A Newly Recognized Pairing Mechanism of the α- and β-Chains of the Chicken Peptide− MHC Class II Complex

This information is current as Lijie Zhang, Xiaoying Li, Lizhen Ma, Bing Zhang, Geng of September 27, 2021. Meng and Chun Xia J Immunol published online 7 February 2020 http://www.jimmunol.org/content/early/2020/02/06/jimmun ol.1901305 Downloaded from

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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 © 2020 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Published February 7, 2020, doi:10.4049/jimmunol.1901305 The Journal of Immunology

A Newly Recognized Pairing Mechanism of the a- and b-Chains of the Chicken Peptide–MHC Class II Complex

Lijie Zhang,* Xiaoying Li,* Lizhen Ma,* Bing Zhang,* Geng Meng,† and Chun Xia*

MHC class II (MHC-II) molecules play a crucial role in cellular and humoral immunity by forming peptide–MHC-II (pMHC-II) complexes. The three-dimensional structures of pMHC-II complexes have been well resolved in humans and mice. However, there is no structural information for pMHC-II complexes in nonmammals. In chickens, there are two closely related and highly polymorphic b-chains and one monomorphic a-chain, and the mechanism by which one monomorphic a-chain combines with two polymorphic b-chains to form a functional heterodimer remains unknown. In this study, we report the crystal structure of a chicken pMHC-II complex (pBL2*019:01) at 1.9-A˚ resolution as the first nonmammalian structure of a pMHC-II complex. The

structure reveals an increase in hydrogen bonding between the a and b main chains at the central interface that is introduced by Downloaded from the insertion of four residues in the a-chain. The residues in the b-chain that form hydrogen bonds with the a-chain are conserved among all b alleles. These structural characteristics explain the phenomenon of only one BLA allele without sequence variation pairing with highly diverse BLB alleles from two loci in the genome. Additionally, the characteristics of the peptide in the peptide- binding groove were confirmed. These results provide a new understanding of the pairing mechanism of the a- and b-chains in a pMHC-II complex and establish a structural principle to design epitope-related vaccines for the prevention of chicken diseases.

The Journal of Immunology, 2020, 204: 000–000. http://www.jimmunol.org/

n jawed vertebrates, the genetic region of the MHC class II The structures of classical MHC-II molecules in humans and (MHC-II) has evolved to encode that are critical mice are well characterized (4, 5). In general, heterodimeric I for the adaptive arm of the immune system (1). The physi- MHC-II complexes are formed by one a-chain and one b-chain. ological function of classical MHC-II is to bind exogenous pep- Each chain (a and b) consists of two domains, a1anda2and tides and present them on the surfaces of APCs for recognition by b1andb2(6).Α1andb1 assemble to form a peptide-binding CD4+ T cells, inducing cytokine secretion for Ab generation and groove (PBG), whereas the two membrane-proximal IgC1-like

immune system regulation (2, 3). domains, a2andb2, support the floor of the a1/b1 U (7). by guest on September 27, 2021 Unlike in MHC-I, the PBG in MHC-II is open ended, with *Department of Microbiology and Immunology, College of Veterinary Medi- increased tolerance for binding peptides ranging from 13 to 25 cine, China Agricultural University, Haidian District, Beijing 100193, China; residues in length (8, 9). Peptides bound to MHC-II adopt an and †Beijing Advanced Innovation Center for Food Nutrition and Human Health, College of Veterinary Medicine, China Agricultural University, Haidian District, extended polyproline type II conformation (10). The canonical Beijing 100193, China conformation of peptides is maintained by a hydrogen-bonding ORCIDs: 0000-0003-0932-8523 (L.Z.); 0000-0003-3335-2283 (G.M.); 0000-0002- network that involves binding of the peptide backbone to res- 3444-0624 (C.X.). idues of MHC-II molecules (7). Studies of peptide–MHC-II Received for publication October 28, 2019. Accepted for publication January 9, (pMHC-II) structures have confirmed the role of four specific 2020. peptide-binding pockets, P1, P4, P6, and P9, with additional This work was supported by the National Natural Science Foundation of China contributions from the P3, P7, and P10 pockets, which control (Grants 31972683 and 31572493). the selection of specific antigenic peptides (11, 12). Further- C.X. designed the study and supervised the project; L.Z. performed experiments and the data analysis; X.L., L.M., and B.Z. assisted experiments; L.Z. and G.M. more, polymorphic MHC-II residues located in areas forming solved the structure; C.X. and G.M. provided guidance on data analysis; C.X. and pockets in pMHC-II structures are key determinants of T epi- L.Z. wrote the paper. tope immunogenicity (13). Overall, structural determination of The crystal structure in this article has been submitted to the Data Bank pMHC-II complexes is crucial for elucidating the molecular (http://www.pdb.org/pdb/home/home.do) under accession number 6KVM. mechanism of the process of peptide loading (14). Address correspondence and reprint requests to Prof. Geng Meng or Prof. Chun The chicken immune system provides an invaluable model Xia, Beijing Advanced Innovation Center for Food Nutrition and Human Health, College of Veterinary Medicine, China Agricultural University, Haidian District, for studies on basic immunology in nonmammalian vertebrates Beijing 100193, China (G.M.) or Department of Microbiology and Immunology, (15). The chicken MHC region includes the chicken MHC region College of Veterinary Medicine, China Agricultural University, Haidian District, Beijing 100193, China (C.X.). E-mail addresses: [email protected] (G.M.) or B (MHC-B) locus and the chicken MHC region Y (MHC-Y) locus [email protected] (C.X.) (16). The MHC-B locus, consisting of highly polymorphic B-F, The online version of this article contains supplemental material. B-L, and B-G regions, is more compact and simple in chickens Abbreviations used in this article: MHC-B, chicken MHC region B; MHC-II, MHC than in mammals and is also arranged in a different manner class II; MHC-Y, chicken MHC region Y; PBG, peptide-binding groove; PDB, Protein (17–19). The highly polymorphic classical MHC-II b-chain is Data Bank; pMHC-II, peptide–MHC-II. encoded by two closely related BLB (BLB1 and BLB2)in This article is distributed under The American Association of Immunologists, Inc., the B-L region. A monomorphic a (BLA) is located out- Reuse Terms and Conditions for Author Choice articles. side of the MHC-B locus and is not linked to nonclassical class Copyright Ó 2020 by The American Association of Immunologists, Inc. 0022-1767/20/$37.50 II b genes in the MHC-Y (18, 20). The two classical BLB genes

