Limitations of Peptide Retro-inverso Isomerization inMolecular Mimicry*□S

Received for publication, February 23, 2010, and in revised form, April 9, 2010 Published, JBC Papers in Press, April 9, 2010, DOI 10.1074/jbc.M110.116814

Chong Li‡§, Marzena Pazgier§, Jing Li§, Changqing Li§, Min Liu§, Guozhang Zou§, Zhenyu Li¶, Jiandong Chen¶,Sergey G. Tarasov�, Wei-Yue Lu‡, and Wuyuan Lu§1

From the ‡School of Pharmacy, Fudan University, Shanghai 201203, China, the §Institute of Human Virology and Department ofBiochemistry and Molecular Biology, University of Maryland School of Medicine, Baltimore, Maryland 21201, the ¶MolecularOncology Program, H. Lee Moffitt Cancer Center, Tampa, Florida 33612, and the �Structural Biophysics Laboratory, NCI-Frederick,National Institutes of Health, Frederick, Maryland 21702

A retro-inverso peptide is made up of D-amino acids in areversed sequence and, when extended, assumes a side chaintopology similar to that of its parent molecule but with invertedamide peptide bonds. Despite their limited success as anti-genic mimicry, retro-inverso isomers generally fail to emu-late the protein-binding activities of their parent peptides ofan �-helical nature. In studying the interaction between thetumor suppressor protein p53 and its negative regulatorMDM2, Sakurai et al. (Sakurai, K., Chung, H. S., and Kahne, D.(2004) J. Am. Chem. Soc. 126, 16288–16289) made a surprisingfinding that the retro-inverso isomer of p53(15–29) retained thesame binding activity as the wild type peptide as determined byinhibition enzyme-linked immunosorbent assay. The authorsattributed the unusual outcome to the ability of the D-peptide toadopt a right-handed helical conformation upon MDM2 bind-ing. Using a battery of biochemical and biophysical tools, wefound that retro-inverso isomerization diminished p53 (15–29)binding to MDM2 or MDMX by 3.2–3.3 kcal/mol. Similarresults were replicated with the C-terminal domain of HIV-1capsid protein (3.0 kcal/mol) and the Src homology 3 domain ofAbl tyrosine kinase (3.4 kcal/mol). CD and NMR spectroscopicas well as x-ray crystallographic studies showed that D-peptideligands ofMDM2 invariably adopted left-handedhelical confor-mations in both free and bound states. Our findings reinforcethat the retro-inverso strategy works poorly in molecular mim-icry of biologically active helical peptides, due to inherent dif-ferences at the secondary and tertiary structure levels betweenan L-peptide and its retro-inverso isomer despite their similarside chain topologies at the primary structure level.

Protein-protein interactions govern a great variety of biolog-ical processes and present important targets for therapeuticintervention (1, 2). Small peptides emulating the activity of onebinding partner to antagonize the other play instrumental rolesin drug screening and design. Despite their ability to bind pro-teins with high affinity and unsurpassed specificity, peptidesthemselves are rarely used as therapeutic agents due primarilyto their poor in vivo stability. Even for in vitro applications,efficacy often necessitates peptide resistance to proteolytic deg-radation. To tackle peptide susceptibility to proteolysis, variouspeptidomimetic chemistries have been developed, involvingthe use of D-amino acids, unnatural amino acids, peptide back-bone modifications, cyclizations, and secondary structure-in-ducing templates, among others (3). Peptide retro-inversoisomerization, pioneered by Chorev and Goodman (4), repre-sents an elegant solution to functional peptides stable underphysiological conditions.It is postulated that a retro-inverso peptide (a peptide of the

reversed sequence made up of D-amino acids, also known as aretro-all-D- or retro-enantio-peptide) assumes a side chaintopology, in its extended conformation, similar to that of itsnative L-sequence, thus emulating biological activities of theparent molecule while fully resistant to proteolytic degradation(4). Some success has been achieved immunologically in usingretro-inverso peptides toward antigenic mimicry of their par-ent L-peptides (5). Failures, however, have also been noted forretro-inverso isomers to elicit antibodies that cross-react withnative immune epitopes (6). In fact, retro-inverso peptides arenot isofunctional to their parent L-peptide molecules withrespect to binding energetics even in some successful immuno-logical applications of antigenic mimicry (7). This mixed out-come comes as no surprise, because antibody-antigen recogni-tion is notoriously lenient at the structural level to tolerateconformational plasticity (8).What surprises, however, is different outcomes arising from

well characterized peptide-protein interacting systems whereconformational rigidity or stability of a helical peptide ligand iskey to its high affinity binding to the preformed cavity of afolded protein (9–11). Limited digestion of ribonuclease A bysubtilisin generates S peptide (residues 1–20) and S protein(residues 21–124), which can reassociate with high affinity toform enzymatically active ribonuclease S (12). Two recentreports on the extensively studied S peptide-S protein interact-ing system have shown that it is structurally impossible for the

* This work was supported, in whole or in part, by National Institutes ofHealth (NIH) Grants AI072732 and AI061482 (to W. L.) and the NIH Intra-mural Research Program (to S. G. T.). This work was also supported byAmerican Cancer Society Research Scholar Grant CDD112858 (to W. L.), theChina Scholarship Council, and National Basic Research Program of ChinaGrant 2007CB935800 (to C. L.).

The atomic coordinates and structure factors (code 3LRY) have been deposited inthe Protein Data Bank, Research Collaboratory for Structural Bioinformatics,Rutgers University, New Brunswick, NJ (http://www.rcsb.org/).

