Conformational changes in the metal-binding sites of cardiac troponin C induced by calcium binding.

Isotope labeling of recombinant normal cardiac troponin C (cTnC3) with 15N-enriched amino acids and multidimensional NMR were used to assign the downfield-shifted amide protons of Gly residues at position 6 in Ca(2+)-binding loops II, III, and IV, as well as tightly hydrogen-bonded amides within the short antiparallel beta-sheets between pairs of Ca(2+)-binding loops. The amide protons of Gly70, Gly110, and Gly146 were found to be shifted significantly downfield from the remaining amide proton resonances in Ca(2+)-saturated cTnC3. No downfield-shifted Gly resonance was observed from the naturally inactive site I. Comparison of downfield-shifted amide protons in the Ca(2+)-saturated forms of cTnC3 and CBM-IIA, a mutant having Asp65 replaced by Ala, demonstrated that Gly70 is hydrogen bonded to the carboxylate side chain of Asp65. Thus, the hydrogen bond between Gly and Asp in positions 6 and 1, respectively, of the Ca(2+)-binding loop appears crucial for maintaining the integrity of the helix-loop-helix Ca(2+)-binding sites. In the apo- form of cTnC3, only Gly70 was found to be shifted significantly downfield with respect to the remaining amide proton resonances. Thus, even in the absence of Ca2+ at binding site II, the amide proton of Gly70 is strongly hydrogen bonded to the side-chain carboxylate of Asp65. The amide protons of Ile112 and Ile148 in the C-terminal domain and Ile36 in the N-terminal domain data-sheets exhibit chemical shifts consistent with hydrogen-bond formation between the pair of Ca(2+)-binding loops in each domain of Ca(2+)-saturated cTnC3.(ABSTRACT TRUNCATED AT 250 WORDS)     

The effects of Ca2+ binding on the dynamic properties of a designed Ca2+-binding protein

The effects of Ca(2+) binding on the dynamic properties of Ca(2+)-binding proteins are important in Ca(2+) signaling. To understand the role of Ca(2+) binding, we have successfully designed a Ca(2+)-binding site in the domain 1 of rat CD2 (denoted as Ca.CD2) with the desired structure and retained function. In this study, the backbone dynamic properties of Ca.CD2 have been investigated using (15)N spin relaxation NMR spectroscopy to reveal the effect of Ca(2+) binding on the global and local dynamic properties without the complications of multiple interactive Ca(2+) binding and global conformational change. Like rat CD2 (rCD2) and human CD2 (hCD2), residues involved in the recognition of the target molecule CD48 exhibit high flexibility. Mutations N15D and N17D that introduce the Ca(2+) ligands increase the flexibility of the neighboring residues. Ca(2+)-induced local dynamic changes occur mainly at the residues proximate to the Ca(2+)-binding pocket or the residues in loop regions. The beta-strand B of Ca.CD2 that provides two Asp for the Ca(2+) undergoes an S(2) decrease upon the Ca(2+) binding, while the DE-loop that provides one Asn and one Asp undergoes an S(2) increase. Our study suggests that Ca(2+) binding has a differential effect on the rigidity of the residues depending on their flexibility and location within the secondary structure.     