www.jimmunol.org/cgi/doi/10.4049/jimmunol.1901305 2 THE FIRST STRUCTURE OF THE CHICKEN pMHC-II COMPLEX exhibit tissue-specific expression rather than dominant ex- After mixing with the reservoir buffer at a 1:1 ratio, the concentrated pression, and BLB2 is more highly expressed at the RNA level pBL2*019:01 complex was crystallized according to the sitting-drop inthespleenthanisBLB1 (21). The most striking feature of vapor diffusion method at 18˚C. After 7 d, pBL2*019:01 crystals were obtained with solution no. 17 from the Crystal Screen 1 Kit (100 mM the chicken MHC system is that the MHC-B is associated with Tris base/hydrochloric acid [pH 8.5], and 200 mM lithium sulfate) resistance and susceptibility to various infectious diseases, (Hampton Research). The 1.9-A˚ diffraction data for the pBL2*019:01 including viruses, bacteria, and parasites (22–24). However, crystal were collected at 100 K; the data collection was performed at knowledge regarding the pMHC-II complex in chickens and the Shanghai Synchrotron Radiation Facility using beamline BL17U at a wavelength of 1.5418 A˚ (Shanghai, China) (31). The collected in- other nonmammalian vertebrates is lacking to date, yet stud- tensities were indexed, integrated, corrected for absorption, scaled, and ies in chicken may provide insight into the evolution of the merged using the HKL2000 package (32). MHC-II system. It is worth noting that there are three classical Structure determination and refinement MHC-II isotypes (DR, DP, and DQ) in humans and two iso- types (I-A and I-E) in mice (25). Both the a-andb-chains of The crystal of pBL2*019:01 belongs to the C2 space group, and the DP, DQ, and I-A are polymorphic. In contrast, the b-chains in structure was solved by molecular replacement using MolRep and I-E and DR molecules are polymorphic, but the a-chains are Phaser in the CCP4 package, with the human HLA-DR1 structure ( [PDB] code: 1AQD) as the search model (33–35). monomorphic (26). In chickens, there are two closely related Extensive model building was performed manually with Coot (36), and and highly polymorphic BLB chains and one monomorphic restrained refinement was conducted using REFMAC5 (37). Additional BLA chain (20). Clearly, chicken BLA is a nonmammalian rounds of refinement were carried out using the Phenix refine program homolog of human HLA-DRa. However, the reason why only implemented in the Phenix package together with isotropic atomic displacement parameter refinement and bulk solvent modeling (38). one a-chain is maintained in chicken and the mechanism by The stereochemical quality of the final model was assessed with the Downloaded from which one monomorphic a-chain combines with polymorphic PROCHECK program (39). Detailed information about collection and b-chains to form a functional heterodimer remain unknown. refinement is shown in Table I. To elucidate the pairing mechanism of the monomorphic a-chain Data analysis and polymorphic b-chains and the peptide-binding mode of chicken MHC-II, in this study, the crystal structure of chicken SignalP 5.0 server was used to predict the presence and location of signal peptide cleavage sites (40). Structural illustrations and electron pMHC-II (pBL2*019:01) in complex with a 17-residue peptide http://www.jimmunol.org/ density–related figures were generated using PyMOL (http://www. was the first solved. Moreover, the pairing mechanism of one pymol.org/) and UCSF Chimera (http://www.cgl.ucsf.edu/chimera/). a-chain with several b-chains was elucidated. Additionally, The isotropic B factor was calculated using the equation B =8p2m2. comparison of the pMHC-II structures among chicken, human, Solvent-accessible surface areas were calculated with the Protein Data and mouse provides evolutionary information regarding the Bank in Europe Proteins, Interfaces, Structures and Assemblies web- page (http://www.ebi.ac.uk/pdbe/pisa/picite.html), and comparison of ancestral MHC-II system in jawed vertebrates. amino acid sequences from different proteins was performed using Clustal Omega (http://www.ebi.ac.uk/Tools/msa/clustalo/). The crystal Materials and Methods Preparation of proteins by guest on September 27, 2021 A gene encoding the extracellular residues 1Leu-184Glu of the chicken Table I. Data collection and refinement statistics (molecular MHC-II a-chain, namely, BLA (GenBank accession no. AY357253), was replacement) synthesized and inserted into the expression vector pET-21a (Novagen), followed by transformation into Escherichia coli strain BL21 (DE3) pBL2*019:01 (TransGen Biotech, Beijing, China) (20). When the OD600 of the bacterial culture reached 0.6 at 37˚C, 0.5 mM isopropyl b-D-thiogalactoside was Data collection added to induce expression of BLA for 6 h. The bacteria were harvested by Space group C2 centrifugation at 6000 3 g for 10 min and resuspended in cold PBS. Af- Cell dimensions ter sonication, the sample was centrifuged at 16,000 3 g, and the pellet a, b, c (A)˚ 152.40, 57.30, 58.06 containing inclusion bodies was washed three times with a solution con- a, b, g (˚) 90.00, 110.68, 90.00 sisting of 20 mM Tris-HCl (pH 8.0), 100 mM NaCl, 1 mM EDTA, 1 mM Resolution (A)˚ 71.29–1.90 (2.20–1.90)a DTT, and 0.5% Triton X-100. The inclusion bodies were then dissolved Total reflections 37,303 to 30 mg/ml in guanidinium chloride (Gua-HCl) buffer (27). A construct Unique reflections 37,269 b a for the chicken MHC-II b-chain, namely, BLB2 (GenBank accession no. Rsym or Rmerge 0.097 (0.615) DQ008584), which encodes a fusion protein comprising ribosomal protein I/sI 15.1 (3.5)a RPL30 (PGDSDIIRSMPEQTSEK), connected by a 16-mer peptide poly- Completeness (%) 99.9 (99.9)a Gly/Ser linker (SGGGSLVPRGSGGGGS) to residues 1Thr-190Pro of Redundancy 4.7 (4.8) the BLB2 chain, was cloned into the expression vector pET-21a and then Refinement transformed into E. coli strain BL21 (DE3) (28–30). The endogenous Resolution (A)˚ 30–1.90 peptide RPL30 originating from the ribosomal protein was identified fol- No. of reflections 36,942 c lowing peptide elution from BL2*019:01 (29, 30). RPL30–BLB2 was Rwork/Rfree (%) 18.70/21.62 expressed in inclusion bodies and purified as described above for BLA. R.m.s. deviations Bond lengths (A)˚ 0.004 Assembly of the pBL2*019:01 complex Bond angles (˚) 0.954 For in vitro refolding, purified BLA and RPL30–BLB2 inclusion bodies Average B factor 28.52 were diluted to a final concentration of 40 mg/l each in a refolding solution Ramachandran plot quality (containing 50 mM Tris-HCl, 20% [w/v] glycerol, 0.5 mM EDTA, 3 mM Most favored region (%) 99.21 reduced glutathione, and 0.9 mM oxidized glutathione [pH 8.0]) (28). Allowed region (%) 0.79 After 8 d at 4˚C, the folding solution was concentrated and purified Disallowed (%) 0.00 by chromatography using a Superdex 200 16/60 column (GE Health- aValues in parentheses represent the highest-resolution shell. b care), followed by RESOURCE Q anion-exchange chromatography Rmerge = Ʃi Ʃhkl |Ii (hkl)- , I (hkl) . | /Ʃhkl Ʃi Ii (hkl), where Ii (hkl)isthe (GE Healthcare) as previously described (27). observed intensity, and , I (hkl) . is the average intensity from multiple measurements. c Crystallization and data collection R =Ʃhkl || Fobs | 2 k | Fcalc ||Ʃhkl |Fobs |, where Rfree is calculated for a randomly chosen 5% of reflections and Rwork is calculated for the remaining 95% of reflections Purified pBL2*019:01 was concentrated to 8 mg/ml in a buffer con- used for structure refinement. taining 20 mM Tris-HCl (pH 8.0) and 50 mM NaCl for crystallization. R.m.s., root mean-square. The Journal of Immunology 3 structure has been deposited in PDB (http://www.pdb.org/pdb/home/ are dispersed (Fig. 1C). The number of hydrogen bonds be- home.do) under accession number 6KVM. tween the a2andb2 domains of the human and mouse struc- Results tures vary from two to six, and those hydrogen bonds are concentrated in the intermediate region (Table II). In short, all The topological structure of pBL2*019:01 residues in the BLB chains, excluding those involved in hy- The crystal structure of pBL2*019:01 was determined at 1.9-A˚ drogen bonds between the main chains at the central interface, resolution by molecular replacement. The final refinement of interacting with the BLA chain are conserved, regardless of the structure generated R/Rfree factors of 18.7/21.6% (Table I). whether the chain is BLB1 or BLB2. In this structure, only one heterodimer forms by the BLA and The a2andb2 domains are the major regions of interaction BLB2 (BLB2*019:01) chains in the asymmetric unit. The BLA with CD4 in mammals (41). The key residues that interact with chain is split into a1anda2 domains, and the BLB2 chain is CD4 are identical in pBL2*019:01; Valb142-Thrb145,Leub158, split into b1andb2 domains (Fig. 1A). The a1andb1 domains Glub162,Glua91,andArga179 are conserved (Fig. 1D). A sig- contribute approximately equal halves to the PBG, and the nificant change is Argb114, which may not affect natural bind- membrane-proximal a2andb2 domains consist of two Ig-C1 ing to CD4 because of the mutation of CD4 Tyr40, and other domains. Superposition of the pBL2*019:01 structure with the positions display changes in residues of the same nature. The solved human and mouse pMHC-II structures results in a root chicken CD4 structure was modeled based on the HLA-DR1– mean-square difference in a-carbon positions of ,1.7 A˚ CD4 complex structure (PDB code: 3S4S). Because the chicken (Supplemental Fig. 1A, Supplemental Table I). The pBL2*019:01 CD4 molecule has a five-residue deletion at the corresponding structure is more similar to the human HLA-DR2 structure position (positions 55–59) of the human CD4 molecule, the Downloaded from than other resolved pMHC-II structures of human and mouse distance between this region of the chicken CD4 molecule and (Supplemental Table I). pBL2*019:01 is changed, suggesting that interaction between A total of 38 hydrogen bonds form between the BLA and pBL2*019:01 and chicken CD4 is different from the corre- BLB2 chains. Among them, 16 hydrogen bonds between the sponding interaction in known pMHC-II complexes. main chains at the central interface occur between the a1and Hydrogen bonds between the main chain at the bottom of the

