Extended Data Table 1.

Izumo1-Juno binding interface mutations

Technique	Protein	Role of residue	Kd	k a (× 105) (M s) −1	k d (× 10−3) (s) −1
SPR	Wild-type	n/a	48 +/− 4 nM	4.2 +/− 0.2	19.4 +/− 0.9
BLI	Wild-type	n/a	59 +/− 1 nM	1.15 +/− 0.02	6.7 +/− 0.1
Izumo1 mutants
BLI	E71A Izumo122-254	salt bridge with Juno K163; L2 leash	96 +/− 13 nM	1.43 +/− 0.04	13.7 +/− 0.2
BLI	E71K Izumo122-254	salt bridge with Juno K163; L2 leash	119 +/− 19 nM	1.65 +/− 0.04	19.5 +/− 0.3
BLI	D72A Izumo122-254	H-bond to Q130 in unbound state	71 +/− 8 nM	1.78 +/− 0.04	12.6 +/− 0.2
BLI	Q130A Izumo122-254	H-bond to D72 in unbound state	56 +/− 19 nM	1.27 +/− 0.03	6.8 +/− 0.1
BLI	W148A Izumo122-254	conserved interface residue	no binding detected
BLI	H157A Izumo122-254	conserved interface residue	1.8 +/− 1 μM	0.51 +/− 0.05	79 +/− 2
BLI	R160A Izumo122-254	salt bridge with Juno E45	730 +/− 78 nM	0.78 +/− 0.04	56.3 +/− 0.9
BLI	R160E Izumo122-254	salt bridge with Juno E45	2.2 +/− 1.2 μM	0.32 +/− 0.02	51.2 +/− 1.1
Juno mutants
BLI	E45A Juno20-228	salt bridge with Izumo1 R160	681 +/− 141 nM	0.37 +/− 0.01	24.7 +/− 0.5
BLI	E45K Juno20-228	salt bridge with Izumo1 R160	2.8 +/− 0.2 μM	0.23 +/− 0.02	62.4 +/− 1.7
BLI	W62A Juno20-228	conserved interface residue	361 +/− 24 nM	1.14 +/− 0.04	41.2 +/− 0.6
BLI	L81A Juno20-228	conserved interface residue	9.1 +/− 0.4 μM	0.4 +/− 0.3	349 +/− 23
BLI	K163A Juno20-228	salt bridge with Izumo1 E71	97 +/− 9 nM	1.38 +/− 0.03	13.3 +/− 0.2
BLI	K163E Juno20-228	salt bridge with Izumo1 E71	134 +/− 4 nM	1.81 +/− 0.05	24.2 +/− 0.3

^*kinetic and binding affinity values are presented as the mean of technical triplicates (n=3) and errors as the standard deviation of the mean

K_d- dissociation constant

k_a - rate of association

k_a - rate of dissociation