Chromatin core particle obtained by selective cleavage of histones H3 and H4 by clostripain.

A new chromatin core particle characterized by a half-proteolyzed octamer is obtained by controlled digestion of the native core particle by clostripain. The proteolyzed histones correspond to four polypeptide fragments which are tentatively assigned to H2A[4-129], H2B[1-125], H3[27-135] and H4[18-102] on the basis of electrophoretic evidence and the known specificity of clostripain for arginyl residues. Despite the loss of the N-terminal regions of histones H3 and H4, the partially proteolyzed core particle retains the structural conformation of the native one as shown by circular dichroism. As expected, this half-proteolyzed core particle presents an intermediate accessibility to polycations, such as spermidine, in comparison with that observed with the native core particle and a fully proteolyzed core particle. The latter includes the polypeptide fragments H2A[12-129], H2B[21-125], H3[27-135] and H4[20-102].

Two-dimensional 1H nuclear magnetic resonance study of pike pI 5.0 parvalbumin (Esox lucius). Sequential resonance assignments and folding of the polypeptide chain.

The structure of alpha pike 5.0 parvalbumin under its Ca-loaded form (or PaCa2) is studied in solution by two-dimensional 1H nuclear magnetic resonance (n.m.r.) at 360 MHz using a conventional strategy of sequential assignments, which involved correlated spectroscopy, relayed coherence transfer spectroscopy and nuclear Overhauser enhancement spectroscopy. In order to overcome the problem of spectral overlapping due to the presence of 108 residues in the protein, experiments were performed at different pH and temperature values, either in 1H2O or in 2H2O solutions. The amino acid sequence of pike 5.0 parvalbumin is thus fully characterized by nearly the totality of its NH, C alpha H and C beta H resonances originating from the different residues (421 protons assigned among 429 in total). When associated with the remaining side resonances, these sequence-specific assignments provide a basis for establishing the secondary organization and tertiary folding of the polypeptide chain. Pike 5.0 parvalbumin was selected as a characteristic representative of the alpha phylogenic series, for which no crystalline structure is presently available, in contrast with the beta series for which two crystalline structures have been determined. A parvalbumin molecule with a single polypeptide chain of 108 amino acids represents one of the highest molecular weights analyzed so far by two-dimensional n.m.r. spectroscopy. The use of a moderate magnetic field strength, with 1H nuclei resonating at 360 MHz, is justified by the fact that ring current effects are operating favorably in this globular protein with a high phenylalanine content. A three-dimensional structure has been generated by the "distance geometry" or DISGEO computational procedure on the basis of about 450 interproton nuclear Overhauser enhancement connectives (short, medium and long-range) in conjunction with a selection of phi and chi dihedral angle constraints. The coherence of the calculated structure, which displays all the features of the typical folding of a parvalbumin protein, provides a good test of reliability of the n.m.r. data collected so far. Although similar to a beta parvalbumin in the folding of its polypeptide chain, the alpha parvalbumin studied here differs markedly from a beta parvalbumin in the length of its C-terminal F-helix domain, which includes 11 residues instead of ten in the latter.(ABSTRACT TRUNCATED AT 400 WORDS)

The N tails of histones H3 and H4 adopt a highly structured conformation in the nucleosome.

The histone N tails correspond to conserved amino acid sequences that are peripherally located in the nucleosome and undergo a variety of post-synthetic modifications during cell cycle. These N tails have been recently recognized as directly interacting with transcription-related proteins. We show here, based on circular dichroic evidence, that the N tails of both tetrameric histones H3 and H4 are highly organized as DNA-bound polypeptide segments in the nucleosome core particle, with about half of their residues, taken together, being alpha-helical. In contrast, the N tails of both dimeric histones H2A and H2B are found essentially in a random-coil conformation. The implications of these findings on nucleosome structure and recognition are discussed.

Ca2+/Mg2+ exchange in parvalbumin and other EF-hand proteins. A theoretical study.