b1 strands of the PBG (Table II). The remaining 22 hydrogen http://www.jimmunol.org/ bonds are distributed between the main chain and side chains or PBG in pBL2*019:01 are increased in number between the side chains of the BLA and BLB2 chains. All The bottom of the PBG in pBL2*019:01 is composed of eight residues of the BLB2 chain involved in the formation of the antiparallel strands, with the a1domainandb1 domain each 22 hydrogen bonds are conserved in the chicken BLB se- contributing four strands (Fig. 3A). The four antiparallel quences (Fig. 2, Table II). However, those amino acids differ strands of the a1domainandb1 domain were termed a1–a4 from those of human isotypes and other mammalian MHC-II strands and b1–b4 strands, respectively. Based on superposi- sequences (Supplemental Table II). tion of pBL2*019:01 with resolved pMHC-II structures, it was Interactions among the four domains (a1, a2, b1, and b2) of found that the a1strand,a2strand,andb1strandinthePBG pBL2*019:01 were analyzed. Only one hydrogen bond occurs are the longest (Fig. 3A, Supplemental Fig. 1). One reason for by guest on September 27, 2021 between Aspa145 of the a1domainandArgb34 of the b2do- this finding is that, compared with other a-chains, 4 aa (GPDK) main, located in the loop rings (Fig. 1B). pBL2*019:01 shows are inserted in the a1 domain of BL2*019:01 at positions 16–19 in eight hydrogen bonds between the a2andb2 domains, which the bottom of the PBG, which lengthens the a1 strand (Fig. 3B).

