Research Communication

The Phytotoxin Fusicoccin Differently Regulates

14-3-3 Proteins Association to Mode III Targets

Alessandro Paiardini1

Patrizia Aducci2*

Laura Cervoni1

Francesca Cutruzzol�a1,3

Cristina Di Lucente2

Giacomo Janson1

Stefano Pascarella1

Serena Rinaldo1,3

Sabina Visconti2

Lorenzo Camoni2*

1Department of Biochemical Sciences, Sapienza University of Rome,Rome, Italy2Department of Biology, University of Rome “Tor Vergata”, Rome, Italy3Istituto Pasteur-Fondazione Cenci Bolognetti, Rome, Italy

Abstract

Modulation of the interaction of regulatory 14-3-3 proteins to

their physiological partners through small cell-permeant mole-

cules is a promising strategy to control cellular processes

where 14-3-3s are engaged. Here, we show that the fungal

phytotoxin fusicoccin (FC), known to stabilize 14-3-3 associa-

tion to the plant plasma membrane H1-ATPase, is able to sta-

bilize 14-3-3 interaction to several client proteins with a mode

III binding motif. Isothermal titration calorimetry analysis of

the interaction between 14-3-3s and different peptides repro-

ducing a mode III binding site demonstrated the FC ability to

stimulate 14-3-3 the association. Moreover, molecular docking

studies provided the structural rationale for the differential FC

effect, which exclusively depends on the biochemical proper-

ties of the residue in peptide C-terminal position. Our study

proposes FC as a promising tool to control cellular processes

regulated by 14-3-3 proteins, opening new perspectives on its

potential pharmacological applications. VC 2014 IUBMB Life,

00(00):000–000, 2014

Keywords: fusicoccin; 14-3-3 proteins; protein–protein interaction;

molecular docking; drug discovery

IntroductionProtein–protein interactions are highly dynamic processes thatplay pivotal roles in many biological systems. As their dysregu-lation can contribute to human disease, modulation of thesebinding events by small molecules represents a promisingstrategy in drug discovery (1).

A remarkable example of regulatory proteins involvedin protein–protein interactions is represented by 14-3-3proteins, a family of conserved dimeric proteins thatregulate a wide variety of cell processes in eukaryoticorganisms, including cell differentiation and proliferation,intracellular trafficking, and gene transcription (2,3). Giventhe 14-3-3 role in various biological pathways that contrib-ute to human diseases, including cancer progression andneurodegenerative diseases, modulation of their interactionwith different proteins is a main target in pharmacologicalresearch (4).

14-3-3 proteins interact with several phosphorylatedligands through a conserved amphipathic groove present ineach 14-3-3 monomer. Analysis of 14-3-3 binding sequence ofthe targets has allowed to identify common features and con-sequently to propose three main consensus binding motifs.The vast majority of targets bind 14-3-3s through the relatedmotifs RSX(pS/pT)XP (mode I) and RXY/FX(pS/pT)XP (mode II),where X is any amino acid and pS/pT represents phosphoryl-ated Ser or Thr (5).

VC 2014 International Union of Biochemistry and Molecular BiologyVolume 00, Number 00, Month/Month, Pages 00–00

*Address correspondence to: Patrizia Aducci, Department of Biology, Uni-versity of Rome “Tor Vergata”, via della Ricerca Scientifica, 00133 Rome,Italy. or Lorenzo Camoni, Department of Biology, University of Rome “TorVergata”, via della Ricerca Scientifica, 00133 Rome, Italy; Tel., 139 0672594343; Fax, 139 06 2023500.E-mail: aducci@uniroma2.it or E-mail: camoni@uniroma2.it.Received 4 October 2013; Accepted 20 December 2013DOI 10.1002/iub.1239Published online in Wiley Online Library(wileyonlinelibrary.com)

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In a restricted number of targets, the 14-3-3 binding site islocated at their C-terminus. Although these binding sequencesare not structurally related, they have been grouped in modeIII consensus motifs, pS/pT(X0,1,2)-COOH (6).

