Larger Helical Populations in Peptides Derived from the Dimerization

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Larger Helical Populations in Peptides Derived from the Dimerization Helix of the Capsid Protein of HIV-1 Results in Peptide Binding toward Regions Other than the “Hotspot” Interface Rosa Dom
enech,† Rebeca Bocanegra,‡ Rosario Gonz
alez-Mu~niz,§ Javier G
omez,† Mauricio G. Mateu,‡ and Jos
e L. Neira*,†,|| †

Instituto de Biología Molecular y Celular, Universidad Miguel Hern
andez, Elche (Alicante), Spain Centro de Biología Molecular Severo Ochoa (CSIC-UAM), Universidad Aut
onoma de Madrid, Cantoblanco Madrid, Spain § Instituto de Química-M
edica (IQM-CSIC), Madrid, Spain Instituto de Biocomputaci
on y Física de Sistemas Complejos, Zaragoza, Spain

)

‡

bS Supporting Information ABSTRACT: The C-terminal domain (CTD) of the capsid protein (CA) of HIV-1 participates both in the formation of CA hexamers and in the joining of hexamers through homodimerization to form the viral capsid. Intact CA and the CTD are able to homodimerize with similar affinity (∼15 μM); CTD homodimerization involves mainly an R-helical region. We have designed peptides derived from that helix with predicted higher helical propensities than the wild-type sequence while keeping residues important for dimerization. These peptides showed a higher helicity than that of the wild-type peptide, although not as high as theoretically predicted, and proved to be able to self-associate with apparent affinities similar to that of the whole CTD. However, binding to CTD mainly occurs at the last helical region of the protein. Accordingly, most of those peptides are unable to inhibit CA polymerization in vitro. Therefore, there is a subtle tuning between monomer
monomer interactions important for CTD dimerization and the maximal helical content achieved by the wild-type sequence of the interface.

’ INTRODUCTION Inhibition of protein
protein interactions provides new avenues for therapeutic intervention. This inhibition might be usually achieved with a small ligand (peptide or organic molecule). However, such an attractive idea has proved to be challenging, probably because of the relatively large protein
protein interfaces that make it difficult for a small molecule to encompass the whole binding site.1,2 Achieving high enough affinity and specificity between the inhibitor molecule and the protein surface is further hampered by entropic penalties. However, a few specifically designed, small molecules have been shown to disrupt such large and complex interfaces by binding to energetic “hotspots”.2,3 Virus capsid proteins provide biomedically relevant targets for the design of protein
protein interfacial inhibitors based on the fact that: (i) they tend to be conserved even in highly variable viruses that mutate extensively; (ii) they are essential for virus assembly; and (iii) some of them are able to self-assemble in well-controlled assays in vitro. Therefore, by targeting protein
protein interactions for virion assembly and maturation, it may be possible to design new antiviral strategies based on the inhibition of those macromolecular complexes.4 Human immunodeficiency virus (HIV), a retrovirus, is the agent responsible for AIDS. Assembly of the immature HIV type r 2011 American Chemical Society

1 (HIV-1) capsid occurs through the controlled polymerization of the viral Gag-polyprotein, which is transported to the plasma membrane of infected cells, where morphogenesis of the immature, noninfectious virion, occurs. Immediately after budding of the immature virion, maturation is initiated by the cleavage of the Gag protein by a viral protease, which yields the MA (matrix), CA (capsid), NC (nucleocapsid), and p6 proteins as well as two spacer peptides. This maturation process induces conformational changes in CA, which reassembles as an independent protein to form a capsid with a distinctive conical shape.4,5 Because of its critical role during HIV-1 morphogenesis, CA has emerged as a promising target for the development of new anti-HIV drugs based on disruption of capsid assembly,6,7 and a similar approach has been proposed for fighting other viruses. (See Strosberg’s review8 and references therein.) The CA protein is formed by two independently folded domains: the N-terminal domain (NTD) and the C-terminal one (CTD) separated by a flexible linker.9
12 The NTD (residues 1
146 in the numbering of the whole intact protein) is composed of Received: May 27, 2011 Revised: July 1, 2011 Published: July 15, 2011 3252

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Figure 1. Crystal structure of the wild-type CTD dimer. The monomers are depicted in the same color (blue), and the dimerization helix (R-helix 9) is highlighted (gold). The Figure was produced using Pymol (http://www.pymol.org)54 by using the Protein Data Bank file for CTD (accession no. 1A43).10 The different R-helices with the numbering of the whole intact CA are indicated.

five coiled-coil R-helices (corresponding to helices 1
5 of CA), with two additional short R-helices (helices 6 and 7) following an extended proline-rich loop. The CTD domain (residues 147
231) is a dimer both in solution and in the crystal form.10,13 Each CTD monomer is composed of a short 310-helix, followed by a strand and four R-helices (helices 8
11): R-helix 8 (residues 160
172), R-helix 9 (residues 178
191), R-helix 10 (residues 195
202), and R-helix 11 (residues 209
214); these helices are connected by short loops or turn-like structures (Figure 1). At the beginning of CTD, there is a polypeptide region (Asp152Leu172), called the major homology region (MHR), which is highly conserved among retroviruses and whose functions have not been fully elucidated. The dimerization interface of CTD is mainly formed by the mutual docking of R-helix 9 from each monomer, with the side chains of each tryptophan (Trp184) deeply buried in the dimer interface. The two additional aromatic residues in each CTD monomer, Tyr164 and Tyr169, are located in the hydrophobic core of each monomer, far away from the dimer interface. The dissociation constant of CTD is 10 ( 3 μM, similar to that of intact CA, 18.0 ( 0.6 μM;10,14 therefore, the CA dimerization interface is fully contained within the isolated CTD. This observation has paved the way for the search of inhibitors of HIV-1 assembly. Molecules found able to dissociate CTD dimers in solution may be later subjected to further assays for activity as inhibitors of CA assembly and HIV-1 infectivity. The first molecules specifically targeted against the self-association of CTD or even the CTD-NTD interface (which is also important during capsid assembly5) have involved: (i) peptides mimicking the sequence of the dimerization interface15 (the so-called CAC-1); (ii) peptides selected from a combinatorial library by phage display;16 (iii) peptides based on those selected by phage display techniques, which have been further redesigned to increase the helical content;17,18 and, recently, (iv) dendrimers with a large hydrophobic moiety.19 The latter stapled peptide is able to penetrate cells and hampers HIV-1 infectivity by increasing the helical content of the original phage-display-selected peptide, although it does not abolish CTD dimerization completely.17 In this work, we go a step further by testing the CTD binding affinities of short peptides derived from the central part of helix 9 comprising a

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substantial part of the residues found energetically important for CTD dimerization.20 In addition, these peptides have been designed to show an increased helical content with respect to the wild-type helical region. The results with the most helical peptides (as predicted by the program AGADIR21,22) indicate that: (i) the peptides selfassociate with apparent affinities similar to that of whole CTD;10 (ii) the peptides interact with CTD with an affinity similar to that found for CTD dimerization (∼15 μM10); (iii) the peptide
protein interface in all peptides, but one, does not involve the dimerization interface of the protein (helix 9); and, as a consequence, (iv) only one peptide (that showing contacts with helix 9 in CTD) is able to inhibit CA polymerization in vitro to some extent. Therefore, protein dimerization cannot be hampered only by increasing the helical content, while keeping the energetically important dimerization residues. We conclude that there is a subtle tuning between monomer
monomer interactions important for CTD dimerization and the maximal helical content achieved by the wild-type sequence of the dimerization interface.

