Báo cáo khoa học: Solution structure and internal dynamics of NSCP, a compact calcium-binding protein doc

Solution structure and internal dynamics of NSCP, a compact calcium-binding protein Ghada Rabah1, Razvan Popescu1, Jos A Cox2, Yves Engelborghs3 and Constantin T Craescu1 INSERM & Institut Curie, Centre Universitaire, Orsay, France ´ ´ ` Departement de Biochimie, Universite de Geneve, Suisse Katholieke Universiteit, Leuven, Belgium Keywords calcium-binding proteins; molecular dynamics; NMR; nuclear relaxation; solution structure Correspondence C T Craescu, INSERM & Institut Curieˆ Recherche, Centre Universitaire, Batiments 110–112, 91405 Orsay, France Fax: +33 69 07 53 27 Tel: +33 69 86 31 63 E-mail: Gil.Craescu@curie.u-psud.fr (Received 15 December 2004, revised 14 February 2005, accepted 24 February 2005) doi:10.1111/j.1742-4658.2005.04629.x The solution structure of Nereis diversicolor sarcoplasmic calcium-binding protein (NSCP) in the calcium-bound form was determined by NMR spectroscopy, distance geometry and simulated annealing Based on 1859 NOE restraints and 262 angular restraints, 17 structures were generated with a ˚ rmsd of 0.87 A from the mean structure The solution structure, which is highly similar to the structure obtained by X-ray crystallography, includes two open EF-hand domains, which are in close contact through their hydrophobic surfaces The internal dynamics of the protein backbone were determined by studying amide hydrogen ⁄ deuterium exchange rates and 15N nuclear relaxation The two methods revealed a highly compact and rigid structure, with greatly restricted mobility at the two termini For most of the amide protons, the free energy of exchange-compatible structural opening is similar to the free energy of structural stability, suggesting that isotope exchange of these protons takes place through global unfolding of the protein Enhanced conformational flexibility was noted in the unoccupied Ca2+-binding site II, as well as the neighbouring helices Analysis of the experimental nuclear relaxation and the molecular dynamics simulations give very similar profiles for the backbone generalized order parameter (S2), a parameter related to the amplitude of fast (picosecond to nanosecond) movements of NH-H vectors We also noted a significant correlation between this parameter, the exchange rate, and the crystallographic B factor along the sequence Calcium is a universal cellular secondary messenger involved in the regulation of a large variety of vital processes from the fertilization and development of a cell to its death [1] Its great versatility is mainly due to multiple specific interactions within various molecular networks, the precise response being finally determined by the local ion concentration as well as its time and spatial evolution Some proteins (such as enzymes and chaperones) bind Ca2+ and change their own biological activity, whereas others convey the Ca2+ signal through a functional change in another (target) molecule Calmodulin and troponin C, the best studied members of the latter category, belong to the EF-hand superfamily, which is characterized by a well-conserved Ca2+-binding motif (helix–loop–helix) In addition to the mediator, or sensor-type activity, proteins of this family may also interact with the metal ion for buffering, uptake, or transport purposes [2] The ion-binding parameters (affinity, binding kinetics, selectivity, cooperativity) and the structural response on ion binding are strongly related to the biological role played by the protein Thus, Ca2+ buffers (e.g parvalbumin, calbindin D9k) generally have a high affinity (Kd < 10)7 m), may bind Mg2+ equally well, and are conformationally less sensitive to ion binding In contrast, calcium sensors have a lower affinity (Kd ¼ 10)5 to 10)7 m) Abbreviations MD, molecular dynamics; NSCP, Nereis diversicolor sarcoplasmic calcium-binding protein; SCP, sarcoplasmic calcium-binding protein 2022 FEBS Journal 272 (2005) 2022–2036 ª 2005 FEBS G Rabah et al and undergo important ion-induced conformational changes Generally, the precise mechanism underlying the biological function of a protein is determined by its structural and dynamic properties which, in turn, depend on the gene-encoded amino-acid sequence A large number of EF-hand proteins are currently known [3] However, despite global sequence and structural similarities, each member has specific functional properties, acquired during a long evolution period A comparative analysis of a large diversity of sequences and biological functions within the EF-hand family is therefore required to better understand the structural basis of the various biological functions Nereis diversicolor sarcoplasmic calcium-binding protein (NSCP) is an acidic calcium buffer protein, which is very abundant in the sarcoplasmic reticulum from the annelid N diversicolor It belongs to the sarcoplasmic calcium-binding protein (SCP) subfamily, also including the invertebrate functional analogs of the vertebrate parvalbumin [4] Similar to other SCPs, NSCP has four potential ion-binding sites, but only three of them (sites I, III and IV) have a high affinity for Ca2+ or Mg2+ [5] ˚ The 3D crystal structure, solved at A resolution [6], revealed a globular structure in which the two EF-hand pairs, constituting an EF-hand domain, are close to each other, in contrast with the bi-lobal, extended conformation of calmodulin or troponin C Spatial proximity of the binding sites makes functional communication between them possible, measured in terms of a strong positive co-operativity in Ca2+ binding [7] In addition, physicochemical experiments performed in our laboratory revealed that metal binding induces transition from a molten globule state into a well-defined, and highly stable conformation [8,9] A rational understanding of these specific properties requires structure and dynamic characterization in solution of the various functionally relevant states To achieve this aim, we initiated a structural and dynamics analysis of Ca2+-saturated NSCP in solution, based on NMR spectroscopy, nuclear