Interconversion of the ligand arrays in the CD and EF sites of oncomodulin. Influence on Ca2+-binding affinity.

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.

The location of the active site of blood coagulation factor VIIa above the membrane surface and its reorientation upon association with tissue factor. A fluorescence energy transfer study.

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-L-arginyl-fVIIa (Fl-FPR-fVIIa). The location of the active-site 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 phosphatidylserine-dependent 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 A assuming a random orientation of donor and acceptor dyes (kappa2 = 2/3; the orientational uncertainty totals approximately 10%). 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 A 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.

Introduction of a fifth carboxylate ligand heightens the affinity of the oncomodulin CD and EF sites for Ca2+.

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.

Intrathymic distribution of the two avian thymic parvalbumins.

Although parvalbumins are generally viewed as intracellular Ca2- buffers/transporters, avian species express two thymus-specific isoforms that may play alternative biological roles. These proteins, known as avian thymic hormone (ATH) and chicken parvalbumin 3 (CPV3), are conjectured to influence thymopoiesis. In this paper, we compare their intrathymic distributions. Isoform-specific monoclonal antibodies against ATH and CPV3 were labeled with fluorescein and Cy5, respectively, and then used to probe paraffin sections of chicken thymus tissue. Confocal microscopy of the stained sections reveals that ATH and CPV3 are both confined to the thymic cortex and that they are frequently coexpressed by a subset of epithelial cells. However, their expression patterns are not completely superimposible. Cortical epithelial cells are also observed that stain for just one of the two avian parvalbumin isoforms, albeit at lower frequency. Significantly, a subset of cortical thymocytes exhibits peripheral staining for one or both of the proteins. This result may imply the existence of plasma membrane receptors for the two proteins on select T-cell precursors. Alternatively, it may signal low level expression of the thymic parvalbumins by the thymocytes population.

Interconversion of the CD and EF sites in oncomodulin. Influence on the Eu3+ 7F0-->5D0 excitation spectrum.

The appearance of the parvalbumin Eu3+ 7F0-->5D0 spectrum is markedly pH dependent, the result of a hitherto unidentified deprotonation event in the CD ion-binding domain [Treviño, C.L., et al. (1991) J. Biol. Chem. 265, 9694-9700]. We are studying this phenomenon in the mammalian placental parvalbumin called oncomodulin. As in other parvalbumins, the liganding residues in the CD and EF sites of oncomodulin differ at the +z and -x coordination positions: serine and aspartate, respectively, in the CD site; aspartate and glycine in the EF site. We have prepared a series of oncomodulin variants in which the +z and/or -x residue(s) from one site have been replaced by the corresponding residue(s) from the other. We herein characterize the resulting proteins by Eu3+ luminescence spectroscopy. Simultaneous replacement of serine-55 by aspartate and aspartate-59 by glycine affords the CD site with a coordination sphere superficially equivalent to that of the EF site. As observed previously for the S55D mutation [Henzl, M. T., et al. (1992) FEBS Lett. 314, 130-134], the Eu3+ 7F0-->5D0 spectrum of the 55/59 variant is pH independent. Interestingly, replacement of aspartate-94 by serine at the +z position of the EF site of 55/59 imparts pH dependent behavior to the EF site. The identical mutation in the wild-type background likewise imparts pH dependence to the EF site, affording a protein in which both sites display broad signals near 578.2 nm at pH 8. Significantly, a variant in which threonine replaces serine-55 retains the pH dependent spectroscopic signature. These results indicate that the presence of a hydroxyl group at the +z position is sufficient to confer pH dependence on the 7F0-->5D0 spectrum of a parvalbumin EF-hand domain. Importantly, the data also suggest that the component peaks of the low-pH doublet are not site-specific signals, as previously believed. Rather, they probably represent differences in coordination environment arising from differential hydration or conformational heterogeneity. In wild-type oncomodulin, the CD site signal dominates the low-pH spectrum. Since this dominance persists even when serine-55 and aspartate-59 are replaced by the corresponding EF site residues, it appears that the context of the CD binding site, as dictated by the global polypeptide fold, exerts a major influence on the metal ion-binding properties of the site.

Oligomerization of an avian thymic parvalbumin. Chemical evidence for a Ca(2+)-specific conformation.

CPV3, the third parvalbumin isoform to be identified in the chicken, is produced exclusively in the thymus gland. Although parvalbumins are typically cysteine-deficient, CPV3 contains two cysteine residues, at positions 18 and 72. The reported three-dimensional parvalbumin structures suggest that the side chain of cysteine-72 should be solvent-accessible. Accordingly, we find that CPV3 readily forms disulfide-linked oligomers in the absence of reducing agents. The reaction, employing either O2 or ferricyanide ion as the oxidant, is apparently restricted to the Ca(2+)-bound form of the protein. The differing reactivity of the Ca2+, Mg2+, and apo-forms has significant structural implications.

Novel avian thymic parvalbumin displays high degree of sequence homology to oncomodulin.

