| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
|
First published online February 18, 2005; 10.1105/tpc.104.028886 © 2005 American Society of Plant Biologists Structural Basis for the Interaction between Pectin Methylesterase and a Specific Inhibitor Protein
a Department of Biochemical Sciences, University of Rome, 00185 Rome, Italy 2 To whom correspondence should be addressed. E-mail daniela.bellincampi{at}uniroma1.it; fax 39-06-49912446.
Pectin, one of the main components of the plant cell wall, is secreted in a highly methyl-esterified form and subsequently deesterified in muro by pectin methylesterases (PMEs). In many developmental processes, PMEs are regulated by either differential expression or posttranslational control by protein inhibitors (PMEIs). PMEIs are typically active against plant PMEs and ineffective against microbial enzymes. Here, we describe the three-dimensional structure of the complex between the most abundant PME isoform from tomato fruit (Lycopersicon esculentum) and PMEI from kiwi (Actinidia deliciosa) at 1.9-Å resolution. The enzyme folds into a right-handed parallel ß-helical structure typical of pectic enzymes. The inhibitor is almost all helical, with four long  -helices aligned in an antiparallel manner in a classical up-and-down four-helical bundle. The two proteins form a stoichiometric 1:1 complex in which the inhibitor covers the shallow cleft of the enzyme where the putative active site is located. The four-helix bundle of the inhibitor packs roughly perpendicular to the main axis of the parallel ß-helix of PME, and three helices of the bundle interact with the enzyme. The interaction interface displays a polar character, typical of nonobligate complexes formed by soluble proteins. The structure of the complex gives an insight into the specificity of the inhibitor toward plant PMEs and the mechanism of regulation of these enzymes.
Pectin, one of the main components of the plant cell wall, is continually modified and remodeled during plant growth and development (Ridley et al., 2001  ). For example, the pattern of pectin esterification changes during cell expansion, growth, and fruit ripening as well as during infection by phytopathogenic microorganisms (Steele et al., 1997; Willats et al., 2001). After secretion into the wall as a highly methylesterified form, pectin is deesterified in muro by pectin methylesterases (PMEs) (E.C. 3.1.1.11) in a spatially regulated manner during development (Knox et al., 1990). Demethylation leads to the formation of polyuronides aggregating into calcium-linked gels that are important in controlling the porosity and mechanical properties of the wall (Willats et al., 2001). By generating free carboxylic groups, PMEs also affect the wall pH and consequently influence the activity of a wide range of hydrolytic enzymes, including PMEs (Grignon and Sentenac, 1991; Denes et al., 2000; Goldberg et al., 2001). PMEs produced by plants take part in important physiological processes, such as microsporogenesis, pollen growth, seed germination, root development, polarity of leaf growth, stem elongation, fruit ripening, and loss of tissue integrity (Tieman and Handa, 1994; Wen et al., 1999; Micheli et al., 2000; Pilling et al., 2000; Micheli, 2001; Pilling et al., 2004). They have also been reported to play a role in response to fungal pathogens (Wietholter et al., 2003) and are required for the systemic spread of Tobacco mosaic virus through the plant (Dorokhov et al., 1999; Chen et al., 2000; Chen and Citovsky, 2003). PMEs are not only produced by plants but also by microbial pathogens (De Lorenzo et al., 1997) and by symbiotic microorganisms during their interactions with plants (Lievens et al., 2002).
Isoforms of PME differing by molecular weight, pI, and biochemical activity are encoded by large families of genes, either constitutively expressed (Giovane et al., 1994
Although a role of PMEIs in regulating the activity of endogenous PMEs is most likely, a physiological action of these inhibitors toward enzymes derived from different species cannot be excluded. It is known that PMEs and PMEIs are both expressed in flower tissues and pollen grains (Wolf et al., 2003
To date, the structures of only two PMEs, one from carrot (Daucus carota) (PDB code 1GQ8) (Johansson et al., 2002
PMEI from kiwifruit is composed of different isoforms that are not easily separated by biochemical methods (Camardella et al., 2000 ; Mattei et al., 2002). To obtain an amount of homogeneous PMEI suitable for structural characterization, a synthetic gene was generated on the basis of the amino acid sequence of the prevalent PMEI isoform from kiwifruit (Camardella et al., 2000) and expressed in Pichia pastoris. The protein, purified to homogeneity, displayed chemical, physical, and spectral properties identical to those of the prevalent natural isoform from kiwi (Scognamiglio et al., 2003). The enzyme was mixed with a molar excess of inhibitor, and the resulting PME/PMEI complex was purified by ion exchange chromatography. The three-dimensional structure of the complex was determined at 1.9-Å resolution using a combination of single isomorphous replacement and molecular replacement methods. Details about data collection, phasing, and refinement statistics are summarized in Table 1. The model, comprising 317 residues for PME, 151 for PMEI, and 462 water molecules, has been refined to an R factor of 20.0% and an Rfree of 23.1% and has a good stereochemistry, with 99.8% of the residues lying either in the most favored or in the additional allowed regions of the Ramachandran plot (Table 1).
