Báo cáo khoa học: Refined solution structure and backbone dynamics of the archaeal MC1 protein ppt

Refined solution structure and backbone dynamics of the archaeal MC1 protein Franc¸oise Paquet, Karine Loth, Herve ´ Meudal, Franc¸oise Culard, Daniel Genest and Ge ´ rard Lancelot Centre de Biophysique Mole ´ culaire, CNRS UPR 4301, Orle ´ ans, France Introduction DNA-binding proteins play a central role in all aspects of genetic activity within an organism, such as tran- scription, packaging, rearrangement, replication, and repair. Archaeons have a variety of abundant, sequence-independent nucleoid proteins, some of which are able to compact DNA. Among the numerous chro- matin proteins identified in archaeons, only two – histones and Alba homologs – are present in almost all archaeal phyla [1]. Archaeal histones (e.g. HMfa and HMfb) are char- acterized by an a-helical histone fold. Their monomers are not stable, and must form homodimers. In the presence of DNA, dimers assemble into tetramers and, sometimes, hexamers [2]. These archaeal histone tetra- mers wrap $ 90 bp in less than one circle, resulting in a horseshoe-shape assembly. Histones are replaced by other chromatin proteins in archaeons that lack them, namely the hyperthermophilic Crenarchaea and euryar- chaeal Thermoplasma. Sulfolobus species (Crenarchaea) have small monomeric proteins with an SH3-like fold, such as Sac7d and Cren7 [3,4], whereas members of the Thermoplasma genus have the dimeric protein Keywords arm; bulges; DNA-binding protein; molecular dynamics (MD) simulation; NMR relaxation Correspondence F. Paquet, Centre de Biophysique Mole ´ culaire, CNRS UPR 4301, Rue Charles-Sadron, F-45071 Orle ´ ans Cedex 2, France Fax: +33 2 38631517 Tel: +33 2 38257692 E-mail: francoise.paquet@cnrs-orleans.fr Database Structural data are available in the Protein Data Bank database under the accession number 2KHL (Received 26 July 2010, revised 15 September 2010, accepted 20 October 2010) doi:10.1111/j.1742-4658.2010.07927.x The 3D structure of methanogen chromosomal protein 1 (MC1), deter- mined with heteronuclear NMR methods, agrees with its function in terms of the shape and nature of the binding surface, whereas the 3D structure determined with homonuclear NMR does not. The structure features five loops, which show a large distribution in the ensemble of 3D structures. Evidence for the fact that this distribution signifies internal mobility on the nanosecond time scale was provided by using 15 N-relaxation and molecular dynamics simulations. Structural variations of the arm (11 residues) induced large shape anisotropy variations on the nanosecond time scale that ruled out the use of the model-free formalism to analyze the relaxation data. The backbone dynamics analysis of MC1 was achieved by compari- son with 20 ns molecular dynamics trajectories. Two b-bulges showed that hydrogen bond formation correlated with u and w dihedral angle transi- tions. These jumps were observed on the nanosecond time scale, in agree- ment with a large decrease in 15 N-NOE for Gly17 and Ile89. One water molecule bridging NH(Glu87) and CO(Val57) through hydrogen bonding contributed to these dynamics. Nanosecond slow motions observed in loops LP3 (35–42) and LP5 (67–77) reflected the lack of stable hydrogen bonds, whereas the other loops, LP1 (10–14), LP2 (22–24), and LP4 (50–53), were stabilized by several hydrogen bonds. Dynamics are often directly related to function. Our data strongly suggest that residues belonging to the flexible regions of MC1 could be involved in the interac- tion with DNA. Abbreviations CSP, chemical shift perturbation; MC1, methanogen chromosomal protein 1; MD, molecular dynamics; RDC, residual dipolar coupling. FEBS Journal 277 (2010) 5133–5145 ª 2010 The Authors Journal compilation ª 2010 FEBS 5133 HTa, a member of the HU family [5,6]. Sac7d and Cren7 have a small b-barrel with or without an amphi- philic C-terminal a-helix, and HUs have a largely a-helical body capped by b-sheets that extend into two b-ribbon arms. Alba homologs, which were first identified in differ- ent species of Sulfolobus (e.g. Sac10b and Sso10b), bind to both DNA and RNA. The Alba dimer has two extended b-hairpins flanking a central body, sug- gesting three main points of contact with the DNA [7]. They are present in all archaeons except Halobacteria and Methanomicrobia (Euryarchaea). For instance, Methanosarcina sequenced genomes contain one gene coding for a true archaeal histone, HMm, as well as genes coding for structural proteins of the methanogen chromosomal protein 1 (MC1) family [8]. In laboratory growth conditions, MC1 is the most abundant structural protein present in Methanosarcina thermophila CHTI55 [9]. A large number of charged residues (24 basic and 12 acidic amino acids) are dis- tributed all along the protein sequence. This small pro- tein of 93 residues is able to bind and to bend any dsDNA as a monomer. Related to its