1H NMR study on amide proton exchange of calmodulin-mastoparan complex

Amide proton exchange rates of Ca2(+)-saturated calmodulin and Ca2(+)-saturated calmodulin-mastoparan complex were studied by 1H NMR spectroscopy. Exchange rates of Gly25, Gly61, Gly98, Gly134, Ile27, Ile100, and Asn137 were determined for Ca2(+)-saturated calmodulin and for Ca2(+)-saturated calmodulin-mastoparan complex, and were found to be less than 10(-4)s-1. All these residues of which the amide proton resonances appear at lower fields were considered to form hydrogen bonds, based on the results of X-ray analysis. Exchange rates of Ile27 and Asn137 became an order of magnitude smaller when mastoparan bound to Ca2(+)-saturated calmodulin, while those of the four glycines and Ile100 did not change appreciably. The reduction in accessibility of Asn137 to water cased by mastoparan binding suggests that a part of the mastoparan binding site is probably located in or near the hydrophobic cluster of the C-terminal-half domain. The reduction in accessibility of Ile27 also suggests that another part of the mastoparan binding site is located in or near the hydrophobic cleft of the N-terminal-half domain.     

1H NMR study of rabbit skeletal muscle troponin C: Mg2(+)-induced conformational change

Binding of Mg2+ to rabbit skeletal muscle troponin C (TnC) is studied by means of two-dimensional (2D) 1H NMR spectroscopy. Using the sequence-specific resonance assignment method we assign several resonances of TnC in the Mg2(+)-saturated state. Assigned resonances are used as probes of the following titration experiments: (1) Mg2+ titration of apo-TnC, (2) Mg2+ titration of Ca2TnC, and (3) Mg2+ titration of Ca4TnC. In experiment 1, the slow-exchange behavior is observed for resonances of Phe99, Asp107, Gly108, Tyr109, Ile110, Asp111, His125, Gly144, Arg145, Ile146, Asp147, and Phe148 located at the high-affinity Ca2(+)-binding sites in the C-terminal-half domain. In experiments 1 and 2, the fast-exchange behavior is observed for resonances of Gly32, Asp33, Ser35, Gly68, Thr69, and Asp71 located at the low-affinity Ca2(+)-binding sites in the N-terminal-half domain. These results suggest that Mg2+ ions bind to the N domain as well as the C domain. In experiment 3, no spectral change is observed for all above-mentioned residues in the C domain and also for Gly32 and Gly68 in the N domain. It can be concluded that all Ca2(+)-binding sites in both the N and C domains can bind Mg2+ ions. No significant change is observed for resonances of Phe23, Ile34, Val68, and Phe72 in experiments 1 and 2. These results suggest that Mg2+ binding to the N domain does not induce conformational change in the hydrophobic region of the N domain. 2D-NMR spectra and Mg2(+)-titration data suggest that the antiparallel beta-sheet conformation is formed in both the N and C domains when Mg2+ ions bind to the two domains.     

Comparative NMR studies on cardiac troponin C and a mutant incapable of binding calcium at site II

