Báo cáo khoa học: Structural mobility of the monomeric C-terminal domain of the HIV-1 capsid protein pptx

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Structural mobility of the monomeric C-terminal domainof the HIV-1 capsid proteinLuis A. Alcaraz1,*, Marta del A´lamo2, Mauricio G. Mateu2and Jose´L. Neira1,3,*1 Instituto de Biologı´a Molecular y Celular, Universidad Miguel Herna´ndez, Elche (Alicante), Spain2 Centro de Biologı´a Molecular ‘Severo Ochoa’ (CSIC-UAM), Universidad Auto´noma de Madrid, Spain3 Biocomputation and Complex Systems Physics Institute, Zaragoza, SpainDynamic processes in proteins contribute toward defin-ing their structure and function, including protein fold-ing, association and ligand binding [1]. The mainchallenge in all structural and dynamic studies is tofind a relationship between the structural and mobilityresults, as well as protein function. Recent advances inisotopic labelling techniques [2] and NMR spectros-copy [3] have raised interest in protein dynamicsas provided by heteronuclear relaxation measurements[4–6]. Relaxation of the particular backbone amide15N provides details of rotational tumbling, and themovement of the internal N–H bonds [3] allows con-clusions to be drawn on the redistribution of confor-mational entropy upon folding and ⁄ or binding [1].The structural retroviral polyprotein (Gag) ofHIV-1 forms the immature capsid, and is subse-quently cleaved by the viral protease into severalmature proteins: the matrix, the capsid protein ofHIV-1 (p24) (CA), the nucleocapsid and p6, as wellas the spacer peptides p2 and p1 [7–9]. After proteo-lytic cleavage of Gag, CA reassembles to form themature capsid [10]. In vitro, CA spontaneouslyassembles into cylindrical structures and cones resem-bling the viral capsid [11–15]. Dimerization throughits C-terminal domain (CAC) is a driving force invirus assembly [14–17]. Recent studies of the maturecapsid lattice have shown that CAC connectsthrough homodimerization the CA hexamers, whichKeywordsflexibility; human immunodeficiency virus;NMR; structureCorrespondenceJ. L. Neira, Instituto de Biologı´a Molecular yCelular, Edificio Torregaita´n, UniversidadMiguel Herna´ndez, Avenida del Ferrocarrils ⁄ n, 03202 Elche (Alicante), SpainFax: +34 966 658 758Tel: +34 966 658 459E-mail: jlneira@umh.es*These authors contributed equally to thiswork(Received 4 February 2008, revised 22 April2008, accepted 24 April 2008)doi:10.1111/j.1742-4658.2008.06478.xThe capsid protein of HIV-1 (p24) (CA) forms the mature capsid of thehuman immunodeficiency virus. Capsid assembly involves hexamerizationof the N-terminal domain and dimerization of the C-terminal domain ofCA (CAC), and both domains constitute potential targets for anti-HIVtherapy. CAC homodimerization occurs mainly through its second helix,and it is abolished when its sole tryptophan is mutated to alanine. Thismutant, CACW40A, resembles a transient monomeric intermediate formedduring dimerization. Its tertiary structure is similar to that of the subunitsin the dimeric, non-mutated CAC, but the segment corresponding to thesecond helix samples different conformations. The present study comprisesa comprehensive examination of the CACW40A internal dynamics. Theresults obtained, with movements sampling a wide time regime (from pico-to milliseconds), demonstrate the high flexibility of the whole monomericprotein. The conformational exchange phenomena on the micro-to-milli-second time scale suggest a role for internal motions in the monomer–monomer interactions and, thus, flexibility of the polypeptide chain is likelyto contribute to the ability of the protein to adopt different conformationalstates, depending on the biological environment.AbbreviationsCA, capsid protein of HIV-1 (p24); CAC, C-terminal domain of CA, comprising residues 146–231 of the intact protein; CACW40A, mutant ofCAC with Ala instead of Trp at position 184 of CA; CSA, chemical shift anisotropy; Gag, the structural retroviral polyprotein of retroviruses;NOE, nuclear Overhauser effect.FEBS Journal 275 (2008) 3299–3311 ª 2008 The Authors Journal compilation ª 2008 FEBS 3299form the mature capsid, and also interacts with theCA N-terminal domain [18].The CA of HIV-1 is formed by two independentlyfolded domains separated by a flexible linker [19–22].The N-terminal domain (residues 1–146 of the intactprotein) is composed of five coiled-coil a-helices, withtwo additional short a-helices following an extendedproline-rich loop [19–21]. The CAC domain (residues147–231) is a dimer both in solution and in the crystalform [22,23]. Each CAC monomer is composed of ashort 310-helix followed by a strand and four a-helices:a-helix 1 (residues 160–172), a-helix 2 (residues 178–191), a-helix 3 (residues 195–202) and a-helix 4 (resi-dues 209–114), which are connected by short loops orturn-like structures. The dimerization interface isformed by the mutual docking of a-helix 2 from eachmonomer, with the side chains of each tryptophan(Trp184) deeply buried in the dimer interface [22,23].Our previous folding equilibrium analyses indicate thatthe monomeric CAC mutant Trp184Ala, CACW40A,resembles a transient monomeric intermediate formedduring dimerization [24,25]. In the present study, forsake of clarity, the mutant is referred to as CACW40Ato denote the position of the mutation in the C-termi-nal domain; in addition, the amino acids ofCACW40A are numbered from its first residue (i.e. theadded N-terminal methionine is Met1, and the secondresidue is Ser2, which corresponds to Ser146 in thenumbering of the intact CA). The CACW40A proteinis monomeric, and its structure is similar to that of thesubunits in the dimeric, non-mutated CAC, but, inthe monomeric form, the segment corresponding to thesecond helix