The Invertase Inhibitor Nt-CIF from Tobacco: A Highly Thermostable Four-helix Bundle with an Unusual N-terminal Extension

doi:10.1016/j.jmb.2003.10.066

Journal of Molecular Biology

Volume 335, Issue 4, 23 January 2004, Pages 987-995

https://doi.org/10.1016/j.jmb.2003.10.066 Get rights and content

Abstract

Plant invertases are sucrolytic enzymes essential for plant metabolism and development. Enzyme activity is regulated on a posttranslational level via inhibitory proteins, referred to as invertase inhibitors. Ectopic expression of invertase inhibitors in crop plants has high biotechnological potential. However, little biochemical and up to now no detailed structural information is available about this class of plant regulatory proteins. Here, we present the crystal structure of the cell wall-associated invertase inhibitor Nt-CIF from tobacco at a resolution of 1.87 Å. The structural model reveals an asymmetric four-helix bundle with an uncommon N-terminal extension that appears to be critical for the structural integrity of the protein. Structure analysis of a second crystal form grown in the presence of CdCl₂ reveals two metal binding sites. Nt-CIF is highly thermostable and retains full inhibitory activity after cooling to ambient temperatures. The structure of Nt-CIF provides the first three-dimensional information source for the posttranslational regulation of plant invertases. Based on the recently discovered sequence homology between inhibitors of invertases and pectin methylesterases, our structural model is likely to represent a scaffold also used for the regulation of the latter enzymes, which do not share sequence similarity with invertases. Thus, our structural model sets the 3D-stage for the investigation of posttranslational regulation of invertases as well as pectin methylesterases.

