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. 2006 Apr 25;4(5):e142. doi: 10.1371/journal.pbio.0040142

Figure 2. A Properly Placed Lysine within Polyvaline/Polyleucine TM Sequences Is Sufficient for Assembly with DAP12.

Figure 2

(A) TM sequences of the KIR mutants utilized in these experiments. The KIR TM domain was replaced with a polyvaline or polyleucine sequence retaining only the native lysine at position 9. In the KIR-(K→H) mutant, the lysine in the TM domain of KIR was substituted by histidine.

(B) Assembly of the KIR mutants utilized in these experiments with DAP12. Complexes were isolated by two-step snIP targeting the PC and HA tags attached to the two DAP12 chains. Quantification of DAP12-associated KIR is shown for the average of two separate experiments and adjusted for the total amount of KIR proteins prior to IP. WT KIR was set to 100%.

(C and D) Substantial changes in the KIR TM domain do not alter the interaction between the basic TM residue of KIR and the aspartic acid pair of DAP12. Assembly of KIR-WT (C) or KIR-pVal (D) with DAP12 was examined for DAP12 dimers with the WT aspartic acid pair (DD) or mutants in which one or both aspartic acids were substituted by asparagine (N), serine (S), or alanine (A). Each specific DAP12 dimer was isolated by two-step snIP (PC and HA) as in (B). For both KIR-WT and KIR-pVal, no assembly was observed when both DAP12 aspartic acids were substituted (NN, SS, and AA combinations, lanes 3, 5, and 7, respectively). For both KIR forms, a similar pattern was also observed with the set of mutants in which one aspartic acid was substituted (DN, DS, and DA combinations, lanes 2, 4, and 6, respectively). SDS-PAGE was performed under nonreducing conditions. The disulfide-linked DAP12 dimer is labeled as DAP12 CD. A small amount of non-covalent DAP12 dimer (DAP12 ND) was also isolated with this two-step IP method that targeted both DAP12 chains (the two chains dissociate during SDS-PAGE owing to the absence of the interchain disulfide bond). Mixing controls (denoted by asterisk) in lane 8 (C and D) were performed with WT KIR and DAP12. Quantification of DAP12-associated KIR proteins is an average of four (C) and two experiments (D), respectively.