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Structure-Activity Relationship of α Mating Pheromone from the Fungal Pathogen Fusarium oxysporum*

  • Stefania Vitale
    Footnotes
    Affiliations
    Department of Genetics, Campus de Excelencia Internacional Agroalimentario ceiA3, Universidad de Córdoba, 14071 Córdoba, Spain
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  • Angélica Partida-Hanon
    Footnotes
    Affiliations
    Department of Biological Physical Chemistry, Institute of Physical Chemistry Rocasolano, CSIC, Serrano 119, 28006 Madrid, Spain
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  • Soraya Serrano
    Affiliations
    Department of Biological Physical Chemistry, Institute of Physical Chemistry Rocasolano, CSIC, Serrano 119, 28006 Madrid, Spain
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  • Álvaro Martínez-del-Pozo
    Affiliations
    Department of Biochemistry and Molecular Biology I, Faculty of Chemistry, Complutense University, 28040 Madrid, Spain
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  • Antonio Di Pietro
    Affiliations
    Department of Genetics, Campus de Excelencia Internacional Agroalimentario ceiA3, Universidad de Córdoba, 14071 Córdoba, Spain
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  • David Turrà
    Correspondence
    To whom correspondence may be addressed. Tel.: 34-957-218981.
    Affiliations
    Department of Genetics, Campus de Excelencia Internacional Agroalimentario ceiA3, Universidad de Córdoba, 14071 Córdoba, Spain
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  • Marta Bruix
    Correspondence
    To whom correspondence may be addressed. Tel.: 34-915-619400.
    Affiliations
    Department of Biological Physical Chemistry, Institute of Physical Chemistry Rocasolano, CSIC, Serrano 119, 28006 Madrid, Spain
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  • Author Footnotes
    * This work was supported by the Spanish Ministerio de Economía y Competitividad (MINECO) through projects CTQ2014–52633-P, BFU2012–32404, and BIO2013–47870-R. The authors declare that they have no conflicts of interest with the contents of this article.
    1 Both authors contributed equally to the work.
    2 Supported by Marie Curie ITN FUNGIBRAIN (FP7-PEOPLE-ITN-607963) from the European Commission.
    3 Recipient of a predoctoral fellowship 579971 from CONACYT (Consejo Nacional de Ciencia y Tecnologia).
Open AccessPublished:January 18, 2017DOI:https://doi.org/10.1074/jbc.M116.766311
      During sexual development ascomycete fungi produce two types of peptide pheromones termed a and α. The α pheromone from the budding yeast Saccharomyces cerevisiae, a 13-residue peptide that elicits cell cycle arrest and chemotropic growth, has served as paradigm for the interaction of small peptides with their cognate G protein-coupled receptors. However, no structural information is currently available for α pheromones from filamentous ascomycetes, which are significantly shorter and share almost no sequence similarity with the S. cerevisiae homolog. High resolution structure of synthetic α-pheromone from the plant pathogenic ascomycete Fusarium oxysporum revealed the presence of a central β-turn resembling that of its yeast counterpart. Disruption of the-fold by d-alanine substitution of the conserved central Gly6-Gln7 residues or by random sequence scrambling demonstrated a crucial role for this structural determinant in chemoattractant activity. Unexpectedly, the growth inhibitory effect of F. oxysporum α-pheromone was independent of the cognate G protein-coupled receptors Ste2 and of the central β-turn but instead required two conserved Trp1-Cys2 residues at the N terminus. These results indicate that, despite their reduced size, fungal α-pheromones contain discrete functional regions with a defined secondary structure that regulate diverse biological processes such as polarity reorientation and cell division.