The Structure of Tomato PME Exhibits the Typical Fold of Pectic Enzymes PME-1 from tomato belongs to family CE8 of the sequence-based classification of carbohydrate esterases (http://afmb.cnrs-mrs.fr/CAZY). The enzyme folds into a right-handed parallel ß-helix, first observed in pectate lyase C (Yoder et al., 1993 ) and typical of pectic enzymes (Jenkins and Pickersgill, 2001) (Figure 1). The ß-helix consists of seven complete coils, which have different lengths because the number of amino acids located in the loops connecting the ß-strands is variable. Each coil consists of three ß-strands that line up to form three extended parallel ß-sheets called PB1, PB2, and PB3. T1 identifies the stack of turns between PB1 and PB2, T2 the stack between PB2 and PB3, and T3 those between PB3 and PB1. Letters following T identify the position of each turn with respect to the coil of the ß-helix, whereas A corresponds to the first turn in the N-terminal region. Turns T1 (except for TB1) are short and mainly composed of residues in L-conformation and are responsible for the sharp bend between the sheets as observed in other parallel ß-helix structures (Federici et al., 2001; Jenkins and Pickersgill, 2001). Turns T2 and T3 are generally longer and more variable; in particular, TF3 and most of T2 turns protrude from the central parallel ß-helix to form the shallow cleft where the putative active site is located. In contrast with what was reported (Markovic and Jornvall, 1992), no electron density corresponding to the disulphide bridges Cys98-Cys125 and Cys166-Cys200 was observed. The absence of these bridges was confirmed by biochemical analysis, indicating the presence of four thiol groups upon titration with the Ellman's reagent in denaturing conditions (data not shown). The N-terminal region of PME is composed by a short -helix followed by a ß-strand that lines up with PB1. The C-terminal region has an extended conformation in which a long tail and four short and distorted -helices protrude out of the parallel ß-helix flanking PB1.
The putative active site of PME is located on the PB3 sheet in a cleft shaped by TB2, TC2, TF2, and TF3. Many aromatic residues (Phe80, Tyr135, Phe156, Tyr218, Trp223, and Trp248) putatively involved in substrate binding are located in this pocket (Johansson et al., 2002 ). These residues are well conserved in plant PMEs (Markovic and Janecek, 2004). Tyr135, Phe156, and Trp223 are also conserved in PME of E. chrysanthemi (Jenkins et al., 2001). Asp132, Asp153, and Arg 221, located inside the crevice, have been hypothesized to be the catalytic residues (Jenkins et al., 2001). In the putative catalytic site, OD1 of Asp153 is located 2.82 Å from and interacts with the NE of Arg221, whereas OD2 of Asp153 is located 2.85 Å from and interacts with NH2 of Arg221. Moreover, OD2 of Asp153 is at H-bonding distance (2.63 Å) from a water residue (W227) that also forms an H-bond with OD1 of Asp132 (2.76 Å) (Figure 2). In analogy with the proposed mechanism of action of PME from carrot (Johansson et al., 2002), we can infer a mechanism of catalysis in which Asp153, polarized by the proximity with Arg221, performs a nucleophilic attack on the carboxymethyl group of the substrate. The tetrahedral anionic intermediate formed is stabilized by the interaction with two conserved Gln residues (Gln109 and Gln131). Afterwards, Asp132 likely acts as a proton donor in the cleavage step where methanol is released. The resulting carboxylate group of Asp132 then behaves as a base and receives a proton from an incoming water molecule (W227), thus restoring the active site of the enzyme. An alternative hypothesis proposed by Johansson (Johansson et al., 2002) foresees a primary nucleophilic attack performed by the water molecule deprotonated both by Asp132 and Asp153.