relaxation measurements, and molecular dynamics (MD) simulations Using purified wild-type samples, as well as 15 N-labeled samples overexpressed in Escherichia coli, we have assigned the 1H and 15N resonances of the protein (BioMagResBank, accession number 4129) [10], and collected distance and angle restraints for structural determination The solution structure, based on 1859 NOE distance restraints and additional experimental and chemical information, is very similar to the previously reported crystallographic structure [6] In order to better understand the great structural stability FEBS Journal 272 (2005) 2022–2036 ª 2005 FEBS Structure and dynamics of NSCP in solution of the holo form of NSCP, we investigated internal MD using both experimental (hydrogen ⁄ deuterium exchange kinetics, nuclear relaxation) and theoretical (MD simulation) approaches The results reveal the NSCP protein as a compact molecule, with restricted dynamics in the picosecond to nanosecond time scale and no important exchange movements at the microsecond or slower scales The rapid internal dynamics are satisfactorily described by the MD simulation, which enabled us to calculate backbone order parameter profiles that were in close agreement with the experimental parameters Results and Discussion Solution structure We have shown previously that apo-NSCP in solution is highly disordered and gives poor NMR spectra, characterized by a low chemical shift dispersion and absence of NOE interactions [8] In the presence of saturating Ca2+ ions, the spectra extend over 12 p.p.m in the proton dimension and exhibit many interproton dipolar interactions, and the sample is stable enough for long lasting 3D experiments Spin systems for 171 of 174 residues (% 98%) were partially or completely assigned [10] Unassigned resonances correspond to a peptide fragment situated at the C-terminus of the protein (D163–T165) Combined analysis of an ensemble of NMR parameters in the Ca2+-bound state, including short-range and medium-range NOE interactions, Ha secondary chemical shifts, 3JHNHa coupling constants, and amide proton exchange rates, enabled us to delineate eight a-helices consisting of fragments 3–15, 25–38, 45–59, 72–82, 90–103, 113–122, 130–137 and 147–159, representing 56% of the residues The position of the helices corresponds closely to those observed in the crystal structure [6], but the helices are often shorter by 1–3 residues Four short b-strands (22–24, 69–71, 110–112, 144–146) were also identified by the low-field-shifted Ha protons, strong daN(i,i+1) sequential connectivities and large 3JHNHa couplings The strands could be grouped into two antiparallel b-sheets based on the strong dipolar interactions observed between Ha and HN protons from opposite chains A total of 1859 interproton distance restraints and 262 dihedral angle restraints were used to fold these secondary-structure elements into a 3D conformation, using distance geometry and simulated annealing computations Figure 1A shows the backbone superimposition of the final 17 structures, and Fig 1B the ribbon representation of the best representative of the ensemble for 2023 Structure and dynamics of NSCP in solution A B C Fig Global representation of the solution structure of Ca2+-NSCP (A) Backbone stereo view of the 17 final structures superimposed based on the main-chain heavy atoms in regular secondary-structure elements The N-terminal EF-hand domain is shown in red, the C-terminal EF-hand domain is shown in blue, the linker is green, and the C-terminal fragment is in magenta (B) Ribbon representation of the best structure selected as the closest to the ensemble average The color code is the same as in (A) The binding loops are noted from I to IV, and the a-helices and some residue positions are labeled to facilitate the chain-folding pathway The figure was prepared with MOLSCRIPT [55] and RASTER3D [56] (C) Electrostatic potential calculated at the molecular surface of the best structure of Ca2+-NSCP using the GRASP program [57] On the left side the molecule is oriented as in (B), whereas on the right side it is rotated through 180° around the vertical axis The positive and negative potential are conventionally coded in blue and red, respectively the Ca2+-bound NSCP form In contrast with the bilobal aspect of calmodulin, the prototype of the EFhand superfamily, NSCP exhibits a globular shape characterized by a large contact area and multiple side-chain contacts between the two EF-hand domains Consequently, residues from the two terminal fragments are very close to each other The structural 2024 G Rabah et al cohesion of the two EF-hand domains is mainly determined by a highly hydrophobic core including 15 Phe and three Trp side chains contributed roughly equally by the two molecular halves (Fig 2) The structure of individual EF-hand domains is close to the canonical geometry [11], observed for the family of proteins analyzed so far The aqua and procheck-nmr programs [12] were used to assess the quality of the restraints and to determine the geometry regularity of the final structures (Table 1) More than 87% of (F, Y) angle pairs of the 17 final structures lie in the most-favored region of the Ramachandran plot, and about 99% of them lie in the allowed regions The segment 162–165 is mostly found in the disallowed regions of the Ramachandran plot These loop residues are not engaged in a detectable hydrogen bond and presumably undergo some geometry constraint from the neighboring amino acids The global solution structure of NSCP is very close to the previously determined crystal structure [6] The rmsd calculated for the heavy atoms in regular secon˚ dary-structure elements is 1.14 A, with a large asym˚ ˚ metry between the two halves: 1.05 A and 0.77 A for the N-terminal and C-terminal halves, respectively This may partially reflect the lack of metal binding in the second loop and the longer linker region between EF-hand I and II As in aequorin [13], these features may result in increased flexibility of the first half It was generally observed that the relative position