samples different conformations [26](Fig. 1). At the end of this region, several hydrophobicresidues are buried and, as a consequence, the last twohelices are rotated compared to their position indimeric non-mutated CAC. Thus, from a structuralpoint of view, only the dimerization interface hassubstantially changed.To determine whether the apparent dynamic charac-ter of this region is shown by other polypeptidepatches, we have studied the dynamics of monomericCACW40A. Flexibility is often associated with inter-faces, and it is well known that complex formation(either in an oligomer or in a more simple substrate–enzyme reaction) can lead to conformational anddynamic changes at some, if not all, of the residuesinvolved [27]. In our previous description of the struc-ture of CACW40A, we observed a high flexibility inthe region involved in the dimerization interface (asconcluded from the absence of signals in the HSQC)[26]. In addition, millisecond-to-second dynamics wasaddressed by following the hydrogen-exchange behav-iour. In the present study, we have advanced a stepfurther and describe the pico-to-millisecond dynamics.The present study aims to ascertain whether there areregions within the CACW40A that exhibit particularhigh flexibility (i.e. whether the region comprising thedimerization interface in the non-mutated CAC is notthe sole highly mobile region). This would indicate alower energy barrier to structural rearrangementsthroughout the whole structure. The results obtainedindicate not only that the dimerization interface dis-plays a high flexibility, but also that the rest of theprotein is affected by movements on the pico-to-milli-second time regime. This mobility, as shown by thedimeric non-mutated CAC, is important in the viruscycle, as confirmed by structural studies of CAC in thepresence of various molecules and agents [28–31].ResultsRelaxation measurements of CACW40AMean R1(= 1 ⁄ T1, the longitudinal relaxation rate)was 2.95 s)1(range 1.49–3.69) (Fig. 2A) (see supple-mentary Table S1). Residues in the first a -helix pre-sented a mean of 2.90 s)1(range 2.56–3.69); the seconda-helix presented a mean of 3.06 s)1(range 2.79–3.15);the third a-helix presented a mean of 2.78 s)1(range2.26–3.05); and, finally, amino acids in the loop regionpresented a mean of 3.18 s)1(range 2.91–3.47). ThereFig. 1. Structure of CACW40A. UCSF CHIMERA software was used torender the model from the 2JO0 Protein Data Bank depositedstructure: the first a-helix is in blue; the second one in green; andthe last a-helix is shown in yellow. The single turn of a 310-helix atthe N-terminus of the protein is shown in red.Dynamics of monomeric CAC L. A. Alcaraz et al.3300 FEBS Journal 275 (2008) 3299–3311 ª 2008 The Authors Journal compilation ª 2008 FEBSwas no clear correlation between the elements of sec-ondary structure and the values of R1. Similar findingshave been found in proteins of similar size at the samemagnetic field, such as eglin c [32,33], CI2 [34], and theGAL4 domain [35,36].Mean R2(= 1 ⁄ T2, the transversal relaxation rates)was 11.9 s)1(range 6.3–14.7) (Fig. 2B) (see supplemen-tary Table S1). Residues in the first a-helix presented amean of 12.3 s)1(range 9.1–14.0); the second a-helixpresented a mean of 13.2 s)1(range 11.9–14.2); thethird a-helix presented a mean of 11.3 s)1(range 8.19–13.5); and, finally, amino acids in the loop regionpresented a mean of 12.3 s)1(range 9.2–14.7). As withR1, there was no clear correlation between the elementsof secondary structure and the values of R2. However,it is interesting to note that the values of R2inCACW40A were clearly higher than those of other pro-teins of similar size measured at the same magnetic field(eglin c, CI2 or GAL4 with average values of 5.6, 6 and8s)1, respectively [32–36]; GAL4 is the most disorderedprotein, and thus shows the highest values of R2).The mean of the nuclear Overhauser effect (NOE) inCACW40A was 0.60 (range 0.28–0.87) (Fig. 2C; seealso supplementary Table S1). This mean is lower thanthe value of 0.79 expected from theoretical consider-ations at a field strength of 11.7 T [37]. These results(together with those of the R2described above) suggesta high flexibility of the whole backbone of CACW40A;interestingly, a study of dynamics of the C-terminalregion of dimeric CAC also shows low NOE values[38], and extensive signal broadening has been observedin the assignment of dimeric non-mutated CAC [31].The residues with low NOE values (< 0.65) inCACW40A were Ile9 (at the C-cap of the 310-helix);Tyr20 (at the beginning of the first helix); Lys26 andAla30 (at the C-cap of the first helix); Val37 (in the mid-dle of the long disordered loop); Thr44, Val47 andGln48 (at the long disordered loop); Lys55, Thr56, Ile57and Leu58 (at the second helix); Ala60, Gly62 (in thetype II b-turn); Leu67 and Met71 (in the second helix);and Gly78 and Gly81 (at the C terminus of the protein).For the different regions, the first a-helix presented amean of 0.73 (range 0.52–0.94); the second a-helix pre-sented a mean of 0.68 (range 0.60–0.85); and the thirda-helix presented a mean of 0.70 (range 0.61–0.90).These data suggest that the second and third heliceswere slightly more mobile than the first one, which agreequalitatively with the last two helices showing a higherrmsd than the rest of the elements of the secondarystructure [26]. The NOE values of CACW40A were,however, lower than those found in other helicalregions of well-ordered proteins of similar size, such asCI2 and eglin c (within the range 0.7–0.8) [32–34], butthey were slightly higher than the values observed infully unfolded proteins (within the range 0.0–0.3)[36,39–41].Next, we decided to use the model-free formalism[42,43] to obtain further insight into the apparentinternal