Superimposition of the known PME structures of carrot, E. chrysanthemi, and tomato reveals the similarity of the overall folding topologies. The similarity of tomato and carrot PMEs is more extensive with a root mean square deviation (RMSD) value of 0.7 Å calculated for all C atoms (Figure 3A), whereas the bacterial enzyme can be well superimposed to tomato PME only for 284 C atoms out of 317 and with a higher RMSD value of 1.8 Å (Figure 3B). The main differences between the plant and the bacterial enzymes are located on TB2, TC2, TF2, TG3, and TH3; these turns protrude out of the ß-helix and are much longer in the bacterial enzyme, making its putative active site cleft deeper and narrower than that of plant PMEs.
The Inhibitor Folds in an Up-and-Down Four-Helical Bundle PMEI is almost all helical, with four long helices ( 1, 2, 3, and 4) aligned in an antiparallel manner in a classical up-and-down four-helical bundle (Figure 1). The interior of the bundle is stabilized by hydrophobic interactions and by a disulphide bridge between Cys74 and Cys114, which connects helices 2 and 3. The N-terminal region, composed of three short and distorted helices (a, b, and c), extends outside the central domain and lines roughly parallel to the plane defined by the helices 1 and 4. A disulphide bridge between Cys9 and Cys18 connects a and b.
According to sequence-based classification, PMEIs belong to the family PF04043 (Pfam database, http://pfam.wustl.edu/) of invertase inhibitor (INH)/PMEIs and share with INH several structural properties (Scognamiglio et al., 2003
The PME-PMEI Complex PME and PMEI form a stoichiometric 1:1 complex in which the inhibitor covers the shallow cleft of the enzyme where the putative active site is located. The four-helix bundle of PMEI packs roughly perpendicular to the parallel ß-helix of PME, and the three helices 2, 3, and 4, but not 1, interact with the enzyme in proximity of the putative active site (Figures 1 and 5) The relative position of 2 and 3 helices is maintained by a disulphide bridge between Cys74 and Cys114. The N-terminal region of PMEI is poorly involved in the formation of the complex and may play a role in the structural stability of the inhibitor, as proposed for Nt-CIF (Hothorn et al., 2004a). Superimposition of the free Nt-CIF with the complexed PMEI shows a similar fold and orientation of helices in the bundle (Figure 4). Similarly, the structure of the free carrot PME is almost completely superimposable to the structure of tomato PME engaged in the complex with PMEI (Figure 3A). These features suggest that PME and PMEI do not undergo dramatic conformational changes upon formation of the complex.
Upon interaction, PME and PMEI bury 1148 Å2 and 1060 Å2, respectively, of their accessible surface area (ASA). The total of 2208 Å2 buried surface is somewhat larger than the average interface area reported for noncovalent protein complexes (  ASA 1600 ± 400 Å2) (LoConte et al., 1999). The interaction interface displays a surprisingly high polar character. No extended zones of hydrophobic interactions are present, whereas more than half (55%) of buried surface arises from polar and charged atoms (Figure 6). A large number of water molecules is present at the interface and 17 of them mediate intermolecular H-bonds (Figure 6). Such a polar character of the interface is typical of nonobligate complexes formed by soluble proteins, which need to expose a hydrophilic surface in their uncomplexed form (Jones and Thornton, 1996). Fifty residues (23 on PME and 27 on PMEI) establish contacts (Table 2). Twenty-two of these residues are engaged in H-bonds, and four form salt bridges (Table 3). Residues of PME forming contacts are mostly located in the proximity of the putative pectin binding site, particularly on PB3 and on T3 (Figure 2A). A large cluster of interacting residues is present on turns TD3 and TF3 and on the fourth strand of PB3. TB2 also participates with three residues contacting the inhibitor, whereas only one contact is present in the C-terminal tail of the enzyme. The contact residues of PMEI are mainly located on helices 2 and 3, with a continuous surface that extends all along. Ten residues are located on 2, 11 on 3, and four on 4; two residues reside on the N-terminal region of the inhibitor.