FIGURE 1. Structural characteristics of the pBL2*019:01 complex. (A) The overall structure of pBL2*019:01 is shown in cartoon representation with the peptide shown in stick representation. The BLA chain is shown in orange, the BLB chain is shown in violet, and the peptide is shown in green. (B) The cyan rectangle shows the interaction between the a2 and b1 domains in detail. (C) The details of the interaction between the a2 and b2 domains are depicted in the red rectangle. (D) The chicken CD4 structure was modeled based on the HLA-DR1–CD4 complex structure (PDB code: 3S4S). The residues of MHC-II that are in contact with two regions of the D1 domain of the CD4 molecule are shown in stick representation. These residues are marked by triangles. Chicken and human CD4 molecules are shown in green and yellow, respectively. 4 Table II. Interactions among the four domains of MHC-II

Interactions between a1 and b1 Domains (Expected between Interactions between Main Chains Interactions between a2andb2 Allele PDB Code Main Chains) of a1 and b1 Domains Interactions between a1 and b2 Domains Interactions between a2 and b1 Domains Domains pBL2*019:01 6KVM (38) a17G-b4S a80S-b32Y a4H-b17Y a6L-b15C a8Q- a2K-b126E a31A-b149Q a32D- a145D-b34R (1) a96P-b156Q a97A-b156Q a83S-b32Y a83S-b33N b13S a10E-b11A a12Y-b9C b149Q a32D-b155Y a33E-b153W a98E-b120T a100V-b100R a85Q-b34R a84Q-b6F (6) a14R-b7F a16E-b4S a16E- a47R-b151G (7) a101S-b100R a149R-b149Q b5A a17G-b4S (16) a153Y-b150N (8) HLA-DR1 1DLH (34) a3E-b19N a8Q-b78Y a3E-b17F a4H-b17F a6I- a30D-b149Q a32D-b149Q a33E- a143R-b12K a144E-b29R (2) a96T-b156Q a153Y-b150N a20D-b6R a79R-b57D b15C a8Q-b13F a10E-b11L b153W a47R-b151G (5) (2) a79R-b53L a80S-b32Y a12Y-b9W a14N-b7F a85I- a83T-b32Y a83T-b33N b6Ra (14) (11) HLA-DR2 1FV1 (39) a3E-b19N a3E-b20G a8Q- a3E-b17F a4H-b17F a6I- a32D-b153W a32D-b149Q a33E- a143R-b12K a145D-b34Q a146H- a96T-b156Q a149R-b149Q b78Y a20D-b6R a79R- b15C a8Q-b13Y a10E- b153W a47R-b151G (5) b12K a146H-b10Q (4) a153Y-b150N a184D- b57D a79R-b53L a80S- b11D a12Y-b9Q a14N-b7F b105R a184D-b106T (6) b32Y a83T-b32Y a83T- a85I-b6Ra (14) b33N (10) HLA-DR52c 3C5J (26) a8Q-b78Y a20D-b6R a83T- a3E-b17F a4H-b17F a6I- a32D-b149H a32D-b153W a33E- a116T-b34Q a143R-b12K a146H- a153Y-b150N (1) b33N (4) b15C a8Q-b13S a10E-b11L b153W a47R-b151G (4) b34Q (3) a12Y-b9E a14N-b7F a85I- b6Ra (14) HLA-DR14 6ATZ (28) a8Q-b78Y a20D-b6R a3E-b17F a4H-b17F a6I- a32D-b149H a32D-b153W a33E- — a96T-b156Q a153Y-b150N a79R-b53L a79R-b57D b15C a8Q-b13S a10E-b11S b153W a47R-b151G (5) (2) a83T-b33N (7) a12Y-b9E a14N-b7F a85I- COMPLEX pMHC-II CHICKEN THE OF STRUCTURE FIRST THE b6Ra (14) HLA-DP2 3LQZ (34) a8Y-b82N a14T-b6N a14T- a3D-b17A a4H-b17A a6S- a30D-b149R a33E-b153W (2) a88D-b34R a143R-b12R a145D- a96P-b156Q a97K-b121D b7Y a79R-b57D a79R- b15C a8Y-b13Q a10A- b34R a146Y-b29R (6) a97K-b152D a98E-b120T b53L a83T-b32Y a83T- b11G a12V-b9F a14T-b7Y a153Y-b150N a153Y- b33N a84Q-b6N (8) (12) b151G (6) HLA-DP5 3WEX (48) a3D-b19N a14T-b6N a13Q- a3D-b17A a4H-b17A a6S- a30D-b149R a31E-b149R a32D- a88D-b34R a143R-b12R a145D- a96P-b156Q a97K-b121D b6N a14T-b7Y a15H- b15C a8Y-b13Q a10M- b149R a33E-b153W a47H-b151G b34R a146Y-b10Q a146Y-b29R a97K-b152D a97K-b156Q b6N a34Q-b90T a34Q- b11G a12V-b9F a14T-b7Y (6) a146Y-b36E (7) a101E-b100N a153Y- b86D a50E-b93R a82H- (12) b150N (6) b7Y a80S-b32Y a83T- b32Y a83T-b33N a84Q- b6N a79R-b53L a79R- b57E (17) HLA- 1UVQ (41) a3D-b19N a10N-b11F a3D-b18T a3D-b17F a4H- a30D-b149R a32D-b149R a33E- a88E-b34R a143K-b12K a145D- a96S-b156Q a97K-b156Q DQ0602 a15Y-b6D a50E-b93R b17F a6A-b15C a8C-b11G b153W (5) b34R a146H-b10Q a146H-b12K a97K-b121D a97K-b152D a79R-b57D a79R-b53Q a9G-b13G a10N-b11F (5) a153Y-b150N (5) a80Y-b51T a80Y-b53Q a12Y-b9F a14F-b7F a85A- a80Y-b35E a83T-b32Y b6Da (14) a83T-b33N (12) HLA-DQ2 1S9V (41) a8Y-b86E a13Q-b6D a14S- a3D-b17F a4H-b17F a6A- a30D-b149R a32D-b149R a34Q- a143K-b12K a145D-b34R a146H- a96S-b156Q a97K-b152D b6D a15Y-b6D a14S-b7F b15C a8Y-b13G a8G-b13G b153W (4) b10Q a146H-b12K (5) a97K-b121D a153Y-b150N a34Q-b86E a72N-b9Y a10N-b11F a12Y-b9Y (4) a79R-b53L a80S-b32Y a14S-b7F a85A-b6Da (13) a83T-b32Y a83T- b33N a88E-b34R a87N-b3S (15)

