Báo cáo khoa học: The effect of heme on the conformational stability of micro-myoglobin doc

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The effect of heme on the conformational stabilityof micro-myoglobinHong-Fang Ji1,2, Liang Shen1,2, Rita Grandori3and Norbert Mu¨ller21 Shandong Provincial Research Center for Bioinformatic Engineering and Technique, Center for Advanced Study,Shandong University of Technology, Zibo, China2 Institute of Organic Chemistry, Johannes Kepler University, Linz, Austria3 Dipartimento di Biotecnologie e Bioscienze, Universita degli Studi di Milano-Bicocca, Milan, ItalyThe monomeric heme protein myoglobin (Mb) isfound mainly in muscle tissue [1], where its principalphysiological functions are oxygen storage and thefacilitation of oxygen transport to the mitochondriafor oxidative phosphorylation [2,3]. The capability ofMb to bind oxygen depends on the presence of a hemeprosthetic group, in which an iron(II) cation is che-lated by the four nitrogen atoms in the center of aprotoporphyrin ring. The metal ion can form twoadditional coordinative bonds on either side of theheme plane, termed the fifth and sixth coordinationpositions, which are essential for the three-dimensionalstructure and oxygen-binding function of the protein.Mb folds into a globular, single-domain structurewith a high a-helix content. It comprises eight right-handed a-helices (A–H, from the N- to C-terminus),which are linked to each other by short loop regions(Fig. 1) [4].It has long been recognized that the central portionof the globin fold (mid-helix B to mid-helix G) forms acompact subdomain containing almost all the protein–heme contact sites [5]. Recent studies have indicatedthat a fragment corresponding to such a portion of thestructure is capable of folding into a functional heme-binding unit forming a complex with the prostheticgroup with characteristics similar to native Mb [6,7].The deletion product was subcloned and expressed asa 77-amino-acid fragment spanning residues 29–105 ofsperm whale Mb, and was named micro-myoglobin(lMb) [6]. The earlier experimental studies revealedthat, in the absence of heme, this fragment is mostlydisordered, but acquires a native-like a-helix contenton interaction with the cofactor [6]. (We note here that‘native-like’ in the context of this paper refers to thenative fold of holo-Mb.) Holo-lMb, nevertheless, isdifficult to characterize experimentally as it has beenKeywordsheme binding; micro-myoglobin; moleculardynamics simulation; protein stability;unfoldingCorrespondenceN. Mu¨ller, Institute of Organic Chemistry,Johannes Kepler University, 4040 Linz,AustriaFax: +43 732 2468 8747Tel: +43 732 2468 8746E-mail: norbert.mueller@jku.at(Received 28 May 2007, revised 29 October2007, accepted 5 November 2007)doi:10.1111/j.1742-4658.2007.06176.xMicro-myoglobin, the isolated heme-binding subdomain of myoglobin, is avaluable model system for the investigation of heme recognition and bind-ing by proteins, and provides an example of protein folding induced by co-factor binding. Theoretical studies by molecular dynamics simulations onapo- and holo-micro-myoglobin show that, by contrast with the case of thefull-length wild-type protein and in agreement with earlier experimental evi-dence, the apo-protein is not stably folded in a native-like conformation.With the cofactor bound, however, the protein fragment maintains itsfolded conformation over 1.5 ns in molecular dynamics simulations. Fur-ther inspection of the model structures reveals that the role of heme in sta-bilizing the folded state is not only a result of its direct interactions withbinding residues (His93, Arg45 and Lys96), but also derives from its shield-ing effect on a long-range electrostatic interaction between Arg45 andAsp60, which, in the molecular dynamics simulations, apparently triggersthe unfolding process of apo-micro-myoglobin.AbbreviationsMb, myoglobin; MD, molecular dynamics; PDB, Protein Data Bank; lMb, micro-myoglobin.FEBS Journal 275 (2008) 89–96 ª 2007 The Authors Journal compilation ª 2007 FEBS 89found to be prone to aggregation and precipitation athigh concentration. By contrast, a longer Mb-derivedfragment, spanning residues 32–139, and named mini-myoglobin (mini-Mb), is capable of independent fold-ing into a native-like conformation, even in theabsence of heme [8]. Other deletion products and circu-lar permutations have also shown that the structuraldeterminants for heme binding can be segregated