of the two helices in EF-hand motifs changes significantly upon Ca2+ binding [14], from an almost antiparallel configuration to a perpendicular arrangement In a domain containing a pair of EF-hand motifs, this movement creates a large exposed hydrophobic surface which, in the case of regulatory proteins, constitutes the target binding site The values of the interhelix angles in NSCP, measured over the NMR ensemble (Table 2), are similar to those observed in regulatory EF-hand proteins, in the Ca2+-bound state, and are centered around 90° Compared with the calmodulin, where the two lobes are spatially separated, the compact SCPs exhibit a larger variability among the interhelix motifs, with significantly lower values for the first two motifs Indeed, these two EF-hand domains in SCPs are more open, than in other Ca2+ buffer proteins, such as calbindin D9k [15] or parvalbumin [16] These differences may be due to the compact structure and the tight interactions between the two EF-hand domains, inducing supplementary constraints on the interhelical angles As can be seen in Table 2, some binding loops lost the high affinity for the metal ion, but this still mainFEBS Journal 272 (2005) 2022–2036 ª 2005 FEBS G Rabah et al Structure and dynamics of NSCP in solution Fig Stereoview of the aromatic residue cluster in Ca2+-NSCP Phe, Trp and Tyr side chains are shown in red, blue and green, respectively Table Experimental restraints and structural statistics for the 17 simulated annealing structures of (Ca2+)3-NSCP Restraint statistics NOE restraints Intraresidue Sequential Medium range (2 £ |i-j| < 5) Long range (|i-j| ‡ 5) Hydrogen bond restraints Dihedral angle restraints (F,Y) Average no of NOE restraint violations ˚ > 0.5 A ˚ > 0.4 A ˚ > 0.3 A ˚ > 0.2 A 1859 491 559 342 467 188 262 Angle (°) 26.4% 30.1% 18.4% 25.1% None 0.12 ⁄ molecule 0.35 ⁄ molecule 2.35 ⁄ molecule ˚ 0.0045 A ˚ 0.0009 A Average of NOE upper restraint violations Averahe of NOE lower restraint violations ˚ Average rmsd from the average structure (A) Residues 3–15, 22–37, 46–57, 69–82, 0.87 ± 0.13 89–102, 110–122, 129–137, 144–159a Residues 1–174a 1.57 ± 0.16 Ensemble Ramachandran plot Residues in the most-favored region 87.1% Residues in additional allowed regions 10.8% Residues in generously allowed regions 1.4% Residues in disallowed regions 0.8% a Backbone atoms (N, C¢, Ca) tains the corresponding motif in an open conformation The presence of Ca2+ in the three active EF-hand motifs is nonambiguously confirmed by the chemical-shift signature of the Hb protons in the Asp residue occupying the first position in the binding loop D1 [17] Indeed, the close proximity to the conserved Phe residue in position )4 (relative to D1) accounts for FEBS Journal 272 (2005) 2022–2036 ª 2005 FEBS Table Statistics of the interhelix angles within EF-hand motifs in NSCP, related EF-hand proteins and calmodulin An asterisk marks EF-hand motif that had lost Ca2+-binding capacity Helix pair NSCPa (NMR) NSCPb (X-ray) BlSCPc (X-ray) SeCaBPd (NMR) Calmoduline (X-ray) A⁄B C⁄D E⁄F G⁄H 67 69 108 101 60 84* 110 100 59 82 111 96* 78 101* 107 113 89 89 101 95 ± ± ± ± 6* a This work, mean ± SD over the 17 molecule ensemble b [6], 2SCP.pdb c B lanceolatum SCP [53], 2SAS.pdb d Bacterial EF-hand protein, calerythrin [54], 1NYA.pdb e [20], 1CLL.pdb the large upfield shift of one of the b protons in D1: 1.58, 1.73, and 1.97 p.p.m in EF-hand motif I, III, and IV, respectively The chemical shift of the amide nitrogen in the residue occupying position of the Ca2+ loop was shown to be larger in the Ca2+-bound form (124–127 p.p.m.), relative to the apo form (111–120 p.p.m.) of EF-hand proteins [18], because of a decreased electronic density around the amide nitrogen nucleus This parameter was therefore proposed as a sensitive probe for the metal occupancy of a given motif In NSCP(Ca2+)3 the amide 15N chemical shift at the corresponding positions (I23, I70, I111, L145) are 124.7, 122.0, 123.1 and 120.8 p.p.m., respectively, with the metal-bound motif IV showing a smaller value than the empty site II According to the above classification, only the first site shows a chemical shift compatible with a bound loop These observations suggest that the nitrogen elec2025 Structure and dynamics of NSCP in solution tron density is not the unique determining factor for its chemical shift, and question the utilization of this NMR parameter as a probe for the metal binding to a given site The electrostatic potential at the protein surface is dominantly negative (Fig 1C), in agreement with the acidic character of the EF-hand proteins It may be noted that the surface area encompassing the Ca2+binding sites III and IV exhibits a more homogeneous and intense negative potential, as compared with the corresponding area of the first two binding loops This may contribute to the low cation-binding affinity of site II The question may be raised as to how the sequence and structure of EF-hand proteins, classified as calcium buffers or transporters, account for the lack of regulatory capacity, essentially expressed by a Ca2+modulated interaction with cellular targets Structural analysis of calmodulin complexes revealed that the interaction interface is constituted by a large hydrophobic surface created by the opening of the two EF-hand domains on Ca2+ binding In the case of SCPs, the highly apolar surface of the two EF-hand domains exhibit a greater affinity for each other [19], yielding a compact globular fold, which precludes the recognition and binding to the hydrophobic surface of the target proteins This is made possible by extensive bending of the interdomain linker, which is usually found to be almost linear in the crystal structure of calmodulin [20] However, a recent crystallographic study showed that calmodulin can equally form a compact structure [21], as suggested by previous biophysical and biochemical studies in solution [22,23] In fact, the D ⁄ E interhelix angle, calculated as in [14], is very close in the compact calmodulin (1PRW.pdb) and NSCP (solution structure): )111° and )116°, respectively