Alanine-scanning mutations of the BMP-binding domain of recombinant secretory bovine spp24 affect cytokine binding

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Abstract
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 Cys110, Arg111, Ser112, Thr113, Val114, Ser117, Val121, Val124, and Cys128 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 = KD = kd/ka. Replacing Arg111 (a positively charged basic residue), polar residues Thr113 and Ser117, and the nonpolar Cys128 with Ala had little effect on BMP-2 binding. Replacing Val114 or Val121 with Ala increased binding affinity, whereas replacing Cys110, Ser112, Val124, or both Cys110 and Cys128 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.

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