SL9; light blue, two molecules in the asymmetric unit of HLAB*3901HL8; wheat, HLA-B8GGK (PDB 1AGD); chocolate, HLA-B8FLR (PDB 1M05); purple, ELS4–HLA-B*3501EPLP (PDB 2NX5). The Cg-Cz arm of Arg62 in HLA-B*3901NAc-SL9 deflects an angle of 152˚ from that in HLA-B*3901SL9. Translucent light blue sector indicates a region of flexibility of Arg62 in HLA-B*3901HL8 molecules. (C) An on/off knife-switch symbolizing the Arg62 in position 1 and position 2, respectively.
The Journal of Immunology thermal denaturation curves and the volumes of peptide-binding grooves for all three complexes (Fig. 6A, Table IV). The Ntacetylation of NAc-SL9 observably enlarges the groove in both volume and surface area. The volume of pocket A in the HLAB*3901NAc-SL9 is .50% larger than that in the HLA-B*3901SL9 because of the side-chain reorientation of the Arg62 (Fig. 2G, 2H, Table IV). Also, the elimination of the interactions from Arg62 might explain the lowered peptide binding stability. For HLAB*3901HL8, one of the alternative conformations of Arg62 in contact with the peptide main chain seems to compensate the absence of the canonical P1 residue for suboptimal peptidic N-terminal interactions as described above (Fig. 4D). To further test whether Nt-acetylation is universal in decreasing the pMHC stability, we subsequently carried out a series of thermostability measurements with different pMHC sets (Table I). The thermostability of epitopes with Ala, Thr, Cys, Val, and Met (that can be frequently acetylated in vivo) at P1 residues, respectively, bound to HLA-A*1101, -A*3303, -A*2402, -B*3501, and -A*0201
5515 was compared with that of their Nt-acetylated counterparts. Ntacetylation of all these peptides causes dramatic reductions of the pMHC thermostability by 8.8˚C or more in midpoint transition temperature (Tm) (Fig. 6D–I). The similar phenomenon was observed for HLA-A*0201 and H-2Kb when in complex with a peptide containing a P1-Ser (Fig. 6B, 6C). In conclusion, Nt-acetylation decreases the pMHC stability in a class I allele– and epitopeindependent manner. Potential influence of N-acetylation on T cell engagement Nt-acetylation is predicted to have a significant influence on T cell recognition in three ways. First, incorporation of the acetyl group blocks the positive peptide charge, thereby altering the distribution of electrostatic potential and it creates a prominent solvent exposure around the peptide N terminus. This may contribute to putative TCR docking sites, based on previously determined TCR–pHLA complex structures (Fig. 7A, 7B) (36). Second, the conformationally altered central region of the Na-acetylpeptide may have different
Downloaded from http://www.jimmunol.org/ by guest on June 7, 2017
FIGURE 6. Thermostability of acetylpeptide- and nonacetylpeptide-MHC complexes as revealed by CD spectroscopy. The thermostability with an Ntacetylated peptide is indicated by a red line and that with their nonacetylated counterpart with a blue line in all panels. (A) Thermostability measurements of three pHLA-B*3901 complexes with the HLA-B*3901HL8 complex represented by a green line. (B–H) Thermostability measurements of different pMHC sets that were chosen from the Immune Epitope Database (IEDB, www.immuneepitope.org). (I) Temperature reductions at the Tm of the complexes after Ntacetylation of peptides. Differences in mean values were evaluated for statistical significance (p , 0.01) by Student two-tailed t test.
Na-ACETYLPEPTIDE–HLA-B*3901 STRUCTURE AND FUNCTION
5516 Table IV.
Volume and surface area quantitations of the pocket A and the peptide-binding grooves Pocket A
Peptide-Binding Groove
Complex
˚ 2) Surface Area (A
˚ 3) Volume (A
˚ 2) Surface Area (A
˚ 3) Volume (A
NAc-SL9 SL9 HL8
150.8 125.9 132.3
236.1 150.5 167.7
1016.8 867.1 1010.7
1592.7 1360.6 1517.6
Pocket/groove volumes and surface areas were measured using the Computed Atlas of Surface Topography of proteins ˚ (http://sts.bioengr.uic.edu/castp/calculation.php) (39). (CASTp) server with a probe radius 1.4 A
the acetylated P1-Met based on the thermal instability (Fig. 6H). Besides the accommodation of the acetyl moiety, Nt-acetylation is presumed to decrease the stability of the pHLA-B*3901 complex as a result of the conformational switch of the Arg62. Arg62 in the
Downloaded from http://www.jimmunol.org/ by guest on June 7, 2017
interactions with the TCR, and the lift of the peptide main chain may influence the TCR binding and correspondingly the TCR repertoires (41). Third, the conformational changes within the MHC H chain, indirectly caused by the peptide Nt-acetylation, position the negatively charged Glu58 slightly outward; most notably, the positively charged Arg62 is positioned as a predominant surface residue toward the central region of the peptide, thereby shifting the charge footprint of Ag presentation surface even more (Fig. 7C, 7D). HLA consensus Glu58 is retained in most TCR engagement (42), Arg62 and Gln65 invariably interacts with the TCR CDR1a or CDR3a loops whenever present on the MHC (37, 42). These analyses indicated that not only the acetyl moiety but also the altered residues of MHC class I are highly accessible for interaction with CDR loops of an incoming TCR. To further address this possibility, we superposed the previously solved TCRs in complex with other HLA-B molecules (B*3501 and B8) onto the HLA-B*3901NAc-SL9 structure (Fig. 7E, 7F). The modeled complex shows close proximity of the CDR1a and CDR3a loops to the acetyl moiety and the MHC residues near the acetylation site, from which the Arg62 is engulfed by the CDR loops signifying it as a prominent binding target for TCR recognition.