Sequence and structural comparison between calmodulin and three different compact EF-hand proteins (NSCP, Botrychium lanceolatum SCP and Saccharopolyspora erythraea calcium-binding protein) may reveal some factors contributing to the preference for the globular shape A Pro residue between the D and E helices, which exists only in NSCP, could induce a tight turn in this region and render the compact structure more stable More significantly, the number of long-chain hydrophobic residues is distinctly higher in the domains that associate to form compact structures Thus, the total number of Phe, Trp, Leu and Ile residues is 25 in calmodulin and 38, 37, and 35 in NSCP, B lanceolatum SCP and S erythraea calcium-binding protein, respectively Therefore, the collapse of the two EF-hand halves, with the formation of a more stable apolar core (Fig 2), is preferred over the extended, 2026 G Rabah et al highly solvent-exposed structure In addition, a 9–10 residue insertion in the C-helix of NSCP, B lanceolatum SCP and S erythraea calcium-binding protein, including five hydrophobic side chains, ensures a larger and tighter contact surface between the N-terminal and C-terminal domains (Fig 1B) This tendency to form a very stable hydrophobic core is very well illustrated by the fact that the isolated N-terminal and C-terminal halves of NSCP form homodimers, but when mixed, they form a complex with a conformation as of intact NSCP [19] A more detailed structural and thermodynamic investigation of the interdomain interface in the compact EF-hand proteins should be very useful for a quantitative explanation of the conformational preference Conformational flexibility studied by amide exchange kinetics Analysis of the amide exchange kinetics provides a site-specific description of global or local conformational dynamics of a protein in solution Description of the exchange process in terms of protection factors [24] enables us to eliminate the influence of the solution properties (pH, ionic strength, temperature, etc.), and of the chemical environment of amide groups (the sequence context) Therefore, the protection factors may be directly related to the relative attenuation of the hydrogen exchange rate in given main-chain positions of the native structure, relative to the randomcoil state We were able to quantitate this parameter for 72 out of the total of 169 amide protons and for three indole amino groups from Trp side chains Five missing values corresponding to the amide protons with high exchange rates (kex > 10)2 min)1), K19, F35, L49, M122, and V168, are indicated by the downward arrows in Fig The intensity of the remaining peaks could not be accurately measured due to overlap in the HSQC spectra Most of the measured protection factors have relatively high values (mean % 106.5), and are associated with the a-helices and b-strands, except for the empty binding motif II (Fig 3A) The strong protection of the amide protons in Ca2+-saturated NSCP is in good agreement with its high structural stability [9] Thus, the free energy of conformational opening estimated here from hydrogen isotopic exchange measurements are centered around 37.7 kJỈmol)1 (9 kcalỈmol)1) for the three bound EF-hands, which is close to the free energy of the structural stability calculated from denaturation experiments [9] This strongly suggests that the conformational fluctuations, enabling the measured proton exchange, correspond to global dynamics, and FEBS Journal 272 (2005) 2022–2036 ª 2005 FEBS G Rabah et al Structure and dynamics of NSCP in solution Fig Dynamics analysis by amide proton exchange kinetics of Ca2+-NSCP (Top) H ⁄ 2H exchange kinetics expressed as the logarithm of the protection factor [log (P)] and the free energy of isotope exchange (DGex) The mean log (P) is indicated by the horizontal line Downward arrows designate the amino protons with exchange kinetics faster than 10)2 min)1 The exchange parameter of the Trp indole protons (W4, W57 and W170) are shown at the end of the sequence, by the grey bars The secondary-structure elements and occupation of the binding loops are represented schematically at the top of the figure (Bottom) crystallographic B factors of NH atoms determined by the X-ray approach [6] are highly similar to those accompanying the co-operative unfolding of the whole structure Clearly, the number of measurable amide protection factors and their magnitude are smaller for the second, unbound EF-hand motif, but also for the neighboring helices (B and E) The unbound loop induces more rapid conformational fluctuations that extend outside the motif, in both directions of the main chain, as also reflected in the crystallographic B factors of the N atoms (Fig 3) The side-chain Trp protons exhibit remarkably high protection factors, comparable to the backbone amide protons in the more flexible helices and in the Cterminal fragment (Fig 3) Among the three Trp residues, the Ne1 proton of Trp4 belongs to a hydrogen bond with the carbonyl oxygen of Phe158, observed both in the NMR and the previous X-ray structure [6] Owing to the deshielding effect of this interaction, the proton chemical shift is significantly low-field-shifted (10.52 p.p.m.) relative to the random coil value (10.22 p.p.m.) The corresponding proton in Trp170 may form an aromatic hydrogen bond with the side chain of Phe157, as suggested by the structure, and suppor- FEBS Journal 272 (2005) 2022–2036 ª 2005 FEBS ted by the large high-field shift of its proton resonance (7.28 p.p.m.) induced by the phenyl ring current The low exchange rate of the indole protons in these two residues may be explained by the protection provided by the hydrogen-bond formation In contrast, no explanation is actually available for the exchange protection in Trp57, which is largely exposed to the solvent, and shows no detectable intramolecular hydrogen bond Internal MD studied by relaxation measurements Computation of the principal components of the inertia momentum, based on the NMR-derived solution structure, gives (1.00 : 0.83 : 0.66) According to these values, only