The best characterized 14-3-3 target of the mode III sub-family is the plant plasma membrane H1-ATPase, a keyenzyme for generation of the electrochemical gradient acrossthe plasma membrane of plant cells, responsible for the con-trol of fundamental processes (7). 14-3-3 proteins associate tothe C-terminal sequence YpTV-COOH, leading to enzyme acti-vation. This interaction displays a remarkable trait, beingstrongly stabilized by fusicoccin (FC), a phytotoxic terpenoidproduced by the fungus Phomopsis amygdali (8). Despite thefungus being host-specific, FC is active in all higher plants,where it affects a number of biochemical and physiologicalprocesses, as a consequence of H1-ATPase activation (9).

FC mode of action has been clarified at the molecular level:the toxin binds to the preformed H1-ATPase/14-3-3 complex,thus greatly stabilizing the interaction and maintaining theenzyme in its activated state (10–12). Crystal structure of the ter-nary complex between FC, 14-3-3, and a phosphopeptide repro-ducing the H1-ATPase binding sequence showed that the toxininserts into the conserved amphipathic cavity of 14-3-3 andmakes molecular contacts also with the C-terminal end of thepeptide, thus promoting mutual stabilization of both ligands (13).

FC does not increase the affinity of 14-3-3 proteins tomode I or II targets (14,15), because in these interactions theFC binding site of 14-3-3 is engaged in peptide binding, thushampering FC binding in the groove. Conversely, structuresimilarity between the H1-ATPase and other mode III targetsmakes conceivable that 14-3-3 interaction with other mode IIItargets could be stabilized by FC. According to this hypothesis,we recently demonstrated the FC ability to stabilize 14-3-3association to human GPIba (15), a platelet glycoprotein whichis part of GPI-IX-V, a protein complex that mediates the adhe-sion of circulating platelets to arteries and capillaries sub-endothelium (16). As a consequence, FC promotes plateletadhesion and subsequent aggregation (15). This finding pro-poses FC as a drug-like molecule potentially exploitable to con-trol a number of physiological processes where 14-3-3 clientswith mode III motifs take part (17).

To clarify the structural requisites of 14-3-3 targets toallow the FC regulatory function, the FC effect on the interac-tion between 14-3-3 proteins and phosphopeptides reproduc-ing a mode III binding sequence has been investigated in thisarticle.

Experimental Procedures

ChemicalsFC was prepared according to Ballio et al. (18). The following pep-tides: p27Kip1 (VEQTPKKPGLRRRQpT), IL9-R (MLLPSVLSKARSWpTF), KCNK3 (SLSTFRGLMKRRSpSV), GPR15 (HAED-FARRRKRSVpSL), HAP1 (GHPPASGTSYRSSpTL), and maize PLDd

(KILGASTSLPDSLpTM) were synthesized both in free and in biotin-bound form by JPT Peptide Technologies (Berlin, Germany). MaizeH1-ATPase (MHA2 isoform) peptide (LKGLDIDTIQQNYTpV) wassynthesized by Neosystem (Strasbourg, France).

Glutathione sepharose 4B was from GE Healthcare Bio-sciences (Pittsburgh, PA). Protein kinase A, streptavidin–aga-rose magnetic beads, and the other chemicals were fromSigma–Aldrich (St. Louis, MO).

Expression and Purification of 14-3-3f

Human 14-3-3f was expressed in Escherichia coli as fusionprotein with the glutathione-S-transferase (GST) using pGEX-2TK vector, following the procedure described by Camoniet al. (15).

This expression vector produces a GST-fused protein con-taining a cAMP-dependent protein kinase phosphorylation siteand a thrombin site between the two polypeptides. The fusionprotein was immobilized onto glutathione sepharose beadsand 14-3-3f purified by treating the immobilized protein withthrombin.

32P Labeling of 14-3-3 Proteins14-3-3f was labeled with [32P]-ATP on the phosphorylation sitepresent at junction between GST and the cloned protein usingthe catalytic subunit of PKA as already described (15). Specificactivity of 32P-14-3-3f was about 3 MBq/mg.

Binding of 14-3-3f to Resin-Bound PhosphopeptidesBiotinylated peptides (0.4 nmol) were immobilized onto 40 mLof streptavidin–agarose magnetic beads. Beads were incubatedfor 60 Min at RT in 50 mL of buffer H (20 mM Hepes-OH, 75mM KCl, 5 mM MgCl2, 0.1 mM EDTA, pH 7.5) containing0.04% Tween-20 and 10 mM of 32P-labelled 14-3-3f in thepresence of 10 or 100 mM FC (19). Resin-bound radioactivitywas measured in a liquid scintillation b-counter.