One- and two-dimensional NMR techniques were used to study both the influence of mutations on the structure of recombinant normal cardiac troponin C (cTnC3) and the conformational changes induced by Ca2+ binding to site II, the site responsible for triggering muscle contraction. Spin systems of the nine Phe and three Tyr residues were elucidated from DQF-COSY and NOESY spectra. Comparison of the pattern of NOE connectivities obtained from a NOESY spectrum of cTnC3 with a model of cTnC based on the crystal structure of skeletal TnC permitted sequence-specific assignment of all three Tyr residues, as well as Phe-101 and Phe-153. NOESY spectra and calcium titrations of cTnC3 monitoring the aromatic region of the 1H NMR spectrum permitted localization of six of the nine Phe residues to either the N- or C-terminal domain of cTnC3. Analysis of the downfield-shifted C alpha H resonances permitted sequence-specific assignment of those residues involved in the beta-strand structures which are part of the Ca(2+)-binding loops in both the N- and C-terminal domains of cTnC3. The short beta-strands in the N-terminal domain of cTnC3 were found to be present and in close proximity even in the absence of Ca2+ bound at site II. Using these assignments, we have examined the effects of mutating Asp-65 to Ala, CBM-IIA, a functionally inactive mutant which is incapable of binding Ca2+ at site II [Putkey, J.A., Sweeney, H. L., & Campbell, S. T. (1989) J. Biol. Chem. 264, 12370]. Comparison of the apo, Mg(2+)-, and Ca(2+)-bound forms of cTnC3 and CBM-IIA demonstrates that the inability of CBM-IIA to trigger muscle contraction is not due to global structural changes in the mutant protein but is a consequence of the inability of CBM-IIA to bind Ca2+ at site II. The pattern of NOEs between aromatic residues in the C-terminal domain is nearly identical in cTnC3 and CBM-IIA. Similar interresidue NOEs were also observed between Phe residues assigned to the N-terminal domain in the Ca(2+)-saturated forms of both cTnC3 and CBM-IIA. However, chemical shift changes were observed for the N-terminal Phe residues in CBM-IIA. This suggests that binding of Ca2+ to site II alters the chemical environment of the residues in the N-terminal hydrophobic cluster without disrupting the spatial relationship between the Phe residues located in helices A and D.     

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].     

An NMR and spin label study of the effects of binding calcium and troponin I inhibitory peptide to cardiac troponin C.

The paramagnetic relaxation reagent, 4-hydroxy-2,2,6,6-tetramethylpiperidinyl-1-oxy (HyTEMPO), was used to probe the surface exposure of methionine residues of recombinant cardiac troponin C (cTnC) in the absence and presence of Ca2+ at the regulatory site (site II), as well as in the presence of the troponin I inhibitory peptide (cTnIp). Methyl resonances of the 10 Met residues of cTnC were chosen as spectral probes because they are thought to play a role in both formation of the N-terminal hydrophobic pocket and in the binding of cTnIp. Proton longitudinal relaxation rates (R1's) of the [13C-methyl] groups in [13C-methyl]Met-labeled cTnC(C35S) were determined using a T1 two-dimensional heteronuclear single- and multiple-quantum coherence pulse sequence. Solvent-exposed Met residues exhibit increased relaxation rates from the paramagnetic effect of HyTEMPO. Relaxation rates in 2Ca(2+)-loaded and Ca(2+)-saturated cTnC, both in the presence and absence of HyTEMPO, permitted the topological mapping of the conformational changes induced by the binding of Ca2+ to site II, the site responsible for triggering muscle contraction. Calcium binding at site II resulted in an increased exposure of Met residues 45 and 81 to the soluble spin label HyTEMPO. This result is consistent with an opening of the hydrophobic pocket in the N-terminal domain of cTnC upon binding Ca2+ at site II. The binding of the inhibitory peptide cTnIp, corresponding to Asn 129 through Ile 149 of cTnI, to both 2Ca(2+)-loaded and Ca(2+)-saturated cTnC was shown to protect Met residues 120 and 157 from HyTEMPO as determined by a decrease in their measured R1 values. These results suggest that in both the 2Ca(2+)-loaded and Ca(2+)-saturated forms of cTnC, cTnIp binds primarily to the C-terminal domain of cTnC.     

NMR study of the phosphate-binding loops of Thermus thermophilus elongation factor Tu.

The phosphoryl-binding loops in the guanosine diphosphate binding domain of elongation factor Tu were studied by 15N heteronuclear proton-observe NMR methods. Five proton resonances were found below 10.5 ppm. One of these was assigned to the amide group of Lys 24, which is a conserved residue in the phosphoryl-binding concensus loop of purine nucleotide binding proteins. The uncharacteristic downfield proton shift is attributed to a strong hydrogen bond with a phosphate oxygen. The amide protons from the homologous lysines in N-ras p21 [Redfield, A.G., & Papastavros, M.Z. (1990) Biochemistry 29, 3509-3514] and the catalytic domain of Escherichia coli elongation factor Tu [Lowry, D.F., Cool, R.H., Redfield, A.G., & Parmeggiani, A. (1991) Biochemistry 30, 10872-10877] also resonate downfield in similar positions. We propose that the downfield shift of this lysine amide proton is a spectral marker for this class of proteins. We also have studied the temperature dependence of the downfield resonances and find a possible conformation change at 40 degrees C.     

