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C T Craescu

Publications and source records attributed to C T Craescu.

At least 19 recordsLinked to original sources
PubMed2001

Solution structure and backbone dynamics of the defunct domain of calcium vector protein.

CaVP (calcium vector protein) is a Ca(2+) sensor of the EF-hand protein family which is highly abundant in the muscle of Amphioxus. Its three-dimensional structure is not known, but according to the sequence analysis, the protein is composed of two domains, each containing a pair of EF-hand motifs. We determined recently the solution structure of the C-terminal domain (Trp81-Ser161) and characterized the large conformational and dynamic changes induced by Ca(2+) binding. In contrast, the N-terminal domain (Ala1-Asp86) has lost the capacity to bind the metal ion due to critical mutations and insertions in the two calcium loops. In this paper, we report the solution structure of the N-terminal domain and its backbone dynamics based on NMR spectroscopy, nuclear relaxation, and molecular modeling. The well-resolved three-dimensional structure is typical of a pair of EF-hand motifs, joined together by a short antiparallel beta-sheet. The tertiary arrangement of the two EF-hands results in a closed-type conformation, with near-antiparallel alpha-helices, similar to other EF-hand pairs in the absence of calcium ions. To characterize the internal dynamics of the protein, we measured the (15)N nuclear relaxation rates and the heteronuclear NOE effect in (15)N-labeled N-CaVP at a magnetic field of 11.74 T and 298 K. The domain is mainly monomeric in solution and undergoes an isotropic Brownian rotational diffusion with a correlation time of 7.1 ns, in good agreement with the fluorescence anisotropy decay measurements. Data analysis using a model-free procedure showed that the amide backbone groups in the alpha-helices and beta-strands undergo highly restricted movements on a picosecond to nanosecond time scale. The amide groups in Ca(2+) binding loops and in the linker fragment also display rapid fluctuations with slightly increased amplitudes.

Amino Acid Sequence↗
PubMed2001

Backbone dynamics of the regulatory domain of calcium vector protein, studied by (15)N relaxation at four fields, reveals unique mobility characteristics of the intermotif linker.

UNLABELLED: CaVP is a calcium-binding protein from amphioxus. It has a modular composition with two domains, but only the two EF-hand motifs localized in the C-terminal domain are functional. We recently determined the solution structure of this regulatory half (C-CaVP) in the Ca(2+)-saturated form and characterized the stepwise ion binding. This paper reports the (15)N nuclear relaxation rates of the Ca(2+)-saturated C-CaVP, measured at four different NMR fields (9.39, 11.74, 14.1, and 18.7 T), which were used to map the spectral density function for the majority of the amide H(N)-N vectors. Fitting the spectral density values at eight frequencies by a model-free approach, we obtained the microdynamic parameters characterizing the global and internal movements of the polypeptide backbone. The two EF-hand motifs, including the ion binding loops, behave like compact structural units with restricted mobility as reflected in the quite uniform order parameter and short internal correlation time (< 20 nsec). Comparative analysis of the two Ca(2+) binding sites shows that site III, having a larger affinity for the metal ion, is generally more rigid, and the amide vector in the second residue of each loop is significantly less restricted. The linker fragment is animated simultaneously by a larger amplitude fast motion and a slow conformational exchange on a microsecond to millisecond time scale. The backbone dynamics of C-CaVP characterized here is discussed in relation with other well-characterized Ca(2+)-binding proteins. SUPPLEMENTAL MATERIAL: See www.proteinscience.org

Animals↗
PubMed2000

Ion-induced conformational and stability changes in Nereis sarcoplasmic calcium binding protein: evidence that the APO state is a molten globule.