A remarkable conformational rearrangement occurs upon Ca2+/Mg2+ exchange in the C-terminal EF-hand site (labelled site EF or EF-4) of parvalbumin, as initially established by X-ray crystallography. Such a conformational rearrangement is characterised as follows: (i) the co-ordination number decreases from seven oxygen atoms in the Ca-loaded form to six oxygen atoms in the Mg-loaded form, the heptaco-ordination of Ca2+ corresponding with a skewed pentagonal bipyramid configuration of the seven oxygen atoms, whereas the hexaco-ordination of Mg2+ corresponds with a regular octahedral configuration of the six oxygen atoms; and (ii) Glu101, at the relative position 12 in the EF-hand loop sequence (labelled "Glu12"), acts as a bidentate ligand in the Ca-loaded form and as a monodentate ligand in the Mg-loaded form. As part of the conformational rearrangement, the chi1 dihedral angle undergoes a gauche(+) to gauche(-) transition upon substitution of Ca2+ by Mg2+, whereas the chi2 angle remains practically unchanged and the chi3 angles in both forms adopt a nearly mirror image relationship. In order to understand the molecular mechanisms underlying such a conformational rearrangement, we undertook a theoretical study using the free energy perturbation (FEP) method, starting from high-resolution crystal structures of the same parvalbumin (pike 4. 10 isoform) differing by the substitution of their two cationic sites EF-3 (or CD) and EF-4 (or EF), i.e. the 1pal structure with EF-3(Ca2+) and EF-4(Ca2+), the 4pal structure with EF-3(Ca2+) and EF-4(Mg2+). When Mg2+ is "alchemically" transformed into Ca2+ within the EF-4 site of 4pal, the conformational rearrangement of Glu12 is correctly predicted by the FEP calculation. When Ca2+ is transformed into Mg2+ within the EF-3 site of 4pal, the FEP calculation predicts the topology of the fully Mg-loaded form for which no crystallographic data is presently available. As expected, Glu62 (at the relative position 12 in EF-3 loop) is predicted to be a monodentate residue within a regular octahedral arrangement of six oxygen atoms around Mg2+. We also investigated the behaviour during Ca2+/Mg2+ exchange of two other typical EF-hand proteins, troponin C (TnC) and calmodulin (CaM), for which no three-dimensional structure of their Mg-loaded forms is available so far. It is also predicted that the EF-3 site of TnC and the EF-1 site of CaM have their invariant Glu12 residues switching from the bidentate to the monodentate configuration when Ca2+ is substituted by Mg2+, with six oxygen atoms being observed in the co-ordination sphere of the alchemically generated Mg2+ cation.

Crystal structure determination and refinement of pike 4.10 parvalbumin (minor component from Esox lucius).

The crystal and molecular structure of the minor component of pike parvalbumins has been determined at 1.93 A resolution by molecular replacement (1 A = 0.1 nm). The crystals are orthorhombic, space group P2(1)2(1)2 with a = 59.62 A, b = 59.83 A and c = 26.35 A. A location of the secondary cation binding site is proposed for this parvalbumin of the beta phylogenetic series.

Crystal structure of the unique parvalbumin component from muscle of the leopard shark (Triakis semifasciata). The first X-ray study of an alpha-parvalbumin.

The three-dimensional structure of parvalbumin from leopard shark (Triakis semifasciata) with 109 amino acid residues (alpha-series) is described at 1.54 A resolution. Crystals were grown at 20 degrees C from 2.9 M-potassium/sodium phosphate solutions at pH 5.6. The space group is P3(1)21 and unit cell dimensions are a = b = 32.12 A and c = 149.0 A. The structure has been solved by the molecular replacement method using pike 4.10 parvalbumin as a model. The final structure refinement resulted in an R-factor of 17.3% for 11,363 independent reflections at 1.54 A resolution. The shark parvalbumin shows the main features of all parvalbumins: the folding of the chain including six alpha-helices, the salt bridge between Arg75 and Glu81, and the hydrophobic core. Compared to the structure of beta-parvalbumins from pike and carp, one main difference is observed: the chain is one residue longer and this additional residue, which extends the F helix, is involved through its C-terminal carboxylate group in a network of electrostatic contacts with two basic residues, His31 in the B helix and Lys36 in the BC segment. Furthermore, hydrogen bonds exist between the side-chains of Gln108 (F helix) and Tyr26 (B helix). There is therefore a "locking" of the tertiary structure through contacts between two sequentially distant regions in the protein and this is likely to contribute to making the stability of an alpha-parvalbumin higher in comparison to that of a beta-parvalbumin. The lengthening of the C-terminal F helix by one residue appears to be a major feature of alpha-parvalbumins in general, owing to the homologies of the amino acid sequences. Besides the lengthening of the C-terminal helix, the classification of the leopard shark parvalbumin in the alpha-series rests upon the observation of Lys13, Leu32, Glu61 and Val66. As this is the first crystal structure description of a parvalbumin from the alpha-phylogenetic lineage, it was hoped that it would clearly determine the presence or absence of a third cation binding site in parvalbumins belonging to the alpha-lineage. In beta-pike pI 4.10 parvalbumin, Asp61 participates as a direct ligand of a third site, the satellite of the CD site. In shark parvalbumin, as in nearly all alpha-parvalbumins, one finds Glu at position 61. Unfortunately, the conformation of the polar head of Glu61 cannot be inferred from the X-ray data.(ABSTRACT TRUNCATED AT 400 WORDS)

Ionic interactions with parvalbumins. Crystal structure determination of pike 4.10 parvalbumin in four different ionic environments.