capacity to introduce strong DNA conformational changes, MC1 is able to discriminate between different deformations of the DNA double helix. Thus, MC1 recognizes and strongly binds to four-way junctions [10] and to mini- circles [11,12]. In addition, MC1 is easily able to recog- nize flexible DNA sequences [13]. Visualization of the linear DNA molecules by electron microscopy reveals that the binding of MC1 induces sharp kinks with an angle value of 116° [14]. We have previously solved the three-dimensional structure of this architectural protein extracted from the M. thermophila strain CHTI55 by using 1 H-NMR spectroscopy only [15]. The overall fold of MC1, characterized by its b–b–a–b–b–b link- ing, is different from those of other known DNA-bind- ing proteins. Site-directed mutagenesis showed that two residues belonging to the loop b4–b5 (Trp74 and Met75) are involved in DNA binding [16]. Further- more, hydroxyl radical footprinting, together with a dystamycin competition experiment, suggested that the monomeric MC1 binds to DNA through the minor groove, and that the binding site, covering at least 15 bp, is composed of two areas of contact separated by nearly 10 bp [13]. The static structure, previously described, could not explain this particular behavior [15]. We therefore decided to continue the structural study of MC1 with a recombinant 13 C, 15 N-labeled pro- tein expressed in Escherichia coli. In this article, we report heteronuclear NMR experiments that have enabled us to assign all side chains and to introduce dihedral angle restraints (u and w angles). Residual dipolar couplings (RDCs) were also measured in a par- tially aligned sample with radially compressed poly- acrylamide gel to add constraints, particularly in the arm. In addition to the structural study, a qualitative analysis of the NMR relaxation and molecular dynam- ics (MD) simulation data was carried out. Results and Discussion Refined NMR structures of MC1 Chemical shift assignments for MC1 were obtained for 97% of N, H N ,H a ,C a ,C b and C¢ nuclei (Table S1). The refined structures of MC1 were determined by using NOE distances, dihedral angles, hydrogen bonds, and 1 D NH RDC restraints (Table 1). The global fold consists of a pseudobarrel with an extension of the b-sheet (b4–b5) forming an arm (Fig. 1A,B). The sec- ondary structure elements, namely an a-helix, a1 (25– 32), and five b-strands, b1 (4–9), b2 (15–21), b3 (43– 48), b4 (55–65), and b5 (79–90), are all antiparallel and packed with each other as previously described [15]. An antiparallel b-bulge (B1), composed of Leu8, His16, and Gly17, is present in seven structures, and another antiparallel b-bulge (B2), composed of Val57, Glu87, and Arg88, is observed for all the structures. The secondary structure elements are connected by loops LP1 (10–14), LP2 (22–24), LP3 (35–42), LP4 (50–53), and LP5 (67–77), referred to as ‘arm’ in the text. The latter now appears to be remote from the protein core, whereas it was previously described as pulled down on the a-helix. Superimposition of the 15 best structures of MC1 clearly shows that the regions with the largest degree of structural variations include the N-terminus, C-terminus, and loops LP1, LP3, LP4, and, especially, LP5 (Fig. 1A). Its rmsd value is large (11 A ˚ ), in agreement with the extensive conformational space swept by its residues (Table 1), whereas the rmsd values of the other loops fall between 2 and 2.7 A ˚ . MC1 can no longer be considered as a spherical pro- tein, but rather as an anisotropic structure defined by the ratio of the principal components of the inertia tensor. This ratio differs within the 15 models of MC1, 1.00 : (0.85–0.94) : (0.34–0.43), according to the posi- tion of the arm. Although this new fold is completely different from those of other known proteins, it has similarities to the small architectural proteins Sac7d and Cren7 belonging to the Sulfolobus strains of the Crenarchaeota subdo- main (Fig. 2) [3,4]. All possess a triple-stranded b-sheet (b3–b4–b5). Sac7d and Cren7 cause a single-step sharp kink in DNA ($ 60° and $ 53°, respectively) through the intercalation of hydrophobic side chains. Despite NMR structure and backbone dynamics of MC1 F. Paquet et al. 5134 FEBS Journal 277 (2010) 5133–5145 ª 2010 The Authors Journal compilation ª 2010 FEBS their similarity in overall structure, these two SH3-like proteins differ in the DNA-binding surface. Cren7 shows a substantially larger binding site ($ 8 bp) than Sac7d (4 bp), as it possesses a long loop of seven resi- dues between b3 and b4 in the DNA binding surface [17]. Loop b3–b4 of Cren7 undergoes a significant con- formational change upon binding of the protein to DNA, suggesting its critical role in the stabilization of the proteinÆDNA complex. The arm of MC1 can also be compared with the DNA-binding b-hairpin arms of HUs, which showed high mobility relative to the core (Fig. 2). The b-ribbon arms wrap around