      Introduction

      Mating pheromone α from bakers' yeast Saccharomyces cerevisiae has served as a model for studying the interaction of small peptides with G protein-coupled receptors (GPCRs)
      The abbreviations used are: GPCR
      G protein-coupled receptor
      r.m.s.d.
      root mean square deviation
      WA
      water-agarose
      CFW
      calcofluor white
      TOCSY
      total correlated spectroscopy
      HSQC
      heteronuclear single quantum coherence
      TFE
      tetrafluoroethylene
      ChFP
      cherry fluorescent protein.
      (
      • Naider F.
      • Becker J.M.
      The α-factor mating pheromone of Saccharomyces cerevisiae: a model for studying the interaction of peptide hormones and G protein-coupled receptors.
      ). Like most ascomycete fungi, S. cerevisiae cells of different mating types secrete small peptide pheromones (α and a) that function as sexual chemoattractants and are sensed by the cognate plasma membrane GPCRs Ste2 and Ste3, respectively (
      • Jones Jr., S.K.
      • Bennett R.J.
      Fungal mating pheromones: choreographing the dating game.
      ). Ligand binding to the receptor elicits a range of cellular responses including G1 cell cycle arrest, formation of a polarized cell projection (known as a shmoo), and chemotropic growth toward the pheromone of the opposite mating type (
      • Arkowitz R.A.
      Chemical gradients and chemotropism in yeast.
      ,
      • Segall J.E.
      Polarization of yeast cells in spatial gradients of α mating factor.
      • Bücking-Throm E.
      • Duntze W.
      • Hartwell L.H.
      • Manney T.R.
      Reversible arrest of haploid yeast cells in the initiation of DNA synthesis by a diffusible sex factor.
      ).
      The mature α-pheromone of S. cerevisiae is a 13-residue peptide with the sequence WHWLGLKPGQPMY (
      • Kurjan J.
      • Herskowitz I.
      Structure of a yeast pheromone gene (MF α): a putative α-factor precursor contains four tandem copies of mature α-factor.
      ). The central residues Pro8-Gly9 were proposed to form a Type II β-turn necessary to orient the N- and C-terminal ends during the interaction with the Ste2 receptor and to adapt to the conformational changes when the receptor switches to an active state (
      • Naider F.
      • Becker J.M.
      The α-factor mating pheromone of Saccharomyces cerevisiae: a model for studying the interaction of peptide hormones and G protein-coupled receptors.
      ,
      • Venkatachalam C.M.
      Stereochemical criteria for polypeptides and proteins. V. Conformation of a system of three linked peptide units.
      ,
      • Chou P.Y.
      • Fasman G.D.
      Prediction of β-turns.
      ). Alanine replacement of these central residues, which is expected to destabilize the Type II β-turn, leads to a reduction in pheromone-receptor affinity (
      • Naider F.
      • Becker J.M.
      The α-factor mating pheromone of Saccharomyces cerevisiae: a model for studying the interaction of peptide hormones and G protein-coupled receptors.
      ,
      • Shenbagamurthi P.
      • Kundu B.
      • Raths S.
      • Becker J.M.
      • Naider F.
      Biological activity and conformational isomerism in position 9 analogues of the des-1-tryptophan,3-β-cyclohexylalanine-α-factor from Saccharomyces cerevisiae.
      ). Besides this central region, the residues close to the C terminus are important for physical interaction with Ste2, as replacement by alanines caused an up to 3000-fold decrease in receptor affinity and loss of growth arrest (
      • Naider F.
      • Becker J.M.
      The α-factor mating pheromone of Saccharomyces cerevisiae: a model for studying the interaction of peptide hormones and G protein-coupled receptors.
      ). Similarly, in the dimorphic human pathogen Candida albicans, α-pheromone tridecapeptides with di-alanine substitutions at C-terminal positions 10–12 largely lost the ability to induce pheromone-mediated processes such as mating and biofilm formation (
      • Alby K.
      • Bennett R.J.
      Interspecies pheromone signaling promotes biofilm formation and same-sex mating in Candida albicans.
      ). On the other hand, the N terminus of S. cerevisiae α-factor plays a major role in receptor activation and downstream signaling events. Alanine scanning of this region resulted in modified peptides, which still bound strongly to Ste2, but failed to exert biological activity. Interestingly, these analogs function as antagonists of α-pheromone in shmoo formation, growth arrest, and gene induction assays (
      • Naider F.
      • Becker J.M.
      The α-factor mating pheromone of Saccharomyces cerevisiae: a model for studying the interaction of peptide hormones and G protein-coupled receptors.
      ).
      The function of α pheromones in sexual development appears to be broadly conserved in ascomycetes and has been experimentally demonstrated for a number of species (
      • Pöggeler S.
      5 Function and Evolution of Pheromones and Pheromone Receptors in Filamentous Ascomycetes.
      ). Interestingly, α-pheromone peptides from filamentous ascomycetes share low to no sequence similarity with yeast α-pheromone, tend to be significantly shorter (typically 10 amino acids), and often carry conserved Trp1-Cys2 and Gly6-Gln7 residues at their N terminus and central region, respectively (
      • Turrà D.
      • El Ghalid M.
      • Rossi F.
      • Di Pietro A.
      Fungal pathogen uses sex pheromone receptor for chemotropic sensing of host plant signals.
      ,
      • Martin S.H.
      • Wingfield B.D.
      • Wingfield M.J.
      • Steenkamp E.T.
      Causes and consequences of variability in peptide mating pheromones of Ascomycete fungi.
      ). The structure-function relationship for this type of α-pheromones has not been investigated so far.
      Here we present an extensive biophysical characterization and high resolution structure of synthetic α-pheromone of Fusarium oxysporum, a highly destructive ascomycete plant pathogen that attacks over a hundred different crop species and has also been reported as an emerging pathogen of humans (
      • Dean R.
      • Van Kan J.A.
      • Pretorius Z.A.
      • Hammond-Kosack K.E.
      • Di Pietro A.
      • Spanu P.D.
      • Rudd J.J.
      • Dickman M.
      • Kahmann R.
      • Ellis J.
      • Foster G.D.
      The Top 10 fungal pathogens in molecular plant pathology.
      ). F. oxysporum was recently shown to encode a predicted α-pheromone peptide with chemoattractant activity (
      • Turrà D.
      • El Ghalid M.
      • Rossi F.
      • Di Pietro A.
      Fungal pathogen uses sex pheromone receptor for chemotropic sensing of host plant signals.
      ). We show that, similar to its yeast counterpart, F. oxysporum α-pheromone adopts a β-turn structure in water that becomes more globular in the presence of intracellular-like cosolvents. Using di-alanine substitution as well as a scrambled derivative of the peptide, we demonstrate a crucial role for the central Gly6-Gln7 residues in α-pheromone bending and chemoattractant activity. We further show that α-pheromone inhibits hyphal growth of F. oxysporum and that the inhibitory function is independent of the plasma membrane GPCR Ste2 and of the central Gly6-Gln7 residues but instead requires the conserved Trp1-Cys2 residues at the N terminus.