Nereis sarcoplasmic Ca(2+)-binding protein (NSCP) is a calcium buffer protein that binds Ca(2+) ions with high affinity but is also able to bind Mg(2+) ions with high positive cooperativity. We investigated the conformational and stability changes induced by the two metal ions. The thermal reversible unfolding, monitored by circular dichroism spectroscopy, shows that the thermal stability is maximum at neutral pH and increases in the order apo < Mg(2+) < Ca(2+). The stability against chemical denaturation (urea, guanidinium chloride) studied by circular dichroism or intrinsic fluorescence was found to have a similar ion dependence. To explore in more detail the structural basis of stability, we used the fluorescent probes to evaluate the hydrophobic surface exposure in the different ligation states. The apo-NSCP exhibits accessible hydrophobic surfaces, able to bind fluorescent probes, in clear contrast with denatured or Ca(2+)/Mg(2+)-bound states. Gel filtration experiments showed that, although the metal-bound NSCP has a hydrodynamic volume in agreement with the molecular mass, the volume of the apo form is considerably larger. The present results demonstrate that the apo state has many properties in common with the molten globule. The possible factors of the metal-dependent structural changes and stability are discussed.

Animals↗
PubMed2000

Sequential calcium binding to the regulatory domain of calcium vector protein reveals functional asymmetry and a novel mode of structural rearrangement.

Calcium vector protein (CaVP) from amphioxus is a two-domain, calcium-binding protein (18.3 kDa) of the calmodulin superfamily. Only two of the four EF-hand motifs (sites III and IV) have a significant binding affinity for calcium ions. We determined the solution structure of the domain containing these active sites (C-CaVP: W81-S161), in the Ca(2+)-saturated state, using NMR spectroscopy and restrained molecular dynamics. The tertiary structure is similar to other Ca(2+)-binding domains containing a pair of EF-hand motifs. The apo state has spectroscopic and thermodynamic characteristics of a molten globule, with conserved secondary structure but highly fluctuating tertiary organization. Titration of C-CaVP with Ca(2+) revealed a stepwise ion binding, with a stable equilibrium intermediate in which only site III binds a calcium ion. Despite a highly fluctuating structure of the free site IV, the calcium-bound site III has a persistent structure, with similar secondary elements but different interhelix angle and hydrophobic packing relative to the fully calcium-saturated state.

Amino Acid Sequence↗
PubMed2000

Cation- and peptide-binding properties of human centrin 2.

Centrin and calmodulin (CaM) are closely related four-EF-hand Ca(2+)-binding proteins. While CaM is monomeric, centrin 2 is dimeric and binds only two Ca(2+) per dimer, likely to site IV in each monomer. Ca(2+) binding to centrin 2 displays pronounced negative cooperativity and a [Ca(2+)](0.5) of 30 microM. As in CaM, Ca(2+) binding leads to the exposure of a hydrophobic probe-accessible patch on the surface of centrin 2. Provided Ca(2+) is present, centrin 2 forms a 1:1 peptide:monomer complex with melittin with an affinity of 100 nM. The complex binds four instead of two Ca(2+). Our data point to surprising differences in the mode of activation of these homologous proteins.

Calcium↗
PubMed1999

A new subfamily of short bacterial adenylate kinases with the Mycobacterium tuberculosis enzyme as a model: A predictive and experimental study.

The adk gene from Mycobacterium tuberculosis codes for an enzyme of 181 amino acids. A sequence comparison with 52 different forms of adenylate kinases (AK) suggests that the enzyme from M. tuberculosis belongs to a new subfamily of "short" bacterial AKs. The recombinant protein, overexpressed in Escherichia coli, exhibits a low catalytic activity and an unexpectedly high thermal stability (Tm = 64.8 degrees C). Based on various spectroscopic data, on the known three-dimensional structure of the AK from E. coli and on secondary structure predictions for various sequenced AKs, we propose a structural model for AK from M. tuberculosis (AKmt). Proteins 1999;36:238-248.

Adenylate Kinase↗
PubMed1999

Multinuclear magnetic resonance studies of Escherichia coli adenylate kinase in free and bound forms. Resonance assignment, secondary structure and ligand binding.

