Structure and interaction site of the regulatory domain of troponin-C when complexed with the 96-148 region of troponin-I.

The structure of the regulatory domain of chicken skeletal troponin-C (residues 1-90) when complexed with the major inhibitory region (residues 96-148) of chicken skeletal troponin-I was determined using multinuclear, multidimensional NMR spectroscopy. This complex represents the first interaction formed between the regulatory domain of troponin-C and troponin-I after calcium binding in the regulation of muscle contraction. The stoichiometry of the complex was determined to be 1:1, with a dissociation constant in the 1-40 microM range. The structure of troponin-C in the complex was calculated from 1039 NMR distance and 111 dihedral angle restraints. When compared to the structure of this domain in the calcium saturated "open" form but in the absence of troponin-I, the bound structure appears to be slightly more "closed". The troponin-I peptide-binding site was found to be in the hydrophobic pocket of calcium saturated troponin-C, using edited/filtered NMR experiments and chemical shift mapping of changes induced in the regulatory domain upon peptide binding. The troponin-I peptide (residues 96-148) was found to bind to the regulatory domain of troponin-C very similarly, but not identically, to a shorter troponin-I peptide (region 115-131) thought to represent the major interaction site of troponin-I for this domain of troponin-C.

Site-specific mutants of oncomodulin. 1H NMR and optical stopped-flow studies of the effect on the metal binding properties of an Asp59----Glu59 substitution in the calcium-specific site.

High resolution 1H nuclear magnetic resonance spectroscopy and optical stopped-flow techniques have been used to study the metal binding properties of a site-specific mutant of bacterial recombinant oncomodulin in which glutamate has replaced a liganding aspartate at position 59 in the CD calcium-binding site. In particular we have followed the replacement of calcium by lutetium in bacterial recombinant oncomodulin and D59E oncomodulin to provide a measure of the protein's preferences for metal ions of different ionic radii. The result of the Asp----Glu substitution is to make the mutant oncomodulin more similar to rat parvalbumin in terms of its relative CD- and EF-domain affinities for lutetium(III), that is to increase its affinity for metal ions with smaller ionic radii. This finding supports the original hypothesis that the presence of Asp at sequence position 59 is an important factor in the reduced preference of the CD site of oncomodulin for smaller metals such as magnesium (Williams, T. C., Corson, D. C., Sykes, B. D., and MacManus, J. P. (1987) J. Biol. Chem. 262, 6248-6256). However, our studies show that both the CD and the EF sites are affected by this single residue substitution suggesting that many factors play a role in the metal binding affinity and interaction between the two sites.

Oncomodulin. 1H NMR and optical stopped-flow spectroscopic studies of its solution conformation and metal-binding properties.

As deduced from its 1H NMR spectrum, oncomodulin's solution conformation is very similar to the tertiary structure of other single domain 2-site calcium-binding proteins of the troponin C class. Despite its extensive amino acid sequence homology with parvalbumins, however, oncomodulin differs significantly from these proteins in its Ca(II)----Ln(III) exchange characteristics. Although the relative affinity of Lu(III) for the EF site of Ca2-oncomodulin was normal, beta Lu:EF/beta Ca:EF being 175 +/- 15, displacement of Ca(II) from the CD site was not favored, beta Lu:CD/beta Ca:CD being 1.2 +/- 0.1. Lineshape analyses of several 1H NMR resonances generated by the Lu(III) titration of Ca2-oncomodulin indicated that Ca(II)----Ln(III) exchange at the CD site was 15-20 s-1, approximately 100 times faster than exchange at the CD site of parvalbumins. Analyses of the distribution of metal-bound oncomodulin species showed that Ca(II)----Lu(III) exchange was cooperative, the coefficient of cooperativity being estimated as 5 +/- 1. The kinetics of the release of Yb(III) from oncomodulin as measured by optical stopped-flow techniques corroborated the observed cooperativity in metal binding; the off-rate constant of Yb(III) from the EF site of Yb2-oncomodulin was 0.0036 s-1, approximately 19 times slower than the release of Yb(III) from the EF site of Ca1Yb1-oncomodulin. We attribute part of the reduced preference of small Ln(III)s for the CD site of oncomodulin to a combination of this site's inherent incompressibility (Williams, T.C., Corson, D.C. & Sykes, B.D. (1984) J. Am. Chem. Soc. 106, 5698-5702) and the Glu----Asp substitution at sequence position 59, the residue which chelates metal at the -X coordination position. Like the CD site in oncomodulin, site III in troponin C has not only a lower affinity for calcium relative to the CD site of parvalbumins but also aspartic acid at its -X position; a water molecule bridges the gap between bound metal and the carboxyl group of the relatively short side chain of Asp-114 (Herzberg, O. & James, M. N. G. (1985) Biochemistry 24, 5298-5302). Hence, we suggest that Asp-59 in oncomodulin binds metal only indirectly through an intervening water molecule, a proposal which is consistent with the CD site's reduced affinity for ions the size of Ca(II) or smaller.