Discussion Our results here provide the structural and thermodynamic insights into the presentation of Nt-acetylated peptides by MHC class I molecules. The structure of the Na-acetylpeptide in complex with HLA-B*3901 outlines a molecular interpretation of the reduced stability of MHC class I–bound Nt-acetylated peptides and also highlights a potential influence of Nt-acetylation on antigenic identity and T cell recognition. In addition, the structure elucidation of HLA-B*3901, the predominant B39 subtype, also is valuable in studying immune diseases associated with this MHC allele. In a previous report, the Nt-formyl group on an Nt-formylated peptide binds to the bottom of the peptide-binding groove of the murine MHC class I H2-M3 playing an anchoring role for MHC class I binding (Supplemental Fig. 2A) (5). In our study, the methyl and carbonyl groups of the acetyl are rotated upwards like two arms that push the peptide-binding groove open (Fig. 2G, Supplemental Fig. 2B), thereby altering its immunogenicity at the expense of the pMHC stability. The thermostability we tested from seven human and one murine complexes indicates a general feature of Na-acetylpeptide in weakening the binding affinity to MHC class I, which could be revealed by the gel-filtration chromatography of pMHC refolding assays as well (Supplemental Fig. 3). Their instability would partially explain why, as yet, such epitopes are rarely found. Within N-terminal residues of eukaryotic proteins, Ser is the most frequently acetylated in vivo (11). The Ala, Thr, Cys, and Val residues can also be Nt-acetylated and have small side chains like Ser. Thus, the rotation of P1 residues observed in the pHLA-B*3901 complex with an acetylated P1-Ser could very well be a general mode in Na-acetylpeptide binding. In contrast, the long side chain of Met precludes it from being rotated into pocket A, but a certain reorientation is presumed to take place in
FIGURE 7. Potential influence of Na-acetylpeptide presentation on antigenic identification and TCR recognition. (A and B) Surface representation of HLA-B*3901 bound with SL9 (A) and NAc-SL9 (B). The peptides are shown in surface representation with red/blue chargesmoothed potential [red, negative; blue, positive; calculated with GRASP (40)]. The residues of the H chains highlighted in purple indicate the putative TCR footprint area, based on the ELS4 TCR–HLA-B*3501EPLP structure (PDB 2NX5). Red arrows point to the peptide acetylation sites. (C and D) Electrostatic potential molecular surface representation of HLAB*3901 H chains bound with SL9 (C) and NAc-SL9 (D). Black dashes encircle the regions composed of Glu58, Arg62, and Gln65. (E and F) Proximity of CDR loops to the Nt-acetylation site. Overlay of CDR loops (red, CDR1a; blue, CDR3a) of the TCRs: ELS4 (PDB 2NX5), LC13 (PDB 1MI5), CF34 (PDB 3FFC), and RL42 (PDB 3SJV) onto HLAB*3901NAc-SL9 complex by superposition of the HLA a1 helices. (F) is consistent with (E) but viewing along the peptide from the N terminus to the C terminus. Peptide-binding grooves are shown both in cartoon and translucent molecular surface representation. The side chains of critical residues (green, Glu58/Arg62/Gln65; yellow, NAc-Ser) that potentially influence TCR engagement are shown in stick representation. Yellow loops indicate NAc-SL9 main chain; ball-and-sticks colored black and red indicate acetyl groups (Ace).
The Journal of Immunology Table V.
5517 Polymorphism of amino acids in position 62 of HLA class I protein sequences Frequency (%)a
Amino Acid in Position 62
Arg Gln Glu Others
HLA-A (2579)
HLA-B (3285)
HLA-C (2133)
Total HLA Class I (7997)
17.37 37.73 16.83 18.07
94.67 0.09 — 5.24
99.20 0.28 — 0.52
70.95 12.28 5.43 11.34
The data statistics of HLA alleles is based on the international ImMunoGeneTics information system (IMGT/HLA) database (http://www.ebi.ac.uk/ipd/imgt/hla/). a Numbers in parentheses are the amounts of alleles.
