Alanine-scanning mutations of the BMP-binding domain of recombinant secretory bovine spp24 affect cytokine binding.
Secreted phosphoprotein 24 kDa (spp24) is a bone morphogenetic protein (BMP)/transforming growth factor-β cytokine-binding protein. The spp24 BMP-2-binding/transforming growth factor receptor II homology-1 (TRH1) domain is a highly conserved N-to-C terminally disulfide-bonded 19-amino acid residue loop similar to those in fetuin and the BMP receptor II. TRH1 domains exhibit a characteristic BTB or β-pleated sheet/turn/β-pleated sheet secondary structure. Our objective was to identify amino acid residues in the spp24 TRH1 domain that bind BMP-2, starting with the nine invariant mammalian residues. Alanine scanning (substitution of Ala for a native residue) was conducted for Cys(110), Arg(111), Ser(112), Thr(113), Val(114), Ser(117), Val(121), Val(124), and Cys(128) of recombinant bovine spp24 (residues 24-203). Binding to rhBMP-2 was assessed by surface plasmon resonance, and the equilibrium binding constants were calculated assuming 1:1 binding between spp24 or its mutants and rhBMP-2, so that affinity = K(D) = k(d)/k(a). Replacing Arg(111) (a positively charged basic residue), polar residues Thr(113) and Ser(117), and the nonpolar Cys(128) with Ala had little effect on BMP-2 binding. Replacing Val(114) or Val(121) with Ala increased binding affinity, whereas replacing Cys(110), Ser(112), Val(124), or both Cys(110) and Cys(128) with Ala decreased it. The kinetics of spp24 binding to BMP-2 can be manipulated by replacing invariant TRH1 residues. Decreasing the relative degree of hydrophobicity in the β-pleated sheet secondary structural motif of the TRH1 domain by replacing key Val residues with Ala increased the affinity for BMP-2 whereas altering the composition of the α-helical turn did not. Thus, the β-pleated sheets play a greater role in BMP-2 binding than the α-helical turn.
Sun
Argus
A
Research Service, VA Greater Los Angeles Health Care System, Sepulveda, California 91343, USA.
Murray
Samuel S
SS
Simon
Robert J
RJ
Jawien
Janusz
J
Behnam
Keyvan
K
Miller
Timothy A
TA
Brochmann
Elsa J
EJ
D000409:Alanine / Q000235:genetics*; D000595:Amino Acid Sequence; D000818:Animals; D055396:Bone Morphogenetic Protein 2 / Q000235:genetics* / Q000378:metabolism; D002417:Cattle; D016207:Cytokines / Q000378:metabolism*; D008297:Male; D051379:Mice; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation / Q000235:genetics*; D010750:Phosphoproteins / Q000235:genetics* / Q000378:metabolism*; D011485:Protein Binding / Q000235:genetics; D017433:Protein Structure, Secondary / Q000235:genetics; D017434:Protein Structure, Tertiary / Q000235:genetics; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012756:Sheep; D013552:Swine
C525719:Bmp2 protein, mouse; D055396:Bone Morphogenetic Protein 2; D016207:Cytokines; D010750:Phosphoproteins; D011994:Recombinant Proteins; C091340:secreted phosphoprotein 24; D000409:Alanine