(Table continues)

Downloaded from from Downloaded http://www.jimmunol.org/ by guest on September 27, 2021 27, September on guest by The Journal of Immunology 5

The lengthened strands are accompanied by an increase in the

2 number of hydrogen bonds in the main chains. In pBL2*019:01, b there are 16 hydrogen bonds between the main chains of the a1 104T 121D 156Q 156Q b b b

2and strand and the b1 strand (Fig. 3C). Only 10–13 hydrogen bonds a at the central interface are present in the resolved human and 96S- b 97K- 97K- 185E- a a a a 150N (5) 150N (6) mouse MHC-II structures, regardless of the number of hydro- b b

Domains gen bonds formed between the a-andb-chains (Fig. 3C, 3D, 156Q 152D 156Q 152D b b b 153Y- 153Y- Table II). b a a In addition, the lengthening of the a1 strand results in in- 97R- 96P- 97K- 97R- a a a Interactions between a teraction between the bottom of the PBG and the sidewall of the a1 helix, because BLA is more dispersed (Fig. 4A–D). The hydrogen bond between Glua16 and Argb82 in BL2*019:01 is

145D- located at the C terminus of the PBG and does not exist in the a 10Q

1 Domains human and mouse structures. Additionally, there are more in- b b

12K teractions between the a1 helix on the side of the PBG and the 28R (5) b b 146H- 2 and strands at the bottom of the a1 domain, reaching six hydrogen 34R (5) a a b bonds, with only one to four hydrogen bonds in the human 144D- 145D- a 12K

a structure (Fig. 4A–D). This result might indicate that the sta- b 145D- Downloaded from

a bility of invariant chicken BLA itself is enhanced. Importantly, 12K b 12K

b the four-residue insertion at positions 16–19 of the a1domain 143N- a is found in avian and reptiles, suggesting that the increase in 143R-

a main-chain hydrogen bonding caused by the insertion may be conserved in these animal groups (Fig. 3B). 33E- 33E- A novel interaction is identified between the a1 and a2 helices http://www.jimmunol.org/ a a

2 Domains Interactions between of the PBG, which lack a salt bridge in pBL2*019:01 b 149R 149R Analysis of the interactions between the a1 helix of the a1 b b 1 strand. (3) b 1 and

a domain and the a2 helix of the b1 domain reveals no direct 32D- 32D- interaction between the two helices in pBL2*019:01. Instead, a a 153W 153W (4) b b the two helices connect indirectly through a molecule of water a79 b53 149R 149R

1 strand and and P10-Ser (Fig. 5A). Similarly, Asn and Leu are con- b b a nected by a water molecule. Comparison of the structures shows 30D- 30D- a difference in the direction and distance of the key residues a a (Fig. 5B). Thus, the condition for forming the salt bridge is by guest on September 27, 2021 absent. The main reason for this change is that the key residue a79 11F 6I- 11C 6G- Arg , which is completely conserved in the human and mouse 85A- b a b a a pMHC-II structures, is replaced by Asna79 in pBL2*019:01 7F (13) 10T- 17F 7F 10E- b a79 17F a b

a (Fig.5D).TheArg residues in humans and mice always b

(13) b53 1 Domains Interactions between

14L- form hydrogen bonds with conserved Leu (Fig. 5C). Be- 4H- b a b 4H- 14S- 13S 13G a a a a b