fromthose for protein stability and solubility [9]. Thus,lMb represents a valuable and, so far, unique modelsystem for the investigation of heme recognition byproteins and its possible role as a structural scaffoldfor the minimal heme-binding subdomain.Molecular dynamics (MD) simulations can aid inthe understanding of the physical basis of the struc-tural and functional features of biological macromole-cules by providing details concerning intra- andintermolecular motions as a function of time.Although results of MD simulations do not alwaysand easily translate to assessment of conformationalstability in a thermodynamic sense, they can be usedto address specific questions about the properties of amodel system, particularly for comparison of mole-cular variants under controlled conditions. Althoughfinding the thermodynamically most stable form of amultiparticle system (and proving it) through MDalone is generally impossible, instability revealed inMD simulations is more straightforward to interpret[10]. In this study, we performed MD simulations on‘heme-excised’ apo-lMb and holo-lMb with the fol-lowing aims: (a) to compare the stability of native-likestructural models for apo-lMb and holo-lMb; (b) toinvestigate the mechanisms by which heme affectsprotein conformation and dynamics; and (c) to probethe transitions of each individual helix during the ini-tial step of thermal unfolding.Results and DiscussionApo-lMbSnapshots of the MD simulation trajectory of apo-lMb at 0.0, 0.25, 0.5, 0.75, 1.0, 1.25 and 1.5 ns havebeen extracted and visualized in Fig. 2 by ribbonFig. 1. Ribbon representation of the crystallographic structure ofsperm whale myoglobin. The eight helices of the myoglobin foldare labeled A–H from the N- to C-terminus (PDB entry 1A6N).Fig. 2. Snapshots of apo-lMb (residues 29–105) and holo-lMbsimulations extracted from the MD trajectories at 400 K. Red,a-helix; green, random coil.Stability of micro-myoglobin H F. Ji et al.90 FEBS Journal 275 (2008) 89–96 ª 2007 The Authors Journal compilation ª 2007 FEBSdrawings. It can be seen that apo-lMb quickly movesaway from the initial native-like conformation, i.e. itunfolds easily over the timescale of the simulation.Therefore, we can conclude that the native-like fold isnot even a metastable conformation of this protein. Tocompare the unfolding of the individual helices of apo-lMb, the average rmsd of each helix after 1.5 ns ofsimulation was calculated over the backbone atomsrelative to the initial energy-minimized conformation.The values are 1.87 A˚for helix B, 1.95 A˚for helix C,1.69 A˚for helix D, 5.23 A˚for helix E, 1.92 A˚for helixF and 2.07 A˚for helix G. A close look at Fig. 2reveals that helix G is the first to unfold, which isunderstandable because it spans only five residuesdespite its relatively low rmsd (2.07 A˚). However, it isinteresting to find that, although helix E is the longestand is positioned close to the core of the sequence, itis the second to unfold, immediately following helix G,as indicated in Fig. 2. This stimulated us to furtherinspect the unfolding process of helix E. Previousinvestigations on native apo-Mb have indicated thathelix E is the first to unfold (or the last to fold)[11,12]. However, according to the present study inapo-lMb, helix G unfolds first. This difference mayFig. 3. Variation of hydrogen-bond distancesin helix E during the 1.5 ns simulation ofapo-lMb at 400 K. The distances are mea-sured between CO(i) and NH(i+4), wherei ranges from residue 58 to residue 73.H F. Ji et al. Stability of micro-myoglobinFEBS Journal 275 (2008) 89–96 ª 2007 The Authors Journal compilation ª 2007 FEBS 91arise from the fact that helix G is truncated in lMb,which allows it to unfold faster than helix E, which iscomplete in the fragment. It should be noted, however,that helix F has been found to be disordered in nativeapo-Mb under neutral and equilibrium conditions [13].A close look at Fig. 2 reveals that helix E of apo-lMb begins to unfold from its N-terminus at 0.5 ns,and approximately one-half of the helix is disorderedat 1.0 ns. Within the first 1.5 ns, helix E disappearscompletely. To describe helix E unfolding, 14 hydro-gen-bond distances [CO(i)–NH( i + 4)] that character-ize the a-helix between residues 58 and 77 weretracked throughout the MD simulation (Fig. 3).The first hydrogen bond that breaks is that betweenCO(60) and NH(64), at the N-terminus of the helix.Starting at 0.3 ns, the CO(60)–NH(64) distance beginsto