Mechanism of the Ca2+-induced enhancement of the intrinsic factor VIIa activity

The intrinsic activity of coagulation factor VIIa (FVIIa) is dependent on Ca(2+) binding to a loop (residues 210-220) in the protease domain. Structural analysis revealed that Ca(2+) may enhance the activity by attenuating electrostatic repulsion of Glu(296) and/or by facilitating interactions between the loop and Lys(161) in the N-terminal tail. In support of the first mechanism, the mutations E296V and D212N resulted in similar, about 2-fold, enhancements of the amidolytic activity. Moreover, mutation of the Lys(161)-interactive residue Asp(217) or Asp(219) to Ala reduced the amidolytic activity by 40-50%, whereas the K161A mutation resulted in 80% reduction. Hence one of these Asp residues in the Ca(2+)-binding loop appears to suffice for some residual interaction with Lys(161), whereas the more severe effect upon replacement of Lys(161) is due to abrogation of the interaction with the N-terminal tail. However, Ca(2+) attenuation of the repulsion between Asp(212) and Glu(296) keeps the activity above that of apoFVIIa. Altogether, our data suggest that repulsion involving Asp(212) in the Ca(2+)-binding loop suppresses FVIIa activity and that optimal activity requires a favorable interaction between the Ca(2+)-binding loop and the N-terminal tail. Crystal structures of tissue factor-bound FVIIa(D212N) and FVIIa(V158D/E296V/M298Q) revealed altered hydrogen bond networks, resembling those in factor Xa and thrombin, after introduction of the D212N and E296V mutations plausibly responsible for tethering the N-terminal tail to the activation domain. The charge repulsion between the Ca(2+)-binding loop and the activation domain appeared to be either relieved by charge removal and new hydrogen bonds (D212N) or abolished (E296V). We propose that Ca(2+) stimulates the intrinsic FVIIa activity by a combination of charge neutralization and loop stabilization.     

Synthetic peptide analogs of skeletal troponin C: fluorescence studies of analogs of the low-affinity calcium-binding site II

Two 12-residue peptides were synthesized by the solid-phase method as structural analogs of a Ca2+-binding loop of rabbit skeletal troponin C. The sequence of the analogs corresponds to the binding loop of the Ca2+-specific low affinity binding site II (residues 63-74) but with two amino acid substitutions. In one analog, Phe-72 was replaced by tyrosine. In the other Gly-66 was substituted by serine and Phe-72 by tyrosine. The intrinsic fluorescence of the peptides was enhanced upon addition of Tb3+ or large excess of Ca2+. From the enhancement of Tb3+ emission association constants in the range (2-3) X 10(5) M-1 and a binding stoichiometry of 1 were determined for Tb3+ binding to the peptides. Large excess of Ca2+ displaced Tb3+ from the Tb3+-peptide complexes and from these results apparent stability constants of 500-700 M-1 were deduced for Ca2+ binding. Preliminary proton nuclear magnetic resonance results on one of the peptides indicated that La3+ induced considerable perturbation of the amide proton resonances of several residues, including the aspartate at position 3, the tyrosine at position 10, and the two glutamates at the C-terminus. The results suggest involvement of these residues in cation coordination.     

Structural changes in the C-terminus of Ca2+-bound rat S100B (beta beta) upon binding to a peptide derived from the C-terminal regulatory domain of p53.

S100B(beta beta) is a dimeric Ca2+-binding protein that interacts with p53, inhibits its phosphorylation by protein kinase C (PKC) and promotes disassembly of the p53 tetramer. Likewise, a 22 residue peptide derived from the C-terminal regulatory domain of p53 has been shown to interact with S100B(beta beta) in a Ca2+-dependent manner and inhibits its phosphorylation by PKC. Hence, structural studies of Ca2+-loaded S100B(beta beta) bound to the p53 peptide were initiated to characterize this interaction. Analysis of nuclear Overhauser effect (NOE) correlations, amide proton exchange rates, 3J(NH-H alpha) coupling constants, and chemical shift index data show that, like apo- and Ca2+-bound S100B(beta beta), S100B remains a dimer in the p53 peptide complex, and each subunit has four helices (helix 1, Glu2-Arg20; helix 2, Lys29-Asn38; helix 3, Gln50-Asp61; helix 4, Phe70-Phe87), four loops (loop 1, Glu21-His25; loop 2, Glu39-Glu49; loop 3, Glu62-Gly66; loop 4, Phe88-Glu91), and two beta-strands (beta-strand 1, Lys26-Lys28; beta-strand 2, Glu67-Asp69), which forms a short antiparallel beta-sheet. However, in the presence of the p53 peptide helix 4 is longer by five residues than in apo- or Ca2+-bound S100B(beta beta). Furthermore, the amide proton exchange rates in helix 3 (K55, V56, E58, T59, L60, D61) are significantly slower than those of Ca2+-bound S100B(beta beta). Together, these observations plus intermolecular NOE correlations between the p53 peptide and S100B(beta beta) support the notion that the p53 peptide binds in a region of S100B(beta beta), which includes residues in helix 2, helix 3, loop 2, and the C-terminal loop, and that binding of the p53 peptide interacts with and induces the extension of helix 4.     