Use of amide 1H-nmr titration shifts for studies of polypeptide conformation

This paper shows that backbone amide proton titration shifts in polypeptide chains are a very sensitive manifestation of intramolecular hydrogen bonding between carboxylate groups and backbone amide protons. The population of specific hydrogen-bonded structures in the ensemble of species that constitutes the conformation of a flexible nonglobular linear peptide can be determined from the extent of the titration shifts. As an illustration, an investigation of the molecular conformation of the linear peptide H-Gly-Gly-L -Glu-L -Ala-OH is described. The proposed use of amide proton titration shifts for investigating polypeptide conformation is based on 360-MHz ¹H-nmr studies of selected linear oligopeptides in H₂O solutions. It was found that only a very limited number of amide protons in a polypeptide chain show sizable intrinsic intration shifts arising from through-bond interactions with ionizable groups. These are the amide proton of the C-terminal amino acid residue, the amide protons of Asp and the residues following Asp, and possibly the amide proton of the residue next to the N-terminus. Since the intrinsic titration shifts are upfield, the downfield titration shifts arising from conformation-dependent through-space interactions, in particular hydrogen bonding between the amide protons and carboxylate groups, can readily be identified.     

Nuclear magnetic resonance studies of the internal dynamics in Apo, (Cd2+)1 and (Ca2+)2 calbindin D9k. The rates of amide proton exchange with solvent.

The backbone dynamics of the EF-hand Ca(2+)-binding protein, calbindin D9k, has been investigated in the apo, (Cd2+)1 and (Ca2+)2 states by measuring the rate constants for amide proton exchange with solvent. 15N-1H correlation spectroscopy was utilized to follow direct 1H-->2H exchange of the slowly exchanging amide protons and to follow indirect proton exchange via saturation transfer from water to the rapidly exchanging amide protons. Plots of experimental rate constants versus intrinsic rate constants have been analyzed to give qualitative insight into the opening modes of the protein that lead to exchange. These results have been interpreted within the context of a progressive unfolding model, wherein hydrophobic interactions and metal chelation serve to anchor portions of the protein, thereby damping fluctuations and retarding amide proton exchange. The addition of Ca2+ or Cd2+ was found to retard the exchange of many amide protons observed to be in hydrogen-bonding environments in the crystal structure of the (Ca2+)2 state, but not of those amide protons that were not involved in hydrogen bonds. The largest changes in rate constant occur for residues in the ion-binding loops, with substantial effects also found for the adjacent residues in helices I, II and III, but not helix IV. The results are consistent with a reorganization of the hydrogen-bonding networks in the metal ion-binding loops, accompanied by a change in the conformation of helix IV, as metal ions are chelated. Further analysis of the results obtained for the three states of metal occupancy provides insight into the nature of the changes in conformational fluctuations induced by ion binding.     

A joint 2D NMR and theoretical investigation of Ca2+ binding loops III and IV of calmodulin

A 2D NMR NOESY spectrum of integral CaM in water(148 residues) reveals a series of downfield-shifted crosspeaks stemming from the NH protons of the Ca2(+)-binding loops III and IV. Their attribution, with the help of already assigned proton resonances of isolated tryptic fragments, was complemented by means of energy-minimizations on the Ca2+ complexes of loops III and IV. From these calculations, a set of two alternative, related conformations was obtained for each loop. The first type of conformation provides a coordination pattern for Ca2+ that is similar to that found in loop EF of parvalbumin. The computed interproton distances in both loops are fully compatible with the inferences from the sets of NOESY cross-peaks. Evidence is also provided for interloop interactions.     