      Discussion

      Elucidation of three-dimensional-structures has been widely used to study structure-function relationships in small biologically active peptides (
      • Naider F.
      • Becker J.M.
      The α-factor mating pheromone of Saccharomyces cerevisiae: a model for studying the interaction of peptide hormones and G protein-coupled receptors.
      ,
      • Grieco P.
      • Luca V.
      • Auriemma L.
      • Carotenuto A.
      • Saviello M.R.
      • Campiglia P.
      • Barra D.
      • Novellino E.
      • Mangoni M.L.
      Alanine scanning analysis and structure-function relationships of the frog-skin antimicrobial peptide temporin-1Ta.
      ,
      • Cunningham B.C.
      • Wells J.A.
      High-resolution epitope mapping of hGH-receptor interactions by alanine-scanning mutagenesis.
      ). NMR analysis of S. cerevisiae α-pheromone suggested that the residues close to the N terminus are important for receptor activation, whereas those at the C terminus are implicated in receptor binding, and those in the center are required for orienting the signaling and binding domains of the pheromone (
      • Naider F.
      • Becker J.M.
      The α-factor mating pheromone of Saccharomyces cerevisiae: a model for studying the interaction of peptide hormones and G protein-coupled receptors.
      ). Moreover, the central region encompassing residues 7KPGQ10 was shown to form a transient Type II β-turn, which is required for activation of Ste2 (
      • Naider F.
      • Becker J.M.
      The α-factor mating pheromone of Saccharomyces cerevisiae: a model for studying the interaction of peptide hormones and G protein-coupled receptors.
      ). These results revealed that even a relatively short peptide contains regions associated with distinct biological functions. So far, however, the lack of a high atomic resolution structure has prevented atomic modeling of the pheromone-receptor interaction.
      In this work we performed structure-function analysis of the mating pheromone α from the fungal pathogen F. oxysporum. Such information is crucial to understand how this small peptide interacts with and activates its cognate GPCR Ste2, which was recently shown to play a key role in chemotropic growth of F. oxysporum toward both α-pheromone and the host plant tomato (
      • Turrà D.
      • El Ghalid M.
      • Rossi F.
      • Di Pietro A.
      Fungal pathogen uses sex pheromone receptor for chemotropic sensing of host plant signals.
      ,
      • Turrà D.
      • Di Pietro A.
      Chemotropic sensing in fungus-plant interactions.
      ). Similar to most α-pheromones from filamentous ascomycetes (
      • Martin S.H.
      • Wingfield B.D.
      • Wingfield M.J.
      • Steenkamp E.T.
      Causes and consequences of variability in peptide mating pheromones of Ascomycete fungi.
      ), the F. oxysporum peptide is significantly shorter than its yeast counterpart. In addition, F. oxysporum α-pheromone contains two cysteines that could potentially be involved in inter- or intramolecular disulfide bonds, whereas the S. cerevisiae α-factor has two prolines, one of which was shown to be involved in the central β-turn structure (
      • Naider F.
      • Becker J.M.
      The α-factor mating pheromone of Saccharomyces cerevisiae: a model for studying the interaction of peptide hormones and G protein-coupled receptors.
      ).

