Construct design and labeling strategy. (a) Assembly of DAP12 with the NKG2C-CD94 heterodimer in the membrane (left); assembly of DAP12_TM with NKG2C_TM (middle; NKG2C ectodomain and CD94 omitted as they do not participate in the transmembrane assembly); and covalent peptide construct representing the membrane-embedded portion of the trimolecular complex (right), (b-d) The tr-HSQC spectra of trimer samples segmentally labeled with ¹⁵N-²H on the DAP12_TM (D12_TM)-only strand (b) or on the DAP12_TM-NKG2C_TM (2C_TM) strand (c), and of the DAP12_TM homodimer alone (d), recorded for samples prepared in 250 mM tetradecylphosphocholine with 25 mM SDS in 20 mM phosphate buffer, pH 6.8, and recorded at 600 MHz and 303 K. Top row, separate view of glycine region. Amino acid positions are noted with single-letter designations and position number. Data are representative of at least three experiments each.

Structure of the DAP12_TM-DAP12_TM-NKG2C_TM complex, (a) One structure from the ensemble of the fifteen trimer structures of lowest energy (helical portions only), showing surface features of the DAP12_TM dimer (all NKG2C_TM (orange ribbon) side chains omitted for clarity); amino acid and position included for regions of interest (bundle views, sample NOE strips and assigned methyl spectra, Supplementary Figs. 2 and 3). C term, carboxyl terminus; N term, amino terminus. (b,c) In vitro translation–based assay of the effects of DAP12 transmembrane substitutions on its assembly with NKG2C (b) or KIR2DS3 (c), assessed with ³⁵S-labeled proteins extracted in 0.5% digitonin and immunoprecipitated with monoclonal antibody to the hemagglutinin tag (DAP12). Numbers below lanes indicate assembly efficiency, calculated as the ratio of receptor to DAP12 dimer and presented relative to assembly efficiency with wild-type DAP12 (set as 100%). WT, wild-type; T20A, substitution of alanine for threonine at position 20; A24F, substitution of phenylalanine for alanine at position 24; 3GL and 3GF, triple substitution of leucine (3GL) or phenylalanine (3GF) for Gly7, Gly11 and Gly15; D12-D, DAP12 dimer; D12-M, DAP12 monomer. Data are from at least two experiments with similar results (mean). (d,e) Two views of one structure from the ensemble of fifteen DAP12_TM dimer structures (helical portions only, from Gly7 to Leu30): red, side-chain oxygen atoms; yellow, glycine residues at positions 7, 11 and 15, as in a (bundle views, sample NOE strips and assigned methyl spectra, Supplementary Figs. 2 and 3). (f) In vitro translation analysis of DAP12 transmembrane substitutions in homodimer formation as in b,c. IAVA, double substitution of alanine for isoleucine at position 12 and alanine for valine at position 13; D16A, substitution of alanine for aspartic acid at position 16; DATA, double substitution of alanine for aspartic acid at position 16 and alanine for threonine at position 20. Numbers below lanes indicate assembly efficiency, calculated as the ratio of DAP12 dimer to DAP12 monomer and presented relative to assembly efficiency with wild-type DAP12 (set as 100%). Data are from at least two experiments with similar results (mean). (g) Alignment of human DAP12 and NKG2C transmembrane sequences; numbers indicate amino acid positions in the engineered NMR constructs. Letter color and underlining: red, electronegative; blue, electropositive.

Structural comparison of ζζ_TM and DAP12_TM receptor-binding sites, (a) En face view of ribbon structures showing the relative locations of disulfide bonds and side chains that participate in the receptor-binding sites in the ζζ_TM-ζζ_TM dimer (blue ribbons; Protein Data Bank, 2HAC) and DAP12_TM-DAP12_TM dimer (green ribbons); all other side chains omitted for clarity. Numbers below indicate χ1 values for aspartic acid side chains. (b,c) Enlargement of the receptor-binding sites of homodimers of ζζ_TM (b) and DAP12_TM (c); green dotted lines indicate putative hydrogen bonds.

Structure of the electrostatic network at the DAP12_TM-DAP12_TM-NKG2C_TM binding site, (a) Bundle of five selected ribbon structures of the DAP12_TM-DAP12_TM-NKG2C_TM covalent trimer showing possible configurations of the critical binding site (all other side chains omitted for clarity). (b,c) Enlarged views of the electrostatic network at Asp16-Thr20-Lys52, presented in the same orientation as in a (b) and presented in an axial view from above (c); green dotted lines indicate putative hydrogen bonds.

A similar electrostatic network governs the assembly of NKG2C-DAP12, NKG2D-DAP10 and TCR-CD3 complexes, (a-c) Sequence alignment of vertebrate DAP12_TM (a) and DAP10_TM (b) and human and mouse CD3_TM (c); red, acidic and hydroxyl-bearing residues involved in receptor binding; green, conserved and semiconserved aromatic residues that suggest further similarities in helix packing. Homo, Homo sapiens; Mus, Mus musculus; Bos, Bos taurus; Xeno, Xenopus laevis; Danio, Danio rerio; Fugu, Fugu rubripes. (d-f) In vitro analysis of the effect of substitution of alanine for serine or threonine in the transmembrane domains on the assembly of DAP10 with NKG2D (d), TCRα with CD3δ-CD3ε (e) or TCRβ with CD3γ-CD3ε (f), assessed as described above (Fig. 2), with monoclonal antibody to hemagglutinin (DAP10; d) or to CD3 (UCH-T1; e,f) used for immunoprecipitation. (d) Formation of disulfide-linked dimers of wild-type DAP10 (D10: WT) and mutant DAP10 with serine-to-alanine substitution (D10: SA), assessed in nonreducing conditions (without dithiothreitol (–DTT); left) or reducing conditions (because oxidized NKG2D runs as a diffuse band that is difficult to quantify; +DTT; right). (e,f) δ(WT), ε(WT) or γ(WT), wild-type CD3δ, CD3ε or CD3γ; δ(TA) or ε(TA), mutant CD3δ or CD3ε with threonine-to-alanine substitution; γ(SA), mutant CDγ with serine-to-alanine substitution. Numbers above lanes indicate assembly efficiency, calculated as the ratio of DAP10 dimer to DAP10 monomer (b, left), NKG2D to total DAP10 (b, right) or TCR to CD3ε (c) and presented relative to assembly efficiency with wild-type DAP12 (set as 100%). Data are from at least three experiments with similar results (mean).

Proposed model for intramembrane immunoreceptor complex assembly. (a) Dimeric signaling modules (CD3, DAP10 or DAP12; green coils) exist as metastable intermediates in the endoplasmic reticulum membrane, whereas newly synthesized receptor subunits (orange coils) are either incorporated into complexes or rapidly degraded^4,27. The electrostatic network in the signaling dimer (red shading) shows a symmetrical electron distribution and may have multiple opportunities to assemble with basic residues (blue shading) from receptor subunits. K(R), lysine or arginine; T(S), threonine or serine, (b) Once a receptor subunit has associated with an available binding site, an asymmetric redistribution of electronegativity in the network (color gradients) may render the opposite side of the signaling module unable to bind a second receptor. This trimeric assembly therefore represents the fundamental structural unit that organizes immunoreceptor complexes, (c) In a step that is either subsequent to or simultaneous and cooperative with that in b, receptors that form dimers (such as TCR, NKG2D and the mouse Ly49 family) combine two or more trimeric units to form the final complex. The relative orientation of the individual trimeric units cannot be determined from structural or biochemical data available at present.