peptides with the size of MHC class I ligands (8–11 aa) as neoepitopes for CD8+ T cells, represent one of the possible roles of the Nt-acetylated digestion products. The vast armory of intracellular proteins that are frequently Nt-acetylated can create a large pool of Na-acetylpeptides for Ag presentation and T cell surveying. The Nt-acetylation potentially impacts the TCR-MHC interaction in three different aspects: 1) the direct interaction of the solvent-exposed acetyl moiety; 2) the altered conformation of the central region of the peptide main chain; and 3) the conformational switches of the MHC residues. The Nt-acetylation creation of a distinctive pMHC landscape and participation in a potential binding element for TCR engagement
FIGURE 8. Structure-based sequence alignment of HLA-B*3901 and other HLA class I molecules. Above the blocks of sequences, symbols correspond to the secondary structure of B*3901 (squiggles indicate a-helices, arrows indicate b-strands, and “TT” letters indicate strict b-turns). Residues in white characters highlighted on a red background are strictly conserved in the column, whereas those well conserved within a group (with similarity . 70%) are in red characters on a white background, which are all rendered with blue frames. Green numbers below the sequences denote residues that form disulfide bonds. Residues that compose pocket A are marked with blue triangles. The sequences were aligned with ClustalW2 (http://www.ebi.ac.uk/Tools/msa/ clustalw2/) (43) and visualized using ESPript 2.2 (http://espript.ibcp.fr/) (44).
Downloaded from http://www.jimmunol.org/ by guest on June 7, 2017
a1-helix is largely conserved in almost all HLA-B and -C allotypes (Table V). For other HLA class I (Table V, Fig. 8), the long charged side chains of the residues in position 62 (Glu62 of A24 and Gln62 of A11 and so on) also may interact with the acetyl. Hence, the residue in position 62 plays a key role in the interaction between acetyl group and the H chain, which may influence not only the Na-acetylpeptide binding to HLA molecules but also the TCR docking. The discoveries that intracellular proteins with Ac-N-degrons are inhibited from being secreted (13) and then are degraded via ubiquitylation (12) raise many questions on the biological significance of acetylation-mediated proteolysis (14). The Nt-acetylated
5518
Na-ACETYLPEPTIDE–HLA-B*3901 STRUCTURE AND FUNCTION
Acknowledgments We thank Yue Liu for excellent suggestions regarding the study, Joel Haywood for comments on the manuscript, and the staff at the Shanghai Synchrotron Radiation Facility beamline 17U for assistance.
Disclosures The authors have no financial conflicts of interest.
References 1. Rock, K. L., and A. L. Goldberg. 1999. Degradation of cell proteins and the generation of MHC class I-presented peptides. Annu. Rev. Immunol. 17: 739– 779. 2. Engelhard, V. H., M. Altrich-Vanlith, M. Ostankovitch, and A. L. Zarling. 2006. Post-translational modifications of naturally processed MHC-binding epitopes. Curr. Opin. Immunol. 18: 92–97. 3. Doyle, H. A., and M. J. Mamula. 2001. Post-translational protein modifications in antigen recognition and autoimmunity. Trends Immunol. 22: 443–449. 4. Anderton, S. M. 2004. Post-translational modifications of self antigens: implications for autoimmunity. Curr. Opin. Immunol. 16: 753–758. 5. Wang, C. R., A. R. Castan˜o, P. A. Peterson, C. Slaughter, K. F. Lindahl, and J. Deisenhofer. 1995. Nonclassical binding of formylated peptide in crystal structure of the MHC class Ib molecule H2-M3. Cell 82: 655–664. 6. Glithero, A., J. Tormo, J. S. Haurum, G. Arsequell, G. Valencia, J. Edwards, S. Springer, A. Townsend, Y. L. Pao, M. Wormald, et al. 1999. Crystal structures of two H-2Db/glycopeptide complexes suggest a molecular basis for CTL crossreactivity. Immunity 10: 63–74. 7. Speir, J. A., U. M. Abdel-Motal, M. Jondal, and I. A. Wilson. 1999. Crystal structure of an MHC class I presented glycopeptide that generates carbohydratespecific CTL. Immunity 10: 51–61. 8. Mohammed, F., M. Cobbold, A. L. Zarling, M. Salim, G. A. Barrett-Wilt, J. Shabanowitz, D. F. Hunt, V. H. Engelhard, and B. E. Willcox. 2008. Phosphorylation-dependent interaction between antigenic peptides and MHC class I: a molecular basis for the presentation of transformed self. Nat. Immunol. 9: 1236–1243.