mobility of the protein. However, the overalltumbling time of CACW40A, sm, must be estimatedfirst.Estimation of the overall tumbling timeWe used two different experimental approaches toestimate the smto avoid any potential error in thedetermination of the model-free parameters.Fig. 2. Relaxation rates of CACW40A. The relaxation rates areshown for (A) R1, (B) R2and (C)15N-1H NOE for CACW40A at11.7 T. Sample conditions were 293 K, pH 7.0 in 0.1M phosphatebuffer. The cylinders at the top of each panel indicate the threea-helices.L. A. Alcaraz et al. Dynamics of monomeric CACFEBS Journal 275 (2008) 3299–3311 ª 2008 The Authors Journal compilation ª 2008 FEBS 3301We first estimated the smwith tensor2, by using thesubset of rigid residues (see Experimental procedures),yielding a value of 6.4 ± 0.1 ns.The smwas also determined by using the approachdeveloped by Wagner et al. [35,36]. Briefly, thismethod assumes that, if the re-orientation of an inter-nuclear15N-1H vector is a composite function of non-correlated motions, then the corresponding spectraldensity functions can be described as a linear combina-tion of spectral density terms characterizing eachmotion (usually two Lorentzian curves). This assump-tion leads to a third degree equation in s, one of whosesolutions is the sm:2ax2Ns3þ5bx2Ns2þ2 a À 1ðÞs þ 5b ¼ 0where the coefficients of the cubic equation, a and b,are obtained from the coefficients of the linear regres-sion of the experimental J(xN) (i.e. the spectral densityfunction at the Larmor frequency of the15N) versusJ(0) (i.e. the spectral density function at 0 MHz)(Fig. 3). In CACW40A, the positive solutions to thecubic equation lead to 1.28 ± 0.03 ns and7.6 ± 0.6 ns. The first root is assigned to an internalmotion of the protein, and the second is the overalltumbling of the molecule, which is close to the valueobtained previously. As can be observed, only a smallnumber of the experimental points in CACW40A areclose to the crossing point, demarcating the smbound-ary of the theoretical Lorentzian curve for the spectraldensity function. Experimental points close to theboundary imposed by the theoretical curve correspondto residues with fast internal dynamic contributions,whereas those undergoing slower dynamics are locatedat J(0) values above the limit of the correlation time,as occurs in CACW40A (Fig. 3).We also used different theoretical approaches toestimate the sm[44,45], and the results are similar tothose described above (data not shown). The valueused in the model-free formalism (see below) was6.4 ± 0.1 ns. It is important to indicate that relaxationmeasurements of the dimeric, non-mutated CAC havebeen carried out, and the smobtained is much higherthan that reported here [46].Model-free formalismIn CACW40A, the residues with high S2(the orderparameter) values (S2> 0.8) were: Arg18, Asp19,Val21, Arg23, Phe24, Tyr25 and Ly26 (all of thembelonging to the first helix); Asn51 and Cys54 (at theN-cap of the second helix); Ala64 and Ala65 (in theb-turn between the second and third helices); and Thr72and Ala73 (at the C-cap of the third helix) (Fig. 4A).The first a-helix is the secondary structure element thathas the highest number of residues with high S2values.Thus, the high S2values cluster at the regions of well-defined secondary structure with a lower rmsd [26].On the other hand, CACW40A has a large numberof residues with low values of S2, suggesting that thoseresidues are affected by fast movements (relative tosm). The mean ± SD of S2in CACW40A is0.56 ± 0.29 (see supplementary Table S2). This num-ber is significantly lower than the average value of 0.86found in other proteins [47], probably due to the longloop in CACW40A, which is not very well hydrogen-bonded to the rest of the structure [48].None of the residues in CACW40A, except Ala65,could be fitted to the simplest model of tensor2 (seesupplementary Table S2). Residues Glu15, Lys26,Gly62, Ala73 and Gly81 could be fitted to the secondmodel. Amino acids Phe17, Asp19, Arg23 and Gly79could be analysed with the third one, where anexchange contribution, Rex,is included. ResiduesGln11, Thr42 and Thr72 were fitted to the fifth model;and the remaining residues could be analysed accord-ing to the fourth model, where Rexcontributions andfast movements are included. A large number of resi-dues (i.e. those fitted to models three and four) didexperience conformational exchange on a micro-to-millisecond time scale (Fig. 4B).In conclusion, most of the residues in CACW40A,and not only those in the loop region, have a fastinternal mobility. Furthermore, the fast internal corre-Fig. 3. Relationship between J(xN) and J(0). The theoretical varia-tion between both parameters assuming a simple Lorentzian curvefor the spectral density function is also shown. Experimental data(filled squares) were fit to a linear function (y = a + bx) with:a = 0.43 ± 0.04 nsÆrad)1and b = 0.05 ± 0.01 nsÆrad)1, which areused in the third degree equation in s (for details, see text). Bothfunctions intersect at points corresponding to the overall correlationtime (sm) and an internal-motion time (se).Dynamics of monomeric CAC L. A. Alcaraz et al.3302 FEBS Journal 275 (2008) 3299–3311 ª 2008 The Authors Journal compilation ª 2008 FEBSlation time, se, for the majority of amino acids wassimilar to the sm(see supplementary Table S2). Itcould be assumed that those fast sevalues are due to awrong election of the diffusion tensor (e.g. the diffu-sion tensor of CACW40A is fully anisotropic) becauseit is well-known that simplified isotropic models inwhich anisotropy is neglected can wrongly lead toexchange terms [49]. However, similar values of S2, seand Rexto those reported in the supplementary(Table S2) were observed when a fully anisotropicmodel was