Bone morphogenetic proteins (BMPs) belong to the transforming growth factor
-beta (TGF-beta) superfamily of multifunctional cytokines. BMP induces its
signal to regulate growth, differentiation, and apoptosis of various cells
upon trimeric complex formation with two distinct type I and type II recept
ors on the cell surface: both are single-transmembrane serine/threonine kin
ase receptors. To identify, the amino acid residues on BMP type I receptor
responsible for its ligand binding, the structure-activity relationship of
the extracellular ligand-binding domain of the BMP type IA receptor (sBMPR-
IA) was investigated by alanine-scanning mutagenesis. The mutant receptors,
as well as sBMPR-IA, were expressed as fusion proteins with thioredoxin in
Escherichia coli, and purified using reverse phase high performance liquid
chromatography (RP-HPLC) after digestion with enterokinase. Structural ana
lysis of the parent protein and representative mutants in solution by CD sh
owed no detectable differences in their folding structures. The binding aff
inity of the mutants to BMP-4 was determined by surface plasmon resonance b
iosensor. All the mutant receptors examined, with the exception of Y70A, di
splayed reduced affinities to BMP-4 with the rank order of decreases: I52A
(17-fold) approximate to F75A (15-fold) much greater than T64A (4-fold) = T
62A (4-fold) approximate to E54A (3-fold). The decreases in binding affinit
y observed for the latter three mutants are mainly due to decreased associa
tion rate constants while alterations in rate constants both, for associati
on and dissociation, result in the drastically reduced affinities for the f
ormer two mutants. These results allow us to conclude that sBMPR-IA recogni
zes the ligand using the concave face of the molecule. The major ligand-bin
ding site of the BMP type IA receptor consists of Phe75 in loop 2 and Ile52
, Glu54, Thr62 and Thr64 on the three-stranded beta -sheet. These findings
should provide a general basis for the ligand/type I receptor recognition i
n the TGF-beta superfamily. (C) 2001 John Wiley & Sons, Inc.