      Structural Characteristics of F. oxysporum α-Pheromone

      Despite the difference in length, S. cerevisiae and F. oxysporum α-pheromones share a number of common features. Both are cationic peptides with a pI around 8. Both have, at the center of the ordered β-turn, a charged residue (Arg in F. oxysporum and Lys in S. cerevisiae) whose side chains point toward the solvent, suggesting that these residues may act as a molecular antenna, playing a key role in regulating potential intermolecular interactions. An interesting feature that differentiates F. oxysporum α-pheromone from its ortholog in S. cerevisiae is the presence of two cysteines, both of which were found to be reduced in vitro. Although it is currently unknown whether this result reflects the biological context of the pheromone-receptor interaction, the finding that the cysteines are not involved in the formation of intra- or interdisulfide bonds together with the high reactivity and known biological functions of the thiol groups opens the intriguing hypothesis that F. oxysporum α-pheromone may have a previously unreported function in redox regulated processes, whereas S. cerevisiae pheromone, which lacks the cysteines, would not. Cysteines can easily function as nucleophiles and thus could form covalent adducts with different molecules such as lipids or ADP, a hypothesis that might be of interest for future investigations.

      Three-dimensional Structure of α-Pheromone Reveals the Presence of a Central β-Turn Essential for Chemoattractant Activity