Isothermal Titration CalorimetryIsothermal titration calorimetry (ITC) experiments were per-formed using an iTC200 microcalorimeter (MicroCal, GEHealthcare Biosciences, Pittsburgh, PA), by titrating 14-3-3fwith mode III peptides. Two microliters aliquots of 0.5 mMpeptide in buffer H containing 1 mM benzamidine wereinjected into a 50 mM 14-3-3f in the same buffer at 25 �C. Ifindicated, 100 mM FC was added. Data were fitted using the“one-binding-site model” of the MicroCal version of ORIGIN, aspreviously described (20). All measurements were done induplicate.

ModelingStructural superposition of the three-dimensional coordinatesof 14-3-3 proteins was done using the Combinatorial Extensionalgorithm (21), as implemented in PyMod plugin (22). 14-3-3fstructure (PDB Code 1QJB) was chosen as archetypal of the f-isoform. PDB Codes 3IQV, 3P1O, and 1O9F were chosen asrepresentatives of RSX, RRX, and QSX clusters, respectively,and subsequently used as initial structural templates to modelmode III peptides. Each ternary complex was modeled using

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2 Fusicoccin Regulation of 14-3-3-Target Interactions

as structural template the 14-3-3c isoform from Nicotianatabacum (PDB Code 2O98).

Subsequent in silico mutagenesis and energy minimizationrelied on the program Molecular Operating Environment(MOE, 2007). The energy minimization protocol has beenapplied according to Paiardini and Pascarella (23).

Results

FC Has Diverse Effects on 14-3-3 Binding to Phospho-peptides Reproducing Mode III Binding Sequences14-3-3 targets with a mode III motif selected in this study (Fig.1, top panel) are the cyclin-dependent kinase inhibitor p27Kip1

(24), the interleukin 9 receptor alpha chain (IL-9Ra, 25), theheart muscle system potassium channel KCNK3 (26), the Gprotein-coupled receptor GPR15 (27), and the neuronalHuntingtin-associated protein 1 (HAP1, 28). PLDd, a maizephospholipase D with a putative mode III binding sequence(DSLpTM), has also been included. Phosphopeptides reproduc-ing the mode III sequence of these targets and the referenceH1-ATPase peptide were used in interaction experiments per-formed with the prototype 14-3-3f isoform.

The FC effect on 14-3-3/peptide association has been ini-tially screened by measuring 14-3-3 binding to immobilizedbiotinyl-peptides under equilibrium conditions. As shown inFig. 1, the FC effect is considerably different in the variouspeptides assayed. In fact, 14-3-3 binding to p27Kip1 peptide isunaffected by both 10 and 100 mM FC, while IL-9Ra associationis inhibited by 100 mM FC. Conversely, 14-3-3 interaction withKCNK3, GPR15, HAP1, and PLDd peptides is significantlystimulated both by 10 and 100 mM FC, resembling the well-known FC stabilizing effect on 14-3-3 interaction with the H1-ATPase peptide.

As it is known that peptide immobilization to a solidmatrix may greatly hamper the interaction with a macromole-cule such as the dimeric 14-3-3 protein, the affinity of theseinteractions has been quantitatively investigated by means ofITC, an innovative approach that determines the thermody-namic parameters of interactions in solution.

In these experiments, 50 mM 14-3-3f has been titratedwith 0.5 mM peptide solutions in the presence or absence of100 mM FC. As expected for specific binding, integration of thetitration peaks produces sigmoidal enthalpy curves (Fig. 2),whose fit led to determine the stoichiometry of binding, thethermodynamic parameters, and the derived KD for each inter-action (Table 1).

ITC data are in total agreement with the peptide bindingassay performed with immobilized biotinyl-peptides. Similarlyto the H1-ATPase peptide, FC stabilizes 14-3-3 association toKCNK3, GPR15, HAP1, and PLDd peptides, while the interac-tion with p27Kip1 peptide is unaffected and that with IL-9Ra isinhibited by the toxin.

The thermodynamic profiles of the aforementioned bindingevents indicate that, for the most part, protein/peptide interac-tions present both favorable enthalpic and entropic compo-nents, with the exception of p27Kip1 peptide, which displays atypical enthalpy driven interaction.

