Ly-49C bound to H-2Kb (PDB ID: 3C8K) from Mus musculus

Created by Heather Lee

Ly49C (PDB ID 3C8K) is a member of the Ly49 receptor family, which contains 23 known receptors Ly49A-Ly49W (5, 4). Ly49 receptors are MHC class I molecule-binding proteins found on NK cells of animals from the genus species mus musculus (5,4). Through these receptors, NK cells monitor the presence of surface MHC class I molecules on cells throughout the body (3). MHC Class I molecules are ubiquitously expressed on the surface of normal cells (3, 4, 5). Cells with downregulated MHC class I presentation, such as infected, tumorigenic or dying cells, lack inhibitory signals from Ly49 receptors on the NK cell and the NK cell is thereby activated to perform cytolytic effector functioning (5,4). Thus, Ly49 receptors are crucial players in NK cell-mediated clearance of tumors and virally infected cells (4). 

Recent studies have suggested a possible second role of Ly49 inhibitory receptors in “licensing” NK cells for greater effector functioning (3). Engagement of certain inhibitory receptors on an NK cell seems to correspond with higher cytotoxicity through activation of activating receptors on the same NK cell, meaning signaling from an inhibitory receptor is necessary for an NK cell to reach its full effector potential (3, 10). Two inhibitory receptors that have shown this licensing effect are Ly49C bound to MHC class 1 molecule H-2Kb and Ly49A bound to MHC class 1 molecule H-2Dd (3, 10). 

In general, Ly49 receptors are C-type lectin-like homodimeric transmembrane glycoproteins found on the NK cell surface (5,4). Each receptor has a natural killer receptor domain (NKD) linked by 70 residues to the transmembrane and cytoplasmic domains (5). The cytoplasmic domain contains an immunoreceptor tyrosine-based inhibitory motif (ITIM) on the end. When phosphorylated, the ITIM recruits SH2-domain-containing protein tyrosine phosphatase 1 (SHP1), which then leads to the dephosphorylation of the nucleotide exchange factor VAV1 and the subsequent disruption of activation signals (10). See Image 1. The transmembrane stalk has great flexibility to allow MHC class I binding on either the NK cell itself or another cell (5). Ly49 receptors have three domains for MHC recognition: a central binding site similar in all receptors and two variable regions corresponding to MHC molecule specificity (5). Ly49 receptors recognize either both H-2D and H-2K alleles (which encode MHC class I molecules), such as Ly49C, or only H-2D alleles, such as Ly49A (5,4).

Ly49C binds to H-2Kb, H-2Kd, and H-2Dd MHC class I molecules (4). The ExPASY Bioinformatics Resource Portal calculated the Ly49C molecular weight to be 31311.1Da with an isoelectric point of 9.02. The H-2Kb molecular weight is given as 39152.1Da and the isoelectric point as 5.85. Deng. Et. al describe the crystal structure of Ly49C homodimer bound to H-2Kb. The crystal structure shows only the NKD and does not include the transmembrane stalk or cytoplasmic domain.  Each Ly49C monomer can bind one H-2Kb molecule, which has two chains, an alpha chain with subunits a1/a2 and a3 and a beta2-microglobulin (B2M) chain (2, 4, 5).  Subunits a1/a2 and a3 form the heavy chain portion of the MHC class 1 molecules, which serves to hold and present protein peptides for recognition by multiple immune cells, including CD8 T cells (2). The B2M chain is not polymorphic, unlike the alpha chain, and it is thought to help serve as a recognition site for inhibitory receptors on NK cells (2).  

Analysis by the DDSP tool reveals the secondary structural composition of the subunits. The H-2Kb alpha subunits are composed of 25% helical (6 helices; 0 residues), 40% beta sheet (19 strands; 111 residues) while the B2M subunit is 49% beta sheet (11 strands; 49 residues). The Ly49C monomer is 20% helical (3 helices; 26 residues), 36% beta sheet (8 strands; 45 residues). See slide for secondary structure. The secondary structure alpha helices and beta sheets serve to sequester polar regions of the protein. A significant amount of both the receptor and MHC class 1 molecule are not involved in secondary structures, leaving large regions of polar surface that serves to enhance the favorability of binding, as binding can burry polar solvent-accessible regions.  