□S The on-line version of this article (available at http://www.jbc.org) containssupplemental Table S1 and Figs. S1 and S2.

1 To whom correspondence should be addressed: Inst. of Human Virologyand Dept. of Biochemistry and Molecular Biology, University of MarylandSchool of Medicine, 725 W. Lombard St., Baltimore, MD 21201. Tel.: 410-706-4980; Fax: 410-706-7583; E-mail: wlu@ihv.umaryland.edu.

THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 285, NO. 25, pp. 19572–19581, June 18, 2010Printed in the U.S.A.

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retro-inverso isomer of the �-helical S peptide to functionallymimic S protein binding (9, 10). Moreover, the exquisite spec-ificity for recognition of the native S peptide is strictly main-tained in antibody recognition and T cell stimulation (9). Instudying the interaction between the tumor suppressor proteinp53 and its negative regulator MDM2 (13, 14), however, Saku-rai et al. (11) found that p53(15–29) and its retro-inverso iso-mer displayed nearly identical binding affinities for the p53-binding domain of MDM2. To rationalize their unexpectedfinding, Sakura et al. (11) suggested that the retro-all-D-peptideisomer of p53(15–29), like its parent L-peptide, adopted a right-handed helical conformation in the complex.These conceptually conflicting reports motivated us to carry

out a comprehensive study of functional effects of peptideretro-inverso isomerization on the p53-MDM2 interaction,using a combination of biochemical, biophysical, and structuraltools. The interactions of p53(15–29) with MDMX (a MDM2homolog) and of PMI (a high affinity, dual specific peptideligand ofMDM2 andMDMX)withMDM2were also subjectedto investigation. To determine whether or not conclusionsfrom the p53/PMI-MDM2/MDMX interactions are applicableto others, we expanded our study to include two additional, wellcharacterized peptide-protein interacting systems: the C-ter-minal domain of HIV-12 capsid protein (CCA) and the Srchomology 3 (SH3) domain of Abl tyrosine kinase with theirrespective peptide ligands.

MATERIALS AND METHODS

Chemical Synthesis of Peptides and Proteins—Total chemicalsynthesis of highly pure and correctly folded p53-bindingdomains of MDM2 (residues 25–109, referred to hereafter assynMDM2) and MDMX (residues 24–108 or synMDMX) hasbeen described elsewhere (15). Abl SH3 domain was preparedas described (16). Folding of Abl SH3 domain was initiated bydissolving the polypeptide at 3mg/ml in 0.2 M phosphate buffercontaining 6 M guanidine HCl and 1 mM dithiothreitol, pH 7.5,followed by extensive dialysis (molecular weight cut-off 3000)in water at 4 °C. HIV CCA (S146PTSILDIRQG156PKEPFRDYV-D166RFYKTLRAEQ176ASQEVKNWMT186ETLLVQNANP-196DCKTILKALG206PGATLEEMMT216ACQGVGGPGH226

-

KARVL) was made using native chemical ligation (17, 18). Thetwo peptide fragments of CCA, H2N-CA(146–197)�COSR(where R represents CH2CO-Leu-OH) and H2N-CA(198–231)COOH, were individually synthesized on t-butoxycar-bonyl-Leu-OCH2-PAM resin using an optimized HBTU acti-vation/DIEA in situ neutralization protocol developed by Kentand co-workers (19). Crude peptides, after hydrogen fluoridecleavage and deprotection in the presence of 5% p-cresol at0 °C, were precipitated with cold ether and purified by prepar-ative C18 reversed-phase HPLC, and their molecular masseswere ascertained by electrospray ionizationmass spectrometry.The determined molecular mass of 9518.7 Da of the full-length

ligation product agreed with the expected value of 9519.0 Dacalculated on the basis of the average isotopic compositions ofCCA. Spontaneous folding of CCA and formation of the disul-fide bond between Cys198 and Cys218 was achieved through airoxidation in aqueous buffer. The determinedmolecularmass of9517.1� 0.6 Da of oxidized CCA, differing by 2mass units fromthe calculated value of 9519.0 Da of reduced CCA, confirmedthe disulfide formation.End-capped p53(15–29) (Ac-SQETFSDLWKLLPEN-NH2)

and its retro-inverso isomer RI-p53(15–29) (Ac-DNDEDPDLD-LDKDWDLDDDSDFDTDEDQDS-NH2) were synthesized on 4-methyl-benzhydrylamine resin using the above describedt-butoxycarbonyl chemistry. Both peptides were N-terminallyacetylated on resin using acetic anhydride and DIEA (95:5).All other peptides were only C-terminally amidated: PMI(TSFAEYWNLLSP-NH2) and RI-PMI (DPDSDLDLDNDWDY-DEDADFDSDT-NH2), CAI (ITFEDLLDYYGP-NH2) andRI-CAI(DPDGDYDYDDDLDLDDDEDFDTDI-NH2), Y4W-P40 (APT-WSPPPPP-NH2) and RI-Y4W-P40 (DPDPDPDPDPDSDWDTD