In more detail, binding affinity of the H1-ATPase peptideto 14-3-3 is increased 9.0-folds in the presence of FC andinvolves a strong favorable enthalpic variation. Binding affin-ity of KCNK3 to 14-3-3 increases 5.2-folds in the presence ofFC and, under these experimental conditions, the ternarycomplex formation contributes favorably to the enthalpiccomponent. Similarly, PLDd/14-3-3 interaction in the pres-ence of FC involves a predominant favorable enthalpic varia-tion (as compared to the entropic one), with respect to thecorresponding FC-free experiment, and the affinity increases11.2-folds.

The presence of FC also increases the affinity of binding ofGPR15 and HAP1 peptides to 14-3-3, 6.3- and 13.5-folds,respectively. The first interaction mainly involves a favorableentropic change, while the latter presents a similar favorablechange of both the enthalpic and the entropic factors.

Conversely, p27Kip1 peptide binds 14-3-3 with low affinity,and the interaction is not significantly affected by FC. In agree-ment with binding experiments with immobilized biotinyl-peptides, the presence of 100 mM FC negatively affects (3.5-

FC differentially regulates binding of 14-3-3f to mode

III peptides. 14-3-3f binding assay to phosphopepti-

des reproducing the binding sites of p27Kip1, IL-9Ra,

KCNK3, GPR15, HAP1, PLDd, and H1-ATPase. Immo-

bilized peptides were incubated with 32P-14-3-3f in

the absence (white bars) or in the presence of 10 mM

(gray bars) or 100 mM (black bars) FC. Resin-

associated radioactivity was measured by scintilla-

tion counting. *P<0.01 by Student’s t-test (n 5 9).

[Color figure can be viewed in the online issue,

which is available at wileyonlinelibrary.com.]

Fig 1

Paiardini et al. 3

Binding of mode III peptides to 14-3-3f by ITC measurements. 14-3-3f interaction with p27Kip1 (A), IL9-Ra (B), KCNK3 (C), GPR15

(D), HAP1 (E), PLDd (F), and H1-ATPase (G). Analysis was performed by titrating 14-3-3f with each mode III peptide both in the

absence (W) or in the presence of 100 mM FC (w). Experiments were performed in 20 mM Hepes-OH buffer (pH 7.5), 75 mM

KCl, 5 mM MgCl2, 0.1 mM EDTA, and 1 mM benzamidine. Graphs show the integrated energy values normalized for injected

protein. Binding isotherms were fitted using a single binding site model. All measurements were done in duplicate.

Fig 2

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4 Fusicoccin Regulation of 14-3-3-Target Interactions

folds) the affinity of 14-3-3 for peptide IL-9Ra, thus confirmingthe inhibiting activity of the toxin in this interaction.

A Structural Rationale by Molecular ModelingThe analysis of available crystal structures revealed that sev-eral structural features are invariant in target peptides. Thesepositions are represented by: i) the phosphate moiety (position0), interacting via ion-pairs with Lys49, Arg56, Arg127, and ahydrogen-bond with Tyr128 (Fig. 3B, left); ii) the carboxy-terminal moiety (position 11), engaged in electrostatic andhydrogen-bonding interactions with the evolutionarily con-served Lys120 and Asn173 (Fig. 3B, center); iii) the main-chain at position 11, invariably interacting with Asn224 viahydrogen-bonds (Fig. 3, right).

Consequently, different affinities displayed by tested modeIII peptides are mainly due to residues in position 21, 22, and23. Position 21 can accommodate a great variety of residueswith different physicochemical properties, with a slight prefer-ence for aromatic residues (5). The presence of a Trp residuein 21 in IL9-Ra peptide can thus explain the high affinity ofthe IL9-Ra/14-3-3 complex (Table 1).

