Figure 6

Mechanism for affinity switch in BMP-2 L100K/N102D. H-bond network around the conserved serine residue in Act-A (a), the BMP-2 variant L100K/N102D with increased ActR-IIB affinity (b) and wildtype BMP-2 (c). The conserved central H-bond between Ser88 Oγ (Ser90 in Act-A) and Leu61 amide of ActR-IIB is shown as green thick stippled line. The intramolecular H-bond network comprising Lys100, Asp102, Ser88 (Lys102, Asp104 and Ser90 in Act-A) and a nearby structurally conserved water molecule is indicated by stippled lines in magenta. The putative intermolecular H-bond between Lys102 of Act-A and Cys59 backbone carbonyl of ActR-IIB in the structure of the complex Act-A:ActR-IIB_ECD (PDB entry 1S4Y, [42]) is indicated by a thin line (a), as this H-bond is only present on one half of the dimeric complex and its geometrical parameters are close to exclusion cutoff criteria. A comparison of the position of the structurally conserved water molecule in the three structures shows that in wildtype BMP-2 (c) this solvent molecule is located directly above the central H-bond indicating direct accessibility of the H-bond by solvent. (d, e) Surface representation of ActR-IIB color coded by the contribution (ΔΔG in kJ mol^-1) of each residue side chain to the binding free energy for (d) wildtype BMP-2 and (e) Act-A as measured by alanine scanning mutational analysis (see Table 1). For residue L61 the exchange to proline was used to point out the influence of the central conserved H-bond. The ΔΔG values are given in kJ mol^-1. Dark red color marks hot spots of binding with an energy contribution of more than 15 kJ mol^-1. Residues in yellow contribute only little; energy contribution of residues marked in blue is considered insignificant.