-

PDA-NH2), DPMI-� (DTDNDWDYDADNDLDEDKDLDLDR-NH2)and DPMI-� (DTDADWDYDADNDFDEDKDLDLDR-NH2), andRI-DPMI-� (RLLKEFNAYWAT-NH2). All peptides were puri-fied to homogeneity by preparative C18 reversed-phase HPLC,and their molecular masses were ascertained by electrosprayionization mass spectrometry. Peptide and protein quantifica-tion was achieved by UVmeasurements at 280 nm using molarextinction coefficients calculated according to a publishedalgorithm (20).CDandFluorescence Spectroscopy—Far-UVCDspectrawere

obtained on a Jasco J-810 spectropolarimeter at room temper-ature using a 0.1-cm path length. p53(15–29) and RI-p53(15–29) were thoroughly dialyzed (2000 molecular weight cut-offdialysis cassette, Pierce) against 10 mM phosphate buffer, pH7.2, to remove trace amounts of trifluoroacetic acid thatinterferes with CD measurements. Trp fluorescence spectraof p53(15–29) and RI-p53(15–29) in the presence andabsence of synMDM2 were recorded at room temperature inphosphate-buffered saline (PBS) containing 0.5 mM tris(2-carboxyethyl)phosphine (TCEP) on a Varian (Cary) Eclipsefluorometer. TCEPwas added to prevent oxidative dimeriza-tion of synMDM2 via a free Cys residue in the protein. Theexcitation wavelength was 295 nm, and the width of both slitswas set to 5 nm.The binding affinity of Y4W-P40 and RI-Y4W-P40 for Abl

SH3 domain was determined essentially as described (16).Briefly, different concentrations of Y4W-P40 (0–20 �M) orRI-Y4W-P40 (0–200 �M) were incubated with SH3 (5 �M forY4W-P40 and 15 �M for RI-Y4W-P40) for 15 min in 5 mM

phosphate buffer, pH 7.0, and ligand-induced changes of Trpfluorescence of SH3weremeasured at 350 nm in a cuvette. TheKd values were obtained using a four-parameter non-linearregression analysis as previously described (16).Surface Plasmon Resonance (SPR)—Competition binding

kinetics was carried out at 25 °C on a Biacore T100 SPR instru-ment using an N-terminally uncapped p53(15–29) peptideimmobilized on a CM5 sensor chip (17 resonance units (RUs)).The buffer was 10mMHEPES, 150mMNaCl, 0.005% surfactantP20, pH 7.4. 50 nM synMDM2 or 100 nM synMDMX was incu-

2 The abbreviations used are: HIV, human immunodeficiency virus; CA, capsidprotein; CCA, C-terminal domain of CA; SH3, Src homology 3; HPLC, highpressure liquid chromatography; PBS, phosphate-buffered saline; TCEP,tris(2-carboxyethyl)phosphine; SPR, surface plasmon resonance; RU, reso-nance unit; ITC, isothermal titration calorimetry; ELISA, enzyme-linkedimmunosorbent assay; TFE, trifluoroethanol; RI, retro-inverso isomer.

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bated at room temperature for 30 min with varying concentra-tions of peptide and injected at a flow rate of 20 �l/min for 2min, followed by a 4-min dissociation. The concentration ofunbound synMDM2 or synMDMX in solution was deduced,based on p53 association RU values, from a calibration curveestablished by RUmeasurements of different concentrations ofsynMDM2 or synMDMX injected alone. Non-linear regressionanalysis was performed using GraphPad Prism 4 to give rise toKd values using the equation, Kd � [peptide][MDM2]/[com-plex]. For Kd measurements of CAI and RI-CAI with CCA,steady state binding kinetics of CAI (0.78–100 �M) and RI-CAI(6.25–800 �M) on oxidized CCA (500 RUs) immobilized on aCM5 biosensor chip were obtained.Isothermal Titration Calorimetry (ITC)—Direct protein-pep-

tide interactions were quantified at 25 °C using a MicroCal VP-ITC microcalorimeter. A typical experiment involved injectionof 30 aliquots (8 �l each) of 200 �M peptide solution (preparedin PBS containing 0.1 mMTCEP and 0.01%NaN3) into a 1.4-mlITC cell containing 15 �M protein solution in the same buffer.For background subtraction, a reference set of injections ofpeptide was made in a separate experiment into the bufferalone. The integrated interaction heat values were analyzedusing the Origin 7.0-based ITC fitting software provided byMicroCal, yielding the binding affinity, stoichiometry, andother thermodynamic parameters.Inhibition ELISA—GST-MDM2(1–150) and His6-p53 were

expressed in Escherichia coli and purified by binding to gluta-thione-agarose andNi2�-nitrilotriacetic acid beads under non-denaturing conditions. ELISA plates were incubated with 2.5�g/ml His6-p53 in PBS for 16 h. After washing with PBS plus0.1% Tween 20 (PBST), the plates were blocked with PBS plus5% nonfat dry milk plus 0.1% Tween 20 (PBSMT) for 30 min.GST-MDM2(1–150) (5 �g/ml) was mixed with compounds inPBSMT plus 10% glycerol plus 10 mM dithiothreitol and addedto the wells. The plates were washed with PBST after incuba-tion for 1 h at room temperature and incubation with MDM2antibody 5B10 in PBSMT for 1 h, followed by washing andincubation with horseradish peroxidase-rabbit anti-mouse Igantibody for 1 h. The plates were developed by incubation withTMB peroxidase substrate (KPL) and measured by absorbanceat 450 nm.NMR Spectroscopy—All NMR experiments were performed

at 293 K on a Varian INOVA 500 spectrometer operating at a1H resonance frequency of 499.754 MHz. The peptides weredissolved at 5 mM in d3-trifluoroethanol/H2O co-solvent (1:1)with 10%D2O, pH 4.4. The homonuclear DQF-COSY, TOCSY,and NOESY spectra were collected using standard protocols(21). TOCSY spectra were recorded with a mixing time of 80ms, whereas mixing times of 80 and 200 ms were used forNOESY experiments and evaluation of spin diffusion effects.All data were processed with the program NMRPipe (22). Thespin systems of all residues were identified using theWuthrichstrategy (23), aided by the CARA software (24). Structure cal-culation was carried out using a standalone ATNOS/CANDIDprogram (25), combined with the molecular modeling soft-ware XPLOR-NIH (26). A total of 234 (RI-p53(15–29)) or 318(p53(15–29)) meaningful nuclear Overhauser effect upper dis-tance constraints, extracted from a total of 525 (RI-p53(15–29))