a modest degree of anisotropy is expected, which may not influence significantly the microdynamic parameters (at least the order parameters) [25] Owing to cross-peak overlap, reliable analysis of peak intensities and relaxation parameters was limited to 121 HN-N vectors, including 118 amide groups (out of the total of 169 observable amide protons) and three indole amino groups from the Trp side chains 2027 Structure and dynamics of NSCP in solution Figure shows the relaxation parameters (R1, R2, NOE) and their uncertainty plotted as a function of residue number The dynamic analysis started with the determination of the rotational correlation time (sc) of the whole protein using a procedure designed to minimize the effects of heterogeneous local movements [26] For a given arbitrary value of sc, the dynamic parameters (S2 and se) are computed using R1 and heteronuclear NOE for each amide vector within the most rigid segments (84 sites) in the frame of the Lipari-Szabo approach [27] Then, R2 values may be reconstructed (from the spectral density functions) and compared with the experimental counterparts for the selected sites The final value of the correlation time, corresponding to the minimum of G Rabah et al the v2(R2) function, was 6.93 nsỈrad)1 Using a simpler method based on the independence of the R2 ⁄ R1 ratio from S2 and se [28], we obtained a very close value for sr (6.86 ±0.34 ns) The value of the rotational correlation time is indicative of a mainly monomeric form of NSCP With the value for the global correlation time obtained by the first method, we analyzed the relaxation parameters in terms of the simple or extended Lipari-Szabo model-free methods [27,29] A MonteCarlo simulation with 500 steps was used to estimate the standard error of the microdynamic parameters The data for all the studied vectors (except amides in A32 and S112) could be fitted to the simple LipariSzabo procedure giving the generalized order param- Fig Relaxation parameters (R1, R2, g) measured in Ca2+-saturated NSCP at 308 K The elements of secondary structure and the occupancy of Ca2+-binding sites are shown at the top of the first panel The last three values correspond to the Ne1-H vector in Trp side chains (W4, W57, W170) 2028 FEBS Journal 272 (2005) 2022–2036 ª 2005 FEBS G Rabah et al Structure and dynamics of NSCP in solution Fig Generalized order parameters along the sequence of Ca2+-saturated NSCP The experimental values obtained from the NMR relaxation experiments, together with the standard deviations, are shown in red The order parameters estimated from the MD simulations are in black The last three values, at the end of the sequence, correspond to the Trp4, Trp57, and Trp170 indole N-H vectors (boxed) eter (S2), the internal correlation time (se) and the exchange contribution to the transversal relaxation (R2ex) The extended procedure of relaxation analysis did not improve the fitting quality of any amide or amine system The generalized order parameter S2 at a given backbone site is a measure of the amplitude of the fast (picosecond to nanosecond) movement of the HN-N vector Figure shows the order parameters and their standard deviations, estimated by Monte-Carlo simulations, as a function of the residue number The parameter varies between 0.66 and 0.93, with a mean value of 0.83 ± 0.04, which is close to the average value usually observed for native globular proteins [30] Overall, the estimated order parameters indicate that the backbone amide vectors undergo picosecond-tonanosecond movements of low amplitude, reflecting a compact and rigid fold, with well-structured end fragments Larger S2 values (from 0.84 to 0.93) are grouped in the loop I, helix F and helix H, while the intermotif linkers and the unoccupied Ca2+-binding loop exhibit lower S2 values (down to 0.66), attesting to larger amplitude fast movements The end fragments are characterized by high S2 values, meaning that their movement is significantly restricted Of the four EF-hand motifs, the first and the fourth exhibit the most restricted picosecond-to-nanosecond mobility of the backbone vectors The absence of Ca2+ binding to the second EF-hand induces an irregular pattern of S2 values in the loop residues that extends over the neighboring helices It is worth noting that the helices characterized by a larger fast movement amplitude and FEBS Journal 272 (2005) 2022–2036 ª 2005 FEBS a high amide proton exchange (B, C, E) belong to the interface between the two EF-hand domains The relatively high values (0.75–0.78) of S2 observed for the amino group of the Trp side chains indicate that the fast movements of these indole moieties are restricted to a similar extent to the backbone amide vectors (Table 3) Inspection of the calculated structure (Fig 2) shows that the three indole groups not have comparable environments in the 3D structure: whereas W4 and W57 are highly exposed to the solvent at the protein surface, W170 is deeply embedded in the hydrophobic core created by helices E, F and H The order parameters of these side chains appear to be independent of this environmental context The large majority of the residues display fast rate librational motions characterized by an internal correlation time se < 50 ps, with a dozen (mainly localized in linker fragments) having a correlation time in the range 50–100 ps (not shown) Five residues (V5, E40, G67, S90, and D156) exhibit R2ex values between and s)1, reflecting microsecond–millisecond internal motions in their environment Again, they are associated with end fragments (V5, D156), linkers (E40, S90) or the empty calcium-binding loop (G67) Table Experimental (S 2NMR ) and calculated (S 2MD ) order parameters for the Trp side chains Trp S 2NMR S 2MD W4 W57 W170 0.78 0.75 0.78 0.83 0.79 0.83 2029 Structure and dynamics of NSCP in solution Comparison with the crystallographic B factor The crystallographic B factor is considered to reflect mainly the molecular flexibility at the atomic level, but other factors related to static and dynamic disorders in the crystal may give important contributions as