Identification of the ligand-binding site of the BMP type IA receptor for BMP-4

Citation
T. Hatta et al., Identification of the ligand-binding site of the BMP type IA receptor for BMP-4, BIOPOLYMERS, 55(5), 2000, pp. 399-406
Citations number
21
Language
INGLESE
art.tipo
Article
Categorie Soggetti
Biochemistry & Biophysics
Journal title
BIOPOLYMERS
ISSN journal
0006-3525 → ACNP
Volume
55
Issue
5
Year of publication
2000
Pages
399 - 406
Database
ISI
SICI code
0006-3525(2000)55:5<399:IOTLSO>2.0.ZU;2-7
Abstract
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.