Identification by mutational analysis of amino acid residues essential in the chaperone function of calreticulin

Calreticulin is a Ca2+ -binding chaperone that resides in the lumen of the endoplasmic reticulum and is involved in the regulation of intracellular Ca2+ homeostasis and in the folding of newly synthesized glycoproteins. In this study, we have used site-specific mutagenesis to map amino acid residues that are critical in calreticulin function. We have focused on two cysteine residues (Cys(88) and Cys(120)), which form a disulfide bridge in the N-terminal domain of calreticulin, on a tryptophan residue located in the carbohydrate binding site (Trp(302)), and on certain residues located at the tip of the "hairpin-like" P-domain of the protein (Glu(238), Glu(239), Asp(241), Glu(243), and Trp(244)). Calreticulin mutants were expressed in crt(-/-) fibroblasts, and bradykinin-dependent Ca2+ release was measured as a marker of calreticulin function. Bradykinin-dependent Ca2+ release from the endoplasmic reticulum was rescued by wild-type calreticulin and by the Glu(238), Glu(239), Asp(241), and Glu(243) mutants. The Cys(88) and Cys(120) mutants rescued the calreticulin-deficient phenotype only partially ( approximately 40%), and the Trp(244) and Trp(302) mutants did not rescue it at all. We identified four amino acid residues (Glu(239), Asp(241), Glu(243), and Trp(244)) at the hairpin tip of the P-domain that are critical in the formation of a complex between ERp57 and calreticulin. Although the Glu(239), Asp(241), and Glu(243) mutants did not bind ERp57 efficiently, they fully restored bradykinin-dependent Ca2+ release in crt(-/-) cells. This indicates that binding of ERp57 to calreticulin may not be critical for the chaperone function of calreticulin with respect to the bradykinin receptor.     

Chemical modification of bovine prothrombin fragment 1 in the presence of Tb3+ ions. Sequence studies on 3-gamma-MGlu-fragment

Chemical modification of the gamma-carboxyglutamyl (Gla) residues of bovine prothrombin fragment 1 using the formaldehyde-morpholine method in the presence of 100 Kappm Tb3+ ions at pH 5.0 provided a modified protein containing 3 gamma-methyleneglutamyl residues (gamma-MGlu) and 7 Gla residues (bovine 3-gamma-MGlu-fragment 1). The modified protein bound the same number of Ca2+ ions as the native protein (six to seven), exhibited 28Mg2+-binding properties identical to native fragment 1 (five Mg2+ ions bound), exhibited the metal ion-promoted quenching of the intrinsic fluorescence in a manner similar to the native protein, but did not bind to phosphatidylserine (PS)/phosphatidylcholine (PC) vesicles in the presence of Ca2+ ions. Modification of the bovine protein using [14C]formaldehyde-morpholine provided a 14C-labeled 3-gamma-MGlu-fragment 1 suitable for sequence analysis. Edman sequencing of the peptides released by a tryptic digest of the reduced and carboxymethylated bovine [14C]3-gamma-MGlu-fragment 1 indicated that Gla residues at positions 7, 8, and 33 had been converted to [14C]gamma-methyleneglutamyl residues. In addition Lys97 was found to contain a 14C label. Similar analysis of the human [14C]3-gamma-MGlu-fragment 1 indicated that Gla residues at positions 7 and 32 were major modification sites and that Gla residues at positions 6 and 14 were partially modified. Lysine 96 was also modified in the human protein. The incorporation of a 14C label at Lys97 in bovine 3-gamma-MGlu-fragment 1 protein is not responsible for the loss of Ca2+-promoted binding to PS/PC vesicles. We suggest that Gla residues 7, 8, and 33 are elements of the first Ca2+-binding site; occupancy of this site establishes the Ca2+-specific conformation which is essential for the Ca2+-promoted interaction of the bovine protein with PS/PC vesicles. These studies also suggest that the loss of Gla residues at positions 7 and 32 prevents the formation of the initial Ca2+-binding site in the human protein.     