or 642 (p53(15–29)) assignedNOESY cross-peaks, were used asinput for the final structure calculation byXPLOR. 20 conform-ers with the lowest residual target function values from energy-refined cycles were superimposed in the figure.Crystal Structure Determination of Oxidized CCA—Crystals

were grown in 24-well plates at room temperature using thehanging drop, vapor diffusionmethod. 1�l of 10mg/ml CCA inwater was mixed with an equal volume of crystallization solu-tion and equilibrated against 800�l ofmother liquor consistingof 0.1 M HEPES, pH 7.5, 0.8 M sodium phosphate monobasicmonohydrate, and 0.8 M potassium phosphate monobasic.Crystals were soaked briefly in reservoir solution plus 30% (v/v)glycerol as cryoprotectant and subsequently flash-frozen in liq-uid nitrogen.X-ray diffraction data were collected using a rotating anode

x-ray generator Rigaku-MSC Micromax 7 and a Raxis-4��image plate detector (at the X-ray Crystallography Core Facil-ity, University of Maryland, Baltimore, MD) and were inte-grated and scaled with the HKL2000 package (27). The struc-ture was solved by the molecular replacement method with theprogram Phaser from the CCP4 suite (28), based on the ProteinData Bank entry 1A43model (29), and refined with Refmac andcoupled with manual refitting and rebuilding with COOT (30,31). Data collection and refinement statistics are summarizedin supplemental Table S1. Molecular graphics were generatedusing PyMOL (DeLano Scientific LLC, San Carlos, CA).

RESULTS15–29p53 and RI-15–29p53 Differ Structurally in Solution—

The N-terminal transactivation domain of p53 encompassesthe sequence F19S20D21L22W23K24L25L26 (critical residueshighlighted in boldface type) minimally required for effectiveMDM2 binding (14, 32, 33). The p53 transactivation domainitself is unstructured in solution (34). Upon binding to theN-terminal domain of MDM2, however, it acquires a right-handed amphipathic �-helical structure where Phe19, Trp23,and Leu26 bury their hydrophobic side chains inside the p53-binding cavity of MDM2 (14). We recently identified PMI(TSFAEYWNLLSP) by phage display, a potent, dual specificpeptide inhibitor with low nanomolar binding affinity for bothMDM2 and MDMX (15). Shown in Fig. 1A is the synMDM2-PMI complex superimposed with the co-crystal structure ofrecombinant MDM2(17–125) and p53(15–29) (14). The syn-thetic and recombinantMDM2 proteins are nearly identical, asevidenced by a root mean square deviation of 0.7 Å betweentheir equivalent C� atoms. The functionally critical residues,Phe3, Trp7, and Leu10 of PMI, are topologically equivalent toPhe19, Trp23, and Leu26 of p53 in bound states. Notably, wecrystallized synMDM2 in complexwith p53(15–29) but failed tocrystallize the same protein with RI-p53(15–29) (data notshown).Shown in Fig. 1B are the CD spectra of 100 �M p53(15–29)

and RI-p53(15–29) obtained at room temperature. Bothpeptides were unstructured in 10 mM phosphate buffer,pH 7.2. However, in the presence of the �-helix-inducingagent trifluoroethanol (TFE) (60%, v/v), although p53(15–29) became only partially ordered, the retro-inverso isomerreadily adopted a distinct left-handed �-helix, as indicated

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by double maxima at 208 and 222 nm and a strong negativepeak at 195 nm. We further characterized both peptides in50% TFE by NMR spectroscopy at 20 °C. The solution struc-tures of p53(15–29) and RI-p53(15–29) were determined bythe standard two-dimensional homonuclear methods usingTOCSY, NOESY, and DQ-COSY spectra (23). As shown inFig. 1C, except for a less-than-a-full-turn �-helical segment,p53(15–29) is largely disordered, particularly at its N termi-nus. By contrast, the retro-inverso isomer is well structured,adopting a somewhat irregular left-handed helix expectedfor D-peptides, fully consistent with the CD spectroscopicdata. Clearly, p53(15–29) and RI-p53(15–29) differ not onlyin the handedness of helix but also in helical propensity; theD-peptide has a significantly stronger propensity to form aleft-handed helix than the L-peptide to form a right-handedone in solution.

Retro-inverso Isomerization of 15–29p53 Diminishes Its Bind-ing to MDM2 and MDMX by 3.2–3.3 kcal/mol—One of thehallmarks of the p53-MDM2 interaction is the blue shift of Trpfluorescence, resulting from the burial of Trp23 of p53 in thehydrophobic cavity of MDM2 (35). As shown in Fig. 2, Trpfluorescence of p53(15–29) and RI-p53(15–29) emitted maxi-mally at 356 nm in phosphate buffer, suggesting that Trp23 inboth peptideswas fully solvent-exposed (36). The addition of 15�M synMDM2 to 10 �M p53(15–29) accentuated a marked shiftof Trp fluorescence maximum by 28 nm, indicative of a com-plex formed between p53(15–29) and synMDM2. (MDM2 itselflacks Trp fluorescence.) By contrast, when 15 �M synMDM2was added to 10 �M RI-p53(15–29), the Trp fluorescence max-imum shifted by only 1 nm. Amodest shift of 6 nm (from 356 to350 nm) was observed only after the concentrations of bothsynMDM2 and RI-p53(15–29) were increased by 10-fold to