’ EXPERIMENTAL PROCEDURES Materials. Deuterium oxide was obtained from Apollo Scientific (Stockport, U.K.), and the sodium trimethylsilyl [2,2,3,3-2H4] propionate (TSP), HPLC-grade methanol, HPLC-grade 1,1,1,3,3,3hexafluoro-2-propanol (HFIP), and 2,2,2-trifluoroethanol (TFE) were from Sigma (St. Louis, MO). Dialysis tubing with a molecular weight cutoff of 3500 Da was from Spectrapor (Spectrum Laboratories, Ibaraki, Japan). Standard suppliers were used for all other chemicals. Water was deionized and purified on a Millipore system (Millipore, MA). Design and Synthesis of the Peptides. Our starting point was the CAC-1 peptide with the sequence Ac-EQASQEVKNWMTETLLVQNA-NH2, where energetically important residues for dimerization (namely, Val181, Trp184, Met185, and Leu189) are highlighted in bold.20 Therefore, if we wanted to design peptides able to associate with CTD, those residues should be kept in the design. We used the program AGADIR21,22 to quantify the helical content of the wild-type CAC-1. AGADIR predicted a 4.86% of helical structure in aqueous solution, in agreement with the experimental far-UV CD and 1D-NMR results, which suggest that the peptide is mainly disordered15 (Table S1 of the Supporting Information). Next, we started calculating the percentages of helical structure by mutating each of the 20 residues at any of the positions of CAC-1, except at Val181, Trp184, Met185, and Leu189. In parallel, because CAC-1 has a high tendency to aggregate, we tried to reduce the length of the peptide to keep charged residues, decreasing the number of hydrophobic ones. It is important to indicate that Thr188 and Val191 are involved in monomer
monomer contacts in the dimerization interface, but they are not energetically important for dimerization.20 After several cycles of testing the percentages of helicity for the shortest versions of the peptides (Table S1 of the Supporting Information), we arrived to four peptides, which showed wide differences in the predicted helicity. The best of all peptides had a percentage of helicity close to 50% (Table 1) and was obtained by introducing a tyrosine at position 188 (T188Y) and an arginine at position 191 (V191R); the high increase in helicity is probably due to a stacking stabilizing interaction between the two aromatic rings, Trp184 and Tyr188 (located at the same face 3253

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Table 1. Sequences, Molecular Masses, Self-Association Properties, and Percentage of Helical Structure of the Peptidesa % of helical

% of helical structure

% of helical structure

structure (from molar ellipticity

in aqueous solution (from TFE-titrations)

[TFE]50%

D  106 (cm2 s
1)

T2-relaxation time (ms)

KSD

(M)

(Rh, A)d

(molecular mass, kDa)

(μM)e

peptide

MW

(AGADIR)

at 222 nm)b

(ΔGw, kcal mol
1)c

VKNWMTETLLRQ

1517.80

13.90

8.4

12.1 ( 0.8 (1.2 ( 0.6)

1522.82

24.80

5.7

VKNWMTEYLLRQ (P-3)

1579.87

52.88

VKNWMTETLLVQ

1460.75

4.59

17 ( 3

2.2 ( 0.1 (8.4)

102.5 (3.9)

3.8 ( 3.5

0.6 ( 0.2f (4 ( 1)

23 ( 1

1.5 ( 0.1 (12.2)

127.4 (3.1)

36 ( 31

8.2

6.1 ( 0.5 (1.6 ( 0.5)

15 ( 1

1.98 ( 0.02 (9.3)

122.5 (3.3)

5.2 ( 4.1

6.9

1.4 ( 0.8 (2.5 ( 0.3) 20.4 ( 0.8

1.93 ( 0.02 (9.5)

122.5 (3.3)

3.6 ( 0.7

(P-1) VKNWMTEYLLVQ (P-2)

(P-4)

P-4 is the wild-type sequence. b These values were calculated from the molar ellipticty at 222 nm for each of the peptides44 at a concentration of 15 μM. Value within brackets is the free energy of the equilibrium U T F, where U is the peptide conformation in aqueous solution and F is the folded conformation in the presence of TFE.27,28 d Rh values were determined as described.37 Errors in the measurements of D are standard deviations to three different measurements with new samples. e Dissociation constants are for the equilibium M2 T 2 M, as reported from changes in the ellipticity at 222 nm. The value for P-2 was obtained from fitting the ellipticity at 208 nm. Errors are fitting errors to eq 3. f Far-UV CD spectra at different [TFE] did not show an isodichroic wavelength. a c

of the putative helix), and a C-cap helix effect at the C terminus of the putative helix. The Thr-to-Tyr mutation was expected to decrease solubility. The peptide with the second highest helicity keeps the wild-type Val191, and the reduction in the predicted helicity was dramatic (Table 1). Conversely, in this peptide, the Val-to-Arg mutation should increase its solubility. Finally, the peptide with the third highest helicity does keep Thr188 but includes the substitution Val191 to Arg. As a control, we also studied the wild-type peptide to test whether: (i) the residues removed at the N and C termini (when compared with CAC-1) affected the binding ability to CTD; and (ii) the new short design increased the solubility of the original CAC-1 peptide. Solubility of the peptides should follow the order P-1 > P-3 > P-4 > P-2, just the same order as the experimentally determined helicity (fifth column of Table 1). A peptide, derived from pea chloroplast fructose-1,6-biphosphatase and able to bind thioredoxin m from pea (Ac-ESLPDYGDDSDDNTLGTEEQRSIVNVSQ-NH2) was used as a control in the binding to CTD as monitored by HSQC-NMR experiments. Peptides were produced and purified by Genscript corporation (Piscataway, NJ), and they were acetylated and amidated; peptide purity was checked by mass spectrometry and in our own laboratory by gel filtration chromatography. Protein Expression and Purification. The wild-type CTD protein was purified as described.23 The 15N-labeled CTDW184A monomeric mutant protein was obtained by using Bioexpress medium (Cambridge Isotope Laboratories, Andover, MA) using the same protocol described.24 The intact CA protein was expressed and purified as described.25 Protein stocks were run in SDS-PAGE gels and found to be >97% pure. Protein concentration was determined from the absorbance of individual amino acids.26 Fluorescence. Spectra were collected on a Cary Eclipse spectrofluorometer (Varian, Santa Clara, CA) interfaced with a Peltier system. A 1 cm path length quartz cell (Hellma) was used. Experiments were performed at 25
C in phosphate buffer at pH 7.0 (50 mM). Steady State Fluorescence Measurements. Spectra were acquired by excitation at 280 or 295 nm; the emission fluorescence

was collected between 300 and 400 nm. The excitation and emission slits were set to 5 nm, and the response was 1 nm. Binding experiments to CTD were carried out as described.15 In brief, increasing amounts of P-1 to P-4, in the range 0.5
70 μM, were added to a solution of a fixed concentration of wild-type CTD (1.1 μM) in 50 mM sodium phosphate buffer (pH 7.0), and the fluorescence was measured after overnight incubation at 25
C. The apparent dissociation constant of the CTD-peptide complex, KD, was calculated by fitting the fluorescence change versus the concentration of added CTD to Fmeas ¼ F þ ΔFmax ðð½Pi þ ½CTD þ KD Þ qffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi
ð½Pi þ ½CTD þ KD Þ2
4½CTD½PiÞ