Functional consequences of alterations to amino acids located in the catalytic center (isoleucine 348 to threonine 357) and nucleotide-binding domain of the Ca2+-ATPase of sarcoplasmic reticulum

The sequence of 10 amino acids (ICSDKTGTLT357) at the site of phosphorylation of the rabbit fast twitch muscle Ca2+-ATPase is highly conserved in the family of cation-transporting ATPases. We changed each of the residues flanking Asp351, Lys352, and Thr353 to an amino acid differing in size or polarity and assayed the mutant for Ca2+ transport activity and autophosphorylation with ATP or P1. We found that conservative changes (Ile----Leu, Thr----Ser, Gly----Ala) or the alteration of Cys349 to alanine did not destroy Ca2+ transport activity or phosphoenzyme formation, whereas nonconservative changes (Ile----Thr, Leu----Ser) did disrupt function. These results indicate that very conservative changes in the amino acids flanking Asp351, Lys352, and Thr353 can be accommodated. A number of mutations were also introduced into amino acids predicted to be involved in nucleotide binding, in particular those in the conserved sequences KGAPE519, RDAGIRVIMITGDNK629, and KK713. Our results indicate that amino acids KGAPE519, Arg615, Gly618, Arg620, and Lys712-Lys713 are not essential for nucleotide binding, although changes to Lys515 diminished Ca2+ transport activity but not phosphoenzyme formation. Changes of Gly626 and Asp627 abolished phosphoenzyme formation with both ATP and Pi, indicating that these residues may contribute to the conformation of the catalytic center.     

Effects of modifying individual amino or carboxyl groups on the affinity of calmodulin for calcineurin

The effects of modifying individual lysyl, aspartyl, or glutamyl residues in calmodulin on its ability to bind to the neural phosphatase calcineurin have been investigated using a competitive binding method termed "label selection." Samples of calmodulin were radiochemically labeled at a low level (0.03-0.6 group/molecule) by acetylation of amino groups or coupling carboxyl groups with ethanolamine to produce preparations containing predominantly single-site modified and unmodified molecules. These preparations were incubated in a 5-10-fold molar excess with bovine calcineurin under conditions appropriate for complex formation. The bound population was isolated, and the level of modification of each reactive residue was compared with the level in the corresponding group in the intial unselected preparation to determine if molecules modified at specific sites had been selected for or against during the competition for complex formation. Significant selection was observed against molecules modified at Lys21, Asp64, Glu67, Lys75, Glu84, Glu114, Asp118, or Lys148, whereas modification of Glu83 increased binding. The modification of other groups, including components of the four Ca2+-binding sites, had no effect on the interaction. Glu67, a Ca2+-liganding residue in Ca2+-binding site II that may regulate the orientation of this site in relation to the central helix, had the strongest influence on complex formation. Most of the residues identified form a nearly linear array in the three-dimensional structure of calmodulin and indicate the location of an extended surface for interaction with calcineurin and other enzymes.     

Coordination structures of Ca2+ and Mg2+ in Akazara scallop troponin C in solution. FTIR spectroscopy of side-chain COO- groups.