In the article by Hothorn et al. (2004b) , the N-terminal region of At-PMEI1 has been proposed to be crucial for the interaction with PMEs. This model does not fit with our crystallographic structure of the PME-PMEI complex, and we cannot exclude that the mode of interaction of At-PMEI1 to PMEs is somewhat peculiar. Experimental data using chimeras between the N-terminal region of At-PMEI1 and the four-helix bundle of Nt-CIF indicate that  100 times more quantity of the chimera is needed to obtain the same inhibition played by the natural At-PMEI1. This suggests that the four-helix bundle is also important for the interaction of At-PMEI1 and PME.
Whereas the electrostatic potential surface of PMEI shows an acidic patch formed by Glu76, Asp80, and Asp83 on
The stability of the complex is pH dependent, being higher in acidic conditions, typical of the apoplastic environment, and decreasing drastically by raising the pH from 6.5 to 7.5; no formation of the complex occurs at pH 8.5 (D'Avino et al., 2003
Detailed analysis of residues involved in forming the complex reveals that the putative catalytic residues (Asp132, Asp153, and Arg221) do not establish contacts with the inhibitor, neither do Gln109 and Gln131, which are thought to stabilize the anionic intermediate formed after the first nucleophilic attack. Instead, three aromatic residues (Phe80, Tyr135, and Trp223), likely responsible for substrate binding, interact with the inhibitor. Remarkably, Phe80 is one of the residues mostly involved in the interaction, burying an area of 81 Å2 upon formation of the complex. This residue establishes 17 contacts with four different residues of the inhibitor (Thr73, Glu76, Asn77, and Thr113) and a water-mediated hydrogen bond. Trp223 of PME forms three contacts with its interacting counterpart, whereas Tyr135 forms only one contact; moreover, each of them forms a water-mediated hydrogen bond. Upon formation of the complex with the inhibitor, Trp223 buries almost half of its solvent-exposed surface, and this explains the decrease of fluorescence observed in PME upon addition of the inhibitor (D'Avino et al., 2003
The structure of the PME-PMEI complex provides a possible explanation for the lack of inhibition of PMEIs on PMEs from the bacterium E. crysanthemi and the fungus Aspergillus aculeatus (Giovane et al., 2004
PMEI and Nt-CIF exhibit an almost identical fold (Figure 4) but recognize different target enzymes. The structural view of the PME-PMEI complex also provides a possible explanation for the absence of interaction of Nt-CIF with PME. Sequence comparison between the PMEIs characterized so far and Nt-CIF shows that the subset of residues of the kiwi inhibitor, Asn101, Asp109, Thr113 (located on 3), and Asn147 (located on 4), which form intermolecular H-bonds with the enzyme, are conserved only in PMEIs. In addition, an amino acid insertion that produces a distortion of the 2 helix of Nt-CIF is close to residues corresponding to Asp82 and Ser83 in 2 of PMEI, which are involved in H-bonds with PME (Figures 4 and 8). We speculate that the lack of residues important for the formation of the complex as well as the distortion of 2 helix of Nt-CIF compared with that of PMEI are responsible for the lack of interaction between Nt-CIF and PME.
Expression and Purification of PMEI and PME A synthetic AcPMEI gene was designed on the basis of the amino acid sequence of the mature PMEI from kiwifruit (Actinidia deliciosa) (AcPMEI accession number P83326 NCBI database) and expressed in Pichia pastoris. The synthesis of three AcPMEI DNA gene fragments was performed by PCR using PWO DNA Polymerase (Roche, Penzberg, Germany), and the entire gene was obtained by cloning the fragments into a pUC19 plasmid vector using two internal restriction sites designed to facilitate cloning. AcPMEI was amplified from pUC19 and cloned into the pPICZ A expression vector, used to transform P. pastoris strain X-33 (Invitrogen, Carlsbad, CA). The transformed cells were grown 3 d after induction with 0.5% (v/v) methanol; the supernatant of the culture was collected and total proteins precipitated with 80% (w/v) ammonium sulfate. The precipitated fraction was dissolved in 10 mM Tris-HCl, pH 7.5, dialyzed against the same buffer and loaded onto a Mono Q HR 10/10 column (Amersham Pharmacia Biosciences). AcPMEI was eluted by applying a linear gradient of 0 to 0.5 M NaCl in 10 mM Tris-HCl, pH 7.5. The eluted inhibitor was concentrated and loaded onto a HiLoad 16/60 Superdex 75 column (Amersham Pharmacia Biosciences) equilibrated in 10 mM Tris-HCl, pH 7.5, and 0.25 M NaCl. The purified inhibitor, displaying the same amino acid sequence of the prevalent natural isoform from kiwi, exhibited a single band by SDS-PAGE and showed a single peak upon reverse-phase HPLC on a Vydac C4 column. The N-terminal sequence of the protein confirmed its identity and indicated the presence of four additional amino acid residues, a remnant of the yeast signal sequence. These additional amino acids did not impair the inhibitory activity of the protein, and purified PMEI strongly inhibited PME-1 (Ki = 28 nM) (data not shown). The isoform PME-1 of tomato (Lycopersicon esculentum) (SwissProt accession number P14280) was purified as reported (Giovane et al., 1994). The inhibitory activity of AcPMEI against PME-1 was determined by automatic titration as previously described (Giovane et al., 1995). The inhibitory constant was calculated by Dixon plot using 0.3% and 0.05% citrus pectin (63 to 66% methylation degree from Sigma-Aldrich, St. Louis, MO).