6D cause of the residue difference at position b57 in the human b 9E a79 1 and 8Y- 9H 8Q- b b 20G

17F and mouse pMHC-II structures, the conserved Arg forms a a b a b

of hydrogen bonds and/or salt bridges with the b57 residue or 12Y- 2E- 3D- b 12Y- 15C 15C a

a connects via a water molecule. Gln is present at position b57 in a a Interactions between Main Chains b b pBL2*019:01 (Fig. 5E). 1 b The peptide main-chain forms an enhanced hydrogen-bonding 53L 32Y 32Y 53L 93R 18Y b network, and the conformation of the peptide changes b b b 5W (11) 1 and b b a 1 domain, excluding hydrogen bonds between main chain of b

79R- According to the clear electron density, the peptide adopts a type II 80S- 83T- 1K- b 79R- 50E- a a a a a 33N (7) 86A- a polyproline helical conformation and consists of 17 residues, even b a 4R 57S 61Y 57D though nine residues are generally observed in the binding site of 19N 86E b b Main Chains) 83T- b b 33N

a the PBG (Fig. 6A). Peptide side chains that orient away from the b 2E- 8Y- 1 domain and 20D- 79R- b 79R- b 72N- a

a PBG have increased B factors, and the side chains are flexible. a a a a a Domains (Expected between 85D- Interactions between

a The peptide forms a hydrogen-bonding network that extends from the main chain of the peptide to both the main-chain and side- chain residues in the PBG of pBL2*019:01 (Fig. 6B). There are 18 hydrogen bonds between the PBG and the main-chain atoms of the peptide, 7 derive from the BLA chain and 11 from the BLB2 chain 1F3J (34) ) (Fig. 6B). Twelve of the eighteen hydrogen bonds that involve the PBG of pBL2*019:01 are conserved in most human and mouse pMHC-II structures. Asna79 and Glnb57 form four hydro- -chain and the number of hydrogen bonds among the four domains. Continued b gen bonds, and because the peptide to which pBL2*019:01 binds Allele PDB Code has 17 aa, it is those four hydrogen bonds that cause the peptide C Hydrogen bonds between main chain of I-Ag7 I-Ek 1KT2 (38)

The sequence numbers of all amino acida residues in the BLA chain and BLB2 chain are based on the sequence alignment in Supplemental Table II. The number in parentheses indicates the total number of hydrogen bonds formed between the terminus to dip. Thus, differences in some hydrogen bonds lead to -chain and a Table II. ( changes in the direction of the peptide backbone. 6 THE FIRST STRUCTURE OF THE CHICKEN pMHC-II COMPLEX Downloaded from http://www.jimmunol.org/

FIGURE 2. Protein sequence alignment of chicken BLB chains. Arrows above the alignment indicate b strands; cylinders indicate helices. Black dots represent identity shared with the reference BLB2*019:01 allele. Red dots emphasize that those residues of the BLB chains interact with the extra residues in the a-chain. Blue dots emphasize that those residues of the BLB (in addition to those residues highlighted by the red dots) main- chain interact with the BLA chain at the central strand interface. Triangles emphasize that all residues in the BLB chains, excluding those involved in hydrogen bonds between main chains at the central interface, interacting with the BLA chain are conserved, regardless of whether the chain is by guest on September 27, 2021 BLB1 or BLB2. Total amino acid identities between BLB2*019:01 and the listed MHC-II molecules are shown on the left of each sequence; the identities of b1 domains are shown on the right.

The PBG of pBL2*019:01 is more open and interacts with DM pocket in pBL2*019:01 consists of Vala75 and Asnb57;inother molecule in different ways than does the PBG in structures, it is composed of Vala75, b57, and Arga79 (Fig. 7F). mammalian structures Therefore, the P9 pocket of pBL2*019:01 is more open than that Four pockets accommodate the side chains of the peptide at the in other structures. The surface-exposed amino acids at P-5, P-1, P1, P4, P6, and P9 positions, but the PBG of pBL2*019:01 also P3, P5, P8, and P11 may dock TCRs. forms the P7 and P10 clefts (Fig. 7). The P3 and P8 pockets The nonclassical MHC-II protein DM plays a critical role in consist of only BLA-chain amino acids, whereas the P2 pocket the endosomal peptide selection process. Based on the HLA- consists of only BLB2-chain amino acids. The P1, P4, P6, DR1–DM structure (42), the DM molecule mainly contacts the a1 domain of pMHC-II close to the P1 pocket (Fig. 8A). In P7, and P9 pockets comprise both BLA and BLB2 chain amino a41 a43 acids. The P1 pocket is composed of the BLB2-chain amino the DM–DR1 structure, two key residues (Lys and Glu ) b81 b82 b85 b86 b85 of DR are at the interface with DM; these charged residues form acids His ,Asn ,Gly ,andVal (Fig.7B).Gly and a183 Val b86 affect the shape and size of the P1 pocket. b85 Is located an extended hydrogen-bonding network that includes Asp , Arga98,andHisa180 of DM. The importance of this network is on the sidewall of the P1 pocket; b85 is Gly in BL2*019:01, but a43 other b85 residues in chicken are Ile/Val. Therefore, the P1 highlighted by previous mutagenesis data: the mutation Glu in DR results in unresponsiveness to DM. Residues located at pocket sidewall of BL2*019:01 is the lowest and more open. a41 a42 a43 b86 Is located at the bottom of the P1 pocket, and the change in Ala ,Ala ,andGln in the BLA chain are converted to b86 of the BLB chain results in a different depth of the P1 noncharged amino acids, which may hinder the formation of pocket and restricts the size of peptide side chains that might hydrogen bond networks with chicken DM molecule. In addi- be accommodated at this position. The P6 pockets are large but tion, polymorphic residues of the BLB2 chain are almost lo- filled with four water molecules rather than proline (Fig. 7D). cated in areas forming pockets in the pBL2*019:01 structure The water molecules occupy the depth of the pocket; thus, in (Figs. 2, 8B). the absence of those water molecules, amino acids with even larger side chains can be accommodated. The P7 pocket has a Discussion negative charge and is stabilized by a hydrogen-bonding network The pBL2*019:01 structure links nonmammals and mammals involving two water molecules and Argb71,Tyra30,andAsnb73 to provide an understanding of the presence of Ag peptides in (Fig.7E).AsthesidechainofAsna79 is short and cannot par- the PBG as well as activation of T cells to further promote the ticipate in the formation of the P9 pocket, the boundary of the P9 production of specific humoral and cellular immunities in birds. The Journal of Immunology 7 Downloaded from http://www.jimmunol.org/