      The three-dimensional structure of F. oxysporum α-pheromone resembles that of the longer α-factor of S. cerevisiae. Both peptides contain a central β-turn with a cationic amino acid residue, Lys in S. cerevisiae and Arg in F. oxysporum. A low resolution model for S. cerevisiae α-factor bound to Ste2 was previously proposed based on biochemical and biophysical data (
      • Lee B.K.
      • Khare S.
      • Naider F.
      • Becker J.M.
      Identification of residues of the Saccharomyces cerevisiae G protein-coupled receptor contributing to α-factor pheromone binding.
      ). The model places α-factor bent around the Pro-Gly center of the peptide, with the Lys side chain facing away from the transmembrane domains and interacting with a binding pocket formed by the extracellular loops of the receptor. A similar model could be proposed for F. oxysporum α-pheromone in the interaction with its cognate receptor. Although the structure of the peptide in the complex could be modified with respect to the free form (i.e. X-Pro cis/trans equilibrium) by an interacting induced fit, the presence of a preferred conformation in solution should be energetically favorable for the process.
      Previous studies revealed that α-pheromone elicits a robust chemotropic response in germ tubes of F. oxysporum, which is dependent on the cognate GPCR Ste2 (
      • Turrà D.
      • El Ghalid M.
      • Rossi F.
      • Di Pietro A.
      Fungal pathogen uses sex pheromone receptor for chemotropic sensing of host plant signals.
      ,
      • Turrà D.
      • Nordzieke D.
      • Vitale S.
      • El Ghalid M.
      • Di Pietro A.
      Hyphal chemotropism in fungal pathogenicity.
      ). Here we found that chemoattraction requires the Gly6 and Gln7, but not the Trp1 and Cys2 residues of α-pheromone. This is in line with the structural role of Gly6 and Gln7 in the maintenance of the three-dimensional structure and strongly suggests that activation of Ste2-mediated chemotropic growth depends on the secondary β-turn structure of α-pheromone rather than on its amino acid composition or pI. The idea is further supported by the finding that the N-terminal Trp1 and Cys2 residues which play no substantial role in the structure of α-pheromone are not required for chemoattraction. Indeed, alanine substitutions at the N terminus of α-pheromone in S. cerevisiae and C. albicans led to increased pheromone activity, suggesting that this region could have an inhibitory function role in receptor-mediated signaling (
      • Naider F.
      • Becker J.M.
      The α-factor mating pheromone of Saccharomyces cerevisiae: a model for studying the interaction of peptide hormones and G protein-coupled receptors.
      ,
      • Alby K.
      • Bennett R.J.
      Interspecies pheromone signaling promotes biofilm formation and same-sex mating in Candida albicans.
      ).
      Besides chemoattraction, α-pheromone triggers a cell cycle and growth arrest in S. cerevisiae and C. albicans (
      • Manney T.R.
      Expression of the BAR1 gene in Saccharomyces cerevisiae: induction by the α mating pheromone of an activity associated with a secreted protein.
      ,
      • Panwar S.L.
      • Legrand M.
      • Dignard D.
      • Whiteway M.
      • Magee P.T.
      MFα1, the gene encoding the α mating pheromone of Candida albicans.
      ). Here we found that hyphae exposed to α-pheromone contained fewer cell compartments and nuclei, indicating that similar to S. cerevisiae and C. albicans (
      • Bücking-Throm E.
      • Duntze W.
      • Hartwell L.H.
      • Manney T.R.
      Reversible arrest of haploid yeast cells in the initiation of DNA synthesis by a diffusible sex factor.
      ,
      • Jones Jr, S.K.
      • Clarke S.C.
      • Craik C.S.
      • Bennett R.J.
      Evolutionary selection on barrier activity: Bar1 is an aspartyl protease with novel substrate specificity.
      ), α-pheromone inhibits cell division in F. oxysporum. In both S. cerevisiae and C. albicans α-pheromone inhibits cell cycle progression via Ste2-mediated activation of a dedicated signaling cascade (
      • Reneke J.E.
      • Blumer K.J.
      • Courchesne W.E.
      • Thorner J.
      The carboxy-terminal segment of the yeast α-factor receptor is a regulatory domain.
      • Elion E.A.
      • Satterberg B.
      • Kranz J.E.
      FUS3 phosphorylates multiple components of the mating signal transduction cascade: evidence for STE12 and FAR1.
      ,
      • Schaefer D.
      • Côte P.
      • Whiteway M.
      • Bennett R.J.
      Barrier activity in Candida albicans mediates pheromone degradation and promotes mating.
      • Lin C.H.
      • Choi A.
      • Bennett R.J.
      Defining pheromone-receptor signaling in Candida albicans and related asexual Candida species.
      ). Unexpectedly, pheromone-mediated growth inhibition of F. oxysporum germ tubes does not require the cognate receptor Ste2. These results suggest the presence of additional, currently unknown α-pheromone ligands or receptors in this species, with differential roles in regulation of cell growth and chemotropism. Interestingly, the growth inhibitory activity was abolished in the d-Ala1,2 and the scrambled analogs, both of which lack a tryptophan residue at the N or the C terminus, respectively. Aromatic residues, particularly tryptophan, have been reported to undergo specific interactions with lipid moieties resulting in anchoring to the membrane (
      • de Jesus A.J.
      • Allen T.W.
      The role of tryptophan side chains in membrane protein anchoring and hydrophobic mismatch.
      ). The presence of the tryptophan residues Trp1 and Trp10 at the two termini suggests that they could act as a structural clamp during a potential membrane interaction linked to the growth inhibitory activity of the α-pheromone.
      In summary our results establish that F. oxysporum α-pheromone adopts a defined secondary structure and, despite its shorter size, contains discrete regions involved in different biological processes such as polarity reorientation and cell cycle control. We consider it likely that these findings apply to α-pheromones from other ascomycetes. The signaling functions of fungal sex pheromones might thus be more complex than previously anticipated.

      Author Contributions

      S. V. designed, performed, analyzed the experiments in Figs. 1FIGURE 2, FIGURE 3, FIGURE 4, and wrote the paper. A. P.-.H and S. S. performed and analyzed the NMR experiments and structure calculations in FIGURE 5, FIGURE 6. A. M.-P. designed, performed, and analyzed the experiments in Figs. 4 and wrote the paper. A. D. P. coordinated the study and wrote the paper. D. T. conceived and coordinated the study and wrote the paper. M. B. conceived and coordinated the study and wrote the paper. All authors analyzed the results and approved the final version of the manuscript.

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