CPV3 is a novel avian parvalbumin. It displays an isoelectric point of 4.6, intermediate between that of avian thymic hormone (pI = 4.3) and the muscle parvalbumin isoform (pI = 5.2). Expression of CPV3, like that of avian thymic hormone (ATH), is restricted to the thymic stroma. However, the CPV3 content of chicken thymus tissue (120 micrograms/g tissue) is 4 times lower than that of ATH (500 micrograms/g tissue). The polymerase chain reaction (PCR) was used to gain access to the nucleotide sequence of CPV3. A 147-base pair fragment of the coding sequence, corresponding to residues 48-97, was amplified from total chicken cDNA using degenerate PCR primers. A RACE-PCR strategy was then used to extend the known sequence in both the 5' and 3' directions. The cDNA sequence thus obtained includes 671 base pairs. Primer extension analysis suggests that the cloned cDNA corresponds to a full-length transcript. Northern analysis of chicken mRNA indicates that the average CPV3 transcript is approximately 800 nucleotides in length, significantly smaller than the ATH message (approximately 1000 nucleotides). Southern analysis suggests the presence of a single CPV3 gene in the chicken genome. The translated nucleotide sequence, displaying 108 residues between the initiator and termination codons, is that of a beta-parvalbumin. The CPV3 sequence exhibits 58% identity with ATH and 52% identity with the chicken muscle isoform. Interestingly, CPV3 and the mammalian oncodevelopmental parvalbumin called oncomodulin are identical at 73 of 108 residues (68% identity). Correspondingly, flow-dialysis measurements with 45Ca2+ indicate that the Ca(2+)-binding domains are inequivalent, as in oncomodulin. The apparent dissociation constants, at pH 7.4 in 150 mM NaCl, are approximately 10 nM and 80 nM.

Site-specific substitution of glutamate for aspartate at position 59 of rat oncomodulin.

Replacement of the aspartate residue at position 59 of rat oncomodulin by glutamate by oligonucleotide-directed mutagenesis has afforded a protein which more closely resembles rat parvalbumin, at least judged by its interaction with the luminescent lanthanide ion Eu3+. The single-peak 7F0----5D0 spectrum observed at pH 5.0 with the fully bound wild-type protein is replaced by one which clearly shows two features at 5791 and 5796 A, arising from Eu3+ ions bound at the CD and EF sites, respectively. Furthermore, the pH dependence of the spectrum is substantially altered; the pKa observed for the CD domain, in which aspartate 59 residues, is shifted upward from pH 6.0 for the wild-type recombinant protein to pH 6.8 in the D59E mutant. Moreover, the maximum in the high-pH spectrum is shifted from 5781 to 5784 A. All three changes are indicative of a CD binding domain having increased parvalbumin-like character. Interestingly, however, the D59E substitution has only a modest effect on the Ca2+- and Mg2+-binding properties of the CD domain. For the wild-type protein, KCa = 7.8 x 10(-7) M and KMg = 3 x 10(-3) M. These affinities are more than an order of magnitude weaker than those seen for various parvalbumins and substantiate previous claims for calcium specificity made for the oncomodulin CD domain. Replacement of aspartate 59 by glutamate resulted in minor increases in affinity of the CD domain for Ca2+ (KCa = 5.5 x 10(-7) M) and Mg2+ (KMg = 1 x 10(-3) M). These findings strongly suggest that residues in oncomodulin besides aspartate 59 are important determinants of the observed calcium specificity of the CD calcium-binding domain. The consequences of the substitution at residue 59 appear to be confined to the CD domain. For the EF site in wild-type recombinant oncomodulin, KCa = 4.2 x 10(-8) M and KMg = 1.6 x 10(-4) M. The corresponding values for the D59E site-specific variant are identical within experimental error (KCa = 4.2 x 10(-8) M and KMg = 1.8 x 10(-4) M).

Lanthanide-binding properties of rat oncomodulin.

Oncomodulin, the parvalbumin-like calcium-binding protein frequently expressed in tumor tissue, was isolated from Morris hepatoma 5123tc and studied using the luminescent lanthanide ions, Eu3+ and Tb3+. Titrations of the apoprotein - whether monitored by indirect excitation of bound Tb3+, by direct laser excitation of bound Eu3+, or by quenching of the intrinsic tyrosine fluorescence - all indicated the presence of two high-affinity binding sites for lanthanide ions, as in parvalbumin. Moreover, the appearance of the Eu3+ 7F0----5D0 excitation spectrum of Eu2-oncomodulin was found to be highly pH-dependent, as previously observed with parvalbumin. At pH 5.0, it consists of a single peak centered at 5796 A, having a linewidth of approximately 6 A. At higher pH values, this spectrum is replaced by a broader, more symmetric peak at 5782 A. Oncomodulin, however, was found to differ from parvalbumin in at least one important respect: In contrast to the muscle-associated protein, the affinities of the CD site in oncomodulation for Tb3+ and Ca2+ were found to be rather similar, with KCa/KTb approximately equal to 11 +/- 2.