Epidermal growth factor from the mouse. Physical evidence for a tiered beta-sheet domain: two-dimensional NMR correlated spectroscopy and nuclear Overhauser experiments on backbone amide protons

When H2O-exchanged, lyophilized mouse epidermal growth factor (mEGF) is dissolved in deuterium oxide at low pH (i.e., below approximately 6.0), 13 well-resolved, amide proton resonances are observed in the downfield region of an NMR spectrum (500 MHz). Under the conditions of these experiments, the lifetimes of these amide protons in exchange for deuterons of the deuterium oxide solvent suggest that these amide protons are hydrogen-bonded, backbone amide protons. Several of these amide proton resonances show splittings (i.e., JNH alpha-CH) of approximately 8-10 Hz, indicating that their associated amide protons are in some type of beta-structure. Selective nuclear Overhauser effect (NOE) experiments performed on all amide proton resonances strongly suggest that all 13 of these backbone amide protons are part of a single-tiered beta-sheet structural domain in mEGF. Correlation of 2D NMR correlated spectroscopy data, identifying scaler coupled protons, with NOE data, identifying protons close to the irradiated amide protons, allows tentative assignment of some resonances in the NOE difference spectra to specific amino acid residues. These data allow a partial structural model of the tiered beta-sheet domain in mEGF to be postulated.     

Solution structures of the C-terminal domain of cardiac troponin C free and bound to the N-terminal domain of cardiac troponin I.

The N-terminal domain of cardiac troponin I (cTnI) comprising residues 33-80 and lacking the cardiac-specific amino terminus forms a stable binary complex with the C-terminal domain of cardiac troponin C (cTnC) comprising residues 81-161. We have utilized heteronuclear multidimensional NMR to assign the backbone and side-chain resonances of Ca2+-saturated cTnC(81-161) both free and bound to cTnI(33-80). No significant differences were observed between secondary structural elements determined for free and cTnI(33-80)-bound cTnC(81-161). We have determined solution structures of Ca2+-saturated cTnC(81-161) free and bound to cTnI(33-80). While the tertiary structure of cTnC(81-161) is qualitatively similar to that observed free in solution, the binding of cTnI(33-80) results mainly in an opening of the structure and movement of the loop region between helices F and G. Together, these movements provide the binding site for the N-terminal domain of cTnI. The putative binding site for cTnI(33-80) was determined by mapping amide proton and nitrogen chemical shift changes, induced by the binding of cTnI(33-80), onto the C-terminal cTnC structure. The binding interface for cTnI(33-80), as suggested from chemical shift changes, involves predominantly hydrophobic interactions located in the expanded hydrophobic pocket. The largest chemical shift changes were observed in the loop region connecting helices F and G. Inspection of available TnC sequences reveals that these residues are highly conserved, suggesting a common binding motif for the Ca2+/Mg2+-dependent interaction site in the TnC/TnI complex.     

NMR study of the phosphate-binding elements of Escherichia coli elongation factor Tu catalytic domain

The phosphoryl-binding elements in the GDP-binding domain of elongation factor Tu were studied by heteronuclear proton observe methods. Five proton resonances were found below 10.5 ppm. Two of these were assigned to the amide groups of Lys 24 and Gly 83. These are conserved residues in each of the consensus sequences. Their uncharacteristic downfield proton shifts are attributed to strong hydrogen bonds to phosphate oxygens as for resonances in N-ras-p21 [Redfield, A. G., & Papastavros, M. Z. (1990) Biochemistry 29, 3509-3514]. The Lys 24 of the EF-Tu G-domain has nearly the same proton and nitrogen shifts as the corresponding Lys 16 in p21. These results suggest that this conserved lysine has a similar structural role in proteins in this class. The tentative Gly 83 resonance has no spectral analogue in p21. A mutant protein with His 84 changed to glycine was fully 15N-labeled and the proton resonance assigned to Gly 83 shifted downfield by 0.3 ppm, thereby supporting the assignment.     