FTIR spectroscopy has been applied to study the coordination structures of Mg2+ and Ca2+ ions bound in Akazara scallop troponin C (TnC), which contains only a single Ca2+ binding site. The region of the COO- antisymmetric stretch provides information about the coordination modes of COO- groups to the metal ions: bidentate, unidentate, or pseudo-bridging. Two bands were observed at 1584 and 1567 cm-1 in the apo state, whereas additional bands were observed at 1543 and 1601 cm-1 in the Ca2+-bound and Mg2+-bound states, respectively. The intensity of the band at 1567 cm-1 in the Mg2+-bound state was identical to that in the apo state. Therefore, the side-chain COO- group of Glu142 at the 12th position in the Ca2+-binding site coordinates to Ca2+ in the bidentate mode but does not interact with Mg2+ directly. A slight upshift of COO- antisymmetric stretch due to Asp side-chains was also observed upon Mg2+ and Ca2+ binding. This indicates that the COO- groups of Asp131 and Asp133 interact with both Ca2+ and Mg2+ in the pseudo-bridging mode. Therefore, the present study directly demonstrated that the coordination structure of Mg2+ was different from that of Ca2+ in the Ca2+-binding site. In contrast to vertebrate TnC, most of the secondary structures remained unchanged among apo, Mg2+-bound and Ca2+-bound states of Akazara scallop TnC, as spectral changes upon either Ca2+ or Mg2+ binding were very small in the infrared amide-I' region as well as in the CD spectra. Fluorescence spectroscopy indicated that the spectral changes upon Ca2+ binding were larger than that upon Mg2+ binding. Moreover, gel-filtration experiments indicated that the molecular sizes of TnC had the order apo TnC > Mg2+-bound TnC > Ca2+-bound TnC. These results suggest that the tertiary structures are different in the Ca2+- and Mg2+-bound states. The present study may provide direct evidence that the side-chain COO- groups in the Ca2+-binding site are directly involved in the functional on/off mechanism of the activation of Akazara scallop TnC.     

Hydrogen bonding in the carboxyl-terminal half-fragment 78-148 of calmodulin as studied by two-dimensional nuclear magnetic resonance

The C-terminal half-fragment (residues 78-148) of scallop testis calmodulin was investigated by 500-MHz two-dimensional proton NMR in order to clarify the structure and the structural change accompanying Ca2+ binding. The sequential resonance assignment to individual amino acid residues was made in part (27 out of 71 residues) by a combination of correlated spectroscopy and nuclear Overhauser effect spectroscopy of a 90% H2O solution. In the Ca2+-bound state, resonances of backbone amide protons of Gly-98, Gly-134, Ile-100, Asn-137, and Val-136 appear at extremely low fields. These findings suggest that amide protons of these residues are hydrogen bonded. In the Ca2+-free state, the amide resonances of Ile-100 and Gly-134 disappear into the crowded normal shift region. This observation indicates that two hydrogen bonds of Ile-100 and Gly-134 are destroyed (or weakened) as Ca2+ ions are removed from two Ca2+-binding sites. Chemical shifts of amide and alpha-protons of residues located in the Ca2+-binding loop of domain III are similar to those of domain IV. These results suggest that the conformations of the two loops are very similar. The present results can be interpreted in terms of a structure predicted by Kretsinger [Kretsinger, R.H. (1980) Ann. N.Y. Acad. Sci. 356, 14].     

The cyclopeptides with the multi-His motif as ligands for copper(II)

The coordination properties of cyclic octapeptides with multi-His motif: c(His-Gly-His-Xaa-His-Gly-His-Xaa) where Xaa = Asp or Lys, were investigated. The binding abilities of this peptides towards Cu(II) ions were studied by using different analytic methods as: potentiometry, spectroscopy and mass spectrometry. The obtained results show that the studied peptides in physiological related pH prefer formation of the species with the {4N_Im} binding mode. The efficiency of Cu(II) binding depends on additional side chain groups Asp or Lys. Additionally the analysis of results for His containing cyclopeptides with different numbers of amino acid residues in cyclopeptide ring e.g. four, eight shows that in higher pH in both cases the binding by four amide nitrogens is not observed in the case of α-amino acid peptides.     

Fourier transform infrared spectroscopic study on the binding of Mg2+ to a mutant Akazara scallop troponin C (E142Q)