used (data not shown). All these findingssuggest that the assumptions of the model-freeapproach are no longer valid in CACW40A (i.e. it isnot possible to separate the overall tumbling of themolecule and the local fast movements of each15N-1Hbond). Thus, although the model-free approach is veryintuitive, we decided to use the reduced spectral inten-sity formalism to test whether our results (i.e. largemobility through all the elements of structure) werenot an artifact of the model-free approach.Reduced spectral density approachThis approach provides insights into the motion of theN–H bond vector at three selected frequencies, x0(= 0), xNand 0.87xH(Fig. 5).As in other proteins [32,33,35,36], the J(0) (i.e. thespectral density function at the frequency 0) had thelargest samplings of the three explored frequencies.The J(0) showed a mean of 3.25 nsÆrad)1(range 1.7–4.2 nsÆrad)1) (Fig. 5A; see also supplementaryTable S3). The J(0) is a sensitive probe of the nano-to-milliseconds motion (i.e. very sensitive to the distri-bution of correlation times): low J(0) values indicateenhanced internal mobility on times scales faster thanthe sm. The regions with the lowest values of J(0) inCACW40A were clustered to: (a) the termini of thehelices and (b) the polypeptide patches in between(Fig. 5A). However, it should be noted that J(0) con-tains not only information on the nanosecond motionsfaster than the overall tumbling of the molecules, butalso on the exchange contributions [because it relies onR2; see Eqn (2) in Experimental procedures], whichincrease J(0). In general, values of J(0) above the meanvalue (3.25 nsÆrad)1) are good candidates for showingenhanced mobility in the millisecond time scale. Acomparison of Tables S2 and S3 in the supplementarymaterial shows that all residues with J(0) values higherthan 3.2 ns did show a Rexcontribution in the model-free approach. These residues were Gly12, Lys14,Phe17, Asp19, Tyr20, Val21, Arg23, Tyr24, Thr27,Glu31, Val37, Met41, Thr44, Gln48, Asn49, Ala50,Asp53 to Leu58, Leu67, Met70, Met71 and Gln75.Because J(xN) (i.e. the spectral density function atthe Larmor frequency of the15N) and J(0.87xH) (i.e.the spectral density function at the 0.87 times the Lar-mor frequency of the1H) are independent of R2[seeEqns (3,4) in Experimental procedures] and less sensi-tive than J(0) to the distribution of correlation times,they can provide insights into protein dynamics. Themean value of J(xN) was 0.58 nsÆrad)1(range0.28–0.76 nsÆrad)1) (see supplementary Table S3). Thelowest values of J(xN) belong to residues involved inthe polypeptide patches between the helices, and thehighest ones correspond to the rigid regions. Thevalues of J(0.87xH) were very low and only accountedFig. 4. The model-free approach parame-ters. (A) The order parameter, S2, is shownon the structure of the protein: 0.8 < S2<1(red); 0.6 < S2< 0.8 (orange);0.4 < S2< 0.6 (green) and 0 < S2< 0.4(blue). (B) Residues that show an Rextermare shown on the structure of the protein:10 < Rex<16s)1(red); 5 < Rex<10s)1(orange) and 0 < Rex<5s)1(blue).L. A. Alcaraz et al. Dynamics of monomeric CACFEBS Journal 275 (2008) 3299–3311 ª 2008 The Authors Journal compilation ª 2008 FEBS 3303for a 1% of J(0) (Fig. 5B). The mean value was0.0138 nsÆrad)1(range 0.00138–0.0245) (see supplemen-tary Table S3). The tendency in J(0.87xH) was theopposite to that observed in J(0): the highest values inJ(0.87xH) correspond to the termini of the helices andthe regions in between, indicating efficient picosecondaveraging.In conclusion, using the reduced spectral densityapproach, analysis of the relaxation parameters showsthat the regions between helices are highly mobile, butalso the rest of the structure has a high flexibility (inqualitative agreement with the model-free formalism);the three helices appeared rigid but they showedmobility in the pico-to-nanosecond time scale. Further-more, from the high J(0) values, there was evidence ofenhanced mobility in the millisecond time regime inresidues involved in the protein core and forming thelast two helices, which showed Rexand ⁄ or long sevalues (i.e. within the same order of magnitude thansm) in the model-free formalism (see supplementaryTables S2 and S3). Thus, both approaches qualitativelyagree in demonstrating a high internal flexibility of themolecule.DiscussionWe first discuss the results obtained within the frame-work provided by the structural elements of mono-meric CACW40A. Subsequently, we examine thebiological and thermodynamical implications of such ahigh flexibility.Backbone dynamics and the relationshipto structure in CACW40AOne of the possible uses of15N backbone dynamics isto predict regions of a protein with sufficient potentialflexibility to allow functional events to occur (binding,conformational changes or catalysis). However, experi-ments with several dozens of proteins [27] demonstratethat there is no easy and general correspondencebetween the order parameter (S2), the spectral densityfunction [J(x)] and the secondary structural elementsof a protein. Furthermore, there are no simple rulesfor the interpretation of the exchange rates (Rex)orthe different correlation times (sm, ssor sf).In CACW40A, although the helical elements havethe highest order parameters, there is no relationshipbetween S2and the location of structural elements(Fig. 4). Furthermore, the Rexterms are distributedthroughout the 3D structure of the protein, and mostof them are large (Fig. 4); the exception is Tyr25, withan Rexvalue of 0.5, which indicates that the dynamicsof its15N backbone nuclei is not robustly identified bythe used calculation protocol [50]. Thus, it appearsthat the whole protein is experiencing the same type ofmovements, ranging from pico- to milliseconds.Furthermore, there is no correlation between themotions measured by Rexand the motions probed byhydrogen-exchange [26], where only the residuesinvolved in the helices are protected. For example, thefirst helix, which has the highest S2values and is rela-tively well-ordered in the pico-to-nanosecond timescale, exhibits extensive ‘opening ⁄ closing’ equilibria onFig. 