The crystal structure of the Ca-loaded form of pike 4.10 parvalbumin (minor component from pike muscle belonging to the beta phylogenetic series), with both its primary sites CD and EF occupied by Ca2+ ions and its third site occupied by an ammonium ion, as previously determined at 1.93 A resolution, has now been refined to a resolution of 1.65 A. The crystallization of this parvalbumin in different ionic environments has allowed three novel non-isomorphous crystalline forms to be obtained: (1) a first form, crystallized in the presence of a mixture of ammonium sulphate and manganese sulphate, for which all the cation binding sites in the protein are occupied by Mn2+; (2) a second form crystallized in the presence of MgSO4 as the precipitating agent, only differs from the Ca/NH4 form by the occupation of the third site by Mg2+, whereas the primary sites remain occupied by Ca2+; (3) a third form, also crystallized in the presence of MgSO4, corresponds to a well-defined molecular species with both the primary EF site and the third site occupied by Mg2+, whereas the primary CD site remains occupied by CA2+. The corresponding molecular structures reported here have been determined at resolutions between 1.8 and 2.4 A. The comparison of the different crystal structures allows the structural modifications accompanying the substitution of the primary sites by cations differing significantly in their ionic radii (Ca2+, Mn2+, Mg2+) to be investigated in detail, and it also leads to a precise description of the third site in a typical beta parvalbumin. The substitution Ca2+ by Mg2+ within the primary site EF is characterized by a "contraction" of the co-ordination sphere, with a decrease of the mean oxygen-metal distance by a value of 0.25 A and a decrease of the co-ordination number from 7 to 6, as a consequence of the loss of a bidentate ligand (Glu101), which becomes a monodentate one. Such an adaptation of the co-ordination sphere around a cation of smaller size involves, among others, the transformation of the Glu101 side-chain from the stable gauche(+) form to the less stable gauche(-) form. The third site is clearly described as a satellite of the CD primary site, since both sites possess common protein ligands, such as Asp53 and Glu59. Furthermore, Asp61 appears as a specific ligand of the third site in the different environments investigated in this work. We finally discuss the relevance of the third site to parvalbumin phylogeny.

Crystals of the cell-binding module of fibronectin obtained from a series of recombinant fragments differing in length.

A recombinant fragment corresponding to the cell adhesion module (FNIII10) of human fibronectin has been crystallized at pH 8.6 from solutions containing polyethylene glycol as precipitant. The crystals formed in the space group P2(1) with a = 30.76 A, b = 35.07 A, c = 37.66 A, beta = 106.9 degrees. There is one molecule per asymmetric unit and the crystals diffract beyond 1.75 A resolution. To improve the prospects for successful crystallization of the FNIII10 module, a series of recombinant fragments was produced with minor differences in the length of N or C-terminal segments. Only one of these variants crystallized. Interestingly, the C-terminal residue of this variant formed stable intermolecular contacts with a symmetry-related molecule in the crystal lattice.

Evidence indicating proximity in the nucleosome between the histone H4 N termini and the globular domain of histone H1.

Proteolysis of rat liver chromatin by the Arg-C peptidase, clostripain, is characterized by a progressive fragmentation of the N-terminal segments of the four core histones H2A, H2B, H3 and H4, until a well-defined limit digest is reached. This work addresses the case of histone H4. Two intermediate proteolytic sites are identified for this histone, i.e. Arg3 and Arg17, before the limit digest is achieved through cleavage of the polypeptide chain after Arg19. The accessibility of these intermediate sites depends strongly on the presence or absence of histone H1. When H1 is absent, both intermediate sites of histone H4 are similarly accessible, whereas one of them, Arg3, becomes totally inaccessible in the presence of histone H1. Di- and trinucleosomes were used with the aim of avoiding any interference with superstructural effects which can occur with longer polynucleosomes in the presence of H1. We also investigated the accessibility of the Arg sites of H1 that are located primarily in the central globular domain of this histone. In free histone H1, all the centrally located Arg sites are accessible to clostripain. In contrast, in the chromatin-bound state none of these sites is accessible. Besides the arginyl sites in the central globular domain of H1, two Arg residues are observed with the most abundant H1d variant in rat chromatin, one in the N-terminal region and the other in the C-terminal region. The restricted number of proteolytic fragments observed with chromatin-bound H1 is accounted for by the cleavage of H1 after these Arg residues located on the outside of the globular domain. Our results suggest that mutual steric effects are at play between histones H1 and H4 and indicate that the N termini of both histones H4 in the nucleosome lie in close proximity to the globular domain of H1. Based on these observations and taking into account the known structural features of the nucleosome, we propose a model for positioning the N-terminal segments of both histones H4 at the periphery of the nucleoprotein structure. In this model both H4 segments are located within the expanded DNA minor grooves, at periods +/- 1, symmetrically disposed relatively to the nucleosome dyad axis. This arrangement brings the amino ends of both H4 molecules in close contact with the H1 globular domain thus accounting for the observed inaccessibility of the Arg3 site of H4 in the presence of H1.