9. Petersen, J., S. J. Wurzbacher, N. A. Williamson, S. H. Ramarathinam, H. H. Reid, A. K. Nair, A. Y. Zhao, R. Nastovska, G. Rudge, J. Rossjohn, and A. W. Purcell. 2009. Phosphorylated self-peptides alter human leukocyte antigen class I‑restricted antigen presentation and generate tumor-specific epitopes. Proc. Natl. Acad. Sci. USA 106: 2776–2781. 10. Beltrami, A., M. Rossmann, M. T. Fiorillo, F. Paladini, R. Sorrentino, W. Saenger, P. Kumar, A. Ziegler, and B. Uchanska-Ziegler. 2008. Citrullination-dependent differential presentation of a self-peptide by HLA-B27 subtypes. J. Biol. Chem. 283: 27189–27199. 11. Polevoda, B., and F. Sherman. 2003. N-terminal acetyltransferases and sequence requirements for N-terminal acetylation of eukaryotic proteins. J. Mol. Biol. 325: 595–622. 12. Hwang, C. S., A. Shemorry, and A. Varshavsky. 2010. N-terminal acetylation of cellular proteins creates specific degradation signals. Science 327: 973–977. 13. Forte, G. M., M. R. Pool, and C. J. Stirling. 2011. N-terminal acetylation inhibits protein targeting to the endoplasmic reticulum. PLoS Biol. 9: e1001073. 14. Sriram, S. M., B. Y. Kim, and Y. T. Kwon. 2011. The N-end rule pathway: emerging functions and molecular principles of substrate recognition. Nat. Rev. Mol. Cell Biol. 12: 735–747. 15. Zamvil, S. S., D. J. Mitchell, A. C. Moore, K. Kitamura, L. Steinman, and J. B. Rothbard. 1986. T-cell epitope of the autoantigen myelin basic protein that induces encephalomyelitis. Nature 324: 258–260. 16. He, X. L., C. Radu, J. Sidney, A. Sette, E. S. Ward, and K. C. Garcia. 2002. Structural snapshot of aberrant antigen presentation linked to autoimmunity: the immunodominant epitope of MBP complexed with I-Au. Immunity 17: 83–94. 17. Yag€ue, J., I. Alvarez, D. Rognan, M. Ramos, J. Va´zquez, and J. A. de Castro. 2000. An N-acetylated natural ligand of human histocompatibility leukocyte antigen (HLA)-B39: classical major histocompatibility complex class I proteins bind peptides with a blocked NH2 terminus in vivo. J. Exp. Med. 191: 2083– 2092. 18. Yague, J., A. Marina, J. Vazquez, and J. A. Lopez De Castro. 2001. Major histocompatibility complex class I molecules bind natural peptide ligands lacking the amino-terminal binding residue in vivo. J. Biol. Chem. 276: 43699–43707. 19. Webb, A. I., M. A. Dunstone, W. Chen, M. I. Aguilar, Q. Chen, H. Jackson, L. Chang, L. Kjer-Nielsen, T. Beddoe, J. McCluskey, et al. 2004. Functional and structural characteristics of NY-ESO-1‑related HLA A2‑restricted epitopes and the design of a novel immunogenic analogue. J. Biol. Chem. 279: 23438– 23446. 20. Degano, M., K. C. Garcia, V. Apostolopoulos, M. G. Rudolph, L. Teyton, and I. A. Wilson. 2000. A functional hot spot for antigen recognition in a superagonist TCR/MHC complex. Immunity 12: 251–261. 21. Li, L., and M. Bouvier. 2004. Structures of HLA-A*1101 complexed with immunodominant nonamer and decamer HIV-1 epitopes clearly reveal the presence of a middle, secondary anchor residue. J. Immunol. 172: 6175–6184. 22. Lim, J. B., M. Provenzano, O. H. Kwon, M. Bettinotti, L. Caruccio, D. Nagorsen, and D. Stroncek. 2006. Identification of HLA-A33‑restricted CMV pp65 epitopes as common targets for CD8+ CMV-specific cytotoxic T lymphocytes. Exp. Hematol. 34: 296–307. 23. Oka, Y., A. Tsuboi, T. Taguchi, T. Osaki, T. Kyo, H. Nakajima, O. A. Elisseeva, Y. Oji, M. Kawakami, K. Ikegame, et al. 2004. Induction of WT1 (Wilms’ tumor gene)-specific cytotoxic T lymphocytes by WT1 peptide vaccine and the resultant cancer regression. Proc. Natl. Acad. Sci. USA 101: 13885–13890. 24. Smith, K. J., S. W. Reid, D. I. Stuart, A. J. McMichael, E. Y. Jones, and J. I. Bell. 1996. An altered position of the a2 helix of MHC class I is revealed by the crystal structure of HLA-B*3501. Immunity 4: 203–213. 25. Collins, E. J., D. N. Garboczi, and D. C. Wiley. 1994. Three-dimensional structure of a peptide extending from one end of a class I MHC binding site. Nature 371: 626–629. 26. Garboczi, D. N., D. T. Hung, and D. C. Wiley. 1992. HLA-A2‑peptide complexes: refolding and crystallization of molecules expressed in Escherichia coli and complexed with single antigenic peptides. Proc. Natl. Acad. Sci. USA 89: 3429–3433. 27. Liu, J., S. Zhang, S. Tan, Y. Yi, B. Wu, B. Cao, F. Zhu, C. Wang, H. Wang, J. Qi, and G. F. Gao. 2012. Cross-allele cytotoxic T lymphocyte responses against 2009 pandemic H1N1 influenza A virus among HLA-A24 and HLA-A3 supertype‑positive individuals. J. Virol. 86: 13281–13294. 28. Parkin, S., and H. Hope. 1998. Macromolecular cryocrystallography: cooling, mounting, storage and transportation of crystals. J. Appl. Cryst. 