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),

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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).
Human sucrase enzyme is a key therapeutic target for type 2 diabetes. While sugarcane sucrase inhibitor (sucinh) modulates invertase activity thereby accumulates sucrose. Molecular level understanding of sucinh towards mammalian α-glucosidases is scarce. The interaction of sucinh with human sucrase was identified and the association of these proteins was confirmed using co-purification, co-immunoprecipitation and pull-down assay. In addition, microscale thermophoresis assay showed that sucinh has a tight binding with sucrase (K_d = 4.77 nM) and a better affinity over acarbose. Collectively, in vitro, ex vivo and in silico data revealed that sucinh is selective for intestinal sucrase. The M region (H5/6 loop) of sucinh identified at the protein-protein interface is shown to have high affinity over N and C regions. Whereas, the biolayer luminescent imaging and microscale thermophoresis on the synthetic peptide of 28 amino acids of M region has a weak dose-dependent binding with sucrase. However, the synthetic peptide did not show substantial inhibition of sucrase and amylase activities at low concentration. Naturally derived carbohydrate mimics were shown to have a positive impact at the in vitro conditions. The insights obtained in this study give clues towards a new class of bioactive therapeutic peptides for α-glucosidases. A new horizon towards polypeptides derived from food sources emerge as a promising strategy for dietary interventions for prediabetic conditions.
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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.
Wild tuber-bearing species are commonly used as a source of valuable traits to develop new potato cultivars. One of the trends in potato breeding is elimination of cold-induced sweetening, which depends on the balance between activities of saccharolytic enzymes (vacuolar invertase PAIN-1 and sucrose synthase SUS4 that hydrolyze sucrose) and vacuolar invertase inhibitor INH2 that regulates the PAIN-1 activity. To clarify the mechanisms underlying cold resistance, we investigated INH2 polymorphism and tissue-specific expression, and co-expression profiles of INH2, PAIN-1, and SUS4 in wild tuber-bearing potatoes and Solanum tuberosum cultivars. Novel INH2 homologs were identified in eight wild species and four cultivars. Structural analysis predicted an N-terminal signal peptide and showed that nucleotide and amino acid polymorphisms (13.09% and 18.91%, respectively) did not affect the INH2 tertiary structure, suggesting that the INH2 function may be conserved in potato both at the intra- and inter-specific levels. INH2 and PAIN-1 mRNA were detected in buds, flowers, leaves, stems, roots, and tubers of S. tuberosum cv. Nadezhda, showing the highest (INH2) and lowest (PAIN-1) levels in the roots. During cold storage, tubers of four cultivars accumulated glucose and fructose, while the sucrose content changed insignificantly; INH2, PAIN-1, and SUS4 co-expression patterns in freshly harvested and cold-stored tubers differed among the cultivars. Cold exposure also caused distinct effects on INH2, PAIN-1, and SUS4 co-expression patterns in the leaves of four wild species differing in cold resistance. These data indicate that cold response in potato may depend on specific combinations of INH2, PAIN-1, and SUS4 activities.
Sequence diversity and in silico structure prediction of the vacuolar invertase inhibitor gene from potato (Solanum tuberosum L.) cultivars differing in sugar content
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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).
The aim of this study was to examine the variations in sugar content and identify the polymorphism in vacuolar invertase inhibitor (INH2) gene from Indian non-processing (Kufri Jyoti, Kufri Pukhraj and PU1) and exotic processing (Atlantic and Frito Lay-1533) potato cultivars. Upon cold storage (4 °C) processing cultivars maintained low reducing sugars as compared to non-processing cultivars. Sequencing of the INH2 gene identified four alleles of which three identified as novel alleles. A total twelve SNPs resulted in silent mutations, with five conferring the amino acid substitutions. Phylogenetic analysis suggests a highly conserved nature of the INH2 gene. The 3D predicted structures generated for all the alleles revealed slight variations in the orientation of the helices (α1-3) in N-terminal region. Sequence polymorphism observed in INH2 alleles in processing and non-processing potato cultivars can be correlated with the observed variations in the sugar content suggesting a possible role in cold-induced sweetening.
Functional characterization of a vacuolar invertase from Solanum lycopersicum: Post-translational regulation by N-glycosylation and a proteinaceous inhibitor
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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.
Plant vacuolar invertases, which belong to family 32 of glycoside hydrolases (GH32), are key enzymes in sugar metabolism. They hydrolyse sucrose into glucose and fructose. The cDNA encoding a vacuolar invertase from Solanum lycopersicum (TIV-1) was cloned and heterologously expressed in Pichia pastoris. The functional role of four N-glycosylation sites in TIV-1 has been investigated by site-directed mutagenesis. Single mutations to Asp of residues Asn52, Asn119 and Asn184, as well as the triple mutant (Asn52, Asn119 and Asn184), lead to enzymes with reduced specific invertase activity and thermostability. Expression of the N516D mutant, as well as of the quadruple mutant (N52D, N119D, N184D and N516D) could not be detected, indicating that these mutations dramatically affected the folding of the protein. Our data indicate that N-glycosylation is important for TIV-1 activity and that glycosylation of N516 is crucial for recombinant enzyme stability. Using a functional genomics approach a new vacuolar invertase inhibitor of S. lycopersicum (SolyVIF) has been identified. SolyVIF cDNA was cloned and heterologously expressed in Escherichia coli. Specific interactions between SolyVIF and TIV-1 were investigated by an enzymatic approach and surface plasmon resonance (SPR). Finally, qRT-PCR analysis of TIV-1 and SolyVIF transcript levels showed a specific tissue and developmental expression. TIV-1 was mainly expressed in flowers and both genes were expressed in senescent leaves.
Interaction proteins of invertase and invertase inhibitor in cold-stored potato tubers suggested a protein complex underlying post-translational regulation of invertase
2013, Plant Physiology and Biochemistry
The activity of vacuolar invertase (VI) is vital to potato cold-induced sweetening (CIS). A post-translational regulation of VI activity has been proposed which involves invertase inhibitor (VIH), but the mechanism for the interaction between VI and VIH has not been fully understood. To identify the potential partners of VI and VIH, two cDNA libraries were respectively constructed from CIS-resistant wild potato species Solanum berthaultii and CIS-sensitive potato cultivar AC035-01 for the yeast two-hybrid analysis. The StvacINV1 (one of the potato VIs) and StInvInh2B (one of the potato VIHs), previously identified to be associated with potato CIS, were used as baits to screen the two libraries. Through positive selection and sequencing, 27 potential target proteins of StvacINV1 and eight of StInvInh2B were clarified. The Kunitz-type protein inhibitors were captured by StvacINV1 in both libraries and the interaction between them was confirmed by bimolecular fluorescence complementation assay in tobacco cells, reinforcing a fundamental interaction between VI and VIH. Notably, a sucrose non-fermenting-1-related protein kinase 1 was captured by both the baits, suggesting that a protein complex could be necessary for fine turning of the invertase activity. The target proteins clarified in present research provide a route to elucidate the mechanism by which the VI activity can be subtly modulated.

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^†: Present address: J. A. Marquez, EMBL Outstation Grenoble, BP181, 38042 Grenoble Cedex 9, France.

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Journal of Molecular Biology

The Invertase Inhibitor Nt-CIF from Tobacco: A Highly Thermostable Four-helix Bundle with an Unusual N-terminal Extension

Abstract

Introduction

Section snippets

Results and Discussion

Concluding Remarks

X-ray analysis

Acknowledgements

Trends Plant Sci.

FEBS Letters

J. Mol. Biol.

Cell

J. Mol. Biol.

FEBS Letters

Methods Enzymol.

Invertases. Primary structures, functions, and roles in plant development and sucrose partitioning

Plant Physiol.

A similar dichotomy of sugar modulation and developmental expression affects both paths of sucrose metabolism: evidence from a maize invertase gene family

Plant Cell

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.

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.