the minor groove of the DNA and, at the tip of each arm, the conserved Pro intercalates between base pairs, creating and ⁄ or stabilizing two kinks in the DNA (global cur- vature between 105° and 140°) [18]. This variability is reflected by extensive DNA contacts between 9 bp of DNA and the b-ribbon arms, and variable contacts between additional DNA and the body of the protein [19]. In the case of MC1ÆDNA complexes, we know that the protein covers at least 15 bp and that the binding site is composed of two areas of contact sepa- rated by nearly 10 bp [13]. The arm (loop LP5) seems to be essential to cover such a long sequence. In fact, the arm of MC1 has many hydrophobic residues (Pro68, Pro72, Trp74, Met75, and Pro76), which are conserved in different species of Methanosarcina and Halobacteria. Site-directed mutagenesis showed that two residues belonging to the loop (Trp74 and Met75) are involved in DNA binding [16]. It is clear that the arm of MC1 is essential for DNA binding and bend- ing. The interaction mode of MC1 is probably com- pletely different from those of Sac7d and Cren7, which bind and bend DNA by placing their triple-stranded b-sheet (b3–b4–b5) across the DNA minor groove. Indeed, the electrostatic potential surface of MC1 reveals that one side of the protein has a considerable number of positively charged residues: Arg4, Lys22, Arg25, Lys53, Lys54, His56, Lys69, Arg71, Lys81, Lys85, Lys86, and Lys91 (Fig. 1C). This side, the reverse of the one used by Sac7d and Cren7 of Sulfolo- bus, is a good candidate to interact with the phosphate group of nucleotides. 15 N-NMR Relaxation for MC1 The 15 N-HSQC spectrum of MC1 recorded at 600 MHz showed good dispersion of the crosspeaks (Fig. S1). Relaxation data were obtained for 84 back- bone N–H pairs (93 residues minus Pro24, Pro42, Pro68, Pro72, Pro76, Pro82, Gly51, and the two N-ter- minal residues Ser1 and Asn2) at 600 MHz (R 1 , R 2 , 15 N-NOE) and 800 MHz (R 1 , R 2 ), and, owing to spec- tral overlap, for 79 residues at 500 MHz ( 15 N-NOE). The experimental relaxation data at 600 and 800 MHz are shown in Figs 3 and S2 respectively. The patterns seen for the individual relaxation rate constants at the different field strengths are similar. The average value of R 1 is 1.6 s )1 at 600 MHz and 1.1 s )1 at 800 MHz. R 2 values showed large deviations up to 60% from the mean value (11.5 s )1 at 600 MHz and 13.8 s )1 at 800 MHz). Such variations in R 2 values can result from relatively large-amplitude motions, efficient exchange processes, or shape anisotropy effects. In our experimental conditions, no significant increase in R 2 values was observed for MC1 between 600 and 800 MHz, indicating the absence of efficient exchange processes. We observed that R 2 values decreased sub- stantially for Gly17, Asp66, Lys69, Asn70, Arg71, and Ile89, whereas R 1 values increased, reflecting local Table 1. NMR constraints and structural statistics. NMR constraints Distance restraints Total NOE 1873 Unambiguous 1089 Ambiguous 784 Hydrogen bonds 37 Total dihedral angles F 69 W 69 RDC constraints 57 Structural statistics for the ensemble of the 15 lowest-energy structures Average violations per structure NOEs ‡ 0.5 A ˚ 0 Hydrogen bonds ‡ 0.5 A ˚ 0 Dihedrals ‡ 10° 0 RDC constraints rmsd (Hz) 0.75 Average pairwise rmsd (A ˚ ) Backbone atoms Heavy atoms a1, b1–5 (50 residues) 1.22 ± 0.26 1.85 ± 0.26 LP1 (10–14) 0.44 ± 0.17 1.15 ± 0.43 LP2 (22–24) 0.13 ± 0.05 1.14 ± 0.29 LP3 (35–42) 1.04 ± 0.32 1.51 ± 0.37 LP4 (50–53) 0.49 ± 0.22 1.45 ± 0.44 LP5 (67–77) 1.44 ± 0.57 2.48 ± 0.80 Average rmsd (A ˚ ) after fitting the secondary structure elements (a1, b1–5) as in Fig. 1 LP1 2.65 ± 1.02 3.37 ± 1.30 LP2 1.11 ± 0.46 1.92 ± 0.52 LP3 2.05 ± 0.69 2.46 ± 0.70 LP4 2.32 ± 0.96 2.72 ± 0.75 LP5 10.96 ± 5.00 11.21 ± 4.73 Ramachandran analysis Most favored region (%) 79.4 Allowed region (%) 19.6 Generously allowed (%) 0.7 Disallowed (%) 0.3 F. Paquet et al. NMR structure and backbone dynamics of MC1 FEBS Journal 277 (2010) 5133–5145 ª 2010 The Authors Journal compilation ª 2010 FEBS 5135 motions. Large variations of 15 N-NOE were observed along the sequence at 600 MHz, particularly for Thr3, Arg4, Gly17, Asp66–Ile79, Ile89, and Glu93, for which 15 N-NOE < 0.65; these residues clearly possess con- siderable internal motions on the nanosecond time scale. It is interesting to locate these residues in the structure: they belong to bulges B1 (Gly17) and B2 (Ile89), loop LP5 (Asp66, Ala67, Lys69, Asn70, Arg71, Ala73, Trp74, Met75, Glu77, Lys78, and Ile79), and the termini (Thr3, Arg4, and Glu93). Although the structure of the MC1ÆDNA complex has not yet been solved, relaxation measurements on the complex have been conducted (Fig. S3). Besides six Pro residues, resonance overlap precluded the interpre- tation of relaxation data for seven residues (Phe19, Arg25, Gly51, Asp66, Lys86, Ile89, and Glu90). LP1 LP3 C LP4 LP5 N LP2 β3 β5 180° β1 β2 β4 180° A B C Fig. 