Table I. Combination of amino acid substitutions in BMP-2 double mutant proteins.

Variant	Epitope	BMPR-IA		BMPR-II	ActR-II	ALP(250)
		koff	kon	EQ45			+10 nM BMP-2	+20 nM BMP-2
		(var)/(wt)				(%BMP-2)
BMP-2		1.0	1.0	0.99	1.0	100	320	220
D30A	1	3.0	0.97	1.0	1.0	62	330	220
W31A	1	5.7	0.91	0.43	0.69	50	150	120
D30A/W31A	1/1	30	0.92	0.49	1.0	<0.5	88	99
F49A	1	1.0	0.10	0.81	0.68	17	210	130
P50A	1	0.80	0.09	0.74	0.67	10	420	170
F49A/P50A	1/1	2.6	0.02	0.84	0.60	<0.5	130	120
H39D	2	1.1	0.79	0.24	0.79	18	100	82
S88A	2	1.1	0.78	0.29	0.32	2.4	65	71
L100A	2	1.2	0.81	0.22	0.34	2.0	32	18
H39D/S88A	2/2	1.2	0.88	0.09	0.35	<0.5	34	50
H39D/L100A	2/2	1.1	0.90	0.02	0.33	0.6	2.6	4.2
A34D	2	0.56	0.56	0.06	0.38	<0.5	<0.5	2.9
D53A	1	1.1	1.2	0.99	1.2	130
E109R	2	1.2	0.76	2.2	1.0	150
D30A/A34D	1/2	1.9	0.85	0.02	0.31	<0.5	2.2	7.4
A34D/D53A	2/1	1.1	1.5	<0.02	0.31	<0.5	<0.5	0.7
D53A/E109R	1/2	1.4	1.3	2.7	1.1	130

Additive effects of kinetic rate constants k_on and k_off for association and dissociation of the complex with BMPR-IA ectodomain as well as of equilibrium binding at 45 nM concentrations, EQ₄₅, to BMPR-II and ActR-II ectodomain. ALP activity at 250 nM variant, ALP(250), was measured in the absence and presence of 10 or 20 nM BMP-2.