Purification of the Complex
Crystallization, X-Ray Data Collection, Structure Determination, and Refinement
Initial phases were determined by single isomorphous replacement and anomalous scattering methods. The crystal of form A was soaked in a solution containing 2.5 M NaCl, 0.1 M sodium acetate, pH 4.5, 0.2 M Li2SO4, and 1 mM K2OsO4 for 6 h, cryoprotected as described above, and flash-frozen in liquid nitrogen. Data were collected at the BW7A Beamline of the Deutsches Elektronen Synchrotron (Hamburg, Germany). Oscillation images were integrated, scaled, and merged using DENZO and SCALEPACK (Otwinowski and Minor, 1996
We are grateful to Maurizio Brunori for is encouragement and valuable advice. We thank the staff at the ELETTRA Synchrotron (Trieste, Italy), the Deutsches Elektronen Synchrotron (Hamburg, Germany), and the European Synchrotron Radiation Facility (Grenoble, France) for beam time allocation and technical support. This research was supported by the European Union Gemini project (contract QLK1-2000-00911), the Institute Pasteur-Fondazione Cenci Bolognetti, the Giovanni Armenise-Harvard Foundation, and the Ministero dell'Università e della Ricerca Scientifica (PRIN 2002).
1 Current address: Dipartimento del Territorio e Sistemi Agro-Forestali, Università di Padova, Italy.  The author responsible for distribution of materials integral to the findings presented in this article in accordance with the policy described in the Instructions for Authors (www.plantcell.org) is: Daniela Bellincampi (daniela.bellincampi{at}uniroma1.it). Article, publication date, and citation information can be found at www.plantcell.org/cgi/doi/10.1105/tpc.104.028886. Received October 27, 2004; accepted December 28, 2004.
Adams, P.D., Pannu, N.S., Read, R.J., and Brunger, A.T. (1997). Cross-validated maximum likelihood enhances crystallographic simulated annealing refinement. Proc. Natl. Acad. Sci. USA 94, 5018–5023. Balestrieri, C., Castaldo, D., Giovane, A., Quagliuolo, L., and Servillo, L. (1990). A glycoprotein inhibitor of pectin methylesterase in kiwi fruit (Actinidia chinensis). Eur. J. Biochem. 193, 183–187.[Web of Science][Medline] Camardella, L., Carratore, V., Ciardiello, M.A., Servillo, L., Balestrieri, C., and Giovane, A. (2000). Kiwi protein inhibitor of pectin methylesterase amino-acid sequence and structural importance of two disulfide bridges. Eur. J. Biochem. 267, 4561–4565.[Medline] Chen, M.-H., and Citovsky, V. (2003). Systemic movement of a tobamovirus requires host cell pectin methylesterase. Plant J. 35, 386–392.[CrossRef][Web of Science][Medline] Chen, M.H., Sheng, J., Hind, G., Handa, A.K., and Citovsky, V. (2000). Interaction between the tobacco mosaic virus movement protein and host cell pectin methylesterases is required for viral cell-to-cell movement. EMBO J. 19, 913–920.[CrossRef][Web of Science][Medline] D'Avino, R., Camardella, L., Christensen, T.M., Giovane, A., and Servillo, L. (2003). Tomato pectin methylesterase: Modeling, fluorescence, and inhibitor interaction studies-comparison with the bacterial (Erwinia chrysanthemi) enzyme. Proteins 53, 830–839.[CrossRef][Web of Science][Medline] De Lorenzo, G., Castoria, R., Bellincampi, D., and Cervone, F. (1997). Fungal invasion enzymes and their inhibition. In The Mycota. V. Plant Relationships, Part B, G.C. Carroll and P. Tudzynski, eds (Berlin: Springer-Verlag), pp. 61–83. De Lorenzo, G., D'Ovidio, R., and Cervone, F. (2001). The role of polygacturonase-inhibiting proteins (PGIPs) in defense against pathogenic fungi. Annu. Rev. Phytopathol. 39, 313–335.[CrossRef][Web of Science][Medline] Denes, J.M., Baron, A., Renard, C.M., Pean, C., and Drilleau, J.F. (2000). Different action patterns for apple pectin methylesterase at pH 7.0 and 4.5. Carbohydr. Res. 327, 385–393.[CrossRef][Web of Science][Medline]