well [31] Intuitively, it should be inversely correlated with the order parameters estimated from the relaxation measurements, but in practice the relationship is more complex [32] Overall, in NSCP the regions of highorder parameters exhibit lower B factors for amide nitrogens (Fig 3) As the range of values for the order parameter is about three times lower than that for the B factor, the quantitative correlation between the two parameters along the sequence is only moderate (the correlation coefficient is )0.32) Discrepancies also arise from the fact that S2 reflects only fast motions, whereas the B factor is sensitive to both fast and slow movements [33] In a similar approach for ribonuclease, Mandel et al [34] found similar low values between )0.36 and )0.64, depending on the X-ray structure considered This variability illustrates the dependence of the thermal factors on the crystallization state and the intermolecular contacts within the crystal MD simulation Simulation of the protein internal dynamics under an appropriate physical force field provide a detailed atomic picture of the movements underlying the nuclear relaxation parameters and the corresponding order parameters Only ns (the second half) from the 2-ns very stable trajectory of the Ca2+-saturated form of NSCP was used in the theoretical analysis The mean ± SD temperature was 299.99 ± 4.47 K, and the total energy was 1722.5 ± 21 kJỈmol)1 (411.4 ± kcalỈmol)1) Correlation functions for backbone NH-H vectors were computed from the selected trajectory using an interval of 400 ps, which provides reliable sampling of the fast (% 100 ps) motions [35] Correlation functions were computed from the trajectory for 168 residues from the total of 174 (the N-terminus and the six prolines not have an sp2 NH-H bond) The correlation functions of the internal motions, CI(t) display different patterns along the sequence, and only 132 could be considered completely convergent For 25 other residues, the correlation function exhibits a slow decay towards a lower plateau and oscillatory behavior, suggesting the presence of slower motions, which may not be accounted for correctly by the length of the present trajectory [35] Finally, for 11 residues no plateau value could be reached within 400 ps 2030 G Rabah et al The plateau values of the correlation functions (including both rapidly and slowly convergent functions), which represent the theoretical order parameters S2MD , were computed for 157 amino-acid residues (Fig 5) A total of 151 of the 157 simulated S2MD values are larger than 0.7, confirming the high rigidity of the NSCP backbone structure As shown in Fig 5, there is a good correlation between the order parameter profiles established by theoretical and experimental approaches The average value of S2 obtained by MD (S2ave ¼ 0.80, average over 157 points) is slightly lower than that calculated from nuclear relaxation data (S2ave ¼ 0.83, average over 116 points), as also noted for aponeocarzinostatin [36] It may be noted that there is a remarkable parallelism between the two S2 profiles at the limits between secondary-structure elements and linkers, where this parameter exhibits larger variations Amide vectors in the unoccupied binding loop II, in the linker between helices F and G, as well as in the last loop show markedly decreased order parameters both in the relaxation experiments and the simulation, permitting cross validation of the underlying MD (Fig 5) The MD simulations predict slightly larger amplitude motions in the more flexible protein segments than estimated by relaxation measurements It must be stressed that the overall shift between the NMR and MD values for the order parameter could be decreased by choosing a slightly smaller value for the initially estimated global correlation time [37] Previous comparative studies on the simulation and experimental dynamic parameters of globular proteins [35–37] have always noted some differences in the order parameters determined by the two methods One of the reasons is that both MD and NMR analysis involve several approximations In the case of the MD simulations, these include the parameters of the empirical force field, insufficient sampling because of the finite length of the trajectories, and the simplified treatment of the solvent The main simplification in the NMR approach concerns the relaxation data analysis, usually performed under the assumption of a unique overall correlation time and the independence of movements on different time scales In addition to the amide vector dynamics, we also analyzed the fast movements of the Ne1-H vectors in the three Trp side chains The order parameters of the picosecond motions of these bonds (Table 3) are only slightly lower than the backbone values (as in the relaxation results), indicating that the side chains also move in a highly restricted regime [37] Trp57 senses the molecular environment of the empty binding site and shows significantly larger amplitude than the other FEBS Journal 272 (2005) 2022–2036 ª 2005 FEBS G Rabah et al two tryptophans, both in simulation and experimental results The good qualitative description of the fast dynamics in the Ca2+-bound form of NSCP encouraged us to use a simple dynamics simulation to investigate the initial structural changes accompanying the holo to apo transition A 2-ns MD trajectory, starting with the crystallographic co-ordinates of (Ca2+)3-NSCP [6] from which the three metal ions were removed, was generated at 300 K As estimated from the temperature and co-ordinate rmsd profiles, the apo and holo dynamics are of comparable quality Figure 6A compares the two final simulated structures and the crystal structure, using the pseudo-dihedral angles defined by four successive Ca atoms along the sequence As may be noted from the upper panel, in presence of the bound Ca2+ ions the final protein secondary structure (blue symbols) shows no significant change relative to the crystal conformation (red bars) In contrast, the final structure of the apo simulation (Fig 6A, lower panel) shows significant secondary-structure