The residue in position 22 is mainly responsible for the N-terminal conformation of the backbone of bound peptides. Serand Arg are the most favored residues in this position (29).The small side chain of Ser allows peptide accommodation atthe bottom of the binding cleft, while a bulky residue like Argprojects the main chain away from the floor of the groove.Position 23 is almost invariably occupied by an Arg residue,

Thermodynamic constant parameters of mode III peptides/14-3-3f interaction obtained by ITC

Binding stoichiometry (n) Ka 3 106 (M21) KD (mM) DH (kcal/mol) 2TDS (kcal/mol) DG (kcal/mol)

P27Kip1

2FC 0.82 6 0.03 0.16 6 0.03 6.25 27.10 6 0.64 0.54 6 0.03 26.56

1FC 0.86 6 0.06 0.23 6 0.05 4.35 28.30 6 1.20 0.96 6 1.30 27.34

IL-R9a

2FC 0.73 6 0.05 22.20 6 3.20 0.045 24.00 6 0.14 26.03 6 0.23 210.03

1FC 0.79 6 0.06 6.06 6 0.23 0.16 23.30 6 0.10 25.99 6 0.08 29.29

KCNK3

2FC 1.05 6 0.06 4.51 6 0.39 0.22 26.40 6 0.01 22.62 6 0.05 29.02

1FC 1.08 6 0.01 23.70 6 2.0 0.042 28.50 6 0.22 21.55 6 0.17 210.05

GPR15

2FC 0.96 6 0.06 6.00 6 0.27 0.17 25.90 6 0.24 23.37 6 0.21 29.27

1FC 0.95 6 0.03 37.00 6 9.40 0.03 24.40 6 0.40 25.87 6 0.25 210.27

HAP1

2FC 0.82 6 0.08 1.84 6 0.17 0.54 25.2 6 0.17 23.31 6 0.14 28.51

1FC 0.75 6 0.13 25.00 6 11.00 0.04 25.9 6 0.07 24.10 6 0.23 210.00

PLDd

2FC 0.83 6 0.03 0.27 6 0.05 3.70 22.00 6 0.46 25.44 6 0.57 27.44

1FC 0.84 6 0.02 3.07 6 1.30 0.33 23.80 6 0.27 25.03 6 0.54 28.83

H1-ATPase

2FC 0.81 6 0.02 11.11 6 1.87 0.09 25.43 6 0.64 23.70 6 0.03 29.13

1FC 0.73 6 0.06 99.65 6 29.80 0.01 28.58 6 1.20 21.87 6 1.30 210.45

Dissociation constants (KD 5 1/Ka), Gibbs free energy changes (DG 5 2RTlnKa), and entropy changes (TDS 5 DH 2 DG) were calculated from DH

and Ka.

TABLE 1

Paiardini et al. 5

which is involved in an ion-pair interaction with the phosphatemoiety of the phosphopeptide and in a close stacking interac-tion with the guanidinium ring of Arg60 (30). In the PLDd pep-tide, Arg in 23 is replaced by the acidic residue Asp. This sub-stitution is detrimental for peptide affinity, as supported by theobserved low affinity of PLDd/14-3-3 complex (Table 1).

To provide a structural rationale for the differential FCeffect observed in peptides/14-3-3 interaction experiments,structures of mode III peptide/14-3-3/FC ternary complexes

have been inferred by molecular docking studies. The initialstep of this method is the selection of known peptide/14-3-3structures that will be used as templates for modeling. At thebeginning of this study, different crystal structures of 14-3-3 incomplex with mode I, II, and III targets have been solved(Table 2). The three-dimensional coordinates of peptides pres-ent in those complexes were transferred to the binding cavityof a representative structure of 14-3-3f (Fig. 3A) uponsuperposition.

(A) Superposition of known peptide structures (green ribbons) into the binding site of 14-3-3f (PDB Code 1QJB, gray cartoons).

(B) Conserved polar interactions of target peptides (light gray) in the 14-3-3 binding groove. Residues are numbered according

to 14-3-3f. Left, the phosphate moiety (position 0), interacting via ion-pairs with Lys49, Arg56, Arg127, and a hydrogen-bond

with Tyr128; center, the carboxy-terminal moiety (position 11), engaged in electrostatic and hydrogen-bonding interactions

with Lys120 and Asn173 of 14-3-3f; right, the main-chain at position 11, interacting with Asn224 via hydrogen-bonds. (C)

superposition of the a-chain of peptides used as structural templates for modeling. RRX (PDB Code 31PO), RSX (PDB Code

31QV), and QSX (PDB Code 1O9F) are represented as cyan, pink, and green scaffolds, respectively. [Color figure can be viewed

in the online issue, which is available at wileyonlinelibrary.com.]