5. The reduced spectral density approach. Values of spectraldensity functions: (A) J(0), (B) J(xN) and (C) J(0.87xH) versus theprotein sequence. The cylinders at the top of each panel indicatethe a-helices.Dynamics of monomeric CAC L. A. Alcaraz et al.3304 FEBS Journal 275 (2008) 3299–3311 ª 2008 The Authors Journal compilation ª 2008 FEBSa much slower time regime than the other helices.These equilibria also occur in the other two helices, asshown by the exchange pattern [26], although they areless well-ordered, as judged by the lower S2.The types of movements and the residues involvedare described below.The pico-to-nanosecond dynamicsResidue Ala65 (at the N-terminus of the third helix) isthe sole residue that has restricted internal dynamics(model-free formalism). Fast internal dynamics (i.e.residues with at least another tumbling time) occurs atthe N (Gln11 and Glu15) and at the C-termini of thefirst a-helix (Lys26); in the long disordered loop(Thr42); and at the N- (Gly62, Ala64), and C-terminiof the third helix (Thr72, Ala73). However, it is notpossible to establish any correlation between anystructural parameter of those residues and the fastdynamics observed.The micro-to-millisecond dynamicsMost of the residues in CACW40A required an Rexterm (model-free formalism) or had long J(0) values(reduced-spectral approach); furthermore, most of theresidues in the loop (which forms the second helix inthe dimeric non-mutated CAC protein [22,23]) werebroad beyond detection in the HSQC experiments [26].Although the arguments could be considered as specu-lative, the highest Rexvalues observed in some aminoacids of CACW40A (see supplementary Table S2)might be ascribed to the proximity of the particularresidue to either aromatic or Cys residues, as describedin other proteins [37,50,51]. Residues Val37, Met41and Thr44 belong to the long disordered loop [26],buried within the structure, but only the amide protonof Thr44 is hydrogen-bonded. We do not know howto ascribe the exchange contribution of Val37 andMet41 to any particular dynamic process. In otherproteins, similar micro-to-milliseconds exchange contri-butions have been observed in well-buried protons,and they have been explained as due to buried watermolecules [37]. Finally, it is important to note that notonly were residues belonging to the second helix absentin the NMR spectra of CACW40A, but also they didnot appear in the spectrum of the dimeric wild-typeprotein [29,31], nor did they appear under physiologi-cal conditions in the NMR spectrum of anotherrecently reported monomeric mutant [52]. These find-ings suggest that the reported flexibility in the domainis not a particular characteristic of the mutant, but isan intrinsic feature of the whole dimeric CAC domain.Model-free analysis versus spectral densitymappingOur results indicate that the relaxation data ofCACW40A could not be satisfactorily explained by themodel-free method. In this formalism, the correlationfunction (the function describing the movement) of eachbond vector is decomposed as the product of the corre-lation function for overall (global) and internal (local)motions (i.e. the internal motions of the bond vectorsare independent of the overall rotational movement ofthe molecule). Furthermore, the internal motions ofeach bond vector are independent of each other, but therotational diffusion of the molecule affects each of thosebond vectors identically [42,43]. On the other hand,spectral density mapping makes no assumptions aboutthe nature of the rotational diffusion (i.e. the informa-tion on which oscillations for a particular bond vectorare associated with global molecular rotation or segmen-tal molecular motions is lost). Thus, based on the spec-tral density formalism results, we are unable to discernwhether the movement of each NH bond is due to localinternal or overall tumbling, but we can conclude thatthe CACW40A has an intrinsically high structural mobi-lity (Figs 4 and 5). To support this conclusion, the sesobtained from the model-free approach for most of theresidues are similar (i.e. they are not faster) than theoverall molecular tumbling of the protein; this meansthat we cannot strictly separate the overall tumbling ofthe molecule from the internal motions of each bondvector and, thus, the model-free formalism cannot be rig-orously applied. This is not the sole example where theuse of the model-free formalism has been unsuccessful:this approach cannot be applied on natively unfoldedproteins, proteins at high temperatures [27,39,53–55], or,even recently, in otherwise well-behaving proteins [56].Biological and thermodynamic implicationsOur study on the dynamics of CACW40A indicates thatthe protein is structurally very flexible, while preservingmost of the native scaffold [26]. It could be assumed thatthis flexibility is due exclusively to the mutation; how-ever, although the mutation increases the flexibility(because the quaternary structure is lost), the high flexi-bility is present in the structure of CAC, as suggested byseveral studies. First, similar dynamic results have beenobserved for the C-terminal region of dimeric, non-mutated CAC [38], and in residues belonging to itsdimerization interface [29,31]. Second, it has beenobserved that: (a) CAC is able to form swappeddomains involving the major homology region and thesecond a-helix [28,57]; (b) CAC is able to bind a peptideL. A. Alcaraz