Troponin C (TnC) is the Ca(2+)-binding regulatory protein of the troponin complex in muscle tissue. Vertebrate fast skeletal muscle TnCs bind four Ca(2+), while Akazara scallop (Chlamys nipponensis akazara) striated adductor muscle TnC binds only one Ca(2+) at site IV, because all the other EF-hand motifs are short of critical residues for the coordination of Ca(2+). Fourier transform infrared (FTIR) spectroscopy was applied to study coordination structure of Mg(2+) bound in a mutant Akazara scallop TnC (E142Q) in D(2)O solution. The result showed that the side-chain COO(-) groups of Asp 131 and Asp 133 in the Ca(2+)-binding site of E142Q bind to Mg(2+) in the pseudo-bridging mode. Mg(2+) titration experiments for E142Q and the wild-type of Akazara scallop TnC were performed by monitoring the band at about 1600 cm(-1), which is due to the pseudo-bridging Asp COO(-) groups. As a result, the binding constants of them for Mg(2+) were the same value (about 6 mM). Therefore, it was concluded that the side-chain COO(-) group of Glu 142 of the wild type has no relation to the Mg(2+) ligation. The effect of Mg(2+) binding in E142Q was also investigated by CD and fluorescence spectroscopy. The on-off mechanism of the activation of Akazara scallop TnC is discussed on the basis of the coordination structures of Mg(2+) as well as Ca(2+).     

Salt bridge induced changes in the secondary structure of ionic polypeptides.

The carboxylate-containing homopolypeptides poly(L-glutamate) [poly(Glu)] and poly(L-aspartate) [poly(Asp)] were found to form different types of ordered structures in the presence of poly(L-lysine) [poly(Lys)]. Mixing poly(Glu) with poly(Lys) in aqueous solution at neutral pH results in the instantaneous formation of a gel-like precipitate. The secondary structure of the gel precipitate can be best described as intermolecular antiparallel beta-strands, involving the backbone amide groups, as evidenced by the presence of characteristic amide I bands in the ir spectrum at 1684 and 1612 cm-1. Mixing poly(Asp) with poly(Lys) under identical conditions results in the formation of a fine precipitate with a different morphology. Examination of the ir spectrum of the precipitate revealed that unlike poly(Glu), poly(Asp) did not yield any discrete secondary structure upon precipitation with poly(Lys). Addition of solutions containing Ca2+ or Mg2+ to the poly(Glu)/poly(Lys) aggregates resulted in complete dissolution of the gel, with the disappearance of the ir bands characteristic of the intermolecular hydrogen-bonded network. The results demonstrate the importance of salt bridges in establishing strong hydrogen bonds between the backbone amide groups. Reaggregation occurred upon heating the poly(Glu)/poly(Lys) mixture in the presence of Ca2+, but not in the presence of Mg2+ ions. In the presence of Ca2+ ions, aggregation and formation of an extended hydrogen-bonded network occurred upon heating. The aggregates formed upon heating poly(Glu)/poly(Lys) in the presence of Ca2+ were attributed solely to complexation of Ca2+ to the carboxylate groups of poly(Glu) with poly(Lys) remaining free in solution. Dissolution of the aggregate could be accomplished through addition of Mg2+ at room temperature.     

Calcium and chlorpromazine binding to the EF-hand peptides of neuronal calcium sensor-1

Neuronal calcium sensor-1, a protein of calcium sensor family, is known to have four structural EF-hands. We have synthesised peptides corresponding to all the four EF-hands and studied their conformation and calcium-binding. Our data confirm that the first putative site, a non-canonical one (EF1), does not bind calcium. We have investigated if this lack of binding is due to the presence of non-favoured residues (particularly at +x and -z co-ordinating positions) of the loop. We have mutated these residues and found that after modification the peptides bound calcium. However, these mutated peptides (EF1 and its functional mutants) do not show any Ca(2+) induced changes in far-UV CD. EF2, EF3, and EF4 peptides bind Ca(2+), EF3 being the strongest binder, followed by EF4. Our data of Ca(2+)-binding to individual EF peptides show that there are three active Ca(2+)-binding sites in NCS-1. We have also studied the binding of a neuroleptic drug, chlorpromazine, with the protein as well as with its EF-hands. CPZ binds myristoylated as well as non-myristoylated NCS-1 in Ca(2+)-dependent manner, with dynamic interaction to myristoylated protein. CPZ does not bind to EF1, but binds to functional EF-hand peptides and induces changes in far-UV CD. Our results suggest that NCS-1 could be a target of such antipsychotic and neuroleptic drugs.     

