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MC1 is the most abundant architectural protein present in Methanosarcina thermophila CHTI55 in laboratory growth conditions and is structurally unrelated to other DNA-binding proteins. MC1 functions are to shape and to protect DNA against thermal denaturation by binding to it. Therefore, MC1 has a strong affinity for any double-stranded DNA. However, it recognizes and preferentially binds to bent DNA, such as four-way junctions and negatively supercoiled DNA minicircles. Combining NMR data, electron microscopy data, biochemistry, molecular modelisation and docking approaches, we proposed recently a new type of DNA/protein complex, in which the monomeric protein MC1 binds on the concave side of a strongly bent 15 base pairs DNA. We present here the NMR chemical shifts assignments of each partner in the complex, 1H 15N MC1 protein and 1H 13C 15N bent duplex DNA, as first step towards the first experimental 3D structure of this new type of DNA/protein complex.
Plant defensins (PDF) are cysteine-rich peptides that are major actors in the innate immunity in plants. Besides their antifungal activity, some PDF such as Arabidopsis halleri PDF1.1b confer zinc tolerance in plants. Here we present (i) an efficient protocol for the production of AhPDF1.1b by solid-phase peptide synthesis followed by controlled oxidative folding to obtain the highly pure native form of the defensin and (ii) the three-dimensional (3D) nuclear magnetic resonance structure of AhPDF1.1b, the first 3D structure of plant defensin obtained with a synthetic peptide. Its fold is organized around the typical cysteine-stabilized α-helix ?-sheet motif and contains the ?-core motif involved in the antifungal activity of all plant defensins. On the basis of our structural analysis of AhPDF1 defensins combined with previous biological data for antifungal and zinc tolerance activities, we established the essential role of cis-Pro41 within the ?-core. In fact, the four consecutive residues (Val39-Phe40-Pro41-Ala42) are strictly conserved for plant defensins able to tolerate zinc. We hypothesized that structural and/or dynamic features of this sequence are related to the ability of the defensin to chelate zinc.
In Archaea the two major modes of DNA packaging are wrapping by histone proteins or bending by architectural non-histone proteins. To supplement our knowledge about the binding mode of the different DNA-bending proteins observed across the three domains of life, we present here the first model of a complex in which the monomeric Methanogen Chromosomal protein 1 (MC1) from Euryarchaea binds to the concave side of a strongly bent DNA. In laboratory growth conditions MC1 is the most abundant architectural protein present in Methanosarcina thermophila CHTI55. Like most proteins that strongly bend DNA, MC1 is known to bind in the minor groove. Interaction areas for MC1 and DNA were mapped by Nuclear Magnetic Resonance (NMR) data. The polarity of protein binding was determined using paramagnetic probes attached to the DNA. The first structural model of the DNA-MC1 complex we propose here was obtained by two complementary docking approaches and is in good agreement with the experimental data previously provided by electron microscopy and biochemistry. Residues essential to DNA-binding and -bending were highlighted and confirmed by site-directed mutagenesis. It was found that the Arg25 side-chain was essential to neutralize the negative charge of two phosphates that come very close in response to a dramatic curvature of the DNA.
Mitogaligin is a mitochondrion-targeting protein involved in cell death. The sequence of the protein is unrelated to that of any known pro- or antiapoptotic protein. Mitochondrial targeting is controlled by an internal sequence from residues 31 to 53, and although this sequence is essential and sufficient to provoke cell death, the precise mechanism of action at the mitochondrial membrane remains to be elucidated. Here, by focusing on the [31–53] fragment, we first assessed and confirmed its cell cytotoxicity by microinjection. Subsequently, with the aid of membrane models, we evaluated the impact of the membrane environment on the 3D structure of the peptide and on how the peptide is embedded and oriented within membranes. The fragment is well organized, even though it does not contain a canonical secondary structure, and adopts an interfacial location. Structural comparison with other membrane-interacting Trp-rich peptides demonstrated similarities with the antimicrobial peptide tritrpcidin.
Dimerization of genomic RNA is directly related with the event of encapsidation and maturation of the virion. The initiating sequence of the dimerization is a short autocomplementary region in the hairpin loop SL1. We describe here a new solution structure of the RNA dimerization initiation site (DIS) of HIV-1(Lai). NMR pulsed field-gradient spin-echo techniques and multidimensional heteronuclear NMR spectroscopy indicate that this structure is formed by two hairpins linked by six Watson-Crick GC base pairs. Hinges between the stems and the loops are stabilized by intra and intermolecular interactions involving the A8, A9 and A16 adenines. The coaxial alignment of the three A-type helices present in the structure is supported by previous crystallography analysis but the A8 and A9 adenines are found in a bulged in position. These data suggest the existence of an equilibrium between bulged in and bulged out conformations in solution.
In order to study the binding of the Myb-like domain of the human telomeric protein TRF2 (Myb-TRF2) with different structural components of the t-loop model, we report NMR studies of the binding of Myb-TRF2 protein with two repeats human telomeric DNA under three conformations. Our results showed that Myb-TRF2 binds to the duplex and even to the quadruplex and the random coil G-rich strand. The solution structure of Myb-TRF2 reported here looks like Myb-TRF1 suggesting similar DNA binding mode. As a matter of fact, we have shown that its binding to the human telomeric duplex presents great similarities with this of Myb-TRF1.