31: 945–953. 29. Otwinowski, Z., and W. Minor. 1997. Processing of X-ray diffraction data collected in oscillation mode. Methods Enzymol. 276: 307–326. 30. Collaborative Computational Project, Number 4. 1994. The CCP4 suite: programs for protein crystallography. Acta Crystallogr. D Biol. Crystallogr. 50: 760–763. 31. Emsley, P., and K. Cowtan. 2004. Coot: model-building tools for molecular graphics. Acta Crystallogr. D Biol. Crystallogr. 60: 2126–2132. 32. Adams, P. D., R. W. Grosse-Kunstleve, L. W. Hung, T. R. Ioerger, A. J. McCoy, N. W. Moriarty, R. J. Read, J. C. Sacchettini, N. K. Sauter, and T. C. Terwilliger. 2002. PHENIX: building new software for automated crystallographic structure determination. Acta Crystallogr. D Biol. Crystallogr. 58: 1948–1954. 33. Laskowski, R. A., M. W. Macarthur, D. S. Moss, and J. M. Thornton. 1993. PROCHECK: a program to check the stereochemical quality of protein structures. J. Appl. Cryst. 26: 283–291. 34. Madden, D. R. 1995. The three-dimensional structure of peptide‑MHC complexes. Annu. Rev. Immunol. 13: 587–622.
Downloaded from http://www.jimmunol.org/ by guest on June 7, 2017
described in our results highlights needs for further investigation into the Na-acetylpeptide–specific TCR repertoires. Although the apparent instability of MHC class I molecules for binding Nt-acetylated self-ligands is likely to be a mechanism of immune tolerance, it cannot yet rule out the possibility that Nt-acetylation of some self peptides are not present during the T cell precursor negative selection in the thymus thus allowing autoreactive T cells to migrate to the periphery. Once in the periphery, T cells encounter the Na-acetylpeptides, and it is then viewed as “foreign,” which will result in autoimmunity. Conversely, if some Nt-acetylated self-Ags or peptides arise temporally in the course of T cell development, the unmodified form of such peptides under disordered or pathological conditions will become neoantigens to which the immune system has never been exposed or tolerated in the periphery. Certain processes that induce Nt-acetylation for proteolysis, such as apoptosis, do take place in the thymus (3). Furthermore, there are many examples of viral protein Ntacetylation that normally uses the Nt-acetylation system of the host eukaryotic cells (11). For instance, the N-terminal peptides detected by Hutchinson et al. (45) from PB1, PA, NP, M1, M2, NS1, and NEP of influenza A virus and from PA, NP, M1, NS1 and NEP of influenza B virus can be acetylated as well as eukaryotic proteins. According to our estimate, some other viral Na-acetylpeptides also could be important MHC class I–associated epitopes displayed during virus infections (Supplemental Table I). Thus, an acetylated form of the N-terminal epitope corresponding with a certain viral Ag designed into epitope vaccines may gain an antivirus utility. To identify more acetylated epitopes requires further exploration of the immunopeptidome, and current algorithms of MHC-associated epitopes prediction should be improved to take account of Nt-acetylation. Na-Acetylpeptides represent a library of new potential vaccine candidates that are of considerable interest to be unearthed in the future.
The Journal of Immunology 35. Matsumura, M., D. H. Fremont, P. A. Peterson, and I. A. Wilson. 1992. Emerging principles for the recognition of peptide antigens by MHC class I molecules. Science 257: 927–934. 36. Tynan, F. E., H. H. Reid, L. Kjer-Nielsen, J. J. Miles, M. C. Wilce, L. Kostenko, N. A. Borg, N. A. Williamson, T. Beddoe, A. W. Purcell, et al. 2007. A T cell receptor flattens a bulged antigenic peptide presented by a major histocompatibility complex class I molecule. Nat. Immunol. 8: 268–276. 37. Webb, A. I., N. A. Borg, M. A. Dunstone, L. Kjer-Nielsen, T. Beddoe, J. McCluskey, F. R. Carbone, S. P. Bottomley, M. I. Aguilar, A. W. Purcell, and J. Rossjohn. 2004. The structure of H-2Kb and Kbm8 complexed to a herpes simplex virus determinant: evidence for a conformational switch that governs T cell repertoire selection and viral resistance. J. Immunol. 173: 402–409. 38. Gras, S., P. G. Wilmann, Z. Chen, H. Halim, Y. C. Liu, L. Kjer-Nielsen, A. W. Purcell, S. R. Burrows, J. McCluskey, and J. Rossjohn. 2012. A structural basis for varied ab TCR usage against an immunodominant EBV antigen restricted to a HLA-B8 molecule. J. Immunol. 188: 311–321. 39. Dundas, J., Z. Ouyang, J. Tseng, A. Binkowski, Y. Turpaz, and J. Liang. 2006. CASTp: computed atlas of surface topography of proteins with structural and
5519
40.