et al. Dynamics of monomeric CACFEBS Journal 275 (2008) 3299–3311 ª 2008 The Authors Journal compilation ª 2008 FEBS 3305forming a five-helical bundle [29]; (c) the second andthird helix in CAC appear to be distorted upon bindingto lipids [30]; and (d) the fourth helix in CAC is involvedin binding to lysyl-tRNA synthetase [31]. Thus, thesestudies show that the CAC domain is able to alter itsstructure and promote other interactions in the presenceof an external agent (lipids, peptides, other regions ofthe Gag protein, or even other proteins). In the firstthree examples, the second helix (as in CACW40A) wasthe main element of secondary structure affected; in thelast example, the fourth helix was the element altered.The detection of slow dynamics not only at the dimer-ization interface (residues Glu31 to Ala40), but also inthe rest of the protein implies the presence of a smallpopulation of pre-existing conformers within the native-state ensemble. This population interacts with otherCACW40A monomers forming the dimeric CAC, prob-ably through the side chains of the hydrophobic residuesof the long disordered loop, buried to avoid nonspecifichydrophobic interactions [26]. There are several exam-ples of proteins in which binding residues are involvedin slow-exchange processes [27,58], most likely to facili-tate rapid partner-binding, and the recognition ofseveral ligands. Internal motions allow amino acids toexplore large regions of the conformational space at avery low energetic cost, increasing the chances ofsuccessful binding. However, are those slow-exchangeprocesses responsible, from a thermodynamic point ofview, for the binding of the monomeric species of CAC?We have previously discussed the variation in the freeenergy of binding as a function of the changes in buriedsurface area upon dimer formation [59]. On the otherhand, there are no clear correlations between theenthalpy of binding and the changes in buried surfacearea [60]; thus, the only thermodynamic magnitude thathas not been estimated in CAC is the binding entropychange, DSb. The binding entropy, DSb, can be dividedinto terms defining the solvent (hydrophobic) (DSsol),the conformational flexibility (DScon) and the rotation-translation portion (DSrt) entropies: DSb= DSsol+DScon+ DSrt. The DSrtaccounts for )50 calÆmol)1ÆK)1[61,62]. The solvent portion of the entropy can be calcu-lated as a function of changes in polar and apolarsurface areas of the binding interface, according to:DSsol= DCpln(T ⁄ 385), where DCpis the heat capacitychange of the binding reaction. We have previouslydetermined the DCp()211 ± 10 calÆmol)1ÆK)1permonomer) and DSb()230 ± 10 calÆmol)1ÆK)1permonomer) [59], and then, the contribution from theconformational flexibility to the entire entropy ofbinding will be: DScon= )234 calÆmol)1ÆK)1per mono-mer. Because, on average, the entropy cost per aminoacid for a folding transition is approximately5.6 calÆmol)1ÆK)1[63], the estimated DSconin CAC uponbinding of the two monomers is due to the cost of fixing42 residues. This value is much higher than the numberof residues present in the long loop, which is disorderedin CACW40A (14 residues), and the difference must beassociated with: (a) the movements of the last twohelices, as observed in the monomeric structure of CAC,and (b) the inherent flexibility for the majority of theresidues. Thus, the conformational entropy appears tobe distributed through the whole structure of the mono-meric species, sampling a wider range of dynamic move-ments, and not only located at the residues in theinterface. In summary, we suggest that the inherent flexi-bility of the CAC domain is consistent with the presenceof a low thermodynamic barrier to diverse, template-assisted conformational changes, that allow interactionwith several macromolecules.Experimental proceduresMaterialsDeuterium oxide was obtained from Apollo Scientific(Bredbury Stockport, UK), and the sodium trimethylsilyl[2,2,3,3-2H4] propionate was obtained from Sigma (Madrid,Spain). Dialysis tubing was obtained from Spectrapore(Breda, the Netherlands), with a molecular mass cut-offof 3500 Da. Standard suppliers were used for all otherchemicals. Water was deionized and purified on a Millipore(Barcelona, Spain) system.Protein expression and purificationThe15N-labelled CACW40A protein was expressed inEscherichia coli BL21(DE3) in LB and purified as previ-ously described [26]; the DNA segment used for the mutantprotein encoded for residues 146–231 of CA from HIV-1(strain BH10) and was cloned as described [24]. The proteinconcentration was calculated from A240by using the extinc-tion coefficients of amino acids [64]. Samples were concen-trated at the desired final NMR concentration by usingCentriprep Amicon devices (Millipore), with a molecularmass cut-off of 3500 Da.Protein structure calculationsThe determination of the solvent-accessible surface areawas obtained using the VADAR web server [65].NMR samplesAll NMR experiments were acquired on an Avance BrukerDRX-500 spectrometer (Bruker, Karlsruhe, Germany)Dynamics of monomeric CAC L. A. Alcaraz et al.3306 FEBS Journal 275 (2008) 3299–3311 ª 2008 The Authors Journal compilation ª 2008 FEBSequipped with a triple resonance probe and pulse fieldgradients. Sample temperature was calibrated using a100% methanol standard [66].NMR relaxation measurementsNMR relaxation data were collected at 293 K.15N-T1,15N-T2and1H-15N NOE experiments were acquired usingenhanced sensitivity, gradient pulse sequences developed byFarrow et al. [67]. All spectra were recorded as 128 · 2Kcomplex matrices with 64 scans per F1experiment. Spectralwidths of 1650 and 8000 Hz were used in F1and F2respec-tively.A total of 10 data sets were acquired to obtain15N-T1ratesusing relaxation