Journal of Molecular Biology
The Invertase Inhibitor Nt-CIF from Tobacco: A Highly Thermostable Four-helix Bundle with an Unusual N-terminal Extension
Introduction
Plant acid invertases catalyze the hydrolytic cleavage of the transport sugar sucrose,1 which is the major transport form of carbohydrates in higher plants. Sucrose is exported from the source tissues (leaves) and transported via the phloem to the different sink tissues (roots, stem, reproductive organs and vegetative storage organs). Cells in the target tissue may take up sucrose symplastically or apoplastically, and the sucrose can be hydrolyzed by invertases subsequently. These enzymes reside in the vacuole and the extracellular space, where invertase activity facilitates long-range carbohydrate transport by creating sucrose concentration gradients.2 Sucrose and its hydrolysis products glucose and fructose provide growing tissues with energy and can serve as signals regulating gene-expression.3 Therefore, invertase activity at the wrong time and place can dramatically affect plant viability and development.4 Anti-sense repression of invertase genes distorts seedling morphology and leaf-to-root ratios5 and induces male sterility in tobacco.6 Thus, invertases are tightly regulated in order to maintain normal plant development. While control of invertase activity can occur at the level of transcription,7 the posttranslational regulation appears to be of equal importance. Hereby, activity is modulated via interaction with highly specific inhibitory proteins, a general strategy also found for other enzymes involved in carbohydrate metabolism.8., 9. These invertase inhibitors have been shown to be heat-stable, non-glycosylated monomers, affecting invertase activity in a pH-dependent manner.10 The inhibition process can be modulated in vitro by the substrate sucrose and divalent cations in the millimolar range.10., 11. Biotechnological relevance has been pointed out in the case of cold-induced sweetening of potato tubers. Transgenic expression of invertase inhibitors can interfere with this major food storage problem.12
Recent plant genome sequencing projects have identified a novel protein family formed by invertase inhibitors and the related inhibitors of pectin methylesterase (PME),13 the latter being involved in the control of pectin metabolism. Common features of this plant-specific family include an N-terminal signal peptide, four conserved cysteine residues involved in the formation of two disulfide bridges13., 14. and a total size of about 18 kDa. In vitro assays have confirmed that particular members of the protein family either inhibit invertase or pectin methylesterase activity, but never both.14
The limited availability of pure protein components has been a major obstacle in the biochemical and structural investigation of invertase–inhibitor interaction. As a first step to characterize the inhibition mechanism in detail, we have recently expressed and crystallized a biologically active invertase inhibitor, termed Nt-CIF (Nicotiana tabacum cell wall inhibitor of beta fructosidase),15 previously cloned from tobacco.16 The crystal structure reported here reveals the first three-dimensional model for a member of the inhibitory protein family. In addition, we show that the recombinant protein is heat-stable and retains biological activity upon cooling to ambient temperature. Using site-directed mutagenesis along with protein deletions, we have identified major determinants of the inhibitors' structural integrity. The presented work implies relevance for the posttranslational inhibition of pectin methylesterases, since (based on sequence homology) the structures of their cognate inhibitors are likely to be similar to our Nt-CIF structure.
Section snippets
Results and Discussion
Nt-CIF was expressed, purified and crystallized as described.15 Briefly, overexpression as a thioredoxin A fusion protein in Escherichia coli Origami cells resulted in soluble protein that was purified in a three-step procedure15 and crystallized in four different crystal forms at pH values ranging from 4.6 to 9. The structure was determined by the multiple isomorphous replacement (MIR) method using the crystal form (native 1) grown in the presence of CdCl2 (see Table 1). Using the coordinates
Concluding Remarks
Based on secondary structure prediction and data presented,13., 14. inhibitors of invertase and pectin methylesterase appear to be structurally very similar. The fifth conserved cysteine residue in PMEIs is replaced by serine in Nt-CIF (Figure 1(c)) pointing towards the center of the bundle. Since we have mapped other conserved residues within the protein family to be of predominant structural importance, it seems likely that specificity towards two totally unrelated enzymes is achieved by a
X-ray analysis
Bacterial protein expression, purification and crystallization of the presented inhibitor from tobacco are reported elsewhere.15 Briefly, Nt-CIF was expressed as a thioredoxin A fusion protein in E. coli Origami cells (Novagen), and crystallized by using the hanging-drop method with 4 M sodium formate, in 0.1 M bis-Tris (pH 7) as precipitant. Heavy atom derivatives were prepared by soaking orthorhombic crystals grown in the presence of CdCl2 in crystallization buffer supplemented with 0.5 mM
Acknowledgements
We thank Thomas Rausch for generous support, helpful discussions and critically reading the manuscript, Manuela Lopez de la Paz for discussion, the staff at beam lines ID29 of the European Synchrotron Radiation Facility (ESRF), Grenoble, France, of beam lines BW7A of the Deutsches Elektronen Synchrotron, Hamburg, Germany, for technical support during data collection. We gratefully acknowledge financial support from the Südzucker AG Mannheim (Germany) and the KWS Saat AG, Einbeck (Germany),
References (37)
- et al.
The sucrose-cleaving enzymes of plants are crucial for development, growth and carbon partitioning
Trends Plant Sci.
(1999) - et al.
Sucrose protects cell wall invertase but not vacuolar invertase against proteinaceous inhibitors
FEBS Letters
(1996) - et al.
Protein structure comparison by alignment of distance matrices
J. Mol. Biol.
(1993) - et al.