41. 42.
43.
44. 45.
topographical mapping of functionally annotated residues. Nucleic Acids Res. 34 (Web Server issue): W116‑W118. Nicholls, A., K. A. Sharp, and B. Honig. 1991. Protein folding and association: insights from the interfacial and thermodynamic properties of hydrocarbons. Proteins 11: 281–296. Turner, S. J., P. C. Doherty, J. McCluskey, and J. Rossjohn. 2006. Structural determinants of T-cell receptor bias in immunity. Nat. Rev. Immunol. 6: 883–894. Marrack, P., J. P. Scott-Browne, S. Dai, L. Gapin, and J. W. Kappler. 2008. Evolutionarily conserved amino acids that control TCR-MHC interaction. Annu. Rev. Immunol. 26: 171–203. Larkin, M. A., G. Blackshields, N. P. Brown, R. Chenna, P. A. McGettigan, H. McWilliam, F. Valentin, I. M. Wallace, A. Wilm, R. Lopez, et al. 2007. Clustal W and Clustal X version 2.0. Bioinformatics 23: 2947–2948. Gouet, P., E. Courcelle, D. I. Stuart, and F. Me´toz. 1999. ESPript: analysis of multiple sequence alignments in PostScript. Bioinformatics 15: 305–308. Hutchinson, E. C., E. M. Denham, B. Thomas, D. C. Trudgian, S. S. Hester, G. Ridlova, A. York, L. Turrell, and E. Fodor. 2012. Mapping the phosphoproteome of influenza A and B viruses by mass spectrometry. PLoS Pathog. 8: e1002993.
Downloaded from http://www.jimmunol.org/ by guest on June 7, 2017
Supplemental Data Table S1. Potential viral Nα-acetylepitopes and their binding affinity to HLA class I evaluated by Nt-acetylation site prediction Sequencea SHVAVENALd MDVNPTLLF MEQEQGTPW MEQEQGTPW MERIKELRDL MERIKELRDL MEDFVRQCF MEDFVRQCF ASQGTKRSY ASQGTKRSY SLLTEVETYVL SLLTEVETYV SLLTEVETY SLLTEVETPT MDSNTVSSF MFIFLLFLTL MFIFLLFLTL MESLVLGV MESLVLGV MESLVLGV MIHSVFLLMFL MIHSVFLLMF MIHSVFLLM MIHSVFLL ASPAAPRAVSF MLPFVQERIGL MLPFVQERI MLPFVQER AFSASLFK AFSASLFK MEESLMDV MRVQRPPTLLL MRVQRPPTLLL MRVQRPPTLL MRVQRPPTLL MRVQRPPTL MRVQRPPTL MRVQRPPTL MDYVSLLNQIW MDYVSLLNQIW
Source organism
Antigen protein
Position
HLA restriction
IC50 (nM) b
Nt-acetylation prediction scorec
Homo sapiens Influenza A virus Influenza A virus Influenza A virus Influenza A virus Influenza A virus Influenza A virus Influenza A virus Influenza A virus Influenza A virus Influenza A virus Influenza A virus Influenza A virus Influenza A virus Influenza A virus SARS-CoV SARS-CoV SARS-CoV SARS-CoV SARS-CoV MERS-CoV MERS-CoV MERS-CoV MERS-CoV MERS-CoV MERS-CoV MERS-CoV MERS-CoV MERS-CoV MERS-CoV MERS-CoV MERS-CoV MERS-CoV MERS-CoV MERS-CoV MERS-CoV MERS-CoV MERS-CoV MERS-CoV MERS-CoV
RNA helicase DBX PB1 PB-F2 PB-F2 PB2 PB2 PA PA NP NP M1 M1 M1 M2 NS1/NS2 S protein S protein ORF1ab protein ORF1ab protein ORF1ab protein S protein S protein S protein S protein N protein E protein E protein E protein NS3D protein NS3D protein NS3C protein NS3A protein NS3A protein NS3A protein NS3A protein NS3A protein NS3A protein NS3A protein NS3B protein NS3B protein
2-10 1-9 1-9 1-9 1-10 1-10 1-9 1-9 2-10 2-10 2-12 2-11 2-10 2-11 1-9 1-10 1-10 1-8 1-8 1-8 1-11 1-10 1-9 1-8 2-12 1-11 1-9 1-8 2-9 2-9 1-8 1-11 1-10 1-10 1-10 1-9 1-9 1-9 1-11 1-11
B*3901 B*4402 B*1801 B*4402 B*4001 B*0801 B*1801 B*4402 B*1501 B*1517 A*0201 A*0201 B*1501 A*0201 B*3501 A*0201 A *2402 B*4001 B*4402 B*1801 A*0201 B*1501 B*3501 A*0201 B*1501 A*0201 A*0201 A*6801 A*0301 A*1101 B*4001 B*2705 B*3901 B*2705 B*3901 B*0801 B*2705 B*3901 B*4402 B*5801