Fig 3

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6 Fusicoccin Regulation of 14-3-3-Target Interactions

Dataset used in molecular modeling

PDB code 14-3-3 isoform (organism)

Amino acid sequence of peptides crystallized in complex with 14-3-3 proteins

Amino acid position respect to pS/pT

24 23 22 21 0 11 12

1A37 14-3-3f (Bos taurus) Q R S T pS T P

1IB1 14-3-3f (Homo sapiens) Q R R H pT L P

1O9D 14-3-3c (Nicotiana tabacum) Q S Y pT V

1O9F 14-3-3c (N. tabacum) Q S Y pT V

1QJA 14-3-3f (H. sapiens) R L Y H pS L P

1QJB 14-3-3f (H. sapiens) A R S H pS Y P

1YWT 14-3-3r (H. sapiens) A R S H pS Y P

2B05 14-3-3! (H. sapiens) R A I pS L P

2BR9 14-3-3e (H. sapiens) R R Q R pS A P

2BTP 14-3-3u (H. sapiens) R Q R pS A P

2C1N 14-3-3f (H. sapiens) A R K pS T G

2C63 14-3-3h (H. sapiens) R A I pS L P

2C74 14-3-3h (H. sapiens) R R Q R pS A P

2NPM CF14e (Cryptosporidium parvum) R A I pS L P

2O98 14-3-3c (N. tabacum) Q Q S Y D I

2V7D 14-3-3f (B. taurus) K S A pT T T

2WH0 14-3-3f (H. sapiens) D R S K pS A P

3AXY GF14-c (Oryza sativa) Q R V L pS A P

3CU8 14-3-3f (H. sapiens) R S T pS T P

3E6Y 14-3-3c (N. tabacum) Q S Y pT V

3IQJ 14-3-3r (H. sapiens) Q R S T pS T P

3IQU 14-3-3r (H. sapiens) Q R S T pS T

3IQV 14-3-3r (H. sapiens) Q R S T pS T

3LW1 14-3-3r (H. sapiens) F K pT E G

3M50 14-3-3c (N. tabacum) Q Q S Y D I

3M51 14-3-3c (N. tabacum) Q Q S Y D I

3MHR 14-3-3r (H. sapiens) R A H pS S P

3NKX 14-3-3r (H. sapiens) Q R S T pS T P

3O8I 14-3-3r (H. sapiens) Q R S T pS T P

3P1N 14-3-3r (H. sapiens) K R R K pS V

3P1O 14-3-3r (H. sapiens) K R R K pS V

3P1P 14-3-3r (H. sapiens) K R R K pS V

3P1Q 14-3-3r (H. sapiens) K R R K pS V

3P1R 14-3-3r (H. sapiens) K R R K pS V

3P1S 14-3-3r (H. sapiens) K R R K pS V

3UAL 14-3-3e (H. sapiens) I R S F pS E P

3UBW 14-3-3e (H. sapiens) I R S F pS E P

3UZD 14-3-3! (H. sapiens) I R S P pS L P

TABLE 2

Paiardini et al. 7

Comparison of peptide structures with their position in the14-3-3 binding groove reveals three main structural clusters(Fig. 3C). In the first cluster (hereinafter “RSX”), positions 22and 23 are almost invariantly occupied by Ser and Arg. Thesmall side-chain of Ser in 22 keeps the peptide’s main-chainlying on the cleft of 14-3-3. The second cluster, “RRX,” is char-acterized by two Arg residues in position 22 and 23. In thiscase, the presence of a bulky residue like Arg in position 22projects the main-chain away from the floor of the bindingcleft. Finally, the “QSX” cluster is characterized by the pres-ence of a Ser residue at position 22 and mainly a Gln residue(not Arg) at 23.

Mode III peptides have been therefore modeled startingfrom a structural representative of RSX, RRX, or QSX clusters.In particular, RSX cluster has been selected for GPR15, IL9-R,and HAP1, RRX for KCNK3 and p27Kip1, and QSX for PLDdpeptide. To obtain models of 14-3-3f/peptide/FC complexes, insilico peptide mutagenesis followed by energy minimizationhas been then applied.