ð1Þ

where [Pi] is the concentration of the corresponding peptide, [CTD] is the concentration of CTD, Fmeas is the measured fluorescence intensity at each concentration of peptide, ΔFmax is the half of the change in the fluorescence measured when all CTD is forming the complex CTD-peptide, and F is the fluorescence of the peptide. Circular Dichroism. Spectra were collected on a Jasco J810 (Madrid, Spain) spectropolarimeter with a Peltier unit. The instrument was periodically calibrated with (+)-10-camphorsulfonic acid. Spectra were acquired at 25
C in phosphate buffer at pH 7.0 (50 mM). Steady-state far-UV measurements were performed using equimolar amounts of wild-type CTD and the corresponding peptide (20 or 200 μM) in 0.1 cm path length quartz cells (Hellma). Steady-State Measurements. Spectra were acquired with a response time of 2 s and averaged over six scans, with a scan speed of 50 nm/min. The step resolution was 0.2 nm, and the bandwidth was 1 nm. Experiments to determine the self-association of the peptides were carried out by following the changes in ellipticity as the concentration of peptide was increased. The changes at a selected wavelength were fitted to the chemical equilibrium: 2 M T M2, where M is the monomeric peptide. We assumed that peptide association involved a dimeric species based in the T2-relaxation measurements (see below). At any given peptide concentration, 3254

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Biomacromolecules [Pi], the fraction of monomeric peptide, fM, is given by pffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi ð
KSD þ K2SD þ 8½PiKSD Þ fM ¼ 4½Pi

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ð2Þ

where KSD is the self-dissociation constant of the peptide. By taking into account the fact that the far-UV CD spectrum is the sum of the spectra of M and M2 species, it is easy to reach pffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi ð
KSD þ K2SD þ 8½PiKSD Þ CDmeas ¼ CD þ ΔCDmax 4½Pi ð3Þ where CDmeas is the ellipticity measured at any chosen wavelength, CD is the value of the ellipticity when the M2 species is fully formed (that is, at high peptide concentrations), and ΔCDmax is the change in the ellipticity between the low and high peptide concentrations. Experiments in the presence of TFE, methanol, and HFIP were carried out at 15 μM of the corresponding peptide because at this concentration the ellipticity at 222 nm in all peptides but P-2 did not show any additional titration (see below). We prepared samples containing increasing concentrations of TFE, methanol, or HFIP (from 0 to 80%); then, the resulting sigmoidal curves for TFE were fitted to a two-state model, as described.27,28 This procedure used to estimate the helical percentage of peptides is based on the assumption that peptide helical formation follows a two-state transition (which must be tested by confirming that all the far-UV CD spectra have an isodichroic wavelength27,28). This approach is similar to that used to estimate the free energy of unfolding of proteins in aqueous solution (that is, in the absence of denaturant): a titration followed by any spectroscopic technique is carried out by using different concentrations of chemical denaturants (urea or guanidinium hydrochloride) and then extrapolated back to zero denaturant concentration to obtain the percentage of unfolded protein.29 Thermal Denaturation. Thermal denaturations in the presence of 20 μM of wild-type dimeric CTD with 20 μM of each peptide were carried out at 60
C/h with a response of 8 s and by following the changes in ellipticity at 222 nm. All transitions were reversible. The isolated peptides did not show a sigmoidal transition when heated (data not shown). We further carried out thermal denaturations by using 20 μM monomeric mutant CTDW184A with 80 μM corresponding peptide. Isothermal Titration Calorimetry. ITC measurements were performed using a VP-ITC isothermal titration calorimeter (MicroCal, Northampton, MA) at 25
C in phosphate buffer 12 mM (pH 7.0). Sample cell (1.4 mL) was loaded with wild-type dimeric CTD at 40 μM (for experiments with the P-1 and P-3 peptides) and with 40 μM corresponding peptide (for experiments with peptides P-2 and P-4). The related peptide was loaded into the syringe (for experiments with the peptides P-1 and P-3) at a concentration of 400
800 μM. For experiments with peptides P-2 and P-4, the syringe was loaded with 560 μM CTD. This alternative experimental device was used because peptides P-2 and P-4 have a larger tendency to aggregate at high concentrations than P-1 and P-3. Twenty-eight injections of 10 μL were added sequentially to the sample cell after a 400 or 210 s spacing to ensure that the thermal power returned to the baseline before the next injection. The amount of power required to maintain the reaction cell at constant temperature after each injection was monitored as a function of time.

The isotherms (differential heat upon binding versus the molar ratio [peptide]/[wild-type CTD] (in the experiments for P-1 and P-3) and molar ratio [wild-type CTD]/[peptide] (in experiments for P-2 and P4)) were fitted to a single-site model (analogous to eq 1, see above) assuming that the CTDpeptide complex had a 1:1 stoichiometry. Data were analyzed with software developed in our laboratory, implemented in the software package Origin 7.0 (OriginLab). As control experiments, the individual dilution heats for the intact peptides (in experiments with P-1 and P-3) were determined under the same experimental conditions by carrying out identical injections of the corresponding peptide into the sample cell, which contained only buffer. Dilution experiments with CTD were carried out in a similar manner. Nuclear Magnetic Resonance Spectroscopy. The NMR experiments were acquired at 20
C (unless it is stated) on a Avance DRX-500 spectrometer (Bruker GmbH, Karlsruhe, Germany) equipped with a triple resonance probe and z-pulse field gradients. TSP was used as the internal chemical shift reference in the homonuclear experiments.15N chemical shifts of monomeric CTDW184A were referenced following the method of Wishart and coworkers.30 All experiments were carried out at pH 7.0, 50 mM phosphate buffer. Values of the pH reported here represent apparent values of pH without correction for isotope effects. Two-Dimensional 15N-HSQC-NMR Spectra. All spectra were acquired in the phase-sensitive mode. Frequency discrimination in the indirect dimensions was achieved by using the echo/ antiecho-TPPI method. The 2D-15N-HSQC experiments31 were acquired with 4 K data points in the 1H dimension and 200 scans in the 15N axis. The spectral widths were 15 and 35 ppm in the 1H and 15N dimensions, respectively. Water was suppressed with the WATERGATE sequence.32 The concentration of the CTDW184A protein was 120 μM in all cases, and the corresponding peptide concentration was four times higher. We also carried out a control experiment with P-3 (for which we had a larger amount) at a concentration five-times higher; the results indicated that the signals affected were the same as those at the lower concentration, although the changes were slightly larger (data not shown). We used the monomeric mutant CTDW184A protein instead of the wild-type dimeric CTD protein to avoid problems with signal broadening of the residues involved in the dimerization interface;14,18 thus, when using the monomeric CTDW184A we have followed the same reasoning as other authors mapping the peptide-binding regions of CTD.16,18 In turn, this will facilitate the comparison among the results with the different systems. Assignments of the CTDW184A in the HSQC spectra were those previously reported.24,33 To allow for a comparison among the peptides and to account for errors in protein concentration, signal intensities of each peak in the HSQC spectra were calculated by using XWINNMR software and calibrated with respect to the intensity of Leu231 (the C-terminal residue of CTD). A control experiment was carried out by acquiring an HSQC spectra of the mixture of 120 μM of wild-type CTD and 500 μM of the peptide derived from fructose-1-6-biphosphatase. Measurements of the T2 (Transverse Relaxation Time). Measurements of the T2 provide a convenient method to determine the molecular mass of a macromolecule, its complexes, or both. We measured the T2 of the peptides by using the 1
1 echo sequence (at echo times of 2.9 ms and 400 μs).34 Then, because the correlation time, τc, is τc ≈ 1/T235 and, roughly, the M is twice the τc, the mass of the biomolecule or its complexes can be estimated. In the experiments containing the sole peptide, the 3255