Structure of the Mg2+-loaded C-lobe of cardiac troponin C bound to the N-domain of cardiac troponin I: comparison with the Ca2+-loaded structure

Cardiac troponin C (cTnC) is the Ca(2+)-binding component of the troponin complex and, as such, is the Ca(2+)-dependent switch in muscle contraction. This protein consists of two globular lobes, each containing a pair of EF-hand metal-binding sites, connected by a linker. In the N lobe, Ca(2+)-binding site I is inactive and Ca(2+)-binding site II is primarily responsible for initiation of muscle contraction. The C lobe contains Ca(2+)/Mg(2+)-binding sites III and IV, which bind Mg(2+) with lower affinity and play a structural as well as a secondary role in modulating the Ca(2+) signal. To understand the structural consequences of Ca(2+)/Mg(2+) exchange in the C lobe, we have determined the NMR solution structure of the Mg(2+)-loaded C lobe, cTnC(81-161), in a complex with the N domain of cardiac troponin I, cTnI(33-80), and compared it with a refined Ca(2+)-loaded structure. The overall tertiary structure of the Mg(2+)-loaded C lobe is very similar to that of the refined Ca(2+)-loaded structure as evidenced by the root-mean-square deviation of 0.94 A for all backbone atoms. While metal-dependent conformational changes are minimal, substitution of Mg(2+) for Ca(2+) is characterized by condensation of the C-terminal portion of the metal-binding loops with monodentate Mg(2+) ligation by the conserved Glu at position 12 and partial closure of the cTnI hydrophobic binding cleft around site IV. Thus, conformational plasticity in the Ca(2+)/Mg(2+)-dependent binding loops may represent a mechanism to modulate C-lobe cTnC interactions with the N domain of cTnI.     

Disease-causing mutations in cartilage oligomeric matrix protein cause an unstructured Ca2+ binding domain.

Chondrocytes from pseudoachondroplasia (PSACH) and multiple epiphyseal dysplasia (EDM1) patients display an enlarged rough endoplasmic reticulum that accumulates extracellular matrix proteins, including cartilage oligomeric matrix protein (COMP). Mutations that cause PSACH and EDM1 are restricted to a 27-kDa Ca(2+) binding domain (type 3 repeat). This domain has 13 Ca(2+)-binding loops with a consensus sequence that conforms to Ca(2+)-binding loops found in EF hands. Most disease-causing mutations are found in the 11-kDa C-terminal region of this domain. We expressed recombinant native and mutant forms of the type 3 repeat domain (T3) and its 11-kDa C-terminal region (T3-Cterm). T3 and T3-Cterm bind approximately 13 and 8 mol of Ca(2+)/mol of protein, respectively. CD, one-dimensional proton, and two-dimensional (1)H-(15)N HSQC spectra of Ca(2+)-bound T3-Cterm indicate a distinct conformation that has little helical secondary structure, despite the presence of 13 EF hand Ca(2+)-binding loops. This conformation is also formed within the context of the intact T3. 19 cross-peaks found between 9.0 and 11.4 ppm are consistent with the presence of strong hydrogen bonding patterns, such as those in beta-sheets. Removal of Ca(2+) leads to an apparent loss of structure as evidenced by decreased dispersion and loss of all down field resonances. Deletion of Asp-470 (a mutation found in 22% of all PSACH and EDM1 patients) decreased the Ca(2+)-binding capacity of both T3 and T3-Cterm by about 3 mol of Ca(2+)/mol of protein. Two-dimensional (1)H-(15)N HSQC spectra of mutated T3-Cterm showed little evidence of defined structure in the presence or absence of Ca(2+). The data demonstrate that Ca(2+) is required to nucleate folding and to maintain defined structure. Mutation results in a partial loss of Ca(2+)-binding capacity and prevents Ca(2+)-dependent folding. Persistence of an unstructured state of the mutated Ca(2+) binding domain in COMP is the structural basis for retention of COMP in the rough endoplasmic reticulum of differentiated PSACH and EDM1 chondrocytes.     