The crystal structure of Escherichia coli adenylate kinase (AKe) revealed three main components: a CORE domain, composed of a five-stranded parallel beta-sheet surrounded by alpha-helices, and two peripheral domains involved in covering the ATP in the active site (LID) and binding of the AMP (NMPbind). We initiated a long-term NMR study aiming to characterize the solution structure, binding mechanism and internal dynamics of the various domains. Using single (15N) and double-labeled (13C and 15N) samples and double- and triple-resonance NMR experiments we assigned 97% of the 1H, 13C and 15N backbone resonances, and proton and 13Cbeta resonances for more than 40% of the side chains in the free protein. Analysis of a 15N-labeled enzyme in complex with the bi-substrate analogue [P1,P5-bis(5'-adenosine)-pentaphosphate] (Ap5A) resulted in the assignment of 90% of the backbone 1H and 15N resonances and 42% of the side chain resonances. Based on short-range NOEs and 1H and 13C secondary chemical shifts, we identified the elements of secondary structure and the topology of the beta-strands in the unliganded form. The alpha-helices and the beta-strands of the parallel beta-sheet in solution have the same limits (+/- 1 residue) as those observed in the crystal. The first helix (alpha1) appears to have a frayed N-terminal side. Significant differences relative to the crystal were noticed in the LID domain, which in solution exhibits four antiparallel beta-strands. The secondary structure of the nucleoside-bound form, as deduced from intramolecular NOEs and the 1Halpha chemical shifts, is similar to that of the free enzyme. The largest chemical shift differences allowed us to map the regions of protein-ligand contacts. 1H/2H exchange experiments performed on free and Ap5A-bound enzymes showed a general decrease of the structural flexibility in the complex which is accompanied by a local increased flexibility on the N-side of the parallel beta-sheet.

Adenylate Kinase↗
PubMed1998

Structural and energetic factors of the increased thermal stability in a genetically engineered Escherichia coli adenylate kinase.

Several variants of Escherichia coli adenylate kinase, designed to bind a Zn2+ ion, were produced by site-directed mutagenesis. The metal binding and enzymatic properties of the engineered variants have been described (Perrier, V., Burlacu-Miron, S., Bourgeois, S., Surewicz, W. K., and Gilles, A.-M. (1998) J. Biol. Chem. 273, 19097-19101). Here we report the structural properties and stability changes in a 4-Cys variant which binds a Zn2+ ion and has an increased thermal stability. CD studies indicate a very similar secondary structure content in the wild type and the engineered variant. NMR analysis revealed that the topology of the parallel beta-sheet, belonging to the protein core, and of the peripheral antiparallel beta-sheet are also conserved. The small local changes observed in the neighborhood of the substitution sites reflect a more compact state of the metal-binding domain. The Zn2+-bound quadruple mutant shows an increased thermal stability, reflected in a 9 degreesC increase of the mid-temperature of the first cooperative unfolding step. Binding of a bisubstrate analog P1, P5-di(adenosine-5')-pentaphosphate increases, by about 7 degreesC, the midpoint of this transition in both wild type and modified variant. The NMR data suggest that the peripheral domains involved in substrate binding unfold during the first denaturation step. Urea denaturation experiments indicate an increased resistance against chemical unfolding of the Zn2+-binding variant. In contrast, the Gibbs free energy of unfolding (at physiologically relevant conditions) of the quadruple mutant is lower than that of the wild type.

Adenylate Kinase↗
PubMed1998

Engineering, expression and biochemical characterization of the hemoglobin domain of a Erwinia chrysanthemi flavohemoprotein.

An artificial hemoglobin-like domain has been constructed by engineering the gene coding for the multi-domain flavohemoprotein from the bacterium Erwinia chrysanthemi. This domain was designed by molecular modelling, cloned and over-expressed in Escherichia coli. The holo-protein was obtained in large quantities after extraction from inclusion bodies and refolding in presence of alkaline hemin. The purified 140-residue domain was studied and characterized to gain new insights into the biochemical function of the recombinant domain and the biological role of this new flavohemoprotein. The structural and functional features of this domain in solution were studied using far-ultraviolet circular dichroism, resonance Raman, proton-NMR spectroscopy, flash laser photolysis and molecular modelling. The recombinant domain is shown to be folded properly and active. This hemoglobin-like domain is able to bind oxygen and carbon monoxide with very high affinity. It exhibits a rapid auto-oxidation which may explain its tight association with a flavin containing reductase domain. A functional model of this hemoglobin is discussed and compared with the X-ray structures of other hemoproteins.