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Biomacromolecules concentration was 200 μM; in the experiments in the presence of CTDW184A, a peptide/protein concentration of 2:1 was used (with a protein concentration of 100 μM). The line-width of the sole indole residue (all peptides have the key tryptophan, Table 1) was measured in the echo experiments for the isolated peptides and the complexes. DOSY-NMR Experiments. NMR samples for DOSY-NMR spectra were prepared by dissolving the corresponding peptide in 100% D2O. The solution was briefly centrifuged to remove insoluble protein and then transferred to a 5 mm NMR tube. Final peptide concentrations were in the range of 100
150 μM. Translational self-diffusion measurements were performed with the pulsed-gradient spin
echo sequence, as described.36 The duration of the variable gradient was changed between 2.6 and 3 ms, and the time between both gradients was 150 ms. The most upfield shifted methyl groups (those between 0.5 and 1 ppm) were used to measure the intensity changes. The value of the Stokes radius, Rh, was determined as described by Dobson and coworkers,37 assuming that the Rh of dioxane is 2.12 Å. One-Dimensional NMR Experiments. NMR samples for 1DNMR experiments of the isolated peptides were acquired at 5
C and at 150 μM peptide concentration. Exchange experiments were carried out by dissolving lyophilized peptide in 100% D2O, pH 7.0 (50 mM phosphate buffer). Attempts to assign the peptides at 2 mM by using standard protocols38 failed probably because of peptide association; in fact, solutions of peptides P-2 and P-4 at high concentrations were viscous and with a gel-like appearance (as we had previously tested in the ITC experiments, see above), suggesting the presence of aggregation and higher order self-associated forms. For peptide P-3, the use of higher concentrations (1 mM) yielded viscous solutions, which resulted in broad NMR spectra (data not shown). We also tried to determine the self-associated state of the peptides at those NMR concentrations by using gel filtration chromatography in a Superdex G75 HR10/30 running on an AKTA FPLC (GE Healthcare, Barcelona, Spain) and following the absorbance at 280 nm in 150 mM NaCl, pH 7.0 (50 mM Tris buffer). We assayed P-3 (the peptide for which we had a larger amount) at concentrations of 40 and 800 μM; the peptide always eluted at volumes larger than the bed volume of the column (∼19 mL), with a strong “tailing” at the beginning of the peak, in the largest concentration used (data not shown). This similarity of elution volumes at the two concentrations explored is probably due to the dilution observed in the column matrix when the peptide is loaded, but the tailing observed at high concentrations might also suggest the presence of self-associated forms (which could interact with the column). Taken together, we can conclude that at the NMR concentrations the peptides have at tendency to form higher-order selfassociated forms. Kinetic Analysis of CA Polymerization in Vitro. Polymerization of recombinant CA into capsid-like structures requires extremely high concentrations of NaCl39,40 and high concentrations of a macromolecular crowding agent (such as Ficoll).25 The addition of high concentrations of both NaCl and Ficoll leads to a substantial increase in the amount of assembled capsids. Assembly reactions were carried out at 2.25 M NaCl pH 7.4 (50 mM, sodium phosphate buffer) and 20 μM CA in the presence of 100 g/L Ficoll 70 as a macromolecular crowding agent. A volume of CA solution at the appropriate concentration in phosphate buffer was introduced to a spectrophotometer cuvette (10 mm by 2 mm of internal section). The assembly reaction was triggered by adding a solution containing 4 M NaCl, 179 g/L

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Ficoll-70 in 50 mM phosphate buffer to get the final concentrations desired for each component. For inhibition assays, CA was mixed with the appropriate amounts of peptides and incubated for 30 min before triggering the reaction, as indicated above. The components in a final volume of 500 μL were rapidly mixed by repeated inversion of the cell. The pH of the final reaction mixture was checked in a test sample. The time-dependent increase in the optical density at 350 nm as a measure of the light scattered by the assembled particles was monitored at 25
C by using a Shimadzu UV-1603 spectrophotometer with data points collected every six seconds. Traces of the variation in the turbidity were analyzed as described.25 Under these conditions, the formation of tubular polymers resembling authentic mature HIV-1 capsids has been observed.25

’ RESULTS Peptides Have a Tendency to Self-Associate. We first measured the helical structure of the peptides in solution to confirm theoretical predictions by AGADIR. Next, we reasoned that if the peptides were good candidates to bind CTD, then they should have a tendency to self-associate, as has been shown in the CAC-1, which contains the entire R-helix 9 of CTD.15 The DOSY-NMR experiments with the isolated peptides suggest that their hydrodynamic radii, Rh, are ∼9 Å, with that of P-2 being the largest (Table 1). We can compare these experimental values with those theoretically predicted by using:37,41 Rh = 0.27 M0.50 (Å), where M is the molecular weight of the corresponding chain. This equation yields values of ∼10.5 Å for the four monomeric peptides and of ∼14.5 Å for possible dimeric species. That is, for P-1, P-3, and P-4, the experimental values (Table 1) are closer to those predicted for monomeric peptides, but the P-2 has an Rh value halfway between that of the monomeric and dimeric species. We can further elaborate on the meaning of the measured Rh values measured by calculating (i) the theoretical R according to the polymer theory; or (ii) the value of a freely jointed chain.42 The hallmark of random-coil behavior is the power-law relationship between polymer length and the radius of gyration (Rg): Rg = 1.927 N0.598, where N is the number of residues. However, the Rg is the root-mean-square distance of all atoms in the molecule from the protein center of mass, and Rh is the hydrodynamic radius of a sphere that has the same D as the protein molecule;42 thus, the relationship between both parameters is highly dependent on the molecular shape. Keeping in mind these limitations, we estimated that the Rg values of the peptides were close 8.5 Å, smaller than their experimental values (Table 1). For a freely jointed chain, the radius of gyration is42 ÆRg2æ = (l2N/6), where l is the link length set to 4 nm;43 this equation yields a value of 56.5 Å for ÆRg2æ1/2 in any of the monomeric peptides. These calculations suggest that (i) the peptides are not fully disordered; and probably (ii) the peptides in solution behave as a mixture of monomers and dimers. Because the calculations from DOSY-NMR measurements rely on the shape of the molecules (because we must calculate the Rh), we used an alternative approach. To that end, we carried out T2relaxation measurements, which allow us to conclude that the peptides are mainly forming dimeric species in solution (Table 1). We further confirmed the self-association of the peptides by carrying out far-UV CD experiments at different peptide concentrations. As the peptide concentration increased (Figure 2), the value of the ellipticity at 222 nm was increased (in absolute 3256

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Figure 2. Self-association properties of peptides. Titration curves describing the peptides self-association properties by reporting the changes in ellipticity at 222 nm as the concentration of (A) P-3 or (B) P-1 was raised.

value), indicating that self-association raised the helical content. Moreover, all CD spectra of peptides at the different concentrations (except those of P-2) showed an isodichroic wavelength, suggesting that the association equilibrium can be described by a two-state model. The peptide with the largest KSD was P-2, where the sigmoidal self-association curve was very flat (data not shown), probably because of the fact that the self-association reaction did not follow a two-state process. Finally, it is interesting to note that the maxima wavelength of the fluorescence spectra of the peptides were not modified as concentration was increased (as was also reported to occur in CAC-115), suggesting that the sole tryptophan was not completely buried upon selfassociation of the peptides. Taken together, the above results suggest that the four peptides self-associate; therefore, they are potential candidates to bind CTD. Peptides Do Not Show Large Helical Populations. Because we designed the peptides to have a larger helical population than the CAC-1, we wanted to test experimentally whether the theoretical predictions had worked. The far-UV CD and the NMR data suggest that the peptides, even at concentrations as high as ∼1 mM used in the 2D-NMR experiments (Experimental Procedures section), are mainly disordered with broad signals. Furthermore, the estimation of helical populations either by (i) TFE titrations or (ii) measuring the molar ellipticity at 222 nm at 15 μM of peptide44 indicates that the populations of ordered structures were lower than theoretically predicted (Table 1); the differences are largest in P-3,