The three-dimensional structure of methanogen chromosomal protein 1 (MC 1), a chromosomal protein extracted from the archaebacterium Methanosarcina sp. CHT155, has been solved using H-1 NMR spectroscopy. The small basic protein MC1 contains 93 amino acids (24 basic residues against 12 acidic residues). The main elements of secondary structures are an a helix and five ß strands, arranged as two antiparallel ß sheets (a double one and a triple one) packed in an orthogonal manner forming a barrel. The protein displays a largely hydrophilic surface and a very compact hydrophobic core made up by side chains at the interface of the two ß sheets and the helix side facing the interior of the protein.
The genome of all retroviruses consists of two identical copies of an RNA sequence associated in a non-covalent dimer. A region upstream from the splice donor (SL1) comprising a self-complementary sequence is responsible for the initiation of the dimerization. This region is able to dimerize in two conformations : a loop-loop complex or an extended duplex. Here, we solve by 2D NMR techniques the solution structure of a 23-nucleotide sequence corresponding to HIV-1 SL1(Lai) in which the mutation G12—>A12 is included to prevent dimerization. It is shown that this monomer adopts a stem-loop conformation with a seven base pairs stem and a nine nucleotide loop containing the G10 C11 A12 C13 G14 C15 sequence.
Structural and dynamic studies of the lac Operator complexed with the headpiece of the lac Repressor are necessary to establish whether the two partners have pre-required structures or if folding is essential to induce the specific interaction. NOESY spectra of the 1:1 complex of half of the lac Operator 5’d(CGCTCACAATT)-5’d(AATGTGAGCG) sequence (selectively labelled with C-13 at C-1’), with the Inc Repressor N-terminal headpiece 1-51 yielded information about the contact points between the nucleic acid and the protein. The operator was selectively labelled with C-13 at the C-1’ position, allowing the measurement of C-13 relaxation rates as a probe of dynamic behaviour.
The DNA duplex d(CTCTCG*AGTCTC)d(GAGACTC*GAGAG) containing a single trans-diammine-dichloroplatinum(ll) interstrand cross-link (where G* and C* represent the platinated bases) has been studied by two-dimensional NMR, All the exchangeable and non-exchangeable proton resonance lines were assigned (except H5’/H5 ") and the NOE intensities were transformed into distances via the RELAZ program. By combining the NOESY and COSY data (330 constraints) and NMR-constrained molecular mechanics using JUMNA, a solution structure of the cross-linked duplex has been determined.
A 10 base pairs double-stranded oligonucleotide with the sequence d(CCTCG*CTCTC). d(GAGAG*CGAGG) containing a single interstrand cross-link resulting from chelation of the N7 position of two guanine residues on the opposite strands of DNA at the d(G*C/G*C) site by a cis-diammineplatinum(II) residue was analyzed by H-1 NMR spectroscopy. All the exchangeable and nonexchangeable protons resonance lines (except some H5’-H5 ’’) were assigned. NOESY spectra and chemical shifts indicated that the cross-linkage of the guanines of G*5 and G*6 induced extrahelicity of C5 and C6.
In order to study some internal dynamic processes of the lac operator sequence, the C-13-labeled duplex 5’d(C(0)G(1)C(2)T(3)C(4)A(5)C(6)A(7)A(8)T(9)T(10)) d(A(10)A(9)T(8)T(7)G(6)T(5)G(4)A(3)G(2)C(1)G(0))31 was used. The spreading of both the H1’ and Cl’ resonances brought about an excellent dispersion of the (1)Hl’-Cl-13’ correlations. The spin-lattice relaxation parameters R(C-z) R(C-x,C-y) and R(H-z—>C-z) were measured for each residue of the two complementary strands, except for the 3’-terminal residues which were not labeled. Variation of the relaxation rates was found along the sequence. These data were analyzed in the context of the model-free formalism proposed by Lipari and Szabo [(1982) J. Am. Chem. Soc , 104, 4546-4570] and extended to three parameters by Clore et al. [(1990) Biochemistry, 29, 7387-7401 ; and (1990) J : Am. Chem. Sec, 112, 4989-4991].
In order to examine the internal dynamic processes of the dodecamer d(CGCAAATTTGCG)(2), the C-13- enriched oligonucleotide has been synthesized. The three central thymines were selectively C-13-labeled at the C1’ position and their spin-lattice relaxation parameters R(C-Z), R(C-X,C-Y), R(H-Z—>C-Z), R(HZCZ), R(2H(Z)C(X,Y)) and R(H-Z(C)) were measured. Density functions were computed for two models of internal motions. Comparisons of the experimental data were made with the spin-lattice relaxation rates rather than with the density functions, whose values were altered by accumulation of the uncertainties of each relaxation rate measurement. The spin-lattice relaxation rates were computed with respect to the motions of the sugar around the C1’-N1 bond.
Enzyme-catalyzed preparation of beta-2-deoxy-D-glucosides and galactosides including disaccharides has been achieved using the corresponding glycals as substrates.
Chargé de recherche , RMN des biomolécules