increase, reaching 6.0 A˚at 0.4 ns, clearly indicatingthat this hydrogen bond no longer exists. Subse-quently, significant fluctuations in the distances ofother hydrogen bonds are observed. The CO(61)–NH(65) and CO(62)–NH(66) hydrogen-bond distancesstart to increase at 0.5 and 0.7 ns, respectively, whichis accompanied by a further increase in the distancebetween CO(60) and NH(64). Subsequent break-up ofall the other hydrogen bonds within helix E ultimatelyresults in complete unfolding of this helix.Further inspection of the unfolding snapshots ofmodel structures of apo-lMb reveals that, during theunfolding simulation, the distance between the carbox-ylate oxygen of Asp60 (helix E) and the imino nitrogenof Arg45 (helix C) decreases rapidly from 8.0 to 3.0 A˚.This can be interpreted as the formation of a saltbridge between these two residues (Figs 4 and 5). Theoccurrence of a salt bridge at approximately 0.5 nscoincides with the beginning of the unfolding ofhelix E described above (Fig. 2). Therefore, the forma-tion of this salt bridge between Arg45 and Asp60seems to play an important role in triggering apo-lMbunfolding.Previous experimental studies have shown that apo-lMb has a disordered conformation in aqueous solu-tion at ambient temperature in the absence of heme[6], whereas, under the same conditions, the full-length apo-protein can fold into a native-like confor-mation and pre-organize the heme pocket beforemaking contact with the cofactor [13]. For compari-son, MD simulations were also performed on ‘heme-excised’ apo-Mb, under the same conditions andinitial assumptions as for apo-lMb. The averagebackbone rmsd for apo-Mb compared with the initialenergy-minimized conformation is 3.51 A˚, much lowerthan that for apo-lMb: 4.99 A˚(Fig. 6). These datacorroborate the effectiveness of MD simulations inFig. 4. Variation of distances between the amino nitrogen of Arg45and the carboxylate oxygen of Asp60 during the 1.5 ns simulationof apo-lMb (residues 29–105).Fig. 6. Evolution of backbone rmsd during the 1.5 ns simulation ofapo-lMb, ‘heme-excised’ apo-Mb and holo-lMb at 400 K, relativeto the respective initial structures.Fig. 5. Three-dimensional model of the salt bridge between Arg45and Asp60 in apo-lMb based on the structure at approximately0.5 ns of the simulation.Stability of micro-myoglobin H F. Ji et al.92 FEBS Journal 275 (2008) 89–96 ª 2007 The Authors Journal compilation ª 2007 FEBSdiscriminating between stable and unstable proteinconformations. Thus, in agreement with the experi-mental evidence [6], the results presented here showthat a native-like Mb conformation does not repre-sent a stable state for the truncated lMb fragment inthe absence of heme.A comparison of the dynamics of helix E, in par-ticular between apo-Mb and apo-lMb, also showsconsiderable differences. The backbone rmsd betweenthe starting and final structures of the fragment corre-sponding to helix E is 3.18 A˚for apo-Mb and 5.23 A˚for apo-lMb. Inspection of the Mb structure indi-cates that, in apo-Mb, helix E is stabilized by severalsalt bridges formed between helix E side-chains andneighboring residues from helices A and B (Fig. 7),such as Glu4-Lys79, Glu18-Lys77 and Asp27-Lys56,as also pointed out in a previous study [14]. The sta-bilizing effect of these salt bridges in apo-Mb pre-vents fluctuations at the N-terminus of helix E, andapparently also retards the formation of the saltbridge between Asp60 and Arg45, which is an impor-tant factor triggering apo-Mb unfolding. By contrast,these salt bridges are absent in apo-lMb because ofthe absence of the helices A and B, providing a possi-ble explanation for the distinct behavior of helix E inapo-lMb and apo-Mb.Holo-lMbIn order to investigate the role of heme in proteindynamics and stability, MD simulations were per-formed on holo-lMb under the same conditions asemployed for apo-lMb. In Fig. 2, the extracted snap-shots of holo-lMb simulation are shown juxtaposi-tioned to the corresponding positions of apo-lMb. Itis evident that the holo-protein displays much reduceddynamics. By contrast with the almost unfolded struc-ture of apo-lMb at the end of the simulation, mosthelices of holo-lMb are still folded at the end of thesimulation period. This remarkably different behaviorof the protein in the presence and absence of heme isalso reflected by the respective rmsd values from acomparison of the backbone conformations before andafter the MD simulation runs, which are 3.28 A˚forholo-lMb