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which was predicted to be the most helical (52%). Although there is not a quantitative agreement among the theoretical and the experimental percentages of helical structure, there is a qualitative one because the experimental helicity values (except in P-2) are higher in those predicted to be the most helical. These experimental populations are, in any case, small enough to allow for a direct and exact experimental detection by NMR, CD, or both. Furthermore, we believe that even in the presence of 40% TFE (where all TFE titrations have finished) the peptides are self-associating, because T2 measurements with P-1 and P-3 also showed a large molecular weight (data not shown). It could be thought that the percentages of helical structure in aqueous solution are overestimated (Table 1, fifth column) because TFE strengthens helical structure in proteins. However, TFE reinforces the population of helical structure only in those polypeptide patches with an intrinsic helical tendency or even a β-turn propensity.45,46 Because we have designed our peptides by using AGADIR, they must have an intrinsic tendency to adopt helical conformations, even though they self-associate; therefore, the addition of TFE must strengthen the intrinsic helical population. We believe that within the limitations of our approach27,28 TFE titrations allow an assessment of the helical populations in aqueous solution. In fact, the estimation of the helical population from TFE qualitatively agrees with that obtained by using the values of the molar ellipticty at 222 nm44 at 15 μM of peptide concentration (Table 1, fourth column). In this discussion, however, it is important to note that we have not considered that the addition of TFE could also induce higher-order peptide selfassociation at the higher concentrations of cosolvent; this additional equilibrium, which could be more important in P-2, could shift the helical formation. Notwithstanding the above data and to provide additional control experiments, we carried out titrations of the four peptides with methanol and HFIP (Figure 1 of the Supporting Information). As the concentration of cosolvent was increased, the ellipticities of the far-UV CD spectra at 208 and 222 nm were increased (in absolute value), suggesting the strengthening of helical populations in the peptides. The changes did follow a more sigmoidal-like shape for methanol than for HFIP; furthermore, at [HFIP] > 60% (v/v), precipitation was observed. In addition, no isodichroic wavelengths were observed for the far-UV spectra in the presence of methanol or HFIP probably because at the high cosolvent concentrations there are higher-order aggregated species of the peptides. These data suggest that the strengthening of helical structure upon addition of methanol or HFIP does not follow a two-state mechanism; therefore, we cannot extrapolate back to zero concentration of cosolvent to estimate the percentage of helical structure in aqueous solution. Measurement of the Affinity Constant of the Peptides for the Wild-Type, Dimeric CTD. We first determined qualitatively the binding of the peptides to CTD by using the same biophysical techniques as those in CAC-1.15 The Tm of the thermal denaturations for complexes for P-2 and P-4 decreased, suggesting that both peptides have a larger affinity for the denaturation state of CTD than for the folded native state (data not shown); alternatively, the decrease in the Tm might be also due to a lower stability of the CTD
peptide complex (due to partial unfolding of CTD upon peptide binding). The Tm of the thermal denaturations for complexes with P-1 and P-3 did not show any change (data not shown). Moreover, we did not observe any change in the far-UV spectra of the CTD
peptide complex when compared with those obtained by the addition of the spectra of the 3257

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corresponding isolated species (data not shown). These results might suggest that the stabilization induced by the peptides (and given by +RT ln(1 + [L]/KD)) was not large enough. Next, we tried to determine quantitatively the apparent affinity constants by using two complementary biophysical techniques, ITC and fluorescence (Table 2; Figure 3 and Figure 2 of the Supporting Information). Fluorescence values were obtained by using eq 1, and the ITC values were obtained by assuming a 1:1 stoichiometry. (See Experimental Procedures section.) Keeping in mind the different concentrations used of wild-type CTD Table 2. Apparent CTD-Binding Affinities of the Peptidesa ITC peptides VKNWMTETLLRQ (P-1) VKNWMTEYLLVQ (P-2)

a

fluorescence

KD (μM)

KD (μM)

3(1 25 ( 4

7(3 15 ( 8

VKNWMTEYLLRQ (P-3)

3(1

17 ( 7

VKNWMTETLLVQ (P-4)

15 ( 1

10 ( 4

Apparent constants were determined at 25
C. Errors are fitting errors.

(2 and 40 μM (or ∼500 μM) for fluorescence and ITC, respectively), we can conclude that the apparent KD values are similar within the experimental error. In P-2, the binding curve showed an almost lack of a pretransition baseline during the ITC measurements, which led to a large error in the determination of the KD (Figure 3C). Measured either by fluorescence or ITC, the values of the apparent KD were smaller than the value measured for CAC-1 (50 μM).15 Thus, shortening the peptide but increasing the helicity appears to increase the apparent affinity for CTD. In this stage, it is important to keep in mind that the figures in Table 2 are apparent KD values because they report the simultaneous equilibria: CTD
peptide dissociation, CTD dissociation, and the peptide self-association (because the peptides have a tendency to self-associate, Table 1). As we carried out two types of ITC experiments, because of the different solubility of the peptides, we tried several approaches to have an estimate of the real constant for the CTD
peptide dissociation. For the ITC experiments with peptides P-2 and P-4, in which CTD is injected from the syringe, we tried to determine from the CTD dilution experiments47 the values of the dissociation constant of CTD in the presence and in the absence of both peptides. However, the

Figure 3. Binding of CTD and the peptides as determined by fluorescence and ITC. Fluorescence titrations with 2 μM of CTD and the (A) P-1 and (B) P-3 peptides by following the changes in the intensities of the spectra at 315 nm. (Similar values in the dissociation constants were obtained by following the changes in fluorescence at other wavelengths.) (C) Binding of wild-type CTD to P-2 showing the CTD dilution experiment (top panel, green), the binding experiment (middle panel, blue), and the binding curve (lower panel, red). The molar ratio is [wild-type CTD]/[P-2]. 3258

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Table 3. Residues of CTDW184A Most Affected upon Peptide Additiona peptide protein region

VKNWMTETLLRQ (P-1)

VKNWMTEYLLVQ (P-2)

N terminus MHR (152
172),

Ala174,

(160
172)

Lys158

in-between loop

Arg154 (Ala228)

Arg154 (Ala228), Ala174

Arg173 Asn183, Ala184b, Met185

in-between loop helix 10 (195
202) in-between loop

VKNWMTETLLVQ (P-4)

Leu151 (Leu190) Arg154 (Ala228),

and R-helix 8

helix 9 (178
191)

VKNWMTEYLLRQ (P-3)

Val191

Asn183, Thr186, Thr188

Asn183

Lys203

Lys203

Gln192 Asp197 Lys203

Thr200, Ile201, Lys203, Ala204, Gly206

helix 11 (209
214)

Thr210, Glu212

Ala209, Leu211, Glu212, Met214

C terminus

Cys218

Leu211, Met214, Glu213 (Glu175, Glu176)

Cys218, Glu219, Val221

Ala217, Cys218

a Only residues whose changes were larger than that of the corresponding cross-peak in the spectrum of isolated CTDW184A are indicated. The different regions of the protein are those described by X-ray in the structure of each monomer in wild-type CTD.10 Residues such as Arg154, which have another residue partially overlapping with their HSQC signal, are indicated within parentheses. Spectra were acquired at 25
C. Residues in bold are those which are involved in monomer
monomer contacts10,13or energetically important for dimerization.20 b Mutated residue in CTDW184A.