differences relative to the starting (holo-type) conformation, mainly localized to the first binding loop, the N-side of the C and D helices, and the C-end of the F helix Comparison of the tertiary structures including these areas (Fig 6B) shows that removing the metal ion from the first site determines an opening of the binding loop, and induces important structural changes in the first half of the protein The rearrangement of this domain has remote consequences on the F helix, in the second protein half, which may be accounted for by the close contacts existing between the B and F helices (Fig 6B) Indeed, this interhelix space belongs to the large interdomain surface which plays an important role in the structural and functional coupling between the two halves The perturbation propagation pathway, observed in the present simulation, includes the higher flexibility secondary-structure elements, highlighted by the nuclear relaxation and proton exchange experiments A longer, and more complete, MD simulation of apo-NSCP, in an explicit water environment, should provide valuable insight into the metal-induced conformational changes and the characteristics of the apo molten globule state This approach is currently being taken in our laboratory Experimental procedures Protein preparation and labeling Wild-type NSCP was purified from the Nereis muscle by the method of Cox & Stein [5], modified as described by FEBS Journal 272 (2005) 2022–2036 ª 2005 FEBS Structure and dynamics of NSCP in solution Engelborghs et al [38] Recombinant protein and 15N labeling was obtained by overexpression in Escherichia coli [39] The NSCP ORF was amplified by PCR starting from the plasmid pNDner04, cloned into the vector pET22b, and transfected into the host E coli strain BL21(DE3) ⁄ pDIA17 ⁄ pHSP234 (Novagen, Madison, WI, USA) (15NH4)2SO4 (1 gỈL)1) was added to the minimal medium to obtain uniformly labeled samples NMR samples NMR samples (1.0–1.2 mm) were prepared in deuterated 20 mm Tris ⁄ HCl buffer ⁄ mm CaCl2, pH 6.5, in 95% 1H2O ⁄ 5% 2H2O or in 100% 2H2O NMR spectroscopy All NMR spectra were acquired at 308 K on a Varian Unity-500 spectrometer, equipped with a triple-resonance probe and a Z-field gradient Standard methods were used to obtain 2D NOESY and 3D 15N-NOESY-HSQC spectra [40,41], with mixing times of 100 ms The 3D spectra were acquired as 128 (t1) · 32 (t2) · 512 (t3) complex points with a spectral width of 1500 Hz in the nitrogen dimension, 3200 Hz in the amide proton dimension, and 7000 Hz in the all-proton dimension Dihedral angle restraints were obtained by analyzing the HMQC-J spectrum by the method proposed by Wishart & Wang [42] Data processing and restraint collection were performed using felix97 software (Accelrys, San Diego, CA, USA), running on a Silicon Graphics Octane workstation 15 N nuclear relaxation The heteronuclear relaxation experiments were performed at 308 K and 11.74 T (500 MHz proton resonance frequency) The R1 relaxation rate was measured using the inversion recovery method, modified to obtain decreasing signal intensities as a function of the relaxation delay Measurement of the transverse relaxation rate (R2) was based on the Carr–Purcell–Meiboom–Gill pulse sequence with a delay between 15N 180° pulses during the relaxation period of 0.9 ms Recycle delays of 2.5 s were used at the beginning of R1 and R2 pulse sequences Spectra for R1 measurements were acquired using 11.04 (· 2), 55.22, 165.66, 220.88 (· 2), 386.54, 552.2, 662.64, 828.3 and 1104.4 ms as relaxation delays R2 data were recorded with delays of 31.4, 47.1, 62.8, 78.5 (· 2), 125.6, 157, 188.4 and 219.8 ms Steady-state 1H-15N NOE was determined from spectra pairs with and without proton saturation The two experiments start with a s recycle delay, but during the last s of the saturation experiment the protons are irradiated by 120° pulses every ms The pulse sequences used in the present experiments were adapted from those kindly 2031 Structure and dynamics of NSCP in solution G Rabah et al A B Fig Comparison of the 2-ns MD simulations of the holo and apo forms of NSCP (A) The pseudo-dihedral angles between consecutive Ca in the final structure obtained for (Ca2+)3-NSCP (upper panel) and apo-NSCP (lower panel) The red lines in both panels represent the calculated values for the crystallographic (Ca2+)3-NSCP structure, and the blue symbols represent the simulated structures (B) Schematic representation of the two simulated final structures focused on the first half of the protein The green sphere represents the calcium ion bound to the first loop provided by Lewis Kay [43,44] Peak picking and peak height measurement were performed with the appropriate routines of the felix97 package Uncertainties in the peak 2032 intensities were evaluated from the standard deviation of the noise in a few rows from the spectra with various relaxation delays Relaxation rates R1 and R2 and their uncer- FEBS Journal 272 (2005) 2022–2036 ª 2005 FEBS G Rabah et al Structure and dynamics of NSCP in solution tainties were obtained by fitting the peak intensities to a single-exponential function with two parameters, using the physica package Errors in relaxation rates were estimated from the uncertainties in the peak heights and in the exponential fit or by Monte-Carlo simulations The steadystate NOEs were calculated as NOE ¼ Isat ⁄ Inonsat (g ¼ NOE ) 1), where Isat and Inonsat are the steady-state peak intensities measured with and without proton saturation, respectively NOE uncertainties were estimated from the peak height uncertainty and the law of error propagation Hydrogen exchange Amide proton exchange kinetics was performed by acquiring a series of 2D HSQC spectra of a 15N-labeled lyophilized sample, freshly dissolved in 2H2O The progress of the exchange process was followed by observing successive spectra, recorded at 308 K, and measurement of the amide peak intensity The experimental points were fitted to a threeparameter exponential function by a nonlinear least squares routine The protection factors for each backbone site were