The topography of membrane-bound blood coagulation factor VIIa (fVIIa) was examined by positioning a fluorescein dye in the active site of fVIIa via a tripeptide tether to yield fluorescein-D-phenylalanyl-L-prolyl-Larginyl-fVIIa (Fl-FPR-fVIIa). The location of the activesite probe relative to the membrane surface was determined, both in the presence and absence of tissue factor (TF), using fluorescence energy transfer between the fluorescein dye and octadecylrhodamine (OR) at the phospholipid vesicle surface. When Fl-FPR-fVIIa was titrated with phospholipid vesicles containing OR, the magnitude of OR-, calcium ion-, and phosphatidylserinedependent fluorescence energy transfer revealed that the average distance of closest approach between fluorescein in the active site of fVIIa and OR at the vesicle surface is 82 Å assuming a random orientation of donor and acceptor dyes ( 2 ‫؍‬ 2/3; the orientational uncertainty totals ϳ10%). The active site of fVIIa is therefore located far above the membrane surface, and the elongated fVIIa molecule must bind at one end to the membrane and project approximately perpendicularly out of the membrane.When Fl-FPR-fVIIa was titrated with vesicles that contained TF, the efficiency of energy transfer was increased by a TF-dependent translational and/or rotational movement of the fVIIa protease domain relative to the membrane surface. If this movement was solely translational, the height of the active site of fVIIa was lowered by an average of 6 Å after binding to TF. The association of fVIIa with TF on the membrane surface therefore causes a significant reorientation of the active site relative to the membrane surface. This cofactor-dependent realignment of the active-site groove presumably facilitates and optimizes fVIIa cleavage of its membrane-bound substrates.

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The acid-pair hypothesis, proposed by Reid and Hodges [(1980) J. Theor. Biol. 84, 401-444], suggests that the affinity of an EF-hand motif for Ca2+ will be maximal with four acidic ligands, paired along the +x, -x and +z, -z axes. Addition of a fifth anionic ligand is predicted to reduce Ca(2+)-binding affinity, as a consequence of increased electrostatic repulsion. Interestingly, for oncomodulin, we observe that introduction of a fifth carboxylate residue at the +z position in the CD coordination sphere or at the -x position in the EF coordination sphere significantly increases the affinity of those sites for Ca2+. The variants resulting from replacement of serine-55 by aspartate (S55D), glycine-98 by aspartate (G98D), and the combined mutations (55/98) have been examined in Ca(2+)- and Mg(2+)-binding studies, titration calorimetry, and differential scanning calorimetry. The KCa for the CD site is reduced from 800 to 67 nM by the S55D mutation, while KCa for the EF site is reduced from 45 to 4 nM by the G98D mutation. Both mutations destabilize the apo form of the protein and increase the thermal stability on the Ca(2+)-bound state. Interestingly, the S55D mutation also increases the affinity of the oncomodulin CD site for Mg2+, decreasing the dissociation constant from > 1 mM to approximately 30 microM. This increase in affinity is reflected in a substantially increased thermal stability of the Mg(2+)-bound form of the protein. In 0.15 M NaCl, 0.025 M Hepes (pH 7.4), and 0.01 M Mg2+, the wild-type protein denatures at 68.5 degrees C. By contrast, under identical conditions, the S55D mutations denatures at 79.0 degrees C. The increased metal ion-binding affinity displayed by the variant proteins may result in part from preferential destabilization of the apo-protein by the additional carboxylate.

The parvalbumin metal ion-binding sites differ at the +z and -x residues: Whereas the CD site employs serine and glutamate (or aspartate), respectively, the EF site employs aspartate and glycine. Although frequently indistinguishable in Ca2+- and Mg2+-binding assays, the CD and EF sites nonetheless exhibit markedly different preferences for members of the lanthanide series [Williams et al. (1984) J. Am. Chem. Soc. 106, 5698-5702], underscoring an intrinsic nonequivalence. This nonequivalence reaches its pinnacle in the mammalian beta-parvalbumin (oncomodulin). Whereas the oncomodulin EF site exhibits the expected Ca2+/Mg2+ signature, the Ca2+ affinity of the CD site is severely attenuated. To obtain insight into the structural factors responsible for this reduction in binding affinity, oncomodulin variants were examined in which the CD and EF site ligand arrays had been exchanged. Our data suggest that binding affinity may be dictated either by ligand identity or by the binding site environment. For example, the Ca2+ affinity of the quasi-EF site resulting from the combined S55D and D59G mutations is substantially lower than that of the authentic EF site. This finding implies that other local environmental variables (e.g., binding loop flexibility, electrostatic potentials) within the CD binding site supersede the influence of ligand identity. However, the CD site ligand array does not acquire a high-affinity signature when imported into the EF site, as in the D94S/G98D variant. Instead, it retains its Ca2+-specific signature, implying that this constellation of ligands is less sensitive to placement within the protein molecule. The D59G and D94S single mutations substantially lower binding affinity, consistent with removal of a liganding carboxylate. By contrast, the S55D and G98D mutations substantially increase binding affinity, a finding at odds with corresponding data collected on model peptide systems. Significantly, the Ca2+ affinity of the oncomodulin CD site is increased by mutations that weaken binding at the EF site, indicating a negatively cooperative interaction between the two sites.