1. (A) Superimposition of the 15 low- est-energy structures fitted on the second- ary structure elements. (B) Ribbon diagram of the lowest-energy solution structure of MC1. (C) Solvent-accessible surface area of MC1 color-coded by surface charge (blue and red correspond to basic and acidic regions, respectively). AB C D C C N N N N C C C Fig. 2. Structures of some archaeal chroma- tin proteins other than the histones and Alba homologs. (A) Sac7d (Protein Data Bank ID code: 1AZP) and (B) Cren7 (Protein Data Bank ID code: 3LWI) specific to Sulfolobus (Crenarchaea). (C) MC1 (Protein Data Bank ID code: 2KHL) specific to Methanosarcina (Euryarchaea) and (D) HU monomer (Protein Data Bank ID code: 1P71) specific to Thermoplasma (Euryarchaea). NMR structure and backbone dynamics of MC1 F. Paquet et al. 5136 FEBS Journal 277 (2010) 5133–5145 ª 2010 The Authors Journal compilation ª 2010 FEBS Several residues belonging to arm LP5 (Ala67, Lys69, Asn70, Ala73, Met75, and Glu77) exhibit an increase in 15 N-NOE. If we compare the sites that exhibit back- bone chemical shift perturbations (CSPs) upon DNA binding with those that exhibit an increase in NOE upon DNA binding, we can conclude that the arm becomes much less mobile after binding with DNA (Fig. 4). This is reminiscent of the structure and dynamics of the highly mobile b-arms in the free pro- tein HU, which become much less mobile after binding with DNA. In the model proposed by Tanaka, the DNA-binding arms can move as rigid arms, creating sufficient room for accepting DNA [20]. The tips of the arms are highly flexible, and once the DNA has moved inwards, the arms close and the tips of the arms wrap around the DNA. The amplitudes and time scales of the intramolecular motions experienced by the protein backbone are com- monly determined from the 15 N-NMR relaxation data, by using the model-free approach suggested by Lipari and Szabo [21,22] and extended by Clore et al. [23]. This approach is applicable for the case of statistically independent overall tumbling and internal motions. In the case of MC1, large-amplitude internal motions on the same scale as global rotation are detected for at least 16 N–H vectors, ruling out use of the model-free formalism. MD analysis Consistent with experimental observations, the protein core was stable at 300 K during the MD simulation. The average backbone rmsd calculated with the sec- ondary structure atoms of the 2000 snapshots was 2.2 ± 0.1 A ˚ . Such deviations are characteristic of pro- tein simulations carried out in the presence of solvent [24,25]. The backbone rmsd calculated with all of the residue atoms starts at 5.8 A ˚ and increases up to 15 A ˚ during the 20 ns trajectory time, showing large motions of the loops and the arm (Fig. S4). Rotational diffusion Knowing the rotational diffusion tensor is essential for a detailed analysis of intramolecular motions in nonspherical proteins. When the shape of a molecule changes over time, its associated rotational diffusion tensor varies. The eigenvalues of the diffusion tensor 2.5 A B C β1 β2 β3α1 β4 β5 0.5 1.0 1.5 2.0 R 1 (s –1 )R 2 (s –1 )15 N-NOE 0.0 10 15 20 0 5 1 0 0.2 0.4 0.6 0.8 3 8 13 18 23 28 33 38 43 48 53 58 63 68 73 78 83 88 93 Sequence Fig. 3. Backbone 15 N-relaxation data for 1.6 m M free MC1 at 600 MHz. (A) Longitudi- nal relaxation rate. (B) Transverse relaxation rate. (C) Heteronuclear NOE. F. Paquet et al. NMR structure and backbone dynamics of MC1 FEBS Journal 277 (2010) 5133–5145 ª 2010 The Authors Journal compilation ª 2010 FEBS 5137 show variations of 25% on the nanosecond time scale during the 20 ns trajectory (Fig. 5). These variations, resulting from the position of the arm, are correlated with internal motions of the overall protein, as can be observed for the C a –C a or H N –H N distances between Arg71 and Leu92 (Fig. 6). The length of the arm Ala67–Glu77 showed high variation (10.4–16.8 A ˚ ), as indicated by the distance C a –C a between Val65 at the end of strand b4 and Arg71 at the extremity of arm LP5. This stretch was made up of complex motions in the arm, as shown by the variations in C a –C a dis- tances between Ala67–Arg71, Ala67–Lys78, Arg71– Val65, and Arg71–Ile79. The motion of the arm is cen- tered on a hinge composed of Ala67 and Glu77. More- over, loops LP1 and LP3 exhibited substantial conformational changes during the trajectory, as shown by variations in the C a –C a distances between Glu11–Asp43, Gly13–Leu92, and Gly35–Lys62. Dur- ing the trajectory, the location of loop LP1 changed in relation to strands b3 and b5, as indicated by varia- tions in the Glu11–Asp43 and Gly13–Leu92 distances. Internal correlation functions The internal autocorrelation functions are calculated within the molecular reference frame of the superposed structures. Figure 7A shows the time-correlation func- tions for three representative residues in different parts of MC1. The upper N–H vector (Gln26 in the helix) shows a rapid (< 10 ps) decay of C(t) from 1.0 to $ 0.9, arising from vibrational motion. This correla- tion function is typical for residues in relatively rigid parts of MC1, such as the a-helix and the b-strands, excluding bulges. The correlation functions for two residues, Val18 in a bulge and Asn70 in the arm, are also shown. The fast decay of the Val18 N–H vector (S f 2 = 0.85) is followed by a slow motion on a nano- second time scale with an order parameter, S 2 , of 0.55. The third C(t) of Asn70 is composed of three decays. The fast decay (< 10 ps) is followed by an intermedi- ate decay (100–500 ps) that primarily arises from libra- tional motion. This intermediate motion is common to residues belonging to loops LP1, LP3, LP4, and LP5, 180° 180° A B Fig. 4. (A) Residues that exhibit a significant increase in 15 N-NOE upon DNA binding are in blue, and those with an intermediate increase are in marine. (B) Residues that exhibit significant CSP upon DNA binding are in red, and those with intermediate changes are in orange. 1.9 1.3 1.5 1.7 D x (10 7 s –1 ) D y (10 7 s –1 ) D z (10 7 s –1 ) Time (ns) 1.6 1.8 1.2 1.4 2.8 3.0 3.2 2.4 2.6 0 2 8 10121416182046 Time (ns) 0 2 8 10121416182046 Time (ns) 0 2 8 10121416182046 Fig. 5. Fluctuations in the anisotropic rotational diffusion eigen- values D x , D y and D z along the 20 ns trajectory. NMR structure and backbone dynamics of MC1 F. Paquet et al. 5138 FEBS Journal 277 (2010) 5133–5145 ª 2010 The Authors Journal compilation ª 2010 FEBS in the last case with larger amplitude. Finally, a slower decay reaches a plateau value S 2 of 0.10 after 8.3 ns. This time value is close to the average harmonic mean correlation time of 8.6 ± 0.3 ns calculated using hydronmr during the trajectory. The MD-derived order parameters S 2 values for the b-strands and the a-helix are consistent with the exper- imental relaxation data (Fig. 7B). Slow motions were detected for Ser1, Asn2, Leu92, Glu93 (terminal resi- dues), Gly17 to Phe19 (bulge B1), Arg34 to Gly37 (loop LP3), Gly51 and Thr52 (loop LP4), Ala67 to Lys78 (loop LP5), and Glu87 to Ile89 (bulge B2). The largest amplitudes were observed for Ser36 (S 2 = 0.13) and from Ala67 to Asn70 (0.1 < S 2 < 0.16). The resi- dues involved in the two bulges have S 2 values around 0.6, which is consistent with the 15 N-NOE values. The calculated S 2 values in the loops are lower than expected, particularly in loops LP3 and LP4. A recent study provides evidence for a specific link between force field deficiencies and disagreement between experimental and MD order parameters [26]. MD sim- ulations using three MD force fields (comprising amber ff03) overestimate the flexibility of backbone N)H vectors at the borders of secondary structure and in loops. Specific inaccuracies in the treatment of hydrogen bonding could be responsible for increased flexibility in silico. In the case of MC1, the conforma- tional changes observed during the trajectory are consistent with the crosspeaks observed on the NOESY spectra. Low values of S 2 computed with the correlation functions indicated slow motions with large amplitude. However, these values can only be obtained with large uncertainties, as a trajectory of 20 ns allowed us to calculate a correlation function only over 10 ns. Achieving reliable correlation functions requires several repetitions of occurrences on the time scale of the trajectory. An isolated occurrence generates waves on the correlation function that have little significance, as seen for Val18. Correlated motions on the nanosecond time scale The trajectories of some dihedral angles and distances were examined in the two b-bulges and in the loops. For bulge B1, a hydrogen bond was alternately present 12 14 16 13–92 14 16 71–65 6 8 10 12 8 10 35–62 10 12 14 9 11 71–67 Distance ( Å ) Distance (Å) 4 6 8 12 14 11–43 5 7 12 14 71–79 Distance ( Å ) Distance (Å) 6 8 10 9 11 67–78 8 10 12 48 52 71–92 Distance ( Å ) Distance (Å) 3 5 7 40 44 0 5 10 15 20 0 5 10 15 20 Time (ns)Time (ns) Distance ( Å ) Distance (Å) 92 13 11 43 35 62 79 78 65 67 71 92 13 11 43 35 62 79 78 65 67 71 A B Fig. 6. (A) Some C a –C a distances (A ˚ ) along the 20 ns simulation trajectory. (B) Snapshots of MC1 after the equilibrium period at 0 and 17.5 ns. Black lines indicate distances between specific residues that show shape variations during the trajectory. F. Paquet et al. NMR structure and backbone dynamics of MC1 FEBS Journal 277 (2010) 5133–5145 ª 2010 The Authors Journal compilation ª 2010 FEBS 5139 between NH(Leu8) and CO(His16) for 9 ns, and between NH(Leu8) and CO(Val17) for 9.5 ns (Fig. 8A,C). Thus, at least one of the two hydrogen bonds was present for 18.5 ns on the 20 ns trajectory time. The transitions occurred at 1, 2, 10, 14, 15 and 19 ns, and are correlated with the motions of the dihe- dral angles w(His16), u(Gly17), and w(Val18). This flip-flop leads to slow internal motions with large amplitude, as seen for C(t) of NH(Gly17) and