Structural and biological studies on synthetic peptide analogues of a low-affinity calcium-binding site of skeletal troponin C

We have synthesized four oligopeptides that are structural analogues of a low-affinity Ca2+-specific binding site (site II) of rabbit skeletal troponin C. One analogue (peptide 3) was a dodecapeptide with a sequence corresponding to the 12-residue Ca2+-binding loop (residues 63-74 in troponin C), two (peptides 4 and 5) were 23-residue in length, corresponding to residues 52-74 of the protein, and the fourth (peptide 6) was a 25-residue peptide corresponding to residues 50-74. All four peptides had one amino acid substitution within the 12-residue binding loop in which phenylalanine at position 10 was replaced by tyrosine to provide a marker for spectroscopic studies. In addition, peptides 3 and 4 each had a second substitution within the binding loop where glycine at position 6 was replaced by alanine. The second substitution was motivated by the conservation of glycine at the position in the Ca2+-binding loops of all four Ca2+-binding sites in troponin C. The peptides were characterized by their intrinsic fluorescence, ability to enhance the emission of bound Tb3+, affinity for Ca2+ and Tb3+, and circular dichroism. The affinity for Ca2+ was in the range 10-10(2) M-1, and the affinity for Tb3+ was in the range 10(4)-10(5) M-1. The binding constants of the longer peptides were several-fold larger than that of the dodecapeptide. With peptides 4 and 5, substitution of glycine by alanine at position 6 within the 12-residue loop decreased the affinity for Ca2+ by a factor of four, but had little effect on the affinity for Tb3+. However, the mean residue ellipticity of peptide 4 was substantially higher than that of peptide 5. Since peptide 4 differs from peptide 5 only in the substitution of glycine at position 6 in the loop segment, the conservation of glycine at that position may serve a role in providing a suitable secondary structure of the binding sites for interaction with troponin I. Peptides 4 and 6, when present in a large excess, mimic troponin C in regulating fully reconstituted actomyosin ATPase by showing partial calcium sensitivity and activation of the ATPase. Since these peptides are the smallest peptides containing the Ca2+-binding loop of site II, their biological activity suggests that a Ca2+-dependent binding site of troponin C for troponin I could be as short as the segment comprising residues 52-62.     

Identification of peptides in the adenine ring binding domain of glutamate and lactate dehydrogenase using 2-azido-NAD+.

Previously, the synthesis and validation of [32P]2N3NAD+ as an active site directed photoaffinity probe for glutamate dehydrogenase (GDH) was reported (8). This report shows that 2N3NAD+ is also an effective probe for the NAD+ binding site of lactate dehydrogenase (LDH). With the appropriate photolabeling procedures and immobilized boronate column chromatography the active site peptides of GDH and LDH involved in the adenine base binding domain have been isolated and sequenced. With both GDH and LDH a single photolabeled peptide, which contained the majority of the photoinserted radiolabel, was isolated. Additionally, these peptides had UV spectra that were markedly different from the nonphotolabeled peptides. The modified peptide from GDH corresponded to Cys270 through Lys289. Both sequencing and compositional analysis identified Glu275 as the site of photoinsertion. Sequencing of this peptide aborted at Glu275 after five rounds of analysis, indicating that insertion was blocking further progress. Compositional analysis showed that the entire sequence from residues 270 to 289 was present except that the single Glu residue was missing. This is interpreted as indicating that the photoinsertion is into the polypeptide backbone at the Glu site. The peptide isolated from LDH corresponded to Asp82 through Arg90. Sequencing of this peptide could be completed throughout with only the round at Tyr83 giving no identifiable residue. Compositional analysis of this peptide was in agreement with the peptide from Asp82 to Arg90 with the exception that the single Tyr residue was missing. This indicates that the photoinsertion is into the tyrosine side chain. This data was found to be in agreement with X-ray crystallographic results identifying the NAD(+)-binding domains.     