Circular Dichroism↗
PubMed1998

A phosphoglycerate kinase mutant (PGK Herlev; D285V) in a Danish patient with isolated chronic hemolytic anemia: mechanism of mutation and structure-function relationships.

Phosphoglycerate kinase (PGK) is a X-linked enzyme that plays a key role in the glycolytic pathway. Twelve different variants have already been reported. We describe a new PGK variant, PGK Herlev (Asp 285-->Val), in a 69-year-old Danish patient with isolated chronic hemolysis but who had no neurological or muscular disorders. The description of the mutation is based upon PCR amplification of specific regions of the PGK gene, followed by direct sequencing. Although observed in a male patient, this mutated X-linked gene is expressed partially, i.e., both normal and substituted nucleotides are present at the same position in a ratio of approximately 1:9. The most likely explanation for this observation is based on the occurrence of a somatic mutation of the PGK gene. The relationship of structure to function in PGK Herlev, as well as in all known variants, was examined by the use of a computer model based on the known spatial structure of the yeast and horse enzymes. Such an approach can be generalized to any other protein that has been crystallized and for which x-ray diffraction data are available in a species closely related to man.

Aged↗
PubMed1997

Immunosuppressor binding to the immunophilin FKBP59 affects the local structural dynamics of a surface beta-strand: time-resolved fluorescence study.

The interaction of the immunophilin domain of FKBP59 (FKBP59-I) with immunosuppressant drugs was investigated by steady-state and time-resolved fluorescence of tryptophan. One of the two Trp residues present in this protein (W89), conserved in almost all immunophilins, is buried in the hydrophobic core and participates in the immunosuppressant binding. By comparison with the highly homologous protein FKBP12, containing only the buried Trp, it has been concluded that its weak fluorescence is due to an atypical H-bond interaction involving the indole nitrogen and the Phe129 benzene ring. The second Trp residue (W59) in FKBP59-I is located on the external hydrophilic side of the 50-60 beta-sheet [Craescu, C. T., Rouvière, N., Popescu, A., Cerpolini, E., Lebeau, M.-C., Baulieu, E.-E., & Mispelter, J. (1996) Biochemistry 35, 11045-11052] and is responsible for >95% of the fluorescence emission. The long lifetime of the major excited state, the large activation energy of thermal quenching, and the rotational correlation time distribution pattern suggest that its environment is not highly mobile. Binding of the immunosuppressant drugs FK506 and rapamycin leads to a approximately 60% decrease of the fluorescence intensity without any change in the fluorescence emission maximum. Time-resolved measurements show that this "quenching" is due to a conformational change which depletes the long excited-state lifetime population to the profit of a more quenched minor excited state, which becomes prominent in the complexes. This is accompanied by a strong slowing of the indole ring dynamics in the case of FK506 and by a complete immobilization in the case of rapamycin, as shown by two-dimensional (tau, theta) maximum entropy analysis of the polarized fluorescence decays. Binding of the immunosuppressant drugs therefore modifies the structure and the dynamics of the external side of the 50-60 beta-sheet in FKBP59-I, which could be relevant for the formation of ternary complexes with other protein targets.

Acrylamide↗
PubMed1997

Critical role of human bisphosphoglycerate mutase Cys22 in the phosphatase activator-binding site.