FIGURE 3. Structural analysis of specific insertion residues in the BLA chain. (A) Superposition of the PBG of pBL2*019:01 with those of the human and mouse MHC-II structures. The longest a1strand,a2strand,andb1 strand are highlighted in the rectangle. (B) Protein sequence alignment of the BLA chain with a representative MHC-II sequence of mammals, amphibians, and fishes (left). Red markings highlight avian- and reptile-specific amino acid insertions. Protein sequence alignment of the related region in the b-chains that interact with the a-chain at the central interface of the PBG (right). Violet markings highlight residues in the BLB2 chains that interact with the extra residues in the BLA chain. These

conserved residues are marked in green. (C and D) Detailed analysis and comparison of hydrogen bonds between the main chains of the a1andb1 by guest on September 27, 2021 strands of pBL2*019:01 and HLA-DR1, respectively.

It is important that the three dimensional structure of pBL2*019:01, the PBG (Fig. 3). In addition, the residues interacting with the the first nonmammalian pMHC-II crystal structure, is elucidated. chicken BLA chain to form these hydrogen bonds are highly Although the architecture of pBL2*019:01 is similar to that conserved in known polymorphic chicken BLB chains (Fig. 2). of human pMHC-II (Fig. 1A), many unique structural features At the C terminus of the a1anda2 helices, Asna79 in the were found in pBL2*019:01, which may explain the novel a- and chicken structure replaces the conserved basic residue Arga79 b-chain–pairing mechanism. Chickens have only one classical in the human and mouse structures. Furthermore, b57 is a monomorphic BLA gene and two classical highly polymorphic nonpolar Asn residue in the chicken structure, which prevents BLB genes (20). The a1 strand of the BLA chain contains four formation of the salt bridges present in the human and mouse more amino acids than in humans and mice, which results in structures; instead, the two helices employ a novel method to a longer a1–a2 region and increased main-chain hydrogen connect via water molecules or peptide residues (Fig. 5). Based bonding at the central strand interface, stabilizing the bottom of on the premise of the above structure, the chicken BLA chain

FIGURE 4. Analysis of interactions between the bottom and sidewall of the PBG of the a-chain. (A) Superposition of the PBG of pBL2*019:01 with the PBG of the human and mouse MHC-II structures. The residues involved in interactions between the a1 helix and strands of the a1domain are shown with sticks. (B–D) Interactions between the a1 helix and strands of the a1 domain are represented. The BLA chain is colored orange, the BLB2 chain is colored violet, the HLA-DR1 (PDB code: 1DLH) a-chain is colored salmon, the b-chain is colored pale green, the HLA-DP2 (PDB code: 3LQZ) a-chain is colored pale yellow, and the b-chain is colored light blue. Hydrogen bonds are displayed as dashed lines corresponding to the structure color. 8 THE FIRST STRUCTURE OF THE CHICKEN pMHC-II COMPLEX Downloaded from http://www.jimmunol.org/ by guest on September 27, 2021

FIGURE 5. Analysis of interactions between the a1anda2 helices of the PBG. (A) Interactions between the a1anda2 helices of the PBG in pBL2*019:01 were analyzed. The hydrogen bonds in pBL2*019:01 are displayed as violet dashed lines. (B) Superposition of a79, b53, and b57 from HLA-DR1, HLA-DQ2, IAg7, and pBL2*019:01. Yellow dashed lines indicate the corresponding distance between a79 and b53/57 in pBL2*019:01 and are labeled with the length in A.˚ Residues a79 and b57 are displayed in stick representations corresponding to the structure color. (C) Interactions between a79 and b57 and between a79 and Leub53 in HLA-DR1, HLA-DQ2, and IAg7 were analyzed. The hydrogen bonds in HLA-DR1, HLA-DQ2, and IAg7 are displayed as green, gray, and cyan dashed lines, respectively. (D) Sequence alignment of the chicken BLA chain (residues 71–87) with the a-chain of other animals; a79 is highlighted. (E) Sequence alignment of the chicken BLB2 chain (residues 49–63) with the human and mouse chains; b53 and b57 are highlighted. rationally matches all chicken b-chains from BLB loci. In genes can coevolve relatively easily to generate functional di- classical mammalian MHC-II molecules, the a-chain and mers. The BLA locus is located outside of BLB genes in b-chain are encoded by A and B genes, which are generally chickens, and this genetic separation may have allowed the located adjacent to each other (43), and this suggests that these b-chain to be highly polymorphic and forced the a-chain to The Journal of Immunology 9