The crystal structure of the full homodimer reveals that the receptor binds to the molecule symmetrically from both NKDs (5). See Image 2. The binding space of the Ly49C monomer is composed to hydrophilic and polar interactions, including 13 hydrogen bonds and 12 salt bridges, that lead to significant solvent-accessible surface buried during binding (5). The primary binding sites are loop 3 (L3), alpha helix 3 (a3), beta strand 3 (B3), beta strand 4 (B4), loop 5 (L5) and loop 6(L6). Binding at the L3/a3 region gives important stability to the complex as it buries 33% of total solvent accessible surface area in the binding interface (5). Within this L3/a3 region of Ly49C, the primary interactions include Ly49C residues Arg 230, Lys 228, Met 225, Asn 226, and Lys 221 with H-2Kb residues Asp212, Iso 213, Asp 30, Leu 110, Arg 111, and Glu 128 on the (5). Upon binding to H-2Kb, little conformational changes to the receptor take place. Most noticeably, the alpha carbon of Ly49C residue Lys 228 shifts 1.7Å to form a salt bridge with H-2Kb Asp 30 and Asp 212 (5). 

The complex Ly49C bound to H-2Kb shares many similarities with its relative, Ly49A bound to H-2Dd (PDB ID IQO3). However, unlike Ly49C-H-2Kb, symmetrical binding is not seen in Ly49A-H-2Dd, which binds at only one of the sites of Ly49C-H-2Kb and another unique site (9). See Image 3. The Ly49C NKD has a similar structure to Ly49A, but has a unique secondary structure of three alpha helices with two short loops between the second and third helix (5). Ly49C forms 10 hydrogen bonds with H-2Kb, while Ly49A forms 20 bonds with H-2Dd (4).  When superimposing Ly49C and Ly49A, binding sites B3, B4, L5, and L6 are very similar and most differences are in L3 and a3, as well as other regions less involved in binding (5, 9). The residues in a3, a helix characteristic of Ly49C compared to other Ly49 receptors, make crucial interactions with H-2Kb. Met 225 and Asn 226 residues form van der Waals interactions with H-2Kb residues Leu 110, Arg 111, and Glu 128 and Ly49C residue Lys 221 forms a salt bridge with H-2Kb Glu 128 (5). 

Significant interactions also take place between Ly49 receptors and the B2M domains of the H-2 molecules. Ly49A contacts B2M residues Lys 58 and Gln 29 of H-2Dd, whereas Ly49C contacts only Lys-58 of H-2Kb (4). Furthermore, far greater interactions take place near the Ly49-B2M interface of Ly49A-H-2Dd (including 20 hydrogen bonds) than Ly49C-H-2Kb (including 10 hydrogen bonds) (4). Unfortunately, the crystal structure of the Ly49A2 binding complex is not available on the Protein Data Bank. See Image 4. The fewer number of interactions Ly49C has with its ligand may be a possible explanation for the cross reactivity of Ly49C with H-2Kb, H-2Kd, and H-2Dd, given that Ly49A reacts only with H-2Dd and H-2Dk (4).

Advanced algorithmic protein databases were used to analyze Ly49C and H-2Kb. The Position-Specific Integrated Basic Local Assignment Search Tool (PSI-Blast) identified similar primary sequence structure with similarity rated by an E value, with E=0 being an identical match. Similar sequences to Ly49C-H-2Kb predictably include Ly49A with H-2Dd (E = 5e-178). Other similar sequences to H-2Kb include H-2Kk (E=3e-180), H-2Dk (E = 1E-179), H-2Dr (E=6E-175), and H-2Db (E=3E-170) , H-2Kd (E=2E-166). Tertiary structure of the proteins was compared by Dali server that gives a Z score with greater than 2 being similar. Comparison to H-2Kb showed similarities to H-2Db (Z=33.1), H-2Kd (Z = 32.7), H-2Dd (Z = 32.6), H-2Kk (Z = 31.4). The very similar E and Z values of H-2Kk, H-2Dk, and H-2Dd are consistent with the known reactivity of Ly49C to those MHC molecules. PSI-Blast also shows H-2Kb similarity between MHC class I antigens of rattus norvegicus, bos Taurus, critcetulus griseus, bison bison, lemur catta, callithrix jacchus, mesocricetus auratus, pongo pygmaeus, ovis arties, scriurus aberti, and homo sapiens most notably. This suggests that the mechanism of NK cell function in mus musculus is similar to that of other organisms. Thus, the study of Ly49 receptors may further the understanding of human immunology and advance medical knowledge.