1H NMR spectroscopic studies of calcium-binding proteins. 1. Stepwise proteolysis of the C-terminal alpha-helix of a helix-loop-helix metal-binding domain.

A series of modified parvalbumins, differing only in length of alpha-helix F at the C-terminus, was prepared by carboxypeptidase-mediated digestions of the beta-lineage parvalbumin (pI = 4.25) from carp (N; 108 residues). Removal of Ala-108 to form the N-1 derivative (des-Ala108,Lys107-parvalbumin) only slightly alters the protein's ability to chelate Ca(II) or lanthanides(III). Analysis of the kinetics of their Yb(III) off-rates by optical stopped-flow techniques, determination of their Lu(III)-binding constants by high-resolution 1H NMR methods, and inspection of their solution structures by Yb(III)-shifted 1H NMR techniques indicate N-1 and N-2 are very similar to N (0.1-0.2 M KCl; pH 6-7; 23-55 degrees C). However, removal of the next one or two residues, Val-106 or Val-106/Leu-105, to generate the N-3 and N-4 derivatives severely alters the metal ion binding characteristics of the protein. Although two Yb(III) off-rates are observed for N-3, both are faster than that for the unmodified protein: kCD by a factor of 2 and kEF by a factor of 2200. Removal of Ala-104 and Ala-104/Thr-103 to give a mixture of N-5 and N-6 derivatives eliminates the slow-release site altogether, the single observable koff being 20-30 times faster than release of Yb(III) from the CD site of native parvalbumin. Removal of the C-terminal alpha-helix by digestion through Phe-102 to give N-7 destabilizes the entire protein structure as judged both by the random-coil appearance of its 1H NMR spectrum and by its aberrant kinetics. Although one abnormally fast koff is still observed at micromolar concentrations, Ln(III) chelation tends to precipitate N-7 at higher parvalbumin concentrations (1-3 mM). In contrast to the critical instability of the N-3 through N-7 derivatives, the remarkable stability of the N-1 and N-2 forms of carp parvalbumin may be attributed to the maintainance of two key structural features: an ion pair bond between the negatively charged C-terminal carboxyl function and the protonated epsilon-NH3+ of Lys-27 and hydrophobic interactions of the inner side of helix F with residues in the protein's core.

1H NMR spectroscopic studies of calcium-binding proteins. 2. Histidine microenvironments in alpha- and beta-parvalbumins as determined by protonation and laser photochemically induced dynamic nuclear polarization effects.