Binding of levosimendan, a calcium sensitizer, to cardiac troponin C

Levosimendan is an inodilatory drug that mediates its cardiac effect by the calcium sensitization of contractile proteins. The target protein of levosimendan is cardiac troponin C (cTnC). In the current work, we have studied the interaction of levosimendan with Ca(2+)-saturated cTnC by heteronuclear NMR and small angle x-ray scattering. A specific interaction between levosimendan and the Ca(2+)-loaded regulatory domain of recombinant cTnC(C35S) was observed. The changes in the NMR spectra of the N-domain of full-length cTnC(C35S), due to the binding of levosimendan to the primary site, were indicative of a slow conformational exchange. In contrast, no binding of levosimendan to the regulatory domain of cTnC(A-Cys), where all the cysteine residues are mutated to serine, was detected. Moreover, it was shown that levosimendan was in fast exchange on the NMR time scale with a secondary binding site in the C-domain of both cTnC(C35S) and cTnC(A-Cys). The small angle x-ray scattering experiments confirm the binding of levosimendan to Ca(2+)-saturated cTnC but show no domain-domain closure. The experiments were run in the absence of the reducing agent dithiothreitol and the preservative sodium azide (NaN(3)), since we found that levosimendan reacts with these chemicals, commonly used for preparation of NMR protein samples.     

Solution structures of the N-terminal domain of yeast calmodulin: Ca2+-dependent conformational change and its functional implication

We have determined solution structures of the N-terminal half domain (N-domain) of yeast calmodulin (YCM0-N, residues 1-77) in the apo and Ca(2+)-saturated forms by NMR spectroscopy. The Ca(2+)-binding sites of YCM0-N consist of a pair of helix-loop-helix motifs (EF-hands), in which the loops are linked by a short beta-sheet. The binding of two Ca(2+) causes large rearrangement of the four alpha-helices and exposes the hydrophobic surface as observed for vertebrate calmodulin (CaM). Within the observed overall conformational similarity in the peptide backbone, several significant conformational differences were observed between the two proteins, which originated from the 38% disagreement in amino acid sequences. The beta-sheet in apo YCM0-N is strongly twisted compared with that in the N-domain of CaM, while it turns to the normal more stable conformation on Ca(2+) binding. YCM0-N shows higher cooperativity in Ca(2+) binding than the N-domain of CaM, and the observed conformational change of the beta-sheet is a possible cause of the highly cooperative Ca(2+) binding. The hydrophobic surface on Ca(2+)-saturated YCM0-N appears less flexible due to the replacements of Met51, Met71, and Val55 in the hydrophobic surface of CaM with Leu51, Leu71, and Ile55, which is thought to be one of reasons for the poor activation of target enzymes by yeast CaM.     

Assignment of the backbone 1H and 15N NMR resonances of bacteriophage T4 lysozyme

The proton and nitrogen (15NH-H alpha-H beta) resonances of bacteriophage T4 lysozyme were assigned by 15N-aided 1H NMR. The assignments were directed from the backbone amide 1H-15N nuclei, with the heteronuclear single-multiple-quantum coherence (HSMQC) spectrum of uniformly 15N enriched protein serving as the master template for this work. The main-chain amide 1H-15N resonances and H alpha resonances were resolved and classified into 18 amino acid types by using HMQC and 15N-edited COSY measurements, respectively, of T4 lysozymes selectively enriched with one or more of alpha-15N-labeled Ala, Arg, Asn, Asp, Gly, Gln, Glu, Ile, Leu, Lys, Met, Phe, Ser, Thr, Trp, Tyr, or Val. The heteronuclear spectra were complemented by proton DQF-COSY and TOCSY spectra of unlabeled protein in H2O and D2O buffers, from which the H beta resonances of many residues were identified. The NOE cross peaks to almost every amide proton were resolved in 15N-edited NOESY spectra of the selectively 15N enriched protein samples. Residue specific assignments were determined by using NOE connectivities between protons in the 15NH-H alpha-H beta spin systems of known amino acid type. Additional assignments of the aromatic proton resonances were obtained from 1H NMR spectra of unlabeled and selectively deuterated protein samples. The secondary structure of T4 lysozyme indicated from a qualitative analysis of the NOESY data is consistent with the crystallographic model of the protein.     