NH(Val18) (S 2 = 0.55). For bulge B2, a unique hydrogen bond between NH(Val57) and CO(Arg88) was present for 13 ns in periods of 2–4 ns (Fig. 8B,C). At the same time and for 7.5 ns, a water- mediated hydrogen bond between NH(Val57) and CO(Glu87) was formed when the distance was $ 6.4 A ˚ . The two strands were thus completely sepa- rated for 7 ns of the trajectory time, which could explain the greater flexibility of this bulge and the slow internal motions of Glu87, Arg88 and Ile89 with very large amplitude. These motions were correlated with the dihedral angle transitions of w(Glu87), u(Arg88), and u(Ile89). The presence of these hydrogen bonds was consistent with the homonuclear NOEs found in this region [15]. Loop LP1 was stabilized with two hydrogen bonds, NH(Asp10)–CO(Asn14) and NH(Gly13)–CO(Asp10), throughout the trajectory time. Similarly, the hydrogen bond NH(Gln26)–CO(Gln23) stabilized the short loop LP2 for 19 ns. The lack of stable hydrogen bonds in loop LP3 (35– 42) corresponds with large motions of the N)H vectors for Gly35, Ser36, and Gly37. However, this nonstructured loop is probably not important in the DNA binding, because the number of residues between Gly35 and Ile45 (MC1-CHTI55 numbering) varies from 3 to 14 in different species of Halobacteria and Methanomicrobia [15]. Loop LP4 (50–53) was stabilized by two hydrogen bonds, NH(Thr52)–CO(Glu49) and NH(Leu92)– CO(Lys53), binding the loop to strand b5 for 11 ns. Supplementary hydrogen bonds involving the side chains NH 2 (Arg48)–CO(Thr52) and OH(Thr52)– CO(Glu49) contribute to the stiffness of the structure for a short time. Moreover, two NOE crosspeaks, OH(Thr52)–NH(Thr52) and OH(Thr52)–NH(Glu49), were observed on the free protein MC1 NOESY spec- tra, owing to a slower exchange process with water. This could be explained by hydrogen bonds involving the hydroxyl proton of Thr52. In arm LP5 (67–77), Ala67, Lys69 and Asn70 have global S 2 values of $ 0.1, whereas the other resi- dues have S 2 values in the range 0.26–0.46. This A 1.0 Q26 0.4 0.6 0.8 C (t) V18 0.0 0.2 Time (ns) N70 0.6 0.8 1 B β1 β2 β3α1 β4 β5 0 0.2 0.4 0123 45 678910 1 6 11 16 21 26 31 36 41 46 51 56 61 66 71 76 81 86 91 S 2 Sequence Fig. 7. (A) Three representative internal cor- relation functions computed on a trajectory of 20 ns for Gln26 in the a-helix, Val18 in a bulge, and Asn70 in the arm. (B) Residue profile of the MD-derived S 2 . NMR structure and backbone dynamics of MC1 F. Paquet et al. 5140 FEBS Journal 277 (2010) 5133–5145 ª 2010 The Authors Journal compilation ª 2010 FEBS corresponds to a combination of slow motions of large amplitude. In accordance with this, only four hydrogen bonds were observed: NH(Lys71)–CO (Pro68), NH 2 (Lys71)–CO(Pro76), NH(Met75)–CO (Pro72), and NH(Trp74)–CO(Pro72) for 6.5, 11, 16 and 0.3 ns respectively. Summary In summary, the structure of MC1, consisting of a pseudobarrel with an extension of the b-sheet (b4–b5) forming an arm of 11 residues, has been refined. The global fold is now compatible with the biochemical data and a DNA-binding site covering at least 15 bp. The structure features five loops that show a large distribution in the ensemble of 3D structures. Evidence for the fact that this distribution signifies internal mobility on the nanosecond time scale is provided by using 15 N-relaxation and MD simulations. These local conformational changes in MC1 could facilitate DNA binding, with two areas of contact separated by nearly 10 bp. Moreover, the flexibility of MC1 builds up con- formations with large positively charged areas that are highly favorable for binding with the phosphate groups of nucleotides. Some residues belonging to arm LP5 (Ala67, Lys69, Asn70, Ala73, and Met75) and to bulge B2 (Ile89) are involved in motions on the nanosecond time scale, and could be related to the interaction with DNA. A study of a DNAÆ MC1 com- plex is currently underway. 200 300 400 Psi angle (deg) Psi angle (deg) H16 50 150 E87 B A 100 –50 0 100 200 300 Phi angle (deg) Phi angle (deg)Phi angle (deg) G17 50 150 250 R88 100 200 300 Psi angle (deg) V18 50 100 150 200 I89 7.0 NH8 - CO16 8.0 NH57 - CO87 1.0 3.0 5.0 Distance (Å) Distance (Å) Distance (Å) Distance (Å) 5.0 7.0 NH8 - CO17 2.0 4.0 6.0 5.0 NH57 - CO88 1.0 3.0 1.0 3.0 0 2 4 6 8 101214161820 0 2 4 6 8 10 12 14 16 18 20 Time (ns) Time (ns) C Fig. 8. Dihedral angle and distance transi- tions as a function of time of the two bulges. (A) Bulge Leu8, His16 and Gly17. (B) Bulge Val57, Glu87 and Arg88. (C) Schemes of the bulges. The dotted lines and the time characterized the presence of hydrogen bonding during the trajectory. F. Paquet et al. NMR structure and backbone dynamics of MC1 FEBS Journal 277 (2010) 5133–5145 ª 2010 The Authors Journal compilation ª 2010 FEBS 5141 Experimental procedures Preparation of 13 C, 15 N-labeled MC1 The proteins were expressed in BL-21(DE3) cells trans- formed with the pET24a–mc1 plasmid. Protein doubly labeled with 15 N and 