Di Matteo, A., Federici, L., Mattei, B., Salvi, G., Johnson, K.A., Savino, C., De Lorenzo, G., Tsernoglou, D., and Cervone, F. (2003). The crystal structure of PGIP (polygalacturonase-inhibiting protein), a leucine-rich repeat protein involved in plant defense. Proc. Natl. Acad. Sci. USA 100, 10124–10128. Dorokhov, Y.L., Makinen, K., Frolova, O.Y., Merits, A., Saarinen, J., Kalkkinen, N., Atabekov, J.G., and Saarma, M. (1999). A novel function for a ubiquitous plant enzyme pectin methylesterase: The host-cell receptor for the tobacco mosaic virus movement protein. FEBS Lett. 461, 223–228.[CrossRef][Web of Science][Medline]
Federici, L., Caprari, C., Mattei, B., Savino, C., Di Matteo, A., De Lorenzo, G., Cervone, F., and Tsernoglou, D. (2001). Structural requirements of endopolygalacturonase for the interaction with PGIP (polygalacturonase-inhibiting protein). Proc. Natl. Acad. Sci. USA 98, 13425–13430. Gaffe, J., Tiznado, M.E., and Handa, A.K. (1997). Characterization and functional expression of a ubiquitously expressed tomato pectin methylesterase. Plant Physiol. 114, 1547–1556.[Abstract] Giovane, A., Balestrieri, C., Quagliuolo, L., Castaldo, D., and Servillo, L. (1995). A glycoprotein inhibitor of pectin methylesterase in kiwi fruit. Purification by affinity chromatography and evidence of a ripening-related precursor. Eur. J. Biochem. 233, 926–929.[Web of Science][Medline] Giovane, A., Quagliuolo, L., Servillo, L., Balestrieri, C., Laratta, B., and Castaldo, D. (1994). Purification and characterization of three isozymes of pectin methylesterase from tomato fruit. J. Food Biochem. 17, 339–349. Giovane, A., Servillo, L., Balestrieri, C., Raiola, A., D'Avino, R., Tamburrini, M., Ciardiello, M.A., and Camardella, L. (2004). Pectin methylesterase inhibitor. Biochim. Biophys. Acta 1696, 245–252.[Medline]
Goldberg, R., Pierron, M., Bordenave, M., Breton, C., Morvan, C., and Du Penhoat, C.H. (2001). Control of Mung bean pectinmethylesterase isoform activities. Influence of pH and carboxyl group distribution along the pectic chains. J. Biol. Chem. 276, 8841–8847. Grignon, C., and Sentenac, H. (1991). pH and ionic conditions in the apoplastic. Annu. Rev. Plant Physiol. Plant Mol. Biol. 42, 103–128.[CrossRef][Web of Science] Hothorn, M., D'Angelo, I., Marquez, J.A., Greiner, S., and Scheffzek, K. (2004a). The invertase inhibitor Nt-CIF from tobacco: A highly thermostable four-helix bundle with an unusual N-terminal extension. J. Mol. Biol. 335, 987–995.[Medline]
Hothorn, M., Wolf, S., Aloy, P., Greiner, S., and Scheffzek, K. (2004b). Structural insights into the target specificity of plant invertase and pectin methylesterase inhibitory proteins. Plant Cell 16, 3437–3447. Jenkins, J., Mayans, O., Smith, D., Worboys, K., and Pickersgill, R.W. (2001). Three-dimensional structure of Erwinia chrysanthemi pectin methylesterase reveals a novel esterase active site. J. Mol. Biol. 305, 951–960.[CrossRef][Web of Science][Medline] Jenkins, J., and Pickersgill, R. (2001). The architecture of parallel beta-helices and related folds. Prog. Biophys. Mol. Biol. 77, 111–175.[CrossRef][Web of Science][Medline] Johansson, K., El Ahmad, M., Friemann, R., Jornvall, H., Markovic, O., and Eklund, H. (2002). Crystal structure of plant pectin methylesterase. FEBS Lett. 514, 243–249.[CrossRef][Web of Science][Medline]