delays of 50, 100 (· 2), 200, 300, 400, 500,600, 700 (· 2), 850 and 1000 ms, where the experiments at100 and 700 ms were repeated twice. The15N-T2measure-ments were made using delays of 15, 25 (· 2), 50, 100, 150,175, 225 (· 2), 300 and 425 ms. For the T1and T2pulsesequences, the delay between transients was 5 s. The1H-15NNOEs were measured by recording interleaved spectra in thepresence and in the absence of proton saturation. The spec-trum recorded in the presence of proton saturation wasacquired with a saturation time of 5 s. The spectrumrecorded without proton saturation incorporated a relaxa-tion delay of 5 s. Each experiment was repeated twice.Experiments were carried out at two protein concentra-tions (1 mm and 400 lm) to rule out any possible concen-tration-dependent effect on the measured relaxation rates,as has been observed in dimeric non-mutated CAC [46].The measured rates were identical at both concentrationswithin the experimental error (see supplementary Table S1).Data processing and analysis of the NMRrelaxation measurementsThe spectra were zero-filled in the F1dimension four timesand processed by using a shifted sine window function. Thesame window function was used through all the T1and T2experiments. Cross-peaks intensities were measured asvolumes, with the xwinnmr software package (Bruker).The T1and T2values were determined by fitting themeasured peak-heights to a two-parameter function:IðtÞ¼I0expðÀt=T1;2Þ; ð1Þwhere I(t) is the peak intensity after a delay t and I0is theintensity at zero time; errors in the relaxation rates werecalculated from fitting to Eqn (1). The data were fitted toEqn (1) with kaleidagraph software (Abelbeck Software,Reading, PA, USA).The steady-state NOE values were determined from theratios of the peak intensities with and without proton satu-ration (i.e. NOE = Isat⁄ Inonsat). The standard deviation ofthe NOE value was determined on the basis of the measuredbackground noise levels by using the repeated experiments.The T1and T2relaxation times (or, R1=1⁄ T1andR2=1⁄ T2) and the NOE enhancement of an amide15Nnucleus are dominated by the dipolar interaction of the15Nnucleus with its attached proton and by the chemical shiftanisotropy (CSA). The energy of the CSA and the dipolarinteraction has a constant value over all the ensemble of spins[68]. The spectral density function, J(x), expresses how thisenergy is distributed over all the spectrum of possible fre-quencies, x, explored by the spins. The measured rates foreach NH are related to the J(x) at the nuclear spin frequen-cies [68], and they can be approximated as (the so-called‘reduced spectral density mapping approach’) [32,33,69]:Jð0Þ¼ð6R2À3R1À2:72rNHÞ=ð3d2þ4c2Þ; ð2ÞJðxNÞ¼ð4R1À5rNHÞ=ð3d2þ4c2Þ; ð3ÞJð0:87xNÞ¼ð4rNHÞ=ð5d2Þ; ð4ÞandrNH¼ R1ðNOE À 1ÞðcN=cHÞ; ð5Þwhere c =(xN⁄Ö3)(r||– r^) and d = l0hcNcH⁄ (8p2<r > 3), l0is the permeability constant of the free space, cNand cHare the gyromagnetic ratios of15N()2.71 · 107radÆs)1ÆT)1) and1H (2.68 · 108radÆs)1ÆT)1), his the Planck constant, xNis the Larmor frequency of the15N, xHis the Larmor frequency of the1H, <r> is thelength of the amide bond vector (1.02 A˚), and r||and r^are the parallel and perpendicular components of the CSAtensor (r||)r^= )160 p.p.m for a backbone amide [70]).The uncertainties in a particular J(x) are the quadrature-weighted sum derived from Eqns (2–5), assuming thaterrors in the relaxation rate constants are independent.Rotational diffusion tensorAn initial estimation of smand the rotational diffusion ten-sors were obtained with tensor2 [71], from the subset ofresidues which accomplished the following criteria [72]: (a)all residues should have a NOE ‡ 0.65 and (b) the residuesshould satisfy:R2;iÀR2hiR2hiÀR1;iÀR1hiR1hi<1:5rwhere <Rj> and <Rj,i> (where j = 1,2 and i is the residuenumber) are, respectively, the average rates and the indi-vidual R1and R2rates of the subset of remaining residuessatisfying criteria (a), and r is the standard deviation of:R2;iÀR2hiR2hiÀR1;iÀR1hiR1hiThe residues which did not accomplish criterion (a) wereIle9, Lys26, Ala30, Val37, Thr44, Val47, Gln48, Lys55,Thr56, Ile57, Leu58, Ala60, Gly62, Leu67, Met71, Gly78L. A. Alcaraz et al. Dynamics of monomeric CACFEBS Journal 275 (2008) 3299–3311 ª 2008 The Authors Journal compilation ª 2008 FEBS 3307and Gly81; and those which did not satisfy criterion (b)were Gln11, Glu15, Asp19 and Thr42 (see supplementaryTable S1). Furthermore, the cross-peaks of Asp22, Leu28,Gln32, Lys38, Glu43, Leu45, Leu46, Leu61, Thr66, Cys74and Val77 overlapped and they were not used in thedynamic analysis. Thus, a total number of 39 residues wereused to estimate the smand the rotational diffusion tensors.The determination of the tumbling of CACW40A wascarried out with tensor2. The rotational diffusion in theisotropic, axially symmetric or anisotropic schemes wasexplored by using 1000 Monte Carlo steps. Briefly, F-testanalysis was performed to choose between isotropic, axiallysymmetric and fully anisotropic diffusion models. A proba-bility factor of 0.2, which indicates whether the probabilityof improvement in different fits when complexity increasesis coincidental, was calculated for: (a) an isotropic and axi-ally symmetric pair of models and (b) an axially symmetricand a fully anisotropic pair of models. In both cases, theexperimentally determined F-value was lower than that at0.2 of probability, indicating that either the axially symmet-ric and the fully anisotropic model did not improve statisti-cally the fitting. Thus, CACW40A showed isotropictumbling.The model-free approachIn the Lipari–Szabo model-free formalism [42,43], J(x)isdefined in terms of: (a) the overall tumbling time, sm(in theorder of nanoseconds), and the diffusion anisotropy; (b) thetime