Structure of the Rho family GTP-binding protein Cdc42 in complex with the multifunctional regulator RhoGDI
Cell
(2000) - et al.
Three-dimensional structure of Erwinia chrysanthemi pectin methylesterase reveals a novel esterase active site
J. Mol. Biol.
(2001) - et al.
Crystal structure of plant pectin methylesterase
FEBS Letters
(2002) - et al.
Raster3D: Photorealistic molecular graphics
Methods Enzymol.
(1997) Invertases. Primary structures, functions, and roles in plant development and sucrose partitioning
Plant Physiol.
(1999)- et al.
A similar dichotomy of sugar modulation and developmental expression affects both paths of sucrose metabolism: evidence from a maize invertase gene family
Plant Cell
(1996) - et al.
Expression of a yeast-derived invertase in the cell wall of tobacco and Arabidopsis plants leads to accumulation of carbohydrate and inhibition of photosynthesis and strongly influences growth and phenotype of transgenic tobacco plants
Embo J.
(1990)
Antisense repression of vacuolar and cell wall invertase in transgenic carrot alters early plant development and sucrose partitioning
Plant Cell
Induction of male sterility in plants by metabolic engineering of the carbohydrate supply
Proc. Natl Acad. Sci. USA
Sugars modulate an unusual mode of control of the cell-wall invertase gene (Incw1) through its 3′ untranslated region in a cell suspension culture of maize
Proc. Natl Acad. Sci. USA
Structural requirements of endopolygalacturonase for the interaction with PGIP (polygalacturonase-inhibiting protein)
Proc. Natl Acad. Sci. USA
Structural analysis of xylanase inhibitor protein I (XIP-I), a proteinaceous xylanase inhibitor from wheat (Triticum aestivum, var Soisson)
Biochem. J.
A 17-kDa Nicotiana tabacum cell-wall peptide acts as an in vitro inhibitor of the cell-wall isoform of acid invertase
Planta
Ectopic expression of a tobacco invertase inhibitor homolog prevents cold-induced sweetening of potato tubers
Nature Biotechnol.
Kiwi protein inhibitor of pectin methylesterase amino-acid sequence and structural importance of two disulfide bridges
Eur. J. Biochem.
Cited by (46)
A bioactive polypeptide from sugarcane selectively inhibits intestinal sucrase
2020, International Journal of Biological MacromoleculesCitation Excerpt :Multiple sequence alignment of characterized plant invertase inhibitor proteins [8,12,34–36] and sucinh (SP80-3280) was performed using MUSCLE 3.8 [37] (Fig. S1). Consurf (http://consurf.tau.ac.il/2016/) is plotted based on 1RJ1 [38] to represent the evolutionary conservation of residues. Rat, mouse and human intestinal sucrase sequences were aligned using ClustalW 2.1(Fig. S3).
Homologs of vacuolar invertase inhibitor INH2 in tuber-bearing wild potato species and Solanum tuberosum: gene polymorphism and co-expression with saccharolytic enzyme genes in response to cold stress
2020, Scientia HorticulturaeCitation Excerpt :However, the signal peptide sequence in INH2 has not been identified, although INH2 N-terminally labeled by GFP was detected in vacuoles of potato tubers (Brummell et al., 2011). The INH2 N-terminal hairpin was found to influence enzyme structural stability and intermolecular interactions, but its effect on the inhibitor activity was not revealed (Hothorn et al., 2004a). In this study, the presence of a 19-aa signal peptide was predicted for INH2 homologs of all analyzed potato accessions.
Sequence diversity and in silico structure prediction of the vacuolar invertase inhibitor gene from potato (Solanum tuberosum L.) cultivars differing in sugar content
2019, Food ChemistryCitation Excerpt :The secondary protein structure prediction is in agreement with previously identified invertase inhibitor structure from tobacco (Hothorn et al., 2010). Four conserved cysteine residues (at C34, C43, C101 and C142) are the characteristic of plant invertase inhibitors (Hothorn et al., 2010) and these Cys residues have been found to be engaged in disulfide bridge formation (Hothorn, D'Angelo, et al., 2004a; Hothorn, Wolf, Aloy, Greiner, & Scheffzek, 2004b; Rausch & Greiner, 2004). Moreover, they were also found to contribute to the structural stabilization of the protein and it has been observed that the well conserved N-terminal end helical hairpin extension is not only crucial for the structural integrity and activity of the protein but also the conserved C-terminal domain is thought to contribute to the interface stabilisation of the protein (Hothorn, D'Angelo, et al., 2004a; Hothorn, Wolf, et al., 2004b; Rausch & Greiner, 2004).
Functional characterization of a vacuolar invertase from Solanum lycopersicum: Post-translational regulation by N-glycosylation and a proteinaceous inhibitor
2014, BiochimieCitation Excerpt :Indeed, SolyVIF possesses four cysteines typically conserved in PMEI-RP (Fig. S4). Indeed, crystallographic analyses of N. tabacum NtCIF [77], Actinidia deliciosa AcPMEI [78] and A. thaliana AtPMEI-2 [38] have shown that these close homologues are composed of α-helices stabilized by two disulphide bridges [38,77,78]. In order to assess whether SolyVIF was a genuine inhibitor of TIV-1 we analysed their potential interaction by SPR.
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Present address: J. A. Marquez, EMBL Outstation Grenoble, BP181, 38042 Grenoble Cedex 9, France.