A Joint 2D NMR and Theoretical Investigation of Ca2+ Binding Loops III and IV of Calmodulin

Abstract

A 2D NMR NOESY spectrum of integral CaM in water(148 residues) reveals a series of downfield-shifted crosspeaks stemming from the NH protons of the Ca²⁺ -binding loops III and IV. Their attribution, with the help of already assigned proton resonances of isolated tryptic fragments, was complemented by means of energy-minimizations on the Ca²⁺ complexes of loops III and IV. From these calculations, a set of two alternative, related conformations was obtained for each loop. The first type of conformation provides a coordination pattern for Ca²⁺ that is similar to that found in loop EF of parvalbumin. The computed interproton distances in both loops are fully compatible with the inferences from the sets of NOESY cross-peaks. Evidence is also provided for interloop interactions.     

Ca2+-induced conformational transition in the inhibitory and regulatory regions of cardiac troponin I

Cardiac muscle activation is initiated by the binding of Ca(2+) to the single N-domain regulatory site of cardiac muscle troponin C (cTnC). Ca(2+) binding causes structural changes between cTnC and two critical regions of cardiac muscle troponin I (cTnI): the regulatory region (cTnI-R, residues 150-165) and the inhibitory region (cTnI-I, residues130-149). These changes are associated with a decreased cTnI affinity for actin and a heightened affinity for cTnC. Using F?rster resonance energy transfer, we have measured three intra-cTnI distances in the deactivated (Mg(2+)-saturated) and Ca(2+)-activated (Ca(2+)-saturated) states in reconstituted binary (cTnC-cTnI) and ternary (cTnC-cTnI-cTnT) troponin complexes. Distance A (spanning cTnI-R) was unaltered by Ca(2+). Distances B (spanning both cTnI-R and cTnI-I) and C (from a residue flanking cTnI-I to a residue in the center of cTnI-R) exhibited Ca(2+)-induced increases of >8 A. These results compliment our previous determination of the distance between residues flanking cTnI-I alone. Together, the data suggest that Ca(2+) activation causes residues within cTnI-I to switch from a beta-turn/coil to an extended quasi-alpha-helical conformation as the actin-contacts are broken, whereas cTnI-R remains alpha-helical in both Mg(2+)- and Ca(2+)-saturated states. We have used the data to construct a structural model of the cTnI inhibitory and regulatory regions in the Mg(2+)- and Ca(2+)-saturated states.     

Solution structures of Ca2+-CIB1 and Mg2+-CIB1 and their interactions with the platelet integrin alphaIIb cytoplasmic domain

The calcium- and integrin-binding protein 1 (CIB1) is a ubiquitous Ca(2+)-binding protein and a specific binding partner for the platelet integrin αIIb cytoplasmic domain, which confers the key role of CIB1 in hemostasis. CIB1 is also known to be involved in apoptosis, embryogenesis, and the DNA damage response. In this study, the solution structures of both Ca(2+)-CIB1 and Mg(2+)-CIB1 were determined using solution-state NMR spectroscopy. The methyl groups of Ile, Leu, and Val were selectively protonated to compensate for the loss of protons due to deuteration. The solution structure of Ca(2+)-CIB1 possesses smaller opened EF-hands in its C-domain compared with available crystal structures. Ca(2+)-CIB1 and Mg(2+)-CIB1 have similar structures, but the N-lobe of Mg(2+)-CIB1 is slightly more opened than that of Ca(2+)-CIB1. Additional NMR experiments, such as chemical shift perturbation and methyl group solvent accessibility as measured by a nitroxide surface probe, were carried out to further characterize the structures of Ca(2+)-CIB1 and Mg(2+)-CIB1 as well as their interactions with the integrin αIIb cytoplasmic domain. NMR measurements of backbone amide proton slow motion (microsecond to millisecond) dynamics confirmed that the C-terminal helix of Ca(2+)-CIB1 is displaced upon αIIb binding. The EF-hand III of both Ca(2+)-CIB1 and Mg(2+)-CIB1 was identified to be directly involved in the interaction of CIB1 with αIIb. Together, these data illustrate that CIB1 behaves quite differently from related EF-hand regulatory calcium-binding proteins, such as calmodulin or neuronal calcium sensor proteins.