FIGURE 1. p53(15–29) and RI-p53(15–29) are structurally different in solution. A, superposition of the crystal structures of synMDM2-PMI in magenta andgreen and recombinant MDM2(17–125)-p53(15–29) in blue and yellow. The image was created from Protein Data Bank entries 3EQS (15) and 1YCR (14) byPyMOL (DeLano Scientific LLC). Note that only residues 25–109 of MDM2 and 17–29 of p53 are visible in the complex structure due to disordered termini. B, CDspectra of 100 �M p53(15–29) and RI-p53(15–29) in 10 mM phosphate buffer, pH 7.2, with or without 60% (v/v) TFE. The double maxima at 208 and 222 nm anda strong negative peak at 195 nm shown by RI-p53(15–29) (green) are characteristic of left-handed �-helical secondary structure. C, 20 superimposed structuresof p53(15–29) (left) and of RI-p53(15–29) (right) determined at 20 °C in 50% TFE by NMR spectroscopy.

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favor complex formation. Thesedata strongly suggest that RI-p53(15–29) binds to synMDM2 at asignificantly lower affinity than thatof the wild type p53 peptide.To quantify these effects, we used

a previously established, SPR-basedcompetition binding assay (15, 35,37), in which an N-terminallyuncapped p53(15–29) peptide wasimmobilized on a CM5 biosensorchip for kinetic analysis of 50 nMsynMDM2 preincubated in solutionwith varying concentrations ofpeptide. As shown in Fig. 3A, solu-tion peptides competed with immo-bilized p53(15–29) for synMDM2binding in a dose-dependent man-ner, giving rise to aKd value of 255�

FIGURE 2. Trp fluorescence spectra of p53(15–29) and RI-p53(15–29) in the presence and absence ofexcess synMDM2 in PBS containing 0.5 mM TCEP: 10 �M p53(15–29) and 15 �M synMDM2 (left), 10 �M

RI-p53(15–29) and 15 �M synMDM2 (middle), and 100 �M RI-p53(15–29) and 150 �M synMDM2 (right). Adecrease in Trp fluorescence intensity with 100 �M RI-p53(15–29) and 150 �M

synMDM2 (right) was due to minorprecipitation at high peptide/protein concentrations.

FIGURE 3. p53(15–29) and RI-p53(15–29) are functionally different. A, quantification of the interaction of synMDM2 (50 nM) with varying concentrations ofp53(15–29) and RI-p53(15–29) by SPR-based competition assays. Each curve is the mean of three independent measurements at 25 °C in 10 mM HEPES, 150 mM

NaCl, 0.005% surfactant P20, pH 7.4. B, isothermal titration calorimetric measurements of the interaction between synMDM2 and p53(15–29) (left) andRI-p53(15–29) (right) at 25 °C in PBS containing 0.1 mM TCEP and 0.01% NaN3. C, SPR-based quantification of p53(15–29) and RI-p53(15–29) interacting withsynMDMX. Each curve is the mean of three independent measurements. D, determination of the Kd values of PMI and RI-PMI with synMDM2 by competition SPRin three separate experiments.

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5 nM. In sharp contrast, the retro-inverso p53 isomer showed agreatly reduced binding affinity for synMDM2with aKd value of71.6 � 8.6 �M. The 280-fold difference in Kd between p53(15–29) and RI-p53(15–29) was independently verified by ITCmeasurements, which yielded Kd values of 144 � 5 nM and44.1� 25.4�M for p53(15–29) and RI-p53(15–29), respectively(Fig. 3B and Table 1). Importantly, weakened binding of theretro-inverso p53 isomer to MDM2 was due entirely to a hugeloss in enthalpy, suggesting that the binding modes or confor-mations of p53(15–29) and RI-p53(15–29) are significantly dif-ferent in the complexes. In addition, the negative entropicchange for RI-p53(15–29) binding to MDM2 (�7.5 cal/mol/K) was substantially smaller than that for p53(15–29)(�19.8 cal/mol/K).We further compared p53(15–29) and RI-p53(15–29) in a

well established inhibition ELISA using recombinant GST-MDM2(1–150) and His6-tagged p53 (38–40). In this assay,PMI (acid form) inhibited the p53-MDM2 interaction with anIC50 value of 20 nM (40), 6-fold higher than itsKd value (3.2 nM)measured by SPR (37). The IC50 value of p53(15–29) was deter-mined by ELISA to be �2 �M (supplemental Fig. S1), �8-foldhigher than its Kd value. These similar IC50/Kd ratios demon-strate an internal consistency between the two different assaymethods. Not surprisingly, RI-p53(15–29) was barely inhibi-tory at the highest concentration of 50 �M tested. The IC50values of p53(15–29) and RI-p53(15–29) probably differedby orders of magnitude, as inferred by the inhibition curves(supplemental Fig. S1), corroborating the findings from the SPRand ITC measurements as well as the fluorescence spectro-scopic studies.The p53-binding domains of MDM2 andMDMX share over