The microenvironments of the histidines in three isoforms of Ca(II)-bound parvalbumin (carp, pI = 4.25; pike, pI = 5.00; rat, pI = 5.50) have been examined with 1H NMR techniques to probe their protonation characteristics and photochemically induced dynamic nuclear polarizability (photo-CIDNP). The histidine at position 26 (or 25), present in all three of these proteins, shows absolutely no photo-CIDNP enhancement of its C2H or C5H resonances. Nor does this nonpolarizable histidine possess a normal pKa: values range only from 4.20 for carp to 4.32 for pike to 4.44 for rat. The C2H and C5H resonances of the histidine in this carp isoform split into doublets as the pH is lowered. The magnitude of this splitting depends on the magnetic field strength, temperature, and pH; however, the line intensities within each doublet are temperature-independent. Although the crystal structure of carp parvalbumin indicates that His-26 is exposed to solvent [Kretsinger, R. H., & Nockolds, C. E. (1973) J. Biol. Chem. 248, 3313-3326], we conclude that in solution this residue, in its unprotonated state, is part of the hydrophobic core of the protein. In contrast, His-48 in rat parvalbumin and His-106 in pike III parvalbumin show dramatic photo-CIDNP enhancements of their C2H, C5H, and beta-CH2 1H NMR resonances. Combined with its nearly normal pKa, 6.14, and exchange-broadened C2H resonance, the photo-CIDNP enhancement results for His-48 indicate that its microenvironment differs little from random-coil exposure, consistent with its presumed position on the solvent surface of helix C.(ABSTRACT TRUNCATED AT 250 WORDS)

1H NMR spectroscopic studies of calcium-binding proteins. 3. Solution conformations of rat apo-alpha-parvalbumin and metal-bound rat alpha-parvalbumin.

Lacking the extraordinary thermal stability of its metal-bound forms, apo-alpha-parvalbumin from rat muscle assumes two distinct conformations in aqueous solution. At 25 degrees C, its highly structured form predominates (Keq = 5.7; delta G degree = -4.3 kJ X mol-1); as deduced from both 1H NMR and circular dichroism (CD) spectroscopy, this conformation is exceedingly similar to those of its Mg(II)-, Ca(II)-, and Lu(III)-bound forms. The temperature dependences of several well-resolved aromatic and upfield-shifted methyl 1H NMR resonances and several CD bands indicate that the native, highly helical structure of rat apo-alpha-parvalbumin is unfolded by a concerted mechanism, showing no indication of partially structured intermediates. The melting temperature, TM, of rat apo-alpha-parvalbumin is 35 +/- 0.5 degrees C as calculated by both spectroscopic techniques. By 45 degrees C, rat apo-alpha-parvalbumin unfolds entirely, losing the tertiary structure that characterizes its folded form: not only are the ring-current-shifted aromatic and methyl 1H NMR resonances leveled, but the 262- and 269-nm CD bands are also severely reduced. As judged by the decrease in the negative ellipticity of the 222-nm CD band, this less-structured form of rat apo-alpha-parvalbumin shows an approximate 50% loss in apparent alpha-helical content compared to its folded state. Several changes in the 1H NMR spectrum of rat apo-alpha-parvalbumin were exceptionally informative probes of the specific conformational changes that accompany metal ion binding and metal ion exchange. In particular, the line intensities of the ortho proton resonance of Phe-47, the unassigned downfield-shifted alpha-CH resonances from the beta-sheet contacts between the metal-binding loops, the C2H resonance of His-48, and the epsilon-CH3 resonance of an unassigned Met residue were monitored as a function of added metal to determine the stability constants of several metal ion-parvalbumin complexes. We conclude that Mg(II) binds to the CD and EF sites independently, its affinity for the EF site being almost twice that for the CD site. Mg(II)----Ca(II) exchange showed that the CD-site Mg(II) is displaced first, in contrast to Lu(III)'s preferential displacement of the EF-site Ca(II) as determined from the Ca(II)----Lu(III) exchange experiments.(ABSTRACT TRUNCATED AT 400 WORDS)

13C and 113Cd NMR studies of the chelation of metal ions by the calcium binding protein parvalbumin.