The enzymatic activities catalyzed by bisphosphoglycerate mutase (BPGM, EC 5.4.2.4) have been shown to occur at a unique active site, with distinct binding sites for diphosphoglycerates and monophosphoglycerates. The physiological phosphatase activator (2-phosphoglycolate) binds to BPGM at an undetermined site. BPGM variants were constructed by site-directed mutagenesis of three amino acid residues in the active site to identify residues specifically involved in the binding of the monophosphoglycerates and 2-phosphoglycolate. Substitution of Cys22 by functionally conservative residues, Thr or Ser, caused a great decrease in 2-phosphoglycolate-stimulated phosphatase activity and in the Ka value of the activator, whereas it caused no change in other catalytic activities or in the Km values of 2,3-diphosphoglycerate (2,3-DPG) and glycerate 3-phosphate (3-PG, EC 1.1.1.12), indicating that Cys22 is specifically involved either directly or indirectly in 2-phosphoglycolate binding. Kinetic experiments showed that the Ka of the cofactor and the Km of 3-PG were affected by the substitution of Ser23 indicating that this residue is necessary for the fixation of both 3-PG and 2-phosphoglycolate. The R89K variant has previously been shown to have a modified Km value for monophosphoglycerates, however, its affinity for 2-phosphoglycolate is unaltered, suggesting that Arg89 is specifically involved in monophosphoglycerates binding. CD spectroscopic studies of substrates and cofactor binding showed that 2,3-DPG induced structural modifications of normal and mutated enzymes which could be due to protein phosphorylation. Addition of 2-phosphoglycolate to phosphorylated proteins with normal affinity for the cofactor produced spectra with the same characteristics as unphosphorylated species. In summary, monophosphoglycerates and 2-phosphoglycolate have partially distinct binding sites in human BPGM. The specific implication of the Cys22 residue in 2-phosphoglycolate binding is of great significance in the design of analogs of therapeutic benefit.

Binding Sites↗
PubMed1997

Solution structure of Lqh-8/6, a toxin-like peptide from a scorpion venom--structural heterogeneity induced by proline cis/trans isomerization.

Lqh-8/6 is a minor fraction isolated from the venom of the scorpion Leiurus quinquestriatus hebraeus. Here we describe the purification, amino acid sequencing and solution structure determination by NMR and molecular modeling of this peptide. Lqh-8/6 is a small polypeptide (38 residues) which contains 8 half-cystines and is highly similar to another venom component, chlorotoxin. Standard homonuclear methods were used to sequentially assign the proton NMR spectra and to collect spatial restraints for structure determination. Two populations, identified early in the assignment step, are in slow interconversion on the NMR timescale. The two conformers were shown to originate from a cis/trans peptidyl-prolyl isomerization. Using a distance geometry program and simulated annealing protocol under the NMR restraints we obtained 10 final structures for the major conformation (trans isomer). None of the structures showed NOE violations larger than 0.05 nm, and the rmsd value relative to the mean structure (considering the main chain atoms in well-defined secondary structure) is 0.07 nm. The three-dimensional structure contains a short alpha-helix strapped on a small antiparallel beta-strand and an N-terminal extended fragment. The sequence/structure and structure/function relationships of the new scorpion toxin-like peptide are discussed in the context of the present structure determination. This toxin shows a stable, highly populated cis conformer of a peptidyl-prolyl peptide bond.

Amino Acid Sequence↗
PubMed1996

Nereis sarcoplasmic Ca2+-binding protein has a highly unstructured apo state which is switched to the native state upon binding of the first Ca2+ ion.

NSCP, a sarcoplasmic Ca2+/Mg2+-binding protein from Nereis diversicolor, shows an allosteric change during Ca2+ binding and a high positive cooperativity for Mg2+ binding. Here we report the results of CD and NMR experiments aiming to characterize the apo state and the Ca2+-induced conformational changes in this protein. Circular dichroism spectra of the apo form are indicative of a reduced helical structure. In contrast, NMR spectra show no element of regular secondary or tertiary structure. Addition of one Ca2+ determines large spectral changes bringing the molecule in a conformation which is very close to the native three Ca2+ state. Addition of the second and third Ca2+ shifts this equilibrium progressively towards the liganded conformation but affects only minimally the spectrum of the liganded species.

Animals↗