50% sequence identity and are highly similar both structurallyand functionally (15, 41). We also quantified via SPR the bind-ing of p53(15–29) and RI-p53(15–29) to synMDMX. p53(15–29) bound synMDMX at an affinity of 368 nM, 246-fold strongerthan the retro-inverso peptide (Kd � 90.5 �M) (Fig. 3C). Thus,the deleterious effects of retro-inverso isomerization onp53(15–29) binding to MDM2 and to MDMX are nearly iden-tical, equivalent to a loss of binding free energy of 3.2 kcal/mol(a 246-fold increase in Kd) to 3.3 kcal/mol (an increase in Kd by280-fold by SPR or 306-fold by ITC).Retro-inverso Isomerization of PMI Diminishes Its Binding to

MDM2 by 4.9 kcal/mol—The binding affinity of PMI forMDM2 is nearly 2 orders of magnitude higher than that ofp53(15–29) (15). We compared the ability of PMI and RI-PMIto bind synMDM2 using the SPR-based competition assay. Asshown in Fig. 3D, the C-terminally amidated PMI bound tosynMDM2with aKd value of 5.2 nM, whereas RI-PMIwas 4,250-fold weaker (Kd � 22.1 �M). The Kd difference between PMIand RI-PMI amounts to a loss of binding free energy of 4.9kcal/mol.

Retro-inverso Isomerization Diminishes CAI Binding to theC-terminal Domain of HIV-1 Capsid Protein by 3.0 kcal/mol—HIV capsid protein (CA) assembles into a cone-shaped corestructure encasing the viral RNA (42). The C-terminal one-third ofCA (CCA) adopts a four-helix bundle conformation anddimerizes in solution through hydrophobic packing of its sec-ond �-helix (29, 43). CCAmediates viral assembly and matura-tion (44) and is a significant yet largely unexploited antiviraltarget. Sticht et al. (45) identified via phage display a duodeci-mal peptide inhibitor, termed CAI (ITFEDLLDYYGP), thatinhibited assembly of immature- and mature-like particles invitro. CAI bound at micromolar affinity as an amphipathic�-helix to a conserved hydrophobic groove of CCA, forming acompact five-helix bundle with altered dimeric interactions(46).We chemically synthesized CCA via native chemical ligation

and determined its crystal structure at 2.05 Å resolution. Asexpected, dimerization of synthetic CCA is mediated primarilyby hydrophobic interactions between the second �-helices(Fig. 4A). Comparative structural analysis indicates that thesynthetic CCA dimer is nearly identical to the recombinantCA(146–231) dimer (root mean square deviation(C�) � 0.47Å) (29); the latter fits well as a dimeric unit into the cryoelectronmicroscopy map of the full-length HIV CA capsid lattice (47).To quantify interactions of CAI and RI-CAI with CCA, weimmobilized the synthetic protein on a CM5 biosensor chipand obtained steady-state binding kinetics of the two pep-tides at different concentrations. A non-linear regressionanalysis yielded Kd values of 13.6 �M and 2.2 mM for CAI andRI-CAI, respectively (Fig. 4B). Thus, retro-inverso isomer-ization of CAI decreased its binding affinity for CCA by 162-fold or 3.0 kcal/mol.Retro-inverso Isomerization Diminishes Y4W-P40 Binding to

Abl SH3 Domain by 3.4 kcal/mol—SH3 domains are smalleukaryotic protein modules of �60 amino acid residues thatintramolecularly regulate the activity of Src family tyrosinekinases and, more generally, target their parent protein mole-cules to cellular sites of their recognition partners (48, 49).Selectively interfering with SH3-dependent signaling eventswith peptide ligands is of great interest in biology (50, 51).MostSH3 domains recognize proline-rich peptides that adopt anextended, left-handed polyproline II helical conformation inthe complexes (52, 53). The Serrano laboratory previouslyreported a micromolar affinity decapeptide ligand, termed P40(APTYSPPPPP), of the SH3 domain of Abl tyrosine kinase (54).We subsequently improved the binding affinity of P40 for AblSH3 domain through a Y4Wmutation, which was identified byaffinity panning and mass spectrometric decoding of targetedsynthetic peptide libraries (16).For this study, wemeasured the binding affinity of Y4W-P40

and its retro-inverso isomer RI-Y4W-P40 for Abl SH3 domain

TABLE 1Thermodynamic parameters measured by isothermal titration calorimetry at 25 °C in PBS containing 0.1 mM TCEP and 0.01% NaN3

Protein Peptide na Ka Kd �H �S

M�1 nM cal/mol cal/mol/KsynMDM2 p53(15–29) 0.78 � 0.01 (6.96 � 0.22) � 106 144 15,230 � 40 �19.8synMDM2 RI-p53(15–29) 0.96 � 0.59 (2.27 � 1.31) � 104 44,100 �3717 � 3108 �7.5

a Stoichiometry of binding.

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using a previously detailed Trp fluorescence titration method(16). As shown in Fig. 4C, Y4W-P40 bound to Abl SH3 domainat an affinity of 0.79 �M, contrasting with a Kd value of 262 �M

of RI-Y4W-P40 for the same protein under identical condi-tions. Therefore, retro-inverso isomerization weakened Y4W-P40 binding to Abl SH3 domain by 332-fold or 3.4 kcal/mol.Effects of Reverse Retro-inverso Isomerization of a D-Peptide

Ligand of MDM2—Mirror image phage display, a powerfulcombinatorial technique developed by Kim and co-workers(55), is an elegant tool that enables quick identification ofD-peptide ligands of a native protein (56–58). Mirror imagephage display screens phage-expressed peptide libraries againstthe D-enantiomer of a native L-protein of interest, yieldingan L-peptide ligand that binds specifically to the D-protein.After enantiomeric inversion, the resultant D-peptide ligand,for reasons of symmetry, binds to the native L-protein at thesame affinity. Using mirror image phage display, we recentlyidentified a duodecimal D-peptide inhibitor of MDM2,termed DPMI-� (DTDNDWDYDADNDLDEDKDLDLDR-NH2)and its mutant DPMI-� (DTDADWDYDADNDFDEDKDLDLDR-NH2) (59). DPMI-� and DPMI-�, differing by 2 amino acid res-idues, bound to synMDM2 at affinities of 219 and 34.5 nM,respectively (59).We used a reverse retro-inverso strategy, con-verting DPMI-� to its retro-all-L-isomer (RLLKEFNAYWAT-NH2). Quantification of the binding of RI-DPMI-� to synMDM2by competition SPR resulted in a Kd value of greater than 1

mM, at least a 25000-fold decreasein binding affinity (supplementalFig. S2).