10 975 15 20 1954 1672 27 94 97 53 153 7 216 194 683 795 955 129 268 424 375 91 263 413 210 1177 275 66 305 42 250 151 19 53 29 271 338 13 230 212
0.492 — — — — — — —
0.465 0.465 0.507 0.507 0.507 0.507 — — — — — — — — — —
0.468 — — —
0.487 0.487 — — — — — — — — — — 1
MEPVDPRLEPW MGARASVL MGGKWSKSSVI MENRWQVMIVW MENRWQVMIVW MENRWQVMIVW MENRWQVMI MENRWQVMI MENRWQVM MENRWQVM MQPIPIVAIV MEPGRNQLF MEPGRNQLF MEPGRNQLF MGSQVSTQR AARLCCQL MESTTSGFL MESTTSGFL MPLSYQHFRKL MPLSYQHFRKL MPLSYQHFRKL MPLSYQHFRKL MPLSYQHFR MPLSYQHF MPLSYQHF MDIDPYKEF MQLFPLCLII MQLFPLCLII MQLFPLCLI MQLFPLCL STNPKPQRKTK STNPKPQRK STNPKPQRK STNPKPQR STNPKPQR MNNQRKKTG MEDQAYDQYL MEDQAYDQYL MEDQAYDQY MEDQAYDQY MEDQAYDQY MMSLLTPAVLL MMSLLTPAVLL MMSLLTPAVL MMSLLTPAVL MMSLLTPAVL
HIV HIV HIV HIV HIV HIV HIV HIV HIV HIV HIV HIV HIV HIV EV71 HBV HBV HBV HBV HBV HBV HBV HBV HBV HBV HBV HBV HBV HBV HBV HCV HCV HCV HCV HCV Dengue virus Bunyamwera virus Bunyamwera virus Bunyamwera virus Bunyamwera virus Bunyamwera virus Bunyamwera virus Bunyamwera virus Bunyamwera virus Bunyamwera virus Bunyamwera virus
Tat Gag Nef Vif Vif Vif Vif Vif Vif Vif Vpu Env Env Env Polyprotein X protein S protein S protein Polymerase Polymerase Polymerase Polymerase Polymerase Polymerase Polymerase Core antigen Precore protein Precore protein Precore protein Precore protein Polyprotein Polyprotein Polyprotein Polyprotein Polyprotein Polyprotein RNA polymerase L RNA polymerase L RNA polymerase L RNA polymerase L RNA polymerase L NS-S protein NS-S protein NS-S protein NS-S protein NS-S protein
1-11 1-8 1-11 1-11 1-11 1-11 1-9 1-9 1-8 1-8 1-10 1-9 1-9 1-9 1-9 2-9 1-9 1-9 1-11 1-11 1-11 1-11 1-9 1-8 1-8 1-9 1-10 1-10 1-9 1-8 2-12 2-10 2-10 2-9 2-9 1-9 1-10 1-10 1-9 1-9 1-9 1-11 1-11 1-10 1-10 1-10
B*4402 B*3901 B*0801 B*4402 B*5801 B*1801 B*4001 B*4402 B*4001 B*4402 A*0201 A*2402 B*4402 B*1801 A*6801 B*1402 B*4001 B*4402 B*0702 B*0801 B*3501 B*5101 A*6801 B*5101 B*3501 B*4402 A*0201 B*3901 A*0201 B*3901 A*1101 A*0301 A*1101 A*1101 A*6801 B*0801 B*4402 B*4001 B*4402 B*3501 B*1801 A*0201 B*3901 A*0201 B*1501 B*3901
109 773 1563 15 144 477 224 40 356 158 789 1132 837 728 97 1759 14 392 33 35 108 474 29 409 25 394 349 501 127 67 126 301 30 111 93 1671 416 56 113 343 141 364 60 49 38 15
— — — — — — — — — — — — — — —
0.473 — — — — — — — — — — — — — —
0.508 0.508 0.508 0.508 0.508 — — — — — — — — — — — 2
MMSLLTPAV MMSLLTPAV MRILILLLAV MRILILLLAV MRILILLLA MYNVFQMAVW MYRLRRKRAAP MYRLRRKRAA MYRLRRKRA SEENPCPRNIF SEENPCPRNIF SEENPCPRNI MGSPGRGRCTW METLANRL METLANRL SIRAKRRKRAS MHGNRPSL TAPRSRAPTTR TATPLTNLFLR TATPLTNLFLR TATPLTNLF TATPLTNLF SAREPAGR ASYPCHQH VPQALLFVPL VPQALLFVPL MLDPGEVY MLDPGEVY MLDPGEVY MNFLRKIVK MNFLRKIVK SKIFVNPSAIR SKIFVNPSAI SYLRYYNML VYKLVLLFCI
MYSLVFVILM MYKKLITFLF MFMYPEFAR TLVQHVVTIK SMKYLMLLFA a b
Bunyamwera virus Bunyamwera virus Bunyamwera virus Bunyamwera virus Bunyamwera virus HPV-1 HPV-1 HPV-1 HPV-1 HPV-2 HPV-2 HPV-2 HPV-2 HPV-2 HPV-2 HPV-2 HPV-2 HSV-1 HSV-1 HSV-1 HSV-1 HSV-1 HSV-1 HSV-1 Rabies virus Rabies virus Rabies virus Rabies virus Rabies virus Rabies virus Rabies virus Rabies virus Rabies virus Cowpox virus Cowpox virus Vaccinia virus Vaccinia virus Vaccinia virus Vaccinia virus Vaccinia virus