FIGURE 6. Hydrogen-bonding network between the peptide main chain and BL2*019:01. (A) Electron density map of the RPL30 peptide colored according to the B factor in the PBG of pBL2*019:01. (B) Top view of the PBG of pBL2*019:01 shown in cartoon representation with the peptide main chain shown as sticks. The conserved hydrogen bonds between the pep- tide residues and PBG are displayed as violet dashed lines. pBL2*019:01-specific hydro- gen bonds are colored orange. become monomorphic toward an average fit. Thus, only one suborder Galloanserae (Anseriformes [e.g., duck] and Galliformes a-chain remains to best function with all b-chains. In addition, [e.g., chicken]) and probably also Serpentes (snakes) appear to the BLA chain might be the a-chain partner with the non- express only classical a-chains, with which the multiple clas- classical class II b-chains encoded by the MHC-Y (22). HLA- sical b-chains found in the same species likely interact (20, 44). DRa and I-Ea are also monomorphic in humans and mice. Conversely, in mammals, the lineages DP, DQ, and DR stably Downloaded from Evolutionary analysis has shown that the chicken a-chain is coexist and have specific interaction motifs that are conserved highly similar to the HLA-DRa–chain and I-Ea–chain in hu- for each lineage, which may explain why the A and B lineages mans, mice, and other mammals (20). The short a1–a2region do not show productive cross-lineage interactions. found in most MHC-II a1 domains is probably not the original Activation of T cells by the PBG-presenting Ag peptide is the MHC form, as the region tends to be longer in the MHC-I a1 core function of pMHC-II (45). The PBG is more open at both domain (Supplemental Fig. 1). Therefore, the a1–a2regionof ends in the 3D structure of pBL2*019:01 than in other struc- MHC-II may have lost length to reduce sequence-independent tures (Fig. 7). Because the chicken CD4 molecule carries a five- http://www.jimmunol.org/ interstrand interactions and to rely more on specific binding, residue deletion, interaction between BL2*019:01 and CD4 facilitating evolution of A and B pairs that do not share each may be different (Fig. 1D). Key residues of the BLA chain other’s A or B chain. The b strand extensions cause an increase involved in HLA-DM–DR complex interactions have been in main-chain hydrogen bonding, which makes it more difficult substituted, suggesting that the DM–MHC-II binding mode in to select A-to-B binding based on lineage-specific interactions. chickens may be different from that in humans (Fig. 8A). Be- This suggests that in species that show the a1domainregion cause the a-chain is invariant, the diversity of the presented Ag extension, namely birds and reptiles, there is reduced evolu- peptide depends on the b-chain. In the heterodimer, the func-

tionary pressure to maintain multiple classical MHC-II lineages, tion of the a-chain may be to bind with the b-chain to stabilize by guest on September 27, 2021 with A and B molecules that do not cross-react. Indeed, the the conformation, whereas the b-chain may tend to select and

FIGURE 7. Structural analysis of pBL2*019:01 pockets in the PBG. (A) The top view of the PBG is shown on the surface. (B–G) Details of the pBL2*019:01 P1, P4, P6, P7, P9, and P10 pockets are shown. The solvent-accessible surfaces of the a1andb1 domains are shown in orange and violet, respectively, and the residues composing these pockets are shown in labeled stick representations. Residues bound by these pockets are shown in green stick representations. Water molecules are shown as red spheres. The hydrogen bonds between peptide residues and pockets are shown as yellow dashed lines. 10 THE FIRST STRUCTURE OF THE CHICKEN pMHC-II COMPLEX

FIGURE 8. Analysis of the conservation of pBL2*019:01 interactions with the DM molecule and polymorphic residues in the pockets of the pBL2*019:01 b-chain. (A) Based on the HLA-DR1–DM complex structure (PDB code: 4FQX), the key residues of the P1 pocket of pBL2*019:01 that are Downloaded from in contact with the DM molecule are shown as sticks. The orange triangle in the sequence alignment indicates the amino acids that might interact with DM in pBL2*019:01. (B) Polymorphic residues in the pockets of the pBL2*019:01 b-chain are colored and labeled with corresponding abbreviations and sequence numbers shown in the table on the left. The peptide main chain is shown in cartoon representation, and the side chains are shown in stick representation; both are green. http://www.jimmunol.org/ bind peptides. Understandably, the difference in binding pep- with HLA-DR1 suggests a common, polyproline II-like conformation for bound peptides. Proc. Natl. Acad. Sci. USA 93: 734–738. tides leads the a-chain to adjust accordingly. Finally, it must be 11. Zavala-Ruiz, Z., E. J. Sundberg, J. D. Stone, D. B. DeOliveira, I. C. Chan, emphasized that the chicken pMHC-II a-andb-chain–pairing J. Svendsen, R. A. Mariuzza, and L. J. Stern. 2003. Exploration of the P6/P7 mechanism found in this study can be used as a reference for un- region of the peptide-binding site of the human class II major histocompatability complex protein HLA-DR1. J. Biol. Chem. 278: 44904–44912. derstanding the pairing mechanism of human and mouse MHC-II. 12. Zavala-Ruiz, Z., I. Strug, M. W. Anderson, J. Gorski, and L. J. Stern. 2004. A The chicken pMHC-II complex establishes a structural princi- polymorphic pocket at the P10 position contributes to peptide binding specificity ple for designing epitope-related vaccines for the prevention of in class II MHC proteins. Chem. Biol. 11: 1395–1402. 13. Nelson, C. A., and D. H. Fremont. 1999. 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