Jones, S., and Thornton, J.M. (1996). Principles of protein-protein interactions. Proc. Natl. Acad. Sci. USA 93, 13–20. Knox, J.P., Linstead, P.J., King, J., Cooper, C., and Roberts, K. (1990). Pectin esterification is spatially regulated both within cell walls and between developing tissues of root apices. Planta 181, 512–521.[Web of Science] Laskowski, R.A., MacArthur, M.W., Moss, D.S., and Thornton, J.M. (1993). PROCHECK—A program to check the stereochemical quality of protein structures. J. Appl. Crystallogr. 26, 283–291.[CrossRef] Lievens, S., Goormachtig, S., Herman, S., and Holsters, M. (2002). Patterns of pectin methylesterase transcripts in developing stem nodules of Sesbania rostrata. Mol. Plant Microbe Interact. 15, 164–168.[Medline] LoConte, L., Chothia, C., and Janin, J. (1999). The atomic structure of protein-protein recognition sites. J. Mol. Biol. 285, 2177–2198.[CrossRef][Web of Science][Medline] Markovic, O., and Janecek, S. (2004). Pectin methylesterases: Sequence-structural features and phylogenetic relationships. Carbohydr. Res. 339, 2281–2295.[CrossRef][Web of Science][Medline] Markovic, O., and Jornvall, H. (1992). Disulfide bridges in tomato pectinesterase: Variations from pectinesterases of other species; conservation of possible active site segments. Protein Sci. 1, 1288–1292.[Medline] Mattei, B., Raiola, A., Caprari, C., Federici, L., Bellincampi, D., De Lorenzo, G., Cervone, F., Giovane, A., and Camardella, L. (2002). Studies on plant inhibitors of pectin modifying enzymes: Polygalacturonase-inhibiting protein (PGIP) and pectin methylesterase inhibitor (PMEI). In Carbohydrate Bioengineering: Interdisciplinary Approaches, T.T. Teeri, B. Svensson, H.J. Gilbert, and T. Feizi, eds (Cambridge, UK: Royal Society of Chemistry), pp. 160–167. Micheli, F. (2001). Pectin methylesterases: Cell wall enzymes with important roles in plant physiology. Trends Plant Sci. 6, 414–419.[CrossRef][Web of Science][Medline]
Micheli, F., Sundberg, B., Goldberg, R., and Richard, L. (2000). Radial distribution pattern of pectin methylesterases across the cambial region of hybrid aspen at activity and dormancy. Plant Physiol. 124, 191–199. Murshudov, G.N., Vagin, A.A., and Dodson, E.J. (1997). Refinement of macromolecular structures by the maximum-likelihood method. Acta Crystallogr. D Biol. Crystallogr. 53, 240–255.[CrossRef][Medline] Navaza, J. (1994). AMoRe: An automated package for molecular replacement. Acta Crystallogr. D Biol. Crystallogr. 50, 157–163.[CrossRef] Otwinowski, Z., and Minor, W. (1996). Processing of x-ray: Diffraction data collected in oscillation mode. In Methods in Enzymology, C.W. Carter and R.M. Sweet, eds (London and New York: Academic Press), pp. 307–326. Pilling, J., Willmitzer, L., Bucking, H., and Fisahn, J. (2004). Inhibition of a ubiquitously expressed pectin methyl esterase in Solanum tuberosum L. affects plant growth, leaf growth polarity, and ion partitioning. Planta 219, 32–40.[CrossRef][Web of Science][Medline] Pilling, J., Willmitzer, L., and Fisahn, J. (2000). Expression of a Petunia inflata pectin methyl esterase in Solanum tuberosum L. enhances stem elongation and modifies cation distribution. Planta 210, 391–399.[CrossRef][Web of Science][Medline] Raiola, A., Camardella, L., Giovane, A., Mattei, B., De Lorenzo, G., Cervone, F., and Bellincampi, D. (2004). Two Arabidopsis thaliana genes encode functional pectin methylesterase inhibitors. FEBS Lett. 557, 199–203.