FIGURE 4.

Highlight of residues in the Ca²⁺-binding loop of the protease domain in the structures of FVIIa_D212N (A and B; 2.7 Å) and wild-type FVIIa (C; 2.0 Å; PDB code 1DAN). The N terminus is indicated with an arrow and hydrogen bonds shown by dotted lines.2F_o – F_c electron density maps are shown at 1σ (blue) and 2σ (red). The mutant structure shows changes of hydrogen bond networks in the Ca²⁺-binding loop (Ca²⁺ shown as a green sphere), for example, surrounding mutated residue Asn²¹² and Ser²¹⁴ and Glu²⁹⁶. A hydrogen bond is abolished between Asn²¹² and Ser²¹⁴ because of a side chain movement of Ser²¹⁴ in the mutant structure. A hydrogen bond network is introduced between Asn²¹², Glu²⁹⁶, and two strongly defined water molecules. In the wild-type structure, Asp²¹² and Glu²⁹⁶ are not in an electrostatically optimal configuration because of charge repulsion and the conformations of the two residues are slightly changed in the mutant structure. A distinct side chain movement of Asp²¹⁷ can be observed as well. In turn, a hydrogen bond between Lys¹⁶¹ and Asp²¹⁷ is lost, whereas bonding to Asp²¹⁹ is strengthened: Asp²¹⁹ to Lys¹⁶¹ is 2.6 Å in the mutant structure (see B) versus 3.9 Å in the wild-type structure (see C). Root mean square displacements (Cα) of the Ca²⁺-binding loop of the mutant structure versus the wild structure were 0.74 Å compared with an overall of 0.65 Å for the heavy chains.