values of the KD were similar under both conditions, suggesting that (i) the KD of CTD was not affected by the presence of the peptides (and, therefore, there was not binding); (ii) the peptides self-dissociate very slowly and then the self-dissociation reaction is the rate-limiting step; or (iii) the peptides bound with equal affinity to the dimeric and monomeric states of the CTD. Because the dilution curves of CTD in the presence or in the absence of P-2 or P-4 are different (blue and green lines in Figure 3C, and in Figure 2C of the Supporting Information), we favor the last explanation, which makes a rigorous analysis of the binding of P-2 or P-4 to CTD quite difficult. For P-1 and P-3, where because of their larger solubility they could be loaded into the syringe, dilution experiments of peptides into buffer did not yield a good estimate of their dissociation constants, probably because of (i) their small values, as suggested by the CD measurements (Table 1); or (ii) a very slow self-dissociation of the peptides. Therefore, we did not have an estimate of the real binding constants of the CTD
peptide complex. Then, if we wanted to obtain a proper estimate of the equilibrium constant of the reaction CTD-peptide T peptide + CTD, we had to rely on the values determined by CD (this work) or by other techniques for the equilibrium constants of the: (i) CTD2 T 2 CTD, which is ∼15 μM,10 and (ii) peptide2 T 2 peptide (Table 1). The use of both set of values leads to KD values for the reaction CTD-peptide T peptide + CTD of 3 to 8 μM, which are lower than those measured by ITC and fluorescence (Table 2). However, the calculation of such values was obtained under several assumptions. First, we have assumed that peptides self-associate to form dimers (as suggested by the T2 relaxation measurements), but we cannot rule out the presence of large self-associated species that are not observed in NMR because of signal broadening. And second, we have assumed that the peptides dissociate before binding to CTD, but self-associated species could also bind to CTD, as recently shown in stapled peptides.48 Thus, we can only indicate that the apparent KD values for the peptides designed in this work are smaller than those measured for CAC-1,15 but we cannot provide a real value of such constants under the approaches used because of the difficulties in

measuring the different thermodynamic constants for the concomitant equilibria. Determining the Peptide Binding Sites in CTD. The above biophysical probes have shown that there is binding between the peptides and the dimeric, wild-type CTD, but what is the region of the protein involved in binding? Is it the R-helix 9? We tried to map the binding site of peptides in CTD by using 2D 15N-HSQC spectra of monomeric CTDW184A. In this type of spectrum, main chain amide protons are observed with one signal for each amino acid. The use of the mutant monomeric protein will allow (i) an easier comparison with results of peptides which are bound to monomeric mutants of CTD16
18; and (ii) an easier assignment of the residues that are modified by the binding because the wild-type CTD protein shows severe signal broadening due to the dimerization equilibrium.14,18,32 Most of the peaks of CTDW184A did not change their chemical shifts upon peptide addition; however, there were several resonances whose intensities changed in the presence of peptides. The variation of the signal intensity for a backbone amide 1 H
15N cross-peak in the HSQC spectrum indicates that a population of CTD is engaged in peptide binding in a way where the amide group of this residue exhibits broader lines. Furthermore, the absence of changes in chemical shifts and a large signal broadening suggest that the exchange kinetics of the complex formation equilibria are intermediate-slow on the NMR time scale (as suggested by the experiments at higher concentrations with P-3; see the Experimental Procedures section); similar findings have been observed in other binding equilibria followed by NMR.49,50 Furthermore, we also hypothesize that if the peptide self-dissociation has intermediate-slow kinetics, then it could lead to broadening of the CTD signals upon binding. Moreover, although we did not observe large changes in the HSQC and in the far-UV CD spectra (data not shown), we cannot rule out the possibility of local conformational changes in CTD upon peptide binding. These changes could result in NMR signal broadening, as described in binding of self-associated stapled-peptides.48 Finally, it is interesting to note that the signals of the monomer of CTD do not change their chemical shifts upon dimer 3259

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Figure 4. Binding of the peptides to monomeric CTDW184A. The NMR structure of the monomeric mutant CTDW184A is indicated. The residues in sticks are those with the largest changes in the presence of the peptides: P-1 (A), P-2 (B), P-3 (C), and P-4 (D). The Figure was produced using Pymol (http://www.pymol.org)53 using the Protein Data Bank file for CTDW184A (accession no. 2JO0).24 The helix 9, which comprises the majority of the dimer interface in wild-type CTD, is not fully formed, most of the time, in the monomer, and the residues involved in R-helix 9 are shown in magenta (except those that are affected by peptide binding). Residues whose resonances overlap in the HSQC spectrum were not indicated (such as Arg154 and Ala228).

formation (that is, upon addition of the largest ligand (9 kDa): the own CTD, with a dissociation constant of ∼15 μM10), but rather their broadness was dramatically altered,14 wiping out most of the signals involved in the interface. Therefore, the differences in the behavior of the CTD signals upon binding to several partners must rely on the unusually high structural plasticity of the domain and how this flexibility is affected at different extent by the corresponding ligand. The control experiments with the peptide derived from fructose-1,6-biphosphatase did not show changes in any signal of the HSQC spectrum of CTD (data not shown), suggesting that the binding above was specific. Table 3 shows a peptide-by-peptide analysis, indicating the most affected peaks in the presence of the polypeptide. It is interesting to note that (i) P-1 showed the largest number of residues involved in dimerization, whose HSQC signals changed; and (ii) the rest of the peptides showed variations in the HSQC signals of residues mainly belonging to the C-terminal region of CTD. Residues that appear involved in the binding of each peptide are shown on the structure of the monomeric species of the protein24 (Figure 4). It could be thought that because ITC and fluorescence experiments were carried out with the wild-type dimeric protein and the NMR experiments were carried out with the monomeric CTDW184A mutant, the mode of binding could be different for both species. To rule out this hypothesis, we carried out two controls. First, we acquired HSQC experiments with a 15Nlabeled sample of wild-type CTD at 100 μM protein and 200 μM P-1. The residues affected were essentially the same as those in the monomeric protein; however, the overall quality of the spectrum with the wild-type protein was much worse than that of the monomeric CTDW184A because the signals were, in general,

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much broader, and those from residues involved in R-helix 9 were not observed. (Then, we lost the binding information provided by those residues.) And second, we carried out far-UV thermal denaturations with the monomeric CTDW184A mutant under the same conditions as those used in the wild-type dimeric protein; no variation in the Tm of the endotherm was observed in the presence of the corresponding peptide when compared with that of the isolated monomeric protein (that is, the same behavior observed in the dimeric protein, see above). Therefore, we can conclude that the mechanism of peptide binding is the same in the monomeric and dimeric CTD species. In summary, all peptides appear to bind CTD, further supporting the ITC and fluorescence results, but not all of them do so at residues involved in the dimerization helix. Testing the Inhibitory Activity of Peptides on an in Vitro Model for Assembly of the HIV-1 Capsid. From all short CTD derived peptides tested, only P-1 bound close to the CTD dimerization interface and showed signal variations in the largest number of residues involved in CTD dimerization. We have evaluated the possible inhibitory activity of P-1 (and that of the rest of the peptides, as an additional control of the above results) on CA polymerization in vitro.25,38 Under the conditions used, hollow tubular structures resembling mature HIV-1 have been observed by EM. The amount of CA polymers formed was estimated in turbidimetry assays, as implemented by several groups.5,6,25 The results revealed that as expected only P-1 was able to reproducibly and significantly inhibit CA polymerization (Figure 5); P-2, P-3, and P-4 did not inhibit CA polymerization, even at the highest peptide concentrations tested (up to ∼20-fold molar excess of peptide relative to CA) (Figure 5A); P-2 showed nonreproducible results, depending on the peptide concentration used. It is important to indicate that although P-1 did inhibit CA polymerization, this effect required a very high molar excess of peptide over CA. In addition, P-1 decreased the polymerization rate (by ∼35% in the presence of a 20-fold molar excess of P-1, Figure 5B) but did not reduce the overall amount of capsid-like particles formed. If peptide binding to CA under polymerization conditions is weaker than association between CA subunits, binding of free CA subunits to the growing capsid may competitively displace the bound peptide molecules. Under the conditions of the assay, CA depolymerization occurs only at a very slow rate, and thus displacement of bound CA subunits by the peptide may not readily occur. As a result, polymerization will be clearly delayed in the presence of the peptide but not prevented. This phenomenon has been observed also when using some other weak inhibitors of capsid assembly.19 Preliminary EM observations using peptides related to CAC-1 and P1 showed normally assembled capsid-like structures with no evidence of aberrant structures. We consider unlikely but cannot exclude that the particular peptide P1 led to some off-pathway aggregation of CA. However, even if some of the turbidity observed in the presence of P1 was due to unspecific CA aggregation, then this would argue for a more effective inhibitory action of this peptide, leading to the same conclusions.

