Modeling of CD300a structure complexed with PE and PS and mutational studies. (A) Alignment of human CD300a and human TIM-1 and TIM-4. The extra cellular domains of the CD300a receptor and the Ig-V like domain of TIM-1 and TIM-4 were aligned using the multiple sequence alignment program “CLUSTALW.” The yellow box highlights the WFND motif of the TIM proteins and the correspondent WLRD motif in CD300a. The residues in green and numbered in blue were shown to have potential for binding to PE and PS according to the model presented in (C). (B) Binding analysis of CD300a-Ig mutant proteins to dead PBLs. Purified CD300a-Ig mutants were incubated with starved PBLs and their binding to 7-AAD⁺ PBLs after adding anti-human Ig FITC was determined by flow cytometry. LAIR-1-Ig was used as a negative control protein. Numbers in the dot plots are the percentage of 7-AAD⁺ PBLs that bind the fusion proteins. A representative experiment is shown (top panel). Bar graph represents the average ± SEM. Results shown are from 3 independent experiments (bottom panel). (C) Modeling of CD300a IgV-like domain structure complexed with PS (green, top panel) and PE (blue, bottom panel). The aminophospholipids (PS and PE) and the divalent metal ion calcium (Ca²⁺), in violet, are embedded into the CD300a structure. The dotted red lines represent the bonds, with and without coordination by the metal ion calcium (Ca²⁺), between the aminoacids in CD300a and PS or PE. (D) Binding analysis of CD300a-Ig mutant proteins to dead PBLs. The residues proposed to bind Ca²⁺ or directly to PE and PS according to the model described in (C) were mutated and the Ig-fusion proteins were tested for the binding to the dead PBLs. Purified CD300a-Ig mutants were incubated with starved PBLs and their binding to 7-AAD⁺ cells after adding anti–human Ig FITC was determined by flow cytometry. LAIR-1-Ig was used as a negative control protein. Numbers in the dot plots are the percentage of 7-AAD⁺ PBLs that bind the fusion proteins. Data shown is a representative of 3 independent experiments.