and 4.99 A˚for apo-lMb (Fig. 6).These findings provide some clues for the interpreta-tion of the experimental result that apo-lMb is almostunfolded [6], whereas the same fragment folds into apredominantly a-helical secondary structure on interac-tion with heme, thus forming a complex with the pros-thetic group with characteristics similar to the fold ofnative holo-Mb. According to the crystal structure ofholo-Mb [15], the heme ligand allows for coordinationinteraction between the iron ion and an imidazole ringnitrogen of His93, and its propionate groups areengaged in salt bridges with the basic side-chains ofArg45 and Lys96 (Fig. 8). These interactions conceiv-ably also contribute to the conformational stability ofholo-lMb. Consistently, they are maintained over the1.5 ns of the simulation (Fig. 2).A comparison of the hydrogen-bond distances inFigs 3 and 9 indicates that helix E also displaysreduced dynamics in holo-lMb relative to its counter-part in apo-lMb. As mentioned above, in apo-lMb,Arg45 forms a salt bridge with Asp60 in the first0.5 ns of MD simulation, triggering the unfolding ofhelix E and the subsequent global unfolding of thestructure. In holo-lMb, the shielding effect of theheme and the engagement of Arg45 in the salt bridgeFig. 7. Three-dimensional model of salt bridges Glu4-Lys79, Glu18-Lys77 and Asp27-Lys56 in apo-Mb (PDB entry 1A6N).Fig. 8. Three-dimensional model of the salt bridges made by Arg45and Lys96 with the two propionate groups of heme in holo-lMb.H F. Ji et al. Stability of micro-myoglobinFEBS Journal 275 (2008) 89–96 ª 2007 The Authors Journal compilation ª 2007 FEBS 93with the heme propionate prevent the build-up ofattractive electrostatic interactions between Arg45 andAsp60 that are seen to lead to apo-lMb unfolding.Therefore, in addition to direct interaction with bind-ing residues, heme seems to stabilize the lMb foldedstate also by counteracting long-range electrostaticinteractions that would otherwise act as destabilizingforces for native-like conformations.ConclusionsThe comparison of apo- and holo-lMb by MD simu-lations reveals a dramatic effect of the heme cofactor,which is seen to stabilize native Mb-like folded confor-mations of the protein fragment, in agreement with theavailable experimental evidence. The simulation resultssuggest that, in the absence of heme, the unfoldingprocess is triggered by the formation of a non-nativesalt bridge between Arg45 and Asp60. The presence ofheme at the active site counteracts this unfoldingmechanism, in addition to providing stabilizing inter-actions with the folded chain per se. Although thestarting conformation used here for apo-lMb simula-tion has not been observed experimentally, it can betaken as a model of an unstable intermediate in holo-lMb unfolding on loss of the cofactor. The MD simu-lation results provide an explanation of why such aconformation is not a stable state for apo-lMb.Electrostatic interactions, in general, have attractedconsiderable interest in protein folding studies, as ithas been shown that surface charges can play animportant role in protein stability [16]. Nevertheless,Fig. 9. Variation of hydrogen-bond distancesin helix E during the 1.5 ns simulation ofholo-lMb at 400 K. The distances arebetween CO(i) and NH(i+4), wherei ranges from residue 58 to residue 73.Stability of micro-myoglobin H F. Ji et al.94 FEBS Journal 275 (2008) 89–96 ª 2007 The Authors Journal compilation ª 2007 FEBSthe contribution of engineered salt bridges to proteinstability is frequently smaller than predicted on thebasis of theoretical models [17]. This observation hasbeen interpreted to be the result of the survival ofpartially folded structures stabilized by electrostaticinteractions in the denatured state [18]. Indeed, theavailable tools for modeling protein conformations inthe denatured state are still insufficient. However, wehave good reasons to assume that, in the presence ofdenaturants or, even more so, at high temperatures,electrostatic interactions can contribute to maintainresidual structure in the denatured state. The perva-siveness of structured clusters under denaturing condi-tions has also been found experimentally for differentproteins [19–21]. The results reported here strengthenthe notion of the structural complexity of proteins inthe denatured state and provide an example of the roleof non-native ion pairs in protein unfolding.Experimental proceduresThe MD simulations on holo- and apo-lMb were per-formed