Crystal structure of the EF-hand parvalbumin at atomic resolution (0.91 A) and at low temperature (100 K). Evidence for conformational multistates within the hydrophobic core.

Several crystal structures of parvalbumin (Parv), a typical EF-hand protein, have been reported so far for different species with the best resolution achieving 1.5 A. Using a crystal grown under microgravity conditions, cryotechniques (100 K), and synchrotron radiation, it has now been possible to determine the crystal structure of the fully Ca2+-loaded form of pike (component pI 4.10) Parv.Ca2 at atomic resolution (0.91 A). The availability of such a high quality structure offers the opportunity to contribute to the definition of the validation tools useful for the refinement of protein crystal structures determined to lower resolution. Besides a better definition of most of the elements in the protein three-dimensional structure than in previous studies, the high accuracy thus achieved allows the detection of well-defined alternate conformations, which are observed for 16 residues out of 107 in total. Among them, six occupy an internal position within the hydrophobic core and converge toward two small buried cavities with a total volume of about 60 A3. There is no indication of any water molecule present in these cavities. It is probable that at temperatures of physiological conditions there is a dynamic interconversion between these alternate conformations in an energy-barrier dependent manner. Such motions for which the amplitudes are provided by the present study will be associated with a time-dependent remodeling of the void internal space as part of a slow dynamics regime (millisecond timescales) of the parvalbumin molecule. The relevance of such internal dynamics to function is discussed.

Two well-defined motifs in the cAMP-dependent protein kinase inhibitor (PKIalpha) correlate with inhibitory and nuclear export function.

The heat stable inhibitor of cAMP-dependent protein kinase (PKIalpha) contains both a nuclear export signal (NES) and a high affinity inhibitory region that is essential for inhibition of the catalytic subunit of the kinase. These functions are sequentially independent. Two-dimensional NMR spectroscopy was performed on uniformly [15N]-labeled PKIalpha to examine its structure free in solution. Seventy out of 75 residues were identified, and examination of the CaH chemical shifts revealed two regions of upfield chemical shifts characteristic of alpha-helices. When PKIalpha was fragmented into two functionally distinct peptides for study at higher concentrations, no significant alterations in chemical shifts or secondary structure were observed. The first ordered region, identified in PKIalpha (1-25), contains an alpha-helix from residues 1-13. This helix extends by one turn the helix observed in the crystal structure of a PKIalpha (5-24) peptide bound to the catalytic subunit. The second region of well-defined secondary structure, residues 35-47, overlaps with the nuclear export signal in the PKIalpha (26-75) fragment. This secondary structure consists of a helix with a hydrophobic face comprised of Leu37, Leu41, and Leu44, followed by a flexible turn containing Ile46. These four residues are critical for nuclear export function. The remainder of the protein in solution appears relatively unstructured, and this lack of structure surrounding a few essential and well-defined signaling elements may be characteristic of a growing family of small regulatory proteins that interact with protein kinases.

NMR studies of primary and secondary sites of parvalbumins using the two paramagnetic probes Gd (III) and Mn (II).

The binding of cations by parvalbumins was studied by the proton relaxation enhancement (PRE) method using the paramagnetic probes Gd(III) and Mn(II). Gd(III) appears as a specific probe of the primary sites CD and EF with the following binding parameters: n = 2, KdGd = 0.5 x 10(-11) M and epsilon b = 2.3. The low value of epsilon b is the result of a nearly complete dehydration of the protein bound ions. Competition experiments between Gd(III) and various diamagnetic cations show the following order of affinity for the EF and CD sites: Mg2+ less than Zn2+ less than Sr2+ less than Ca2+ less than Cd2+ less than La3+ less than or equal to Gd3+. Mn 2+ is a specific probe of a secondary site with the following binding parameters: n = 1, KdMn = 0.6 x 10(-3) M and epsilon b = 17. The high value of epsilon b suggests that the protein bound Mn(II) has retained most of its hydration shell. Competition experiments between (Mn(II) and different cations show similar affinities for this site: Ca2+ less than or equal to Mg2+ less than or equal to Cd2+ less than or equal to Mn2+. This secondary site is located near the EF primary site.

Photochemical rearrangement of oxaziridines and nitrones in the hexahydroindole series: a convenient synthetic route to 1-azabicyclo[5.2.0]nonan-2-ones as novel RGD mimetics.

[reaction: see text] Photolysis of oxaziridines a or nitrones b provides a convenient synthetic route to fused bicyclic lactams c adequately substituted on both cycles A and B as scaffolds for mimicking conformationally constrained beta-turn peptides as in the tripeptide RGD signaling motif of fibronectin.