DISCUSSION

The tumor suppressor protein p53transcriptionally regulates growthinhibitory and apoptotic responsesto prevent stressed cells from pro-liferating and passing mutations onto the next generation (41, 60, 61).Dubbed the “guardian of thegenome,” p53 is critical for main-taining genetic stability and pre-venting tumor development (62).Not surprisingly, in 50% of humancancers, p53 is either deleted or car-riesmissensemutations primarily inits DNA-binding domain. In manyother tumors harboring wild typep53, the oncoproteinMDM2 and itshomolog MDMX negatively regu-late the activity and stability of thetumor suppressor protein (41, 60),resulting directly in p53 inactivationand malignant progression (41, 60).It has been validated both in vitroand in vivo that MDM2/MDMXantagonists disrupt the p53-MDM2/MDMX interactions andselectively kill tumor cells by reacti-vating the p53 pathway (63, 64). Due

to their high potency and specificity (thus low toxicity), pep-tides and/or peptidomimetics capable of antagonizing MDM2to activate p53 are of potential therapeutic value (65). D-Peptideantagonists are particularly attractive because they are fullyresistant to proteolytic degradation in vivo, thereby ensuingmaximal bioavailability and optimal therapeutic efficacy.Retro-inverso peptides as antigenic mimicry of their parent

L-peptides succeed in some cases yet fail in others (5, 6). Bycontrast, the retro-inverso strategy has garnered a much lessimpressive track record in mimicking small, biologicallyactive peptides that become helical upon target binding (9,10, 66). For these reasons, the report by Sakurai et al. (11)that retro-inverso p53(15–29) retains the same biologicalactivity as its parent L-form is highly unusual and somewhatprovocative. Obviously, if the retro-inverso strategy worksfor the p53-MDM2 interacting system, protease-resistantD-peptide activators of p53 can be readily designed throughenantiomeric conversion of high affinity L-peptide ligands ofMDM2 selected from phage-displayed peptide libraries (15,38). More importantly, this simple but powerful approach, ifborne out, will have a far reaching impact on the design ofpotent and stable D-peptide inhibitors for a great variety oftherapeutic applications.Unfortunately, we completely failed on multiple accounts to

replicate the finding by Sakurai et al. (11) after subjecting fourpeptide-protein interacting systems to painstaking scrutiny

FIGURE 4. Effects of retro-inverso isomerization on peptide binding of HIV CCA and Abl SH3 domain.A, stereo view of superimposed crystal structures of synthetic HIV CCA (green) and recombinant HIV CA(146 –231) (gray; Protein Data Bank code 1A43) (29). Disulfide bonds are displayed as ball-and-stick representations.The overall structures of dimers are very similar, with the root mean square deviation between equivalent C�atoms of 0.47 Å. Dimerization is primarily mediated by hydrophobic interactions between the �2 helices in ananti-parallel fashion, which buries 1,500 –1,800 Å2 of the surface area. B, SPR quantification of direct binding ofCAI and RI-CAI to immobilized synthetic HIV CCA (500 RUs) based on steady-state kinetic assays. C, fluorescencetitration of the synthetic Abl SH3 domain by Y4W-P40 and RI-Y4W-P40 in 5 mM phosphate buffer, pH 7.0.Progressive subtractions of the background signal contributed by the Trp-containing peptides were carriedout as described (16).

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using a battery of biochemical, biophysical, and structural tools.Compelling evidence from themultifaceted experiments allowsus to conclude that retro-inverso isomers are not isofunctionalto their parent L-peptides with respect to target protein bind-ing. As has been shown repeatedly in this work, the energeticpenalty of peptide retro-inverso isomerization amounts to 3.0–4.9 kcal/mol in binding free energy. The lack of general successwith the retro-inverso strategy in molecular mimicry is attrib-uted to the fact that a retro-inverso peptide, with invertedamide peptide bonds, cannot possibly be equivalent to its par-ent sequence at the secondary and tertiary structure levels,despite similar side chain topologies at the primary structurelevel (66).The co-crystal structure of DPMI-�-MDM2(25–109) has

recently been determined (59), providing amuch needed expla-nation for the deleterious functional effect of peptide retro-inverso isomerization. Comparative structural analysis indi-cates that the binding mode of DPMI-� for MDM2 differssignificantly from that of p53 or PMI. As expected and, again,