[CrossRef][Web of Science][Medline] Ridley, B.L., O'Neill, M.A., and Mohnen, D. (2001). Pectins: Structure, biosynthesis, and oligogalacturonide-related signaling. Phytochemistry 57, 929–967.[CrossRef][Web of Science][Medline] Scognamiglio, M.A., Ciardiello, M.A., Tamburrini, M., Carratore, V., Rausch, T., and Camardella, L. (2003). The plant invertase inhibitor shares structural properties and disulfide bridges arrangement with the pectin methylesterase inhibitor. J. Protein Chem. 22, 363–369.[Medline] Steele, N.M., McCann, M.C., and Roberts, K. (1997). Pectin modification in cell walls of ripening tomatoes occurs in distinct domains. Plant Physiol. 114, 373–381.[Abstract] Terwilliger, T.C. (1999). Reciprocal-space solvent flattening. Acta Crystallogr. D Biol. Crystallogr. 55, 1863–1871.[CrossRef][Medline] Terwilliger, T.C. (2000). Maximum-likelihood density modification. Acta Crystallogr. D Biol. Crystallogr. 56, 965–972.[CrossRef][Medline] Tieman, D.M., and Handa, A.K. (1994). Reduction in pectin methylesterase activity modifies tissue integrity and cation levels in ripening tomato (Lycopersicon esculentum Mill.) fruits. Plant Physiol. 106, 429–436.[Abstract]
Wen, F.S., Zhu, Y.M., and Hawes, M.C. (1999). Effect of pectin methylesterase gene expression on pea root development. Plant Cell 11, 1129–1140. Wietholter, N., Graessner, B., Mierau, M., Mort, A.J., and Moerschbacher, B.M. (2003). Differences in the methyl ester distribution of homogalacturonans from near-isogenic wheat lines resistant and susceptible to the wheat stem rust fungus. Mol. Plant Microbe Interact. 16, 945–952.[Web of Science][Medline]
Willats, W.G., Orfila, C., Limberg, G., Buchholt, H.C., van Alebeek, G.J., Voragen, A.G., Marcus, S.E., Christensen, T.M., Mikkelsen, J.D., Murray, B.S., and Knox, J.P. (2001). Modulation of the degree and pattern of methyl esterification of pectic homogalacturonan in plant cell walls. Implications for pectin methyl esterase action, matrix properties and cell adhesion. J. Biol. Chem. 276, 19404–19413. Wolf, S., Grsic-Rausch, S., Rausch, T., and Greiner, S. (2003). Identification of pollen-expressed pectin methylesterase inhibitors in Arabidopsis. FEBS Lett. 555, 551–555.[CrossRef][Web of Science][Medline]
Yoder, M.D., Keen, N.T., and Jurnak, F. (1993). New domain motif: The structure of pectate lyase C, a secreted plant virulence factor. Science 260, 1503–1507. This article has been cited by other articles:
|
|||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| ASPB Publications | THE PLANT CELL | PLANT PHYSIOLOGY | |
|---|---|---|---|
TOP
ABSTRACT
INTRODUCTION
= 90.0°. Single crystals of form B were obtained in drops made up by 1 µL of 11.0 mg/mL of purified complex and 1 µL of well solution consisting of 1.6 M MgSO4, and 0.1 M Mes, pH 6.0. After 1 week, crystals grew to 
, with the X-SOLVATE tool of QUANTA. Only solvent residues with a Bfactor 
45 Å2 and hydrogen bonded to the protein molecule were kept in the structure refinement. Further refinement and rebuilding led to a crystallographic R factor of 20% and an Rfree of 23.1%. The final model contains residues 1 to 317 of chain A (PME), 0 to 150 of chain B (PMEI), and 462 water residues. A total of 91.9% of residues are in the most favored region of the Ramachandran space, with 7.9% in additional allowed regions, 0.2% in generously allowed regions, and none in disallowed regions. Secondary structure assignment and the check of the geometrical quality were performed using PROCHECK (Laskowski et al., 1993