Structures obtained by modeling (Fig. 4) can account forthe different FC effect observed in peptides/14-3-3 interactionsexperiments (Table 1). In fact, side-chains of amino acidsoccupying the 11 position of mode III peptides are predictedto be located in a hydrophobic cleft facing the FC-binding siteof 14-3-3. In absence of FC, part of this position is exposed tothe solvent; upon toxin binding, however, the cleft becomesoccupied by FC hydrophobic rings. p27Kip1 peptide, lacking theresidue in position 11, is not able to establish molecular con-tacts with FC (panel A). This predicted structure consequentlyconfirms our experimental data, which point out the FC inabil-ity to modulate p27Kip1 peptide/14-3-3 association. Conversely,bulky hydrophobic residues could be disfavored in the pres-ence of FC, due to steric hindrance of the latter inside the cleft.This observation could explain the behavior of IL-9Ra, bearinga Phe residue at position 11. In fact, the benzyl group of Phesterically interferes with FC (panel B), which therefore inhibitspeptide association to 14-3-3. Finally, peptides with small,non-polar side-chains at position 11, like KCNK3 (panel C),GPR15 (panel E), HAP1 (panel F), and PLDd (panel D) are steri-cally compatible and able to establish hydrophobic interac-tions, thus explaining the FC-mediated energy stabilizationexperimentally observed.

The FC Effect Depends on the C Terminal Residue ofMode III TargetsAs previously pointed out, pSer/pThr position and consequentlypeptide positioning are almost invariant in the inferred struc-tures. Therefore, FC stimulatory/inhibitory action uniquelydepends on the biochemical features of amino acid in position11. To evaluate how each single amino acid in C-terminalposition can accommodate in the binding cleft and establishmolecular interactions with FC, in silico site-directed mutagen-esis of the C-terminal residue of mode III peptide has beenperformed using the H1-ATPase/14-3-3/FC ternary structureas template. For each analysis, the more stable FC rotamer

has been chosen. As shown in Table 3, positive potential ener-gies of C-terminal amino acids with cyclic rings, like His, Pro,Tyr, Phe, and Trp, indicate that these groups are stericallyincompatible with FC. Therefore, binding of peptides withthese bulky residues at the C terminus is strongly disfavoredby the toxin (Fig. 5, upper panel). Slight positive energy varia-tions associated to Gln and Asn indicate that these residues atthe C terminus weakly hamper peptide binding in the presenceof FC, while null energy variations associated to Gly and Alaindicate that binding of peptides ending with these amino acidsis unaffected by the toxin. Conversely, negative potential ener-gies of remaining residues indicate that they are stericallycompatible and able to establish molecular interactions withFC (Fig. 5, lower panel). As a consequence, 14-3-3 interactionof peptides ending with these amino acids is stimulated by FC.

DiscussionThe identification of small cell-permeable molecules able toselectively modulate 14-3-3 binding with client protein repre-sents a promising strategy to control physiological processeswhere 14-3-3 proteins play a role. In particular, the use ofprotein2protein stabilizers could allow to restore physiologicalprocesses impaired in pathologies correlated to 14-3-3 mal-functioning (4).

In this study, we show that FC, a cell-permeant fungaltoxin whose activity has been so far deeply described only inplants, can represent a useful tool to selectively regulate 14-3-3 interactions with any target containing a mode III motif.

Mode III peptides used in this study bind 14-3-3f with dif-ferent affinity, ranging from 45 nM of IL9-Ra/14-3-3 to 6.25mM of p27Kip1/14-3-3 interactions. Analysis of crystallographicstructures and modeling studies underline that some interac-tions are totally conserved, such as those established by thephosphate moiety, the main chain, and the carboxyl group inposition 11. Therefore, the affinity of a peptide basically arisesfrom the biochemical feature of residues surrounding thepSer/pThr. In particular, positions 23 and 22 mainly contrib-ute to establish molecular connections with the 14-3-3 bindinggroove (5). The optimal motifs Arg-Arg (KCNK3) and Arg-Ser(IL9-Ra and GPR15) can thus explain their high binding affin-ity. Conversely, in PLDd peptide Arg in 23 is replaced by anAsp residue. This could explain the low binding affinity(KD 5 3.7 mM) of the PLDd/14-3-3 complex.

p27Kip1, although sharing the Arg–Arg motif, binds 14-3-3at very low affinity (KD 5 6.25 mM). This could result from thelack of any residue in position 11, which is essential to anchorthe peptide to the 14-3-3 groove. Despite the affinity of eachmode III peptide/14-3-3 complex, the FC effect is solelydependent on the physicochemical features of the residue inposition 11. In fact, FC hampers the association of IL-9Ra (Phein 11), while it greatly stimulates 14-3-3 interactions withKCNK3, GPR15, HAP1, and PLDd, all displaying an hydropho-bic side chain in 11 position. More generally, steric hindranceand limited plasticity of residues with cyclic rings obstruct FC

IUBMB LIFE

8 Fusicoccin Regulation of 14-3-3-Target Interactions

insertion in the 14-3-3 binding groove, thus hampering ternarycomplex formation. In addition, we suggest that different resi-dues, although biochemically diverging, are able to form favor-able connections (Table 3).