in parallel. The starting structure of holo-lMb wasobtained from the highest resolution X-ray crystallographicstructure of sperm whale Mb [Protein Data Bank (PDB)entry 1A6N] [15] [which matches well the 12 structure bun-dle obtained earlier by NMR (PDB entry 1MYF)] [22], byexcision of the corresponding fragment (residues 29–105)whilst retaining the bound heme. The structure of apo-lMbwas obtained by additional excision of the heme ligand.Likewise, the starting geometry for the simulations of apo-Mb were obtained by deleting the heme from the abovecrystal structure. All the simulated structures wereimmersed in two layers (20 A˚for the inner and 15 A˚forthe outer) of explicit water molecules. The inner layer wasdynamic, whereas the outer layer was static, and served asa solvent boundary to prevent the escape of the inner layerwater molecules. All of the simulated structures were calcu-lated for a 1.5 ns MD simulation in a neutral environment.The initial structures were first energy minimized by 1000conjugate gradient steps, and then heated from 2 to 300 Kover 35 ps, with temperature increments of 50 K per 5 ps,and kept at 300 K for 20 ps using the constant pressureand temperature algorithm. The velocity Verlet integratorwas used with an integration step of 2 fs. As it has beenreported that a few nanoseconds are sufficient to assess therelative stabilities of the initial structures [23], the produc-tion MD phase (i.e. the unfolding) was carried out for1.5 ns, which was sufficient to reach the quasi-equilibriumstates of both holo- and apo-lMb, using 2 fs steps and9.5 A˚cut-off, as demonstrated by plateaus in the MD tra-jectories. To accelerate the unfolding processes of both pro-teins, the simulations were performed at 400 K. During thesimulations, the non-bonded interaction cut-off was set to12 A˚, which is sufficiently large to include long-range elec-trostatic interactions. Main-chain hydrogen bonds withina-helices were assigned when the distance between the COgroup of residue i and the NH group of residue i + 4 wasshorter than 2.5 A˚. A salt bridge was identified when thedistance between the nitrogen of the base and the oxygenof the carboxylate was shorter than 5.0 A˚[24]. Structureswere saved every 0.5 ps for a total of 3000 snapshots foreach trajectory. All of the constant volume constant tem-perature MD simulations were performed by the Discovermodule of the insightii program package (Accelys Inc.,San Diego, CA), which has been widely employed in MDsimulation studies [25–28]. The consistent valence force field[29–32] was used for all of the simulations. The calculationswere carried out on an SGI Origin 3800 server (SiliconGraphics Inc., Sunnyvale, CA).AcknowledgementsWe thank Elena Papaleo, Luca De Gioia and StephanSchwarzinger for helpful discussions and critical read-ing of the manuscript. This research was supported inpart by the European Union in the REGINS-INBIOprogram and the Austrian Science Funds (projectP15380). Hardware and software support was providedby the Supercomputing Department at Johannes Kep-ler University, Linz, Austria.References1 Cole RP (1982) Myoglobin function in exercisingskeletal muscle. Science 216, 523–525.2 Wittenberg BA & Wittenberg JB (1989) Transport ofoxygen in muscle. Annu Rev Physiol 51, 857–878.3 Wittenberg BA, Wittenberg JB & Caldwell PB (1975)Role of myoglobin in the oxygen supply to red skeletalmuscle. J Biol Chem 250, 9038–9043.4 Antonini E & Brunori M (1971) Hemoglobin andMyoglobin in their Reactions with Ligands. 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Carbohy-drate compounds and anomeric effects. Biopolymers 45,435–468.31 Peng Z, Ewig CS, Hwang MJ, Waldman M & HaglerAT (1997) Derivation of Class II force fields. 4. van derWaals parameters of alkali metal cations and halideanions. J Phys Chem A 101, 7243–7252.32 Maple JR, Hwang MJ, Jalkanen KJ, Stockfisch TP &Hagler AT (1998) Derivation of Class II force fields. 5.Quantum force field for amides, peptides, and relatedcompounds. J Comput Chem 19, 430–458.Stability of micro-myoglobin H F. Ji et al.96 FEBS Journal 275 (2008) 89–96 ª 2007 The Authors Journal compilation ª 2007 FEBS . the conformational stability of holo-lMb. Consistently, they are maintained over the 1.5 ns of the simulation (Fig. 2).A comparison of the hydrogen-bond. The effect of heme on the conformational stability of micro-myoglobin Hong-Fang Ji1,2, Liang Shen1,2, Rita
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