’ DISCUSSION Predicted-to-Be Highly Populated Helical Peptides Have a High Tendency to Self-Associate and Remain Disordered Most of the Time . In this work, we have characterized four

short, variant peptides derived from the central part of helix 9 in CTD. The peptides were constructed to increase the helical 3260

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Figure 5. Analysis of the inhibitory activity of peptides on the in vitro assembly of the HIV-1 capsid. (A) Polymerization of CA under the conditions described in Experimental Section, either in the absence (black line) or in the presence of P-1 at a 16-fold molar excess over CA (red line), P-2 at a 19-fold molar excess (blue line), P-3 at a 23-fold molar excess (green line), or P-4 at a 15-fold molar excess (orange line). (B) Polymerization of CA in the absence (black line), or presence of P-1 at a 1-fold molar excess (orange line), 5-fold molar excess (blue line), 10-fold molar excess (green line), or a 20fold molar excess (red line).

propensity of peptide CAC-1, by both shortening the sequence and including amino acid substitutions while at the same time preserving a substantial part of the CTD residues that proved to be energetically important for CTD dimerization.20 The specific rationale behind the design of each of the three variant peptides (P-1, P-2, and P-3) was (i) to introduce an Arg at the position of Val191, to compensate the C-cap of the possible helical region, and to favor the formation of a salt bridge between Glu187 and Arg191; and (ii) to introduce a Tyr at position Thr188 to favor possible π
π interactions between the aromatic ring of the newly introduced tyrosine with the indole ring of Trp184. (However, we could also speculate that the new Tyr could interact with its neighbor Trp to stabilize the helix in the peptide but decrease the ability of the Trp in the peptide to interact with Trp184 in the CTD.) Although the peptides were predicted to be highly helical, far-UV CD spectra, the measurements of the molar ellipticity at 222 nm, the TFE-titration, and the 1D NMR experiments indicate that the peptides are most of time disordered (Table 1). We do not know the reason behind the discrepancies between the theoretical and the experimental results because AGADIR has worked well in other systems.21,22 We suspect that this is due to the fact that the peptides have a tendency to self-associate, which, although it favors helical formation (Figure 2) because of peptide-peptide interactions, could hamper the formation of a fixed regular helical structure. Alternatively, at very high peptide concentrations, the unspecific peptide self-association could shift the formation of a folded structure toward an oligomeric species with a lower percentage of helix. Nonetheless, the experimentally obtained percentage of helical structure in the substituted peptides P-1 and P-3 was, as predicted, higher than that in the nonsubstituted peptide (P-4). The results for P-2 could be underestimated due to the fact that the TFE titration did not follow a two-state mechanism (see above); in fact, determination of the helical population by an alternative procedure44 suggests that helicity in P-2 at 15 μM of peptide concentration is similar to that of P-4, and slightly lower than that of P-1 and P-3 (Table 1). In our own experience with other helical peptides, the low correlation of helical content in water with respect to the AGADIR predicted values is probably due to the low solubility of peptides. Normally, for this type of highly hydrophobic peptides, the higher the helical character, the higher the self-association; under these conditions, the helical content experimentally

measured is just due to the soluble nonaggregated fraction, which is lower when the aggregation increases. The peptides have a tendency to self-associate as the CAC-115 (which has the sequence of P-4 plus four residues on each termini), as shown by T2-relaxation and DOSY-NMR measurements (Table 1) and the far-UV CD spectra; furthermore, the T2-relaxation data suggest that the peptides have a tendency to form dimers (at least up to 200 μM because at higher concentrations viscous solutions were observed). The self-association constants were similar, within the fitting errors, for all of the peptides and interestingly enough also similar to that of the whole CTD domain (∼15 μM)10 (Table 1). This result suggests that the peptides could be, in principle, potential candidates to interfere with the dimerization of the whole CTD domain. Furthermore, it also suggests that helical formation and dimerization are not coupled. Peptide-Binding Does Not Occur, In Most Cases, At the Dimerization Interface of CTD. The peptides proved able to bind CTD, with affinity constants within the range of 10 μM (Table 2), which is similar to that measured for the intact CTD.10 The fact that the values measured by fluorescence or ITC are similar suggests that, even with the different protein concentrations used in both techniques, the same binding reaction is being monitored. We have shown above that the apparent estimated constant of the equilibrium complex formation (CTD-monomer-peptide T CTD-monomer + peptide) is 3
8 μM, which is similar to the values of the KSD described in Table 1 (last column). If we assume that peptides adopt a native-like helical conformation upon binding, then, the intrinsic binding constant, KD0, must be larger than the apparent KD estimated by calorimetry and fluorescence because the measured binding constant (Table 2) involves binding and folding; therefore, the structural rearrangement is supposed to penalize the binding energetics. If K = [folded]/[unfolded] is the conformational equilibrium constant for a conformational change coupled to ligand binding, then KD is then given by KD0/(1 + 1/K). Because we have an estimation of K (the fifth column in Table 1), then, assuming a KD in average of 15 μM (Table 2), the values of KD0 go from 1.5 μM in P-1 to 212 nM in P-2, with values of ∼20 nM for P-3 and P-4. These binding constants are in the range of the best described inhibitors of CTD assembly to date,16
18 but, as we have shown, not all peptides described in this work inhibit capsid assembly. Therefore, these estimations of the KD0 must be taken with caution. 3261

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Biomacromolecules Because the peptides could probably acquire a native-like conformation upon binding, is the epitope region of the protein the dimerization interface? The fluorescence studies suggest that the environment around Trp184 changes, which can be due to binding to the neighborhood of the indole moiety or binding to other polypeptide regions, which affect the tryptophan environment. The results of Table 3 and Figure 5 indicate that most of the residues involved in the helix 9 do not intervene in peptide binding, and thus the changes observed by fluorescence in most of the peptides (except in P-1, Table 3) are probably due to changes in the environment of Trp184 as a consequence of conformational changes transmitted along the polypeptide chain upon peptide binding. Previous structural studies on isolated wild-type dimeric CTD10,13 show that residues Thr148 and Ile150 (belonging to the 310 helix); Leu151, Asp152, and Arg154 (belonging to the MHR); Leu172, Glu175, Ala177, and Ser 178 (at helix 8); Glu180, Val181, Trp184, Met185, Glu1187, Thr188, Leu189, Val191, Gln192, and Asn193 (at the helix 9, the dimerization helix); Lys199 (helix 10); and Lys203 and Pro207 are involved in monomer
monomer contacts. Many of those residues are also energetically important for CTD dimerization,20 namely, Trp184, Met185, Leu189, Arg154, Leu151, Val181, Iel150, Leu172, and Glu175 (in decreasing order of energetic importance), and their importance in dimerization has also been addressed in molecular dynamic simulations.51 Interestingly, several of the residues of the dimerization interface of CTD are also involved in the contact with the peptides, specifically, residues Leu151, Arg154, Trp184 (the mutated residue in the CTDW184A), Met185, Thr188, and Lys203 (Table 3, Figure 5). However, residues at the C terminus of the dimerization helix, such as Glu187, Thr188, and Val191 (which are involved in the dimer interface), did not show changes in the HSQC spectra. Moreover, Leu189, which is one of the three energetically most important residues (after Trp184 and Met185) for CTD dimerization, is not involved in binding of any peptide. Therefore, whereas the contact epitope in CTD for binding of the peptides analyzed in this work does overlap with the contact and the energetic epitope for CTD homodimerization, the overlap is rather limited. We think this might be the reason why all but one of the peptides are not able to hamper significantly polymerization of CA. The only peptide that is able to inhibit to some extent CA polymerization is P-1; this peptide shows the largest number of contacts with residues involved in the CTD dimerization interface, or alternatively, residues energetically important for dimerization (residues in bold in Table 3). However, inhibition of capsid assembly by P-1 occurred only at high peptide concentrations, as expected from the observation that the P-1 binding region only partially overlaps with the CTD homodimerization interface, especially if the more restricted, energetic epitope and not the whole contact epitope is considered.20 Peptide P-4 was not able to hamper capsid assembly, although it contains the wild-type sequence. This behavior is probably due to the absence of other residues not energetically important for the interface, which, nevertheless, could be critical in directing the polypeptide chain toward the “hotspot” interface (see below). The peptides did establish a large number of contacts with the C-cap regions of the last two helices of CTD (Table 3). These regions are involved in the binding to lysyl-tRNA synthetase, especially Thr210.52 The peptides probably interact with those helices due to proximity at the C-terminal region of helix 9. However, it would be interesting to monitor whether the