NS-S protein NS-S protein Envelope glycoprotein Envelope glycoprotein Envelope glycoprotein Capsid protein L1 Capsid protein L2 Capsid protein L2 Capsid protein L2 E6 protein E6 protein E6 protein E4 protein Regulatory protein E2 Regulatory protein E2 Capsid protein L2 E7 protein Portal protein UL6 Tegument protein UL55 Tegument protein UL55 Tegument protein UL55 Tegument protein UL55 Tegument protein UL47 Thymidine kinase Glycoprotein G Glycoprotein G Large structural protein Large structural protein Large structural protein Matrix protein Matrix protein Phosphoprotein Phosphoprotein Membrane protein CPXV040 Secreted glycoprotein B7 protein precursor O1 protein K5 protein Growth factor precursor
1-9 1-9 1-10 1-10 1-9 1-10 1-11 1-10 1-9 2-12 2-12 2-11 1-11 1-8 1-8 2-12 1-8 2-10 2-10 2-12 2-10 2-10 2-9 2-9 2-11 2-11 1-8 1-8 1-8 1-9 1-9 2-12 2-11 2-10 2-10 1-10 1-10 1-9 2-11 2-11
A*0201 B*3901 B*2705 B*3901 B*2705 A*2402 B*0801 B*0801 B*0801 B*4001 B*4402 B*4402 B*5801 B*4001 B*4402 B*0801 B*3901 A*6801 A*1101 A*6801 B*3501 B*5801 A*6801 A*1101 B*3501 B*3901 A*0101 B*1501 B*3501 A*0301 A*6801 A*6801 B*3901 A*2402 A*2402 A*2402 A*2402 A*1101 A*1101 A*0201
5 109 83 45 153 180 514 398 45 323 455 130 18 21 468 448 94 159 239 8 57 149 492 241 473 108 41 412 145 855 241 367 330 15 377 548 19 655 206 340
— — — — — — — — —
0.511 0.511 0.511 — — —
0.512 —
0.498 0.498 0.498 0.498 0.498 0.501 0.475 — — — — — — —
0.491 0.491 0.510 — — — —
0.472 0.486
Amino acid residues in bold format are the potential Nt-acetylation sites. Artificial neural network prediction of the peptide binding affinity as IC50 value in nM calculated using the NetMHC 3.4 server
(http://www.cbs.dtu.dk/services/NetMHC/). c
Artificial neural network prediction of the protein Nt-acetylation sites as score calculated using the NetAcet 1.0 server
(http://www.cbs.dtu.dk/services/NetAcet/). The amino acid sequence without Ala, Ser or Thr at positions 1-3 cannot be evaluated by this web server. d
The well-identified Nα-acetylpeptide NAc-SL9 evaluated by the same methods as a reference. 3
Supplemental Figure S1
FIGURE S1. Electron density maps of Glu58, Arg62, and Gln65 residues contoured at 1.0 σ, related to Fig. 5. Dashed lines in different colors indicate H-bonding interactions in HLA-B*3901SL9 (red) and HLA-B*3901NAc-SL9 (blue), respectively.
4
Supplemental Figure S2
FIGURE S2. Comparison between the pockets A of H2-M3Nf-ML9 and HLA-B*3901NAc-SL9. The pockets A are highlighted in green. The residues that compose pocket A are shown in lines. The P1 residues (Nf-M1 and NAc-S1) of peptides are shown in stick representation. The formyl and acetyl groups are colored black and red in ball-and-sticks representation. (A) H2-M3 in complex with Nt-formylpeptide (Nf-ML9). (B) HLA-B*3901 in complex with Nt-acetylpeptide (NAc-SL9).
5
Supplemental Figure S3
FIGURE
S3.
The
representative
curve
alignment
of
the
gel-filtration
chromatography of pMHC complex protein in duplicate assays, which indicates the refolding result comparisons of MHC class I molecule in complex with Nα-acetylpeptides and nonacetylated peptides. The peaks of protein with an elution volume about 90ml would be pMHC complex (45kDa). (A) The HLA-B*3901 molecule refolded with NAc-SL9 and SL9, respectively. (B) The HLA-A*0201 molecule refolded with NAc-ML9 and ML9, respectively.
6