13C NMR spectra are presented for the calcium binding protein parvalbumin (pI 4.25) from carp muscle in several different metal bound forms: with Ca2+ in both the CD and EF calcium binding sites, with Cd2+ in both sites, with 113Cd2+ in both sites, and with 113Cd2+ in the CD site and Lu3+ in the EF site. The different metals differentially shift the 13C NMR resonances of the protein ligands involved in chelation of the metal ion. In addition, direct 13C-113Cd spin-spin coupling is observed which allows the assignment of protein carbonyl and carboxyl 13C NMR resonances to ligands directly interacting with the metal ions in the CD and EF binding sites. The displacement of 113Cd2+ from the EF site by Lu3+ further allows these resonances to be assigned to the CD or EF site. The occupancy of the two sites in the two cadmium species and in the mixed Cd2+/Lu3+ species is verified by 113Cd NMR. The resolution in these 113Cd NMR spectra is sufficient to demonstrate direct interaction between the two metal binding sites.

Structural studies of calcium-binding proteins using nuclear magnetic resonance.

Lanthanide-shifted 1H nuclear magnetic resonance (NMR) spectroscopy has been used to compare the structure in solution of the EF-hand calcium-binding domains of four parvalbumins (isoelectric pH[pI] 3.95, 4.25, and 4.37 from carp, and pI from buffalo fish). These four parvalbumins are shown by NMR to have very similar structures at the level of resolution typical of x-ray structures. At the higher resolution possible by the lanthanide NMR technique, specific differences are noted between the pI 3.95 isoprotein from carp and the other two carp isoproteins, and the buffalo fish parvalbumin is shown to be different from all three carp isoproteins. The differences are estimated to correspond to changes of the order of 0.2 A in the positions of some of the nuclei surrounding the EF calcium site.

Calcium binding proteins: optical stopped-flow and proton nuclear magnetic resonance studies of the binding of the lanthanide series of metal ions to parvalbumin.

Optical stopped-flow techniques have been used to determine the dissociation rate constants (koff) for the lanthanide(III) ions from carp (pI 4.25) parvalbumin. For most of the 13 different lanthanides studied, the release kinetics were diphasic, composed of both a fast phase (whose rate varied across the series, La3+ leads to Lu3+, between the limits -1.2 less than or equal to log kFAST less than or equal to -0.7) and a slower phase (whose rate varied across the series, La3+ leads to Lu3+, between the limits -1.2 greater than or equal to log kSLOW greater than or equal to -2.9). In addition, the La3+- and Lu3+-induced changes in the 270-MHz proton nuclear magnetic resonance spectrum of parvalbumin were used to calculate the dissociation constants for these specific lanthanides from the two high-affinity Ca2+ binding sites. The KD for one site appears to remain constant across the lanthanide series, determined to be 4.8 X 10(-11) M for both La3+ and Lu3+. The other site, however, is evidently quite sensitive to the nature of the bound Ln3+ ion and shows a strong preference for La3+ (KD,La = 2.0 X 10(-11) M; KD,Lu = 3.6 X 10(-10) M). We conclude from these observations that reports of nearly indistinguishable CD/EF binding site affinities for parvalbumin complexes of the middle-weight lanthanides (i.e., Eu3+, Gd3+, and Tb3+) are quite reasonable in view of the crossover in relative CD/EF site affinities across the lanthanide series.

An optical stopped-flow and 1H and 113Cd nuclear magnetic resonance study of the kinetics and stoichiometry of the interaction of the lanthanide Yb3+ with carp parvalbumin.

The rate constants for the dissociation of the lanthanide Yb3+ from the CD and EF calcium-binding sites of carp parvalbumin (isoelectric point, 4.25) have been measured using optical stopped-flow and 1H nuclear magnetic resonance (NMR) methods. The off-rate constants for Yb3+ are 1.5 X 10(-1) and 1.3 X 10(-3) s-1, respectively, at pH 6.6 and 23 degrees C. The relative displacement of Ca2+ from the two sites by Yb3+ was determined from the observed amplitude of the fast and slow kinetic phases. Yb3+-shifted 1H-NMR spectra of parvalbumin are presented as a function of pH, concentration, and H2O:D2O ratio to relate the NMR results to the kinetic and optical results. The displacement of Cd2+ from parvalbumin by Yb3+ was studied using 113Cd NMR. All of the results show the sequential displacement of Ca2+ from the CD and EF sites of parvalbumin by Yb3+. Some results are also presented for Tb3+ and Gd3+.