The Structure of the MHC Class I Molecule of Bony Fishes Provides Insights into the Conserved Nature of the Antigen-Presenting System.

MHC molecules evolved with the descent of jawed fishes some 350-400 million years ago. However, very little is known about the structural features of primitive MHC molecules. To gain insight into these features, we focused on the MHC class I Ctid-UAA of the evolutionarily distant grass carp (Ctenopharyngodon idella). The Ctid-UAA H chain and ß2-microglobulin (Ctid-ß2m) were refolded in vitro in the presence of peptides from viruses that infect carp. The resulting peptide-Ctid-UAA (p/Ctid-UAA) structures revealed the classical MHC class I topology with structural variations. In comparison with known mammalian and chicken peptide-MHC class I (p/MHC I) complexes, p/Ctid-UAA structure revealed several distinct features. Notably, 1) although the peptide ligand conventionally occupied all six pockets (A-F) of the Ag-binding site, the binding mode of the P3 side chain to pocket D was not observed in other p/MHC I structures; 2) the AB loop between ß strands of the α1 domain of p/Ctid-UAA complex comes into contact with Ctid-ß2m, an interaction observed only in chicken p/BF2*2101-ß2m complex; and 3) the CD loop of the α3 domain, which in mammals forms a contact with CD8, has a unique position in p/Ctid-UAA that does not superimpose with the structures of any known p/MHC I complexes, suggesting that the p/Ctid-UAA to Ctid-CD8 binding mode may be distinct. This demonstration of the structure of a bony fish MHC class I molecule provides a foundation for understanding the evolution of primitive class I molecules, how they present peptide Ags, and how they might control T cell responses.

Crystallization and preliminary X-ray diffraction analysis of the two distinct types of zebrafish ß2-microglobulin.

ß(2)-Microglobulin (ß(2)m) noncovalently associates with the heavy chain of major histocompatibility complex class I (MHC I) molecules, which bind foreign antigen peptides to control the cytotoxic T lymphocyte (CTL) immune response. In contrast to mammals, there are distinct types of ß(2)ms derived from two loci in a number of teleost species. In order to clarify the structures of the ß(2)ms, the zebrafish (Danio rerio) ß(2)ms Dare-ß(2)m-I and Dare-ß(2)m-II were expressed in Escherichia coli, purified and crystallized, and diffraction data were collected to 1.6 and 1.9 Å resolution, respectively. Both crystals belonged to space group P2(1)2(1)2(1). The unit-cell parameters were determined to be a = 38.2, b = 50.4, c = 50.9 Å for Dare-ß(2)m-I and a = 38.9, b = 52.7, c = 65.8 Å for Dare-ß(2)m-II. Each asymmetric unit was constituted of one molecule, with Matthews coefficients of 2.22 and 3.01 Å(3) Da(-1) and solvent contents of 45 and 59% for Dare-ß(2)m-I and Dare-ß(2)m-II, respectively. These two ß(2)m structures will provide relevant information for further studies of the structures of the MHC I complex.

Complex assembly, crystallization and preliminary X-ray crystallographic analysis of the bovine CD8αα-BoLA-2*02201 complex.

In order to clarify the structural characteristics of the bovine MHC class I molecule (BoLA-I) complexed with CD8αα (CD8αα-BoLA-I), bovine CD8αα, BoLA-I (BoLA-2*02201) and ß2m were expressed and purified, and were then assembled with a peptide derived from Foot-and-mouth disease virus (FMDV-VP1YY9) and crystallized. The crystal diffracted to 1.7 Šresolution and belonged to space group P21, with unit-cell parameters a=53.9, b=103.8, c=61.8 Å, α=γ=90, ß=96°. The asymmetric unit contained one complex, with a Matthews coefficient of 2.41 Å3 Da(-1) and a solvent content of 48.9%. The rotation-function Z-score and translation-function Z-score for molecular replacement were 3.4 and 8.9, respectively. In addition, SDS-PAGE analysis of CD8αα-BoLA-I crystals showed three bands corresponding to the molecular weights of BoLA-I heavy chain, ß2m and CD8α. The structure of the CD8αα-BoLA-I complex should be helpful in obtaining insight into the interaction between bovine CD8αα and MHC class I molecules. Structure determination of BoLA-2*02201-FMDV-VP1YY9 will be useful in the design of vaccines for foot-and-mouth disease.

Crystal structure of swine major histocompatibility complex class I SLA-1 0401 and identification of 2009 pandemic swine-origin influenza A H1N1 virus cytotoxic T lymphocyte epitope peptides.

The presentation of viral epitopes to cytotoxic T lymphocytes (CTLs) by swine leukocyte antigen class I (SLA I) is crucial for swine immunity. To illustrate the structural basis of swine CTL epitope presentation, the first SLA crystal structures, SLA-1 0401, complexed with peptides derived from either 2009 pandemic H1N1 (pH1N1) swine-origin influenza A virus (S-OIV(NW9); NSDTVGWSW) or Ebola virus (Ebola(AY9); ATAAATEAY) were determined in this study. The overall peptide-SLA-1 0401 structures resemble, as expected, the general conformations of other structure-solved peptide major histocompatibility complexes (pMHC). The major distinction of SLA-1 0401 is that Arg(156) has a "one-ballot veto" function in peptide binding, due to its flexible side chain. S-OIV(NW9) and Ebola(AY9) bind SLA-1 0401 with similar conformations but employ different water molecules to stabilize their binding. The side chain of P7 residues in both peptides is exposed, indicating that the epitopes are "featured" peptides presented by this SLA. Further analyses showed that SLA-1 0401 and human leukocyte antigen (HLA) class I HLA-A 0101 can present the same peptides, but in different conformations, demonstrating cross-species epitope presentation. CTL epitope peptides derived from 2009 pandemic S-OIV were screened and evaluated by the in vitro refolding method. Three peptides were identified as potential cross-species influenza virus (IV) CTL epitopes. The binding motif of SLA-1 0401 was proposed, and thermostabilities of key peptide-SLA-1 0401 complexes were analyzed by circular dichroism spectra. Our results not only provide the structural basis of peptide presentation by SLA I but also identify some IV CTL epitope peptides. These results will benefit both vaccine development and swine organ-based xenotransplantation.

Crystallization and preliminary X-ray crystallographic studies of swine CD8α.

CD8αα homodimers or CD8αß heterodimers form on the T-cell surface, where they are essential as co-receptors for MHC class I molecules in activation of the CTL response. To date, swine have been found to show the highest percentage of lymphocytes with surface expression of CD8α. Crystallographic analysis of swine CD8α (sCD8α) to 1.8 Šresolution revealed that the crystals belonged to space group P3(2)21, with unit-cell parameters a = 80.97, b = 80.97, c = 95.19 Å. The Matthews coefficient and the solvent content were calculated to be 3.23 Å(3) Da(-1) and 61.89%, respectively. These results may aid further structural and functional analyses of sCD8α.