13 C was obtained by using an iso- tope-enriched Celtone-rich medium (Martek Biosciences, Columbia, MD, USA). To obtain 15 N-labeled protein, cells were first grown in LB medium, and then, at a D 600 nm of 0.7, they were collected and resuspended in M9 medium containing 15 NH 4 Cl [27]. In both cases, expression of the protein was performed for 2 h after addition of 0.1 mm isopropyl thio-b-d-galactoside. Purifi- cation of the proteins was performed by SP-Sepharose (GE Healthcare Europe GmbH, Orsay, France) chroma- tography followed by Ultrogel AcA 54 chromatography. The concentration of protein was determined by absorp- tion spectrophotometry, with a molecular absorbance coefficient of 11 000 m )1 Æcm )1 at 280 nm. The NMR protein sample was prepared by concentrating MC1 to 1.6 mm (100 mm acetate buffer, pH 5.1, 800 mm NaCl, 1 mm EDTA, 10% D 2 O). In order to check the pos- sible presence of MC1 oligomers, 15 N-HSQC spectra were obtained with different concentrations of MC1 in the same buffer conditions. Decreasing the protein concentration by a factor of 20 (1.6 mm to 0.08 mm) showed no significant variation in the 1 H and 15 N chemical shifts. The DNA oligonucleotides used for NMR were purchased from Eurogentec (Lie ` ge, Belgium) (OliGold oligonucleotides quality). The single-stranded 15 bp oligodeoxynucleotides were characterized by NMR and annealed at a 1 : 1 ratio. The MC1ÆDNA complex was prepared by slowly adding the 7.5 mm DNA duplex solution (10 mm phosphate buffer, pH 6, 100 mm NaCl, 1 mm EDTA, 10% D 2 O) to the 1.6 mm protein solution (10 mm phosphate buffer, pH 6, 100 mm NaCl, 1 mm EDTA, 10% D 2 O) to give a final complex concentration of $ 1mm. NMR spectroscopy and structure calculations Two-dimensional and three-dimensional NMR experiments were performed on a 600 MHz Varian UNITY INOVA spec- trometer at 299 K. Spectra were processed with nmrpipe [28], and analyzed with nmrview [29]. Backbone and side chain resonance assignments were obtained from the stan- dard triple resonance experiments [30]. 4,4-dimethyl-4-sila- pentane-1-sulfonic acid was used as a 13 C chemical shift reference. Interproton distances were derived from NOESY datasets obtained at mixing times of 100, 150 and 200 ms. Backbone dihedral angle restraints were determined with the talos program [31]. 1 D NH RDCs were measured by using 2D InPhase AntiPhase 1 H– 15 N-HSQC experiments in radially compressed 7% polyacrylamide gel (6.0–4.2 mm) [32,33]. Structures were calculated with NOE distance, hydrogen bond, u and W angle and RDC constraints, using aria2 (version 2.2) [34]. The aria2 protocol (cns 1.1) used simu- lated annealing with torsion angle and Cartesian space dynamics with the default parameters. RDC restraints within the aria2 protocol were incorporated at the last iter- ation with the correct parameters (D a = 15.21 and R = D r ⁄ D a = 0.19). RDC restraints were fitted and ana- lyzed with the module program [35]. Fifteen structures from six independent runs were selected on the basis of total energies and restraint violation statistics, to represent the structure of MC1 in solution. The electrostatic potential was calculated by using the pdb2pqr server (version 1.6) [36] and apbs software [37]. The figures were prepared with pymol [38] or molmol [39]. Determination and analysis of 15 N-relaxation parameters (R 1 , R 2 , and NOE) for MC1 NMR relaxation experiments were measured at 299 K on a Varian 500 MHz (NOE), Varian INOVA 600 MHz (NOE, R 1 and R 2 ) and Varian INOVA 800 MHz (R 1 and R 2 ) equipped with a cryogenic triple resonance probe spec- trometer. On each instrument, 15 N R 1 and R 2 spectra were acquired with 32 scans per t1 point, with a recycle delay of 3.0 s. R 1 relaxation delays of 10, 100, 200, 380, 500, 750, 1000 and 1300 ms were used for data collection. R 2 relaxation delays of 10, 20, 30, 50, 70, 90, 150, 210 and 310 ms were used for data collection at 600 MHz, and R 2 relaxation delays of 10, 20, 30, 50, 70, 90, 110 and 150 ms were used for data collection at 800 MHz. The errors in R 1 and R 2 were determined by generating random distri- butions of the measured volume V within the V ± DV range and by repeating the fit with this procedure 1000 times. The 15 N-NOE spectra were collected at 500 and 600 MHz with a 3 s presaturation period and a 2 s relaxa- tion delay; the reference experiment had an equivalent 5 s delay. The 1 H– 15 N heteronuclear NOE was calculated from the equation NOE = I sat ⁄ I eq , where I sat and I eq were the volumes of a crosspeak in the spectra collected with and without proton saturation. Both were acquired with 64 scans. All experiments were run twice in the same con- ditions. Volumes for the amide 15 N– 1 H crosspeaks were measured by using nmrview software [29]. Uncertainties in the volumes were measured from the duplicate spectra. After obtaining volumes of crosspeaks and their errors, the above time series were fitted from a single exponential decay function. Relaxation experiments for the MC1ÆDNA complex were performed at 600 MHz as described above, with R 1 relaxa- tion delays of 10, 100, 200, 300, 500, 800, 1000 and 1300 ms and R 2 relaxation delays of 10, 30, 50, 70, 90, 110, 130 and 150 ms at 299 K. Backbone CSPs and 15 N-NOE changes upon DNA bind- ing were analyzed with samplex [40]. NMR structure and backbone dynamics of MC1 F. Paquet et al. 5142 FEBS Journal 277 (2010) 5133–5145 ª 2010 The Authors Journal compilation ª 2010 FEBS [...]