confirmed by the CD and NMRspectroscopic studies, the D-pep-tide ligand adopts an amphipathicleft-handed helical conformationin the complex (Fig. 5A), contrast-ing with the right-handed �-helicalp53(15–29) (Fig. 5B). This findingcomes as no surprise because D-pep-tides, contrary to the suggestion bySakurai et al. (11), are never knownto exist in right-handed helical con-formations due to highly unfavor-able energy (67, 68). Superpositionof DPMI-�-MDM2(25–109) andp53(15–29)-MDM2(17–125) unveils asubstantial positional difference atthe binding interface between thetwo helical peptide ligands of oppo-site handedness, despite similaroverall structures of MDM2 fromthe two complexes (root meansquare deviation(C�) � 0.7 Å). Asshown in Fig. 5C, the D-peptideshifts, in relation to p53(15–29), 3.8Å toward the �2 helix of MDM2,accompanying a “close-in” move-ment of the opposite edge of thebinding pocket. It is thereforeinconceivable that RI-p53(15–29)would be able to structurally mimicthe binding of p53(15–29) toMDM2 in the same handedness andwith a similar conformation.The change of helical peptide

handedness alters protein-bindingenergetics as well. Occupying thesame recognition pockets ofMDM2, DTrp3, DLeu7, and DLeu11of DPMI-� are topologically equiva-

lent to Phe19, Trp23, and Leu26 of p53 (Fig. 5D). In p53 andp53-like peptides, Trp23, as a functional hot spot residue, con-tributes the greatest binding free energy to MDM2 association(69–71). In contrast, DPhe7 is most preferred at the equivalentposition of DPMI-�, roughly 6-fold better than DTrp7 (59). Fur-ther, although the hydrophobic triad Phe19/Trp23/Leu26 of p53dominates MDM2 recognition, energetic contributions toMDM2 binding are more evenly distributed among DTrp3,DTyr4, DLeu7, DGlu8, and DLeu11 of DPMI-� (Fig. 5D) (59).Importantly, despite the fact that DPMI-� and p53(15–29) orp53(17–28) had similar binding affinities for MDM2 (15, 59),the retro-inverso isomer of p53(17–28) (DEDPDLDLDKDWDLD-DDSDFDTDE) or RI-p53(15–29) shares no sequence identityand only limited sequence similarity to DPMI-� (DTDNDWDY-DADNDLDEDKDLDLDR). Assuming that RI-p53(17–28) orRI-p53(15–29) binds toMDM2 similarly to DPMI-�, the D-pep-tide isomer would be obligated to present contact residues sig-nificantly different from DTrp3, DTyr4, DLeu7, DGlu8, andDLeu11 of DPMI-�. In the two most probable modes of interac-

FIGURE 5. The p53 binding domain of MDM2 in complex with peptide inhibitors. A, close-up view of thebinding interface of DPMI-� and synMDM2 (Protein Data Bank code 3LNJ) (59). The electrostatic potential isdisplayed on the molecular surface of MDM2 and colored red for acidic, blue for basic, and white for apolarresidues. The energetically most important residues, DTrp3, DLeu7, and DLeu11, are shown as red sticks.B, close-up view of the binding interface of p53(15–29) and MDM2(17–125) (Protein Data Bank code 1YCR) (14).C, superimposition of the overall structures of the synMDM2-DPMI-� and MDM2(17–125)-p53(15–29) com-plexes. synMDM2-DPMI-� is colored gray/cyan, whereas MDM2(17–125)-p53(15–29) is orange/yellow. TheMDM2 molecules are depicted by ribbons, and the side chains of DTrp3 (Phe19), DLeu7 (Trp23), and DLeu11 (Leu26)are shown as ball-and-stick representations. D, close-up view of superimposed DPMI-� and p53(15–29) bindinginterfaces on a molecular surface of MDM2. DTyr4 and DLeu10 side chains are displayed together with theresidues forming DTyr4 and DLeu10 binding pockets.

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tion with MDM2, these corresponding residues would be 1)DLeu, DLeu, DLeu, DAsp, and DThr or 2) DPro, DLeu, DTrp,DLeu, and DPhe. In either case, the binding of the retro-inversopeptide to MDM2 would be decimated due to less favorablesubsite interactions. As was the case with RI-p53(15–29) andp53(15–29) structurally, it is equally implausible that these twopeptides would be functionally equivalent.It remains unclear why a stark discrepancy exists between

our findings and the results reported by Sakurai et al. (11), whobased their conclusion solely on an inhibition ELISA. The IC50values of p53(15–29) and RI-p53(15–29) determined by Saku-rai et al. (11) are 17.4 and 15.4�M, respectively. Their IC50 valueof p53(15–29) is significantly higher than ours (�2 �M). Nota-bly, the Kd values of wild type p53 peptides interacting withMDM2 have been determined by a number of laboratoriesusing various (more accurate) biochemical and biophysicaltechniques. These values, including ours, generally fall between60 to 700 nM (72, 73), depending on the method as well as thelength of peptide/protein constructs. For example, Schon et al.(32) reported a Kd value of 220 nM for p53(15–29) and recom-binant MDM2(2–125) using stopped-flow fluorescence spec-troscopy and of 575 nM using isothermal titration calorimetry.Additional studies are warranted to clarify this important dis-crepancy for both conceptual and practical reasons.In conclusion, although successful use of retro-inverso iso-

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Peptide Retro-inverso Isomerization in Molecular Mimicry

JUNE 18, 2010 • VOLUME 285 • NUMBER 25 JOURNAL OF BIOLOGICAL CHEMISTRY 19581

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Li, Jiandong Chen, Sergey G. Tarasov, Wei-Yue Lu and Wuyuan LuChong Li, Marzena Pazgier, Jing Li, Changqing Li, Min Liu, Guozhang Zou, Zhenyu

Limitations of Peptide Retro-inverso Isomerization in Molecular Mimicry

doi: 10.1074/jbc.M110.116814 originally published online April 9, 20102010, 285:19572-19581.J. Biol. Chem. 

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