scale of internal motions faster than sm, the so-calledeffective internal correlation time, se(in the pico-to-nano-second time scale); and (c) the degree of restriction of thesefast internal motions (which is measured by the square ofthe order parameter, S2). Thus, in residues where the relax-ation mechanism is dominated by the internal motion (i.e.residues highly mobile relative to the overall rotationaltumbling), S2would approach to zero; on the other hand,in residues where relaxation is described only by the globalmotion of the molecule, S2would approach to the unity.Extensions of this formalism have been developed to incor-porate two time scales of internal motions or to accountfor the effects of slow (micro-to-millisecond time scale) con-formational exchange; in these cases, the global orderparameter is defined as S2= S2fS2s, where S2fand S2sarethe order parameters for faster and slower motions,respectively.The calculations of the S2and separameters were carriedout using tensor2, with a Monte Carlo simulation of 1000steps. The program models the internal dynamics of each15N–H bond for which R1, R2and NOE parameters areavailable, with five different models [71,72]: (a) in the firstmodel, the seof each amide proton is very fast and notrelaxation-active; (b) in the second model, the seis relaxa-tion-active; (c) the third model is identical to the first,except the conformational (or chemical) exchange on amicrosecond-to-millisecond time scale is taken into account(by using the Rexparameter); (d) the fourth model is identi-cal to (b), but also includes the Rexterm; and (e) the fifthmodel includes the extension of the formalism, withtwo kinds of internal motions: one very fast and othervery slow.AcknowledgementsWe thank the two anonymous reviewers for their help-ful suggestions and discussions. This work was sup-ported by grants from Ministerio de Sanidad yConsumo (MSC) (FIS 01 ⁄ 0004-02), Ministerio de Edu-cacio´n y Ciencia (MEC) (CTQ2005-00360⁄ BQU) andthe private organization FIPSE (Exp: 36557⁄06) toJ. L. N.; grants from MSC (FIS 01 ⁄ 0004-01) andMEC (BIO2006-00793) and the private organizationFIPSE (Exp: 36557 ⁄ 06) to M. G. M., and by institu-tional grants from Fundacio´n Ramo´n Areces to theCentro de Biologı´a Molecular ‘Severo Ochoa’. We sin-cerely thank May Garcı´a, Marı´a del Carmen Fuster,Javier Casanova and Olga Ruiz de los Pan˜os for theirexcellent technical assistance.References1 Stone MJ (2001) NMR relaxation studies of the role ofconformational entropy in protein stability and ligandbinding. Acc Chem Res 34, 379–388.2 LeMaster DM (1994) Isotope labelling in solution pro-tein assignment and structural analysis. Prog NMRSpectrosc 26, 371–419.3 Kay LE (1998) Protein dynamics from NMR. NatStruct Biol 5, 513–517.4 Ishima R & Torchia DA (2000) Protein dynamics fromNMR. Nat Struct Biol 7, 740–743.5 Fischer MWF, Majumdar A & Zuiderweg ERP (1998)Protein NMR relaxation: theory, applications and out-look. 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Solution structure and backbone dynamics of the pleckstrin homology domain of the human protein kinease B (PKB ⁄ Akt) Interactions with inositol phosphates J Biomol NMR 28, 137–155 52 Wong HC, Shin R & Krishan NR (2008) Solution structure of a double mutant of the carboxy-terminal 3310 53 54 55 56 57 58 59 60 61 62 63 64 65 dimerization domain of the HIV-1 capsid protein Biochemistry 47, 2289–2297 Viles... 104, 4353–4358 Ternois F, Stitch J, Duquerroy S, Krausslich H-G & ¨ Rey FA (2005) The HIV-1 capsid protein C-terminal domain in complex with a virus assembly inhibitor Nat Struct Mol Biol 12, 678–682 ´ ´ Barrera FN, Hurtado-Gomez E, Lidon-Moya MC & Neira JL (2006) Binding of the C-terminal domain of the HIV-1 capsid protein to lipid membranes: a biophysical characterization Biochem J 394, 345–353 Kovaleski... supplementary Table S1) Furthermore, the cross-peaks of Asp22, Leu28, Gln32, Lys38, Glu43, Leu45, Leu46, Leu61, Thr66, Cys74 and Val77 overlapped and they were not used in the dynamic analysis Thus, a total number of 39 residues were used to estimate the sm and the rotational diffusion tensors The determination of the tumbling of CACW40A was carried out with tensor2 The rotational diffusion in the isotropic, axially... RK, Lee BM, Walker J, Summers MF, Yoo S & Sundquist WI (1996) Structure of the amino-terminal core domain of the HIV-1 capsid protein Science 273, 231–235 21 Gamble TR, Vajdos FF, Yoo S, Worthylake DK, Houseweart M, Sundquist WI & Hill CP (1996) Crystal structure of human cyclophilin A bound to the aminoterminal domain of HIV-1 capsid Cell 87, 1285–1294 22 Gamble TR, Yoo S, Vajdos FF, von Schwedler... dominated by the internal motion (i.e residues highly mobile relative to the overall rotational tumbling), S2 would approach to zero; on the other hand, in residues where relaxation is described only by the global motion of the molecule, S2 would approach to the unity Extensions of this formalism have been developed to incorporate two time scales of internal motions or to account for the effects of slow . Structural mobility of the monomeric C-terminal domain of the HIV-1 capsid protein Luis A. Alcaraz1,*, Marta del. 2008)doi:10.1111/j.1742-4658.2008.06478.x The capsid protein of HIV-1 (p24) (CA) forms the mature capsid of the human immunodeficiency virus. Capsid assembly involves hexamerization of the N-terminal
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Xem thêm: Báo cáo khoa học: Structural mobility of the monomeric C-terminal domain of the HIV-1 capsid protein pptx, Báo cáo khoa học: Structural mobility of the monomeric C-terminal domain of the HIV-1 capsid protein pptx, Báo cáo khoa học: Structural mobility of the monomeric C-terminal domain of the HIV-1 capsid protein pptx