The classical description of MHC molecules as cell surface receptors that bind peptide ligands to generate complexes detected by TCR and NK receptors has steadily given way to a more general view that the MHC fold may serve as a structural scaffold for a number of different functions, some immunological and some not. The identification of MHC-like genes in the DNA viruses and the demonstration that several of these encode molecules that behave as immunoevasins has provided the impetus for a more complete biochemical and structural analysis. Although ligands for some of the MHC-Iv molecules have been identified, detailed structural studies have been lacking. The description of the m144 structure reported here offers a tangible view of the evolution of an MHC-Iv molecule that contributes to viral virulence.
Molecules that contain an MHC fold serve a wide variety of functions, and their ligands belong to a number of different molecular families. Although no ligand for m144 has yet been identified, the known function of this molecule with respect to recognition by NK cells suggests that its primary molecular mechanism may be one of two: to be expressed at the cell surface of the MCMV infected cell to serve directly as a ligand for a NK cell inhibitory molecule; or to be expressed intracellularly in the infected cell to bind and sequester molecules, induced by the stress of viral infection, that would serve as ligands for NK activating receptors. It has been shown that the MHC-Iv molecule, m157, functions by the first mechanism,8,16
and m145, m152 and m155 function by the second.19-21,45
m144 can be detected at the surface of MCMV infected cells shortly after infection,46
and functional data suggest that it interferes with NK recognition.24,47,48
A viral mutant bearing an m144 deletion is cleared more efficiently in vivo than its wild-type counterpart.47
This phenotype is dependent upon NK cells of the host, and can be reversed by treating the host with NK cell-depleting anti-GM1 antibodies. In addition, the tumorigenicity of the RMA-S cell line can be enhanced by transfected expression of m144,24
and m144 transfection of target cells partially blocks antibody-dependent cellular cytoxicity by mouse NK cells.48
Cultured myoblasts, normally acutely rejected upon transplantation into normal syngeneic mice, can be tolerated if expressing transfected m144.49
The structure of m144 reveals several features that were predicted by amino acid sequence alignment and three-dimensional structure prediction programs: m144 preserves the MHC fold and it associates with the light chain β2m in a manner similar to other MHC-I molecules. Our biochemical analysis of the in vitro refolded m144 indicates that its assembly with β2m is weak. The structure also confirms the experimental observation that no peptides copurify with the recombinant molecule.22
TAP function, which provides peptides for stable assembly of MHC-I molecules, was not required for cell surface expression of m144. Also, no peptide addition was required for proper refolding of the E. coli expressed molecules. The lack of electron density in the putative peptide binding cleft, and the substitution of several amino acid residues that structurally are required for binding the amino and carboxyl termini of the peptide are further evidence for the lack of peptide ligands. We cannot formally rule out the possibility that other non-peptide ligands such as lipids or carbohydrate might be bound by m144. The lack of electron density in the cleft region and the lack of well defined pockets or channels in the molecule argue against this possibility.
Additional unexpected features of m144 have been elucidated by the determination of its three-dimensional structure. The unique intradomain disulfide bond linking the α1 helix with the β2 strand adds structural stability, and almost certainly explains the observed peptide-free thermal stability of m144.22
Our efforts to directly assess the importance of this unique disulfide bond using in vitro mutagenesis have been thwarted by the decreased solubility of mutants lacking either or both α1 domain cysteine residues. A large deletion encompassing what would be the b8 strand and the amino-terminal end of the region that would be the α2 helix, a unique characteristic of m144, eliminates residues that contribute to the F pocket (which anchors the peptide carboxyl terminus in MHC-I molecules). The lack of bound peptide might suggest that m144 has no physiological requirement to interact with components of the peptide loading complex that facilitate this process for MHC-Ia molecules.50,51
A crucial step in the loading of peptide into MHC-I and the selection of peptide ligands is the bridging of unfolded MHC-I with the peptide transporter, TAP, by tapasin. Amino acid residues of MHC-I that influence the interaction of the peptide-free MHC-I molecule in the endoplasmic reticulum with tapasin include residues 70, 86, 115, 116, 122, 128-136, and 151 in the α1α2 domain, and, in the α3 domain, 219-233.52
Although m144 conserves residues equivalent to MHC-I 70, 86, 116, and 122, its deletion of the b8 strand (133-135 in H-2Kb
) would appear to significantly impair tapasin interaction. In addition, the insertion in m144 of four residues into the region equivalent to the MHC-Ia α3 domain 219-233 loop would also be expected to impede tapasin interaction.
Lack of similarity of m144 in its α3 domain to the CD8 binding site of MHC-I molecules leads to the prediction that this viral molecule cannot bind the CD8 coreceptor. Consideration of the location of the four carbohydrate addition sites of m144, in particular the lack of positions that might block interaction across the α1 and α2 helices, and the presence of the carbohydrate at position 111, suggest that NK receptors of either the Ig or C-type lectin-like families might bind through the α1 and α2 helices. The possibility of an interaction involving the b-sheet floor, the α3 domain, and β2m, similar to the site of the Ly49/H-2 interaction, cannot be eliminated, though we would consider this unlikely.
The region of m144 not visualized in the electron density map, extending from amino acid residues Asp115 to Asp127, most likely represents a region of structural flexibility, and thus would be a candidate for the site of NK receptor interaction. Lack of electron density reveals the absence of a canonical structure in the repeating asymmetric units as propagated in the crystal, and suggests that, despite being tethered at both ends by amino acid residues of well-defined structure, this part of the molecule differs from one instance to another. The best example of an MHC-Ib molecule that has a specific region lacking electron density is that of MICA, which, remarkably, shows no density in a similar region of its α2 helix.53
However, in the complex of MICA with the NKG2D activating receptor, this region of the molecule tightens up, presumably as a result of this interaction, and is then revealed in electron density.25
We suggest that the undefined region between residues 115 and 127 is a flexible region, primarily a-helical, that in the absence of the m144 ligand explores a variety of positions in conformational space, and in the presence of its ligand favors one of these.
Another line of evidence is also consistent with the involvement of this flexible region with a host ligand. Comparison of the amino acid sequence of m144 with its rat viral homolog, r144 (), indicates that this molecule, with 36% identity to m144 over the extracellular domains, preserves the novel disulfide bond between the α1-helix and β2-strand, but has unique predicted N-asparaginyl carbohydrate addition sites (r144 has lost sites at 45, 55, and 111, preserved the one at 82, and has a new site at 121 (m144 numbering)). Strikingly, m144 and r144 differ the most in the region from residue 100 to 132 (only 13% identical, with two residues deleted in r144), the same region that lacks electron density in m144. Because of the apparent rapid evolution of these MHC-Iv molecules, in the context of rapidly evolving host responses,54
such variability further supports the view that this region may be involved in interaction with host NK receptors.
In summary, we describe the X-ray structure of an MHC-Iv molecule, m144, encoded by the murine CMV. Despite low (29%) amino acid sequence identity with MHC-Ia molecules, m144 preserves the MHC-I fold and β2m association, but lacks bound peptide or other identifiable small molecule ligand. A unique disulfide bond between the α1-helix and the β2-strand stabilizes the molecule. Further efforts to identify murine host ligand(s) for m144, and the comparative structure of other MHC-Iv molecules will be helpful in revealing not only the mechanism of action of these molecules, but also the nature of the evolutionary forces that have molded their structure.