## 2 Results
### A Central Binding Groove in LAIR-1 Common for C1q and Generic Collagen Recognition  
LAIR-1 is known to be a collagen receptor and previous studies identified a three-stranded \(\beta\)-sheet as the surface involved in the interaction of LAIR-1 lg-like domain with collagens (in grey in Figure 5A,B). Among the residues forming this interaction groove, two LAIR-1 central amino acids, arginine 59 (R59) and glutamate 61 (E61) are essential for its binding to collagens I, III and IV [22]. Interestingly, these two important residues for collagen interaction are conserved in the homologous glycoprotein VI receptor (GPVI) involved in collagen-induced activation and aggregation of platelets (Figure 5A,B; [23]. Based on the assumption that LAIR-1 interaction with C1q CLR could be similar to the one with collagens I, III and IV, we produced LAIR-1 lg-like domain variants with an alanine residue at positions 59 and 61 (R59A and E61A, respectively). In order to investigate the mutation effects on the interaction of LAIR-1 with a generic collagen used as a positive control, we tested LAIR-1 interaction with a synthetic homo-otrimeric collagen peptide containing a central stabilizing GPKGEQ motif, found in several defense collagen sequences, surrounded by GPO triplets [24; 25]. To do so, we took advantage of the fact that the collagen binding groove in LAIR-1 contains a unique exposed tryptophan residue (W109) (Figure 5B). Indeed, the interaction of the collagen peptide with LAIR-1 may thus change the tryptophan environment and induce a transition in its fluorescence properties that can be detected using nano-differential scanning fluorimetry (nanoDSF). As illustrated in Figure 5C, collagen peptide binding to LAIR-1 was assessed using nanoDSF measurements. This showed that both independent substitutions of R59 or E61 strongly impact LAIR-1 binding to the collagen peptide, with a higher effect for the R59A mutation. Figure 4: Analysis of LAIR-1 interaction with C1 qCLR_nc2 and C1 qstem_nc2. (**A**) Schematic representation of the full length C1 qCLR_nc2 protein (left part) and C1 qstem_nc2 fragment (right part), the C1r2s2 binding site is represented with an orange star. (**B**,**C**) Soluble LAIR-1 lg-like domain was injected at increasing concentrations over immobilized C1 qCLR_nc2 (**B**) and C1 qstem_nc2 (**C**). Fits obtained by a global fitting of the data to a steady-state model are shown in the top right corner of each panel. The results shown are representative of three experiments. SPR experiments on immobilized sC1q, C1 qCLR_nc2 and C1 qstem_nc2 showed that these substitutions of arginine 59 or glutamate 61 into an alanine residue almost completely abolish LAIR-1 interaction with C1q, pointing out that, as for the positive collagen peptide control, these two residues are essential for C1q interaction (Figures 6 and 7B). The E61A mutation effect on LAIR-1 interaction with C1q fragments is slightly weaker than for the R59A mutation, which is in line with the nanoDSF experiments with a collagen peptide (Figures 5C, 6 and 7B). Circular dichroism (CD) experiments confirmed that these mutations do not affect folding, since both R59A and E61A variants displayed similar CD spectra to the WT LAIR-1 Ig-like domain (Figure S2). These results unveil that C1q interaction with LAIR-1 is based on a generic collagen recognition by the two central arginine 59 and glutamate 61 LAIR-1 residues. Previous reports showed that LAIR-1 and its soluble homologue LAIR-2 have different affinities for C1q [14; 26] and comparison of LAIR-1 and LAIR-2 sequences highlights only few differences in the amino acids exposed on the \(\beta\)-sheet forming the interaction groove for collagen binding (Figure 5A). Among them, the isoleucine 102 of LAIR-1, which is in a central position in the interaction groove (Figure 7A), corresponds to a leucine residue in LAIR-2 sequence.