... hydrodynamic theory, the program hydronmr [42] computes the eigenvalues of the anisotropic rotational diffusion tensor The harmonic mean correlation time is calculated from the five rotational relaxation times Internal correlation function To gain insights into the nature of the dynamics, the autocorrelation functions C(t) were evaluated during the first NMR structure and backbone dynamics of MC1 10 ns, using the. .. total of 2000 snapshots with a time increment of 10 ps were analyzed from the final 20 ns of the MD simulation Prior to this, overall translational and reorientational motions were removed by a least squares superposition of the secondary structure backbone atoms of each snapshot on those of the mean snapshot The mean structure is taken as the most central structure among the simulated ones, and corresponds... of the residue atoms during the 20 ns trajectory time, showing large motions of the loops and the arm The structures obtained during the trajectory were aligned by least squares fitting of the secondary structure backbone atoms before evaluation of the rmsd Table S1 15N, 1H and 13C chemical shifts (p.p.m.) for MC1 (1.6 mm) in 800 mm NaCl and 100 mm acetate buffer at pH 5.1 and 26 °C This supplementary... P2[x] is the second Legendre polynomial, and l is the N–H bond vector scaled to unit magnitude When the internal correlation function is made up of three decreasing exponentials, the expression of the internal correlation function is: CðtÞ ¼ S2 þ Af eðÀt=sf Þ þ Am eðÀt=sm Þ þ As eðÀt=ss Þ with Af, Am and As are the amplitudes, and sf, sm andss are the correlation times of the fast, medium and slow... (constant number of atoms, volume, and temperature), during which the temperature was progressively increased from 0 to 300 K, the positions of protein atoms and of counterions being restrained Then, over a period of 200 ps at 300 K, the restraints were progressively removed Equilibration of the system in the NPT ensemble (constant number of atoms, pressure, and temperature) was then performed for 1500 ps... terial chromosomal protein MC1 reveals a new protein fold Biochemistry 43, 14971–14978 Bure C, Goffinont S, Delmas AF, Cadene M & Culard F (2008) Oxidation-sensitive residues mediate the DNA bending abilities of the architectural MC1 protein J Mol Biol 376, 120–130 Zhang Z, Gong Y, Guo L, Jiang T & Huang L (2010) Structural insights into the interaction of the crenarchaeal chromatin protein Cren7 with... time for the 93 NH vectors The autocorrelation function, C(t), of the overall dynamic process is the product of the global, C0(t), and the internal, Ci(t), correlation functions [21–23] For MD simulations where the global motion is eliminated, the autocorrelation function, C(t), is equal to the internal correlation function, Ci(t) Internal correlation functions were then calculated according to the equation:... central structure among the simulated ones, and corresponds to the one at 12.01 ns in the present work During the analysis, the rmsd between two structures was evaluated as:   X 1 2 1 ðr1 À r2 Þ2 rmsd ¼ N where N is the number of atoms taken into consideration, r1 and r2 are the position vectors of an atom in both structures, respectively, and the summation is performed over N atoms Rotational diffusion... perturbed and unperturbed regions of proteins and complexes BMC Bioinformatics 11, 51–58 NMR structure and backbone dynamics of MC1 41 Case DA, Darden TE, Cheatham TE III, Simmerling CL, Wang J, Duke RE, Luo R, Merz KM, Pearlman DA, Crowley M et al (2006) AMBER 9 University of California, San Francisco 42 Garcia de la Torre J, Huertas ML & Carrasco B (2000) HYDRONMR: prediction of NMR relaxation of globular... reduced growth and genomic transcription Mol Microbiol 67, 662–671 9 Chartier F, Laine B & Sautiere P (1988) Characterization of the chromosomal protein MC1 from the thermophilic archaebacterium Methanosarcina sp CHTI 55 and its effect on the thermal stability of DNA Biochim Biophys Acta 951, 149–156 10 Paradinas C, Gervais A, Maurizot JC & Culard F (1998) Structure- specific binding recognition of a methanogen . between 9 bp of DNA and the b-ribbon arms, and variable contacts between additional DNA and the body of the protein [19]. In the case of MC1 DNA complexes,. highly flexible, and once the DNA has moved inwards, the arms close and the tips of the arms wrap around the DNA. The amplitudes and time scales of the intramolecular motions