Our studies open new perspectives in the control of cellu-lar processes where mode III targets take part. A recurring

outcome of 14-3-3 binding is the promotion of target deliveryto the plasma membrane. A selective stabilizer of 14-3-3/targetinteraction could significantly affect levels of protein deliveredto the plasma membrane, bringing about a strong perturbationof the physiological/pathological processes controlled by theproteins. Notably, it has been recently shown that a FC

Modeling of mode III peptides bound to 14-3-3f. (A) Close-up view of p27Kip1 (purple) modeled with FC (cyan) in the binding

groove of 14-3-3f (gray a-helices). (B) IL-9Ra/14-3-3f complex. FC was also modeled to show the predicted steric clash between

Phe in position 11 and the toxin. (C–F) Predicted ternary complexes with KCNK3 (C), GPR15 (D), HAP1 (E), and PLDd (F). [Color

figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

Fig 4

Paiardini et al. 9

derivative enhances the surface expression of the mode III tar-get TASK-3 (KCNK6) by stabilizing its interaction with 14-3-3proteins (31).

In eukaryotes, elevated 14-3-3 expression has been associ-ated to cellular transformation and proliferation (32).Thedevelopment of inhibitors of 14-3-3 interactions is therefore amajor target in cancer research (33). However, in the treat-ment of some diseases, the alternative approach of drug-mediated stabilization of 14-3-3 protein/target interactionscould be preferable (34). In this respect, FC action on mode IIItargets could allow to selectively modulate specific pathwayswithout influencing the complex network between 14-3-3 andtheir targets in the cell.

Very recently, a novel regulatory mechanism of estrogenreceptor a (ERa), a master regulator of tumor cell proliferationexpressed in the majority of breast tumors, has been discov-ered (35). Binding of 14-3-3 proteins to the extreme ER C ter-

minus (sequence ApTV-COOH) reduces dimerization andreceptor activation. Intriguingly, FC stabilizes 14-3-3/ERa com-plex and reduces the estradiol-stimulated ERa dimerization,thus acting as an antiestrogenic compound with antiprolifera-tive activity. Future work will address the potential pharmaco-logical application of FC as a drug directed to 14-3-3/targetcomplexes.

AcknowledgementsThis work has been supported by a grant from Regione Lazio,Fondo per lo Sviluppo Economico, la Ricerca e l’Innovazione(P.A.), by the Italian Ministry of Education, University and

In silico site-directed mutagenesis of residue in

position 11

Residue Potential energy (kcal/mol)

His 27.03

Pro 25.22

Tyr 23.97

Phe 20.05

Trp 15.76

Gln 0.77

Asn 0.68

Gly 0.00

Ala 0.00

Leu 21.81

Ile 22.95

Cys 23.85

Lys 23.86

Thr 23.88

Glu 24.15

Met 24.24

Arg 24.35

Ser 24.38

Asp 24.38

Val 25.37

Internal strain energy of the best rotamer plus hydrogen bonding and

electrostatic energy is shown.

Inferred position of each amino acid substituting the

residue in position 11 in the H1-ATPase phospho-

peptide. Panel A, residues inhibiting the formation of

the H1-ATPase/FC/14-3-3 ternary complex (red) and

neutral residues (orange). Panel B, residue stimulat-

ing the interaction (green). [Color figure can be

viewed in the online issue, which is available at

wileyonlinelibrary.com.]

TABLE 3

Fig 5

IUBMB LIFE

10 Fusicoccin Regulation of 14-3-3-Target Interactions

Research (20094BJ9R7 to F.C., RBFR10LHD1 to S.R.), and bySapienza, University of Rome (F.C. and A.P.). The authorsthank Prof. Haian Fu for providing the 14-3-3f cDNA.

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