calculated using the procedure proposed by Bai et al [24], designed to eliminate the dependence on the physicochemical properties of the solution and on the chemical environment of the site For the indole Ne1 protons of the Trp residues, only the temperature and the pH corrections were measured Under the EX2 exchange conditions (in which the rate of conformational change is much larger than the rate of proton exchange), the exchange rate (kex) for an amide proton is calculated from the relation [24]: kex ¼ Kop kch where kch is the chemical exchange rate in the given physicochemical conditions, and Kop the equilibrium constant between the open (exchange-compatible) and closed (exchange-incompatible) conformations The free energy of the opening process, enabling the isotopic exchange (DGex), is calculated from the formula: DGex ¼ ÀRT ln Kop The decrease in the exchange rate of a proton in the native structure, relative to the random coil (kch ⁄ kex) defines the so-called protection factor, P ¼ ⁄ Kop [24] Structure determination Spin systems for 171 of 174 residues (% 98%) were partially or completely assigned [10] Unassigned resonances correspond to a polypeptide fragment situated in an irregular secondary-structure region (D163–T165) at the C-terminal site of the sequence A total number of 1859 NOE distance restraints were identified in the 2D and 3D NOESY spectra, corresponding to an average of 10.7 restraints per residue In addition, 188 hydrogen-bond restraints were identified from the analysis of hydrogen-exchange FEBS Journal 272 (2005) 2022–2036 ª 2005 FEBS experiments, and 262 dihedral angle restraints were generated based on 3JHNHa coupling constant measurements and preliminary secondary-structure delineation NOE distance restraints were classified into three categories, 1.8–2.9, 2.9– ˚ 3.7 and 3.7–5.0 A except for daN(i,i +2) and dNN(i,i +2) distances in regular helical fragments which fall into 4.2–4.6 ˚ and 4.0–4.4 A, respectively No restraints involving calcium ions were available However, several hydrogen bonds within the Ca2+-binding loop, confirmed by the low hydrogen exchange rate and a large downfield chemical shift [45,46], were considered for the binding loop I, III and IV The restraint statistics are detailed in Table The aqua and procheck-nmr programs [12] were used to analyze the restraint violations and to estimate the precision and quality of the structures obtained Molecular dynamics MD simulations were performed using the program charmm and the extended atom force field PARAM19 [47] in which the polar hydrogen atoms are treated explicitly The initial set of atomic co-ordinates was obtained from the crystal structure of the NSCP [6] completed with the polar hydrogen co-ordinates [48] Co-ordinates for the apo form of the NSCP were generated by removing the three Ca2+ ions from the holo-NSCP structure Both apo-NSCP and holo-NSCP structures were then energy-minimized in vacuo by 6000 steepest descent steps, followed by 3000 Newton–Raphson steps The minimized structures were heated to 300 K for 12 ps, equilibrated at 300 K for 18 ps, and finally two production runs of ns were generated for both the holo and apo forms The nonbonded interactions were truncated to ˚ zero by applying a switching function between and A ˚ and updated [49] The nonbond list was cut off at 10 A every 10 steps An ‘r’ relationship was considered for the dielectric constant (r is the interatomic distance) For the MD simulations, the Newton equations of motion were integrated using the VERLET algorithm with an integration step of fs The heavy atom–hydrogen bond lengths were constrained with the SHAKE algorithm [50] The co-ordinates were saved every 100 fs to the trajectory file Correlation functions and order parameter computation from MD trajectories Assuming that internal motion and the overall tumbling are independent, the reorientation of the NH-H vector, which contributes to the 15N relaxation, can be described by the angular autocorrelation function: CðtÞ ¼ ð1=5ÞCo ðtÞCI ðtÞ where the correlation functions Co(t) and CI(t) are related to the overall rotational tumbling of the molecule and to the internal dynamics, respectively The internal correlation 2033 Structure and dynamics of NSCP in solution functions describing the dynamics of an NH-H bond are computed from the trajectory using an autocorrelation function of the unit vector upon the direction of NH-H ^ atoms, lNH ðsÞ [51,52]:   cos2 hNH tị CI tị ẳ hP2 cos hNH ịi ẳ t ẳ TX MD t TMD t sẳ1 3ẵ^NH sị^NH s ỵ tފ2 À l l where P2(coshNH) is the second rank Legendre polynomial, ^ lNH the unit vector along the NH-H direction, TMD the total number of MD frames, and s the index of MD frames for the computed correlation function (the maximum value of the ‘t’ parameter should be lower than TMD ⁄ 3) The generalized order parameters (S2) were evaluated for amide NH-H bonds and Ne1-H bonds of Trp side chains, by fitting the calculated CI(t): l l S2 ẳ lim CI tị ẳ lim < P2 ½^NH ð0Þ^NH ðtފ > t!1 t!1 for one-third of the whole trajectory [35] to a single exponential (‘model-free’ approach) Accession number Co-ordinates corresponding to the 17 structures of the Ca2+-saturated NSCP have been 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Methods Enzymol 277, 505–524 Nicholls A, Sharp KA & Honig B (1991) Protein folding and association: insights from the interfacial and thermodynamic properties of hydrocarbons Proteins 11, 281–296 FEBS Journal 272 (2005) 2022–2036 ª 2005 FEBS ... structure and dynamic characterization in solution of the various functionally relevant states To achieve this aim, we initiated a structural and dynamics analysis of Ca2+-saturated NSCP in solution, ... Oobatake M & Kanaya S (1995) Characterization of the internal motions of Escherichia coli ribonuclease HI by a combination of 15N-NMR relaxation analysis and molecular dynamics simulation: examination... AK, Majumdar A & Udgaonkar JB (2000) Backbone dynamics of barstar: a 15N NMR relaxation study Proteins 41, 460–474 34 Mandel AM, Akke M & Palmer AG III (1995) Backbone dynamics of Escherichia