Identification of single Mn(2+) binding sites required for activation of the mutant proteins of E.coli RNase HI at Glu48 and/or Asp134 by X-ray crystallography

Escherichia coli RNase HI has two Mn(2+)-binding sites. Site 1 is formed by Asp10, Glu48, and Asp70, and site 2 is formed by Asp10 and Asp134. Site 1 and site 2 have been proposed to be an activation site and an attenuation site, respectively. However, Glu48 and Asp134 are dispensable for Mn(2+)-dependent activity. In order to identify the Mn(2+)-binding sites of the mutant proteins at Glu48 and/or Asp134, the crystal structures of the mutant proteins E48A-RNase HI*, D134A-RNase HI*, and E48A/D134N-RNase HI* in complex with Mn(2+) were determined. In E48A-RNase HI*, Glu48 and Lys87 are replaced by Ala. In D134A-RNase HI*, Asp134 and Lys87 are replaced by Ala. In E48A/D134N-RNase HI*, Glu48 and Lys87 are replaced by Ala and Asp134 is replaced by Asn. All crystals had two or four protein molecules per asymmetric unit and at least two of which had detectable manganese ions. These structures indicated that only one manganese ion binds to the various positions around the center of the active-site pocket. These positions are different from one another, but none of them is similar to site 1. The temperature factors of these manganese ions were considerably larger than those of the surrounding residues. These results suggest that the first manganese ion required for activation of the wild-type protein fluctuates among various positions around the center of the active-site pockets. We propose that this fluctuation is responsible for efficient hydrolysis of the substrates by the protein (metal fluctuation model). The binding position of the first manganese ion is probably forced to shift to site 1 or site 2 upon binding of the second manganese ion.     

Loss of a metal-binding site in gelsolin leads to familial amyloidosis-Finnish type.

Mutations in domain 2 (D2, residues 151-266) of the actin-binding protein gelsolin cause familial amyloidosis-Finnish type (FAF). These mutations, D187N or D187Y, lead to abnormal proteolysis of plasma gelsolin at residues 172-173 and a second hydrolysis at residue 243, resulting in an amyloidogenic fragment. Here we present the structure of human gelsolin D2 at 1.65 A and find that Asp 187 is part of a Cd2+ metal-binding site. Two Ca2+ ions are required for a conformational transition of gelsolin to its active form. Differential scanning calorimetry (DSC) and molecular dynamics (MD) simulations suggest that the Cd2+-binding site in D2 is one of these two Ca2+-binding sites and is essential to the stability of D2. Mutation of Asp 187 to Asn disrupts Ca2+ binding in D2, leading to instabilities upon Ca2+ activation. These instabilities make the domain a target for aberrant proteolysis, thereby enacting the first step in the cascade leading to FAF.     

Using protein design to dissect the effect of charged residues on metal binding and protein stability

Ca2+ controls biological processes by interacting with proteins with different affinities, which are largely influenced by the electrostatic interaction from the local negatively charged ligand residues in the coordination sphere. We have developed a general strategy for rationally designing stable Ca2+- and Ln3+-binding proteins that retain the native folding of the host protein. Domain 1 of cluster differentiation 2 (CD2) is the host for the two designed proteins in this study. We investigate the effect of local charge on Ca2+-binding affinity based on the folding properties and metal-binding affinities of the two proteins that have similarly located Ca2+-binding sites with two shared ligand positions. While mutation and Ca2+ binding do not alter the native structure of the protein, Ca2+ binding specifically induced changes around the designed Ca2+-binding site. The designed protein with a -5 charge at the binding sphere displays a 14-, 20-, and 12-fold increase in the binding affinity for Ca2+, Tb3+, and La3+, respectively, compared to the designed protein with a -3 charge, which suggests that higher local charges are preferred for both Ca2+ and Ln3+ binding. The localized charged residues significantly decrease the thermal stability of the designed protein with a -5 charge, which has a T(m) of 41 degrees C. Wild-type CD2 has a T(m) of 61 degrees C, which is similar to the designed protein with a -3 charge. This decrease is partially restored by Ca2+ binding. The effect on the protein stability is modulated by the environment and the secondary structure locations of the charged mutations. Our study demonstrates the capability and power of protein design in unveiling key determinants to Ca2+-binding affinity without the complexities of the global conformational changes, cooperativity, and multibinding process found in most natural Ca2+-binding proteins.