## 2 Results
### Exploring the Distal LAIR-1 Residues Involved in C1q Binding  
Interestingly, in the selected model shown in Figure 8B, two glutamate residues at positions 72 and 111 of LAIR-1 (E72 and E111) are facing two positively charged C1q residues: lysine 65 of C1q B chain (B_K65) and arginine 72 of C1q A chain (A_R72), respectively. These four residues might likely lock the specific recognition of the central C1q hydroxyproline residues by LAIR-1 R59 and E61. Indeed, these two pairs of charge-opposed residues are located at each side of the collagen binding groove of LAIR-1 and could introduce more specificity toward C1q than generic collagen recognition (Figures 7A and 8B). In order to test the engagement of the two LAIR-1 glutamate residues, we generated two LAIR-1 variants. The SPR results show that both mutations reduce LAIR-1 binding to C1q and CLR_nc2 constructions, with a stronger effect for E72K than for E111K mutation (Figure 7B). However, these two mutations on LAIR-1 have no effect on LAIR-1 interaction with the control collagen peptide (Figure 7B, lower part). This suggests that the substitution of these two LAIR-1 glutamate residues more specifically impact the recognition of C1q by LAIR-1, possibly through C1q_A_R72 and B_K65 basic residues. In order to test the engagement of these two basic C1q residues in the interaction with LAIR-1, we generated two C1 qCLR_nc2 variants containing the substitution of lysine 65 on C1q B chain and arginine 72 on C1q A chain into glutamate residues (B_K65E and A_R72E,  
Figure 8: Model of LAIR-1 Ig-like domain in complex with a C1q collagen stem. (**A**) Sequences of C1q A, B and C chains corresponding to the C1q collagen-like region shown in B. The three lysine residues constituting the C1r\({}_{2}\)s\({}_{2}\) binding site (A_K59, B_K61 and C_K58) are in bold and the two essential lysines are surrounded with a dashed orange ellipse. The putative LAIR-1 binding OGXO motifs in this region are underlined. The remarkable residues are labeled with their position numbers. The three residues facing the LAIR-1 I102 in each alternative model are labeled in black. The “O” in the sequences stands for proline residues modified into hydroxyprolines, as determined in the literature [27; 28]. (**B**) Model of LAIR-1 Ig-like domain (blue, PDB code: 3 KGR) in interaction with the C1q CLR triple helix (A chain in cyan, B chain in pink and C chain in green). The collagen-binding groove of LAIR-1 is shown in grey and the side chains of the identified key binding residues are shown and labeled. Mutated lysine 65 and arginine 72 of C1q B and A chains, respectively, are labeled. The residues facing LAIR-1 I102 in each model are colored in black. The side chains of the identified OGXO motifs are shown in lines. respectively). SPR analyses showed that the B_K65E mutation did not have any effect on LAIR-1 interaction in comparison with the C1 qCLR_nc2 WT (Figure 9A and Table 1). Regarding the ClqCLR_nc2 A_R72E variant, the K\({}_{\text{D}}\) value obtained for LAIR-1 interaction was slightly higher than for ClqCLR_nc2 WT, suggesting that the arginine 72 on C1q A chain, albeit not essential, is likely involved in LAIR-1 interaction (Figure 9B and Table 1). These results can be related to the interaction behavior of LAIR-1 mutants of the two E72 and E111 residues. Indeed, the LAIR-1 E72K and E111K mutations exhibited weaker inhibitory effect on Clq interaction than the mutations in the central LAIR-1 binding site (i.e., R59 and E61; Figure 7). It is therefore consistent that the mutation of the suggested CLR residues facing the E72 and E111 residues of LAIR-1 triggers a weak effect on the global affinity of ClqCLR_nc2/LAIR-1 interaction, explained by their distal position from the central binding site of LAIR-1 (Figure 7B). We suggest that these two residues are not involved in the direct binding with LAIR-1 but lock and give the specificity of the interaction after initial LAIR-1 docking through its R59 and E61 residues.