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presence of the peptides is able to obstruct, to some extent (with apparent dissociation constants on the order of micrometers; see above), the interaction between CTD and the tRNA synthetase (which is on the order of nanometers). The peptides used in this work did include a substantial part of the CTD residues that are energetically important for CTD homodimerization; in addition, they were designed to increase the helical propensity. Moreover, one energetically very important residue, Glu180, was not included in the peptides; it might be that such a residue could act as an anchor point between the isolated helical peptide and the protein, favoring binding. Nevertheless, it could be argued that P-1 does not have Glu180 at its N terminus either and yet it is able to bind CTD and even hamper CA assembly to some extent (Figure 4). However, P-1 has an Arg at the C terminus, which could serve as a potential anchoring point to the negatively charged C terminus of helix 9 in CTD, overcoming the absence of the N-terminal region of such helix. The presence of this Arg in P-3 seems not to be essential to promote binding to the dimerization interface of CTD, and we do not know the exact reasons, although it is tempting to suggest that they might be due to the presence of the tyrosine in this peptide. The introduction of the mutation of Thr188 to Tyr might hamper to some extent packing of the peptide helical region against the well-formed helix 9 of CTD probably due to steric constraints (either because of the large volume of the tyrosine residue or to the interaction between the two aromatic residues within the peptide); although, the experimental increase in helical propensity for P-2 with respect to the P-1 sequence is insignificant (Table 1); then, the presence of the tyrosine residue does not seem to contribute noticeably to helical packing. Furthermore, the presence of Tyr188 (together with Val191) might be the reason why: (i) the TFE titration curves do not follow a two-state model in P-2 and (ii) the self-association curve, as detected by CD is very flat (data not shown), leading to a large error in the determination of the KSD. Interestingly enough, P-2 is the sole peptide with the largest number of contacts in the highly hydrophobic region comprising helices 10 and 11 (Table 3) and the one that shows a negative binding enthalpy during the ITC measurements, probably suggesting the presence of different binding sites to CTD. We hypothesize that the presence of the highly hydrophobic tyrosine, together with the absence of a positively charged residue at the C terminus of the peptide, could be a driving-force to reorient the peptide toward other hydrophobic polypeptide regions of CTD.53 A final question may be raised: how do the results obtained with peptides P-1 to P-4 compare with those obtained with other molecules able to bind CTD? Several peptides able to bind at or close to the CTD
CTD dimerization interface or the NTD
CTD interface in CA of HIV-1 have been described.6,7,16,18 These two interfaces, together with the NTD
NTD hexamerization interface, are important during viral assembly.5 In the examples investigated, there is also an overlap between these interfaces and the binding sites recognized by the peptides:16,18 Ser149, Arg162, Val165, Asp166, Phe168, Tyr169, Leu172; Arg173, Leu151, Glu175, Gln176, Ala177, Ser178, Gln179, Glu180, Val181, Lys182, Asn183 Trp184, Met185, Thr186, Glu187, Thr188, Leu189, Leu190, Pro207, Ala209, Thr210, Leu211, Met214, and Met215. We have also recently mapped the binding of dendrimeric organic molecules to CTD, which showed affinity constants similar to those described in this work for the peptides;19 one of these dendrimers was, like peptide P-1, 3262

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Biomacromolecules able to inhibit CA polymerization in vitro, although only to a limited extent and only when very high concentrations were used. Interestingly, the CTD residues involved in binding of the dendrimers and those involved in binding to the peptides analyzed in this work overlapped to a very significant extent.19 In contrast, the majority of the residues of CTD involved in peptide binding in the other models16,18 do not appear to intervene in the binding of the peptides described in this work (Table 3), and only residues Leu151, Asn183; Ala184, Met185, Thr186, Thr188, Thr210, Leu211, and Met214 are in common. The results, taken together, indicate that inhibition of CA polymerization may directly correlate with the overlap between the peptide binding sites in the protein and the dimerization interface, as determined by NMR.

’ CONCLUSIONS Increased helical propensity achieved, by rationally shortening and substituting some residues of a peptide mimic of a region of a helical protein
protein interface, led to unexpected binding of the peptides to other regions of the protein. Thus, helicity and selfassociation are mutually and intrinsically modulated in the CTD. ’ ABBREVIATIONS CA, intact capsid protein of HIV-1 CAC-1, peptide comprising residues 178 to 191 of CTD CTD, C-terminal domain of CA CTDW184A, monomeric mutant of CTD with an alanine at the position of the sole tryptophan HFIP, 1,1,1,3,3,3-hexafluoro-2-propanol HSQC, heteronuclear single quantum coherence ITC, isothermal titration calorimetry MA, matrix protein MHR, major homology region NC, nucleocapsid protein NTD, N-terminal domain of CA Rh, hydrodynamic radius TFE, 2,2,2-trifluorotehanol TSP, sodium trimethylsilyl-[2,2,3,3-2H4]-propionate Tm, thermal denaturation midpoint UV, ultraviolet ’ ASSOCIATED CONTENT

bS

Supporting Information. All explored mutations in the peptides designed and HFIP and methanol titrations for selected peptides and the ITC and fluorescence binding experiments for the peptides. This material is available free of charge via the Internet at http://pubs.acs.org.

’ AUTHOR INFORMATION Corresponding Author

*Address: Instituto de Biología Molecular y Celular, Universidad Miguel Hern
andez, Avda. del Ferrocarril s/n, 03202, Elche (Alicante), Spain. Tel: + 34 966658459. Fax: +34 966658758. E-mail: [email protected].

’ ACKNOWLEDGMENT We deeply thank May García, María del Carmen Fuster, Javier Casanova, and Alicia Rodríguez-Huete for technical assistance.

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This work was supported by FIPSE foundation (exp: 36557/06) to J.L.N. and M.G.M., Spanish Ministerio de Ciencia e Innovaci
on (SAF2008-05742-C02-01 to J.L.N. and J.G., SAF200909323 to R.G.M., and BIO2009-10092 to M.G.M.), Generalitat Valenciana (ACOMP2011/113 to J.L.N. and J.G.), and an Institutional grant from Fundaci
on Ramon Areces to the Centro de Biología Molecular “Severo Ochoa”. M.G.M. is an associate member of the Institute for Biocomputation and Physics of Complex Systems (BIFI), Zaragoza, Spain.

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