Structural insights into the pH-controlled targeting of plant cell-wall invertase by a specific inhibitor protein

Invertases are highly regulated enzymes with essential functions in carbohydrate partitioning, sugar signaling, and plant development. Here we present the 2.6 Å crystal structure of Arabidopsis cell-wall invertase 1 (INV1) in complex with a protein inhibitor (CIF, or cell-wall inhibitor of β-fructosidase) from tobacco. The structure identifies a small amino acid motif in CIF that directly targets the invertase active site. The activity of INV1 and its interaction with CIF are strictly pH-dependent with a maximum at about pH 4.5. At this pH, isothermal titration calorimetry reveals that CIF tightly binds its target with nanomolar affinity. CIF competes with sucrose (Suc) for the same binding site, suggesting that both the extracellular Suc concentration and the pH changes regulate association of the complex. A conserved glutamate residue in the complex interface was previously identified as an important quantitative trait locus affecting fruit quality, which implicates the invertase–inhibitor complex as a main regulator of carbon partitioning in plants. Comparison of the CIF/INV1 structure with the complex between the structurally CIF-related pectin methylesterase inhibitor (PMEI) and pectin methylesterase indicates a common targeting mechanism in PMEI and CIF. However, CIF and PMEI use distinct surface areas to selectively inhibit very different enzymatic scaffolds.

[1]  C. Chiang,et al.  Crystal Structures of Aspergillus japonicus Fructosyltransferase Complex with Donor/Acceptor Substrates Reveal Complete Subsites in the Active Site for Catalysis* , 2010, The Journal of Biological Chemistry.

[2]  B. González,et al.  Structural and Kinetic Analysis of Schwanniomyces occidentalis Invertase Reveals a New Oligomerization Pattern and the Role of Its Supplementary Domain in Substrate Binding* , 2010, The Journal of Biological Chemistry.

[3]  G. Seifert,et al.  Irritable Walls: The Plant Extracellular Matrix and Signaling1 , 2010, Plant Physiology.

[4]  S. Smeekens,et al.  Sugar perception and signaling--an update. , 2009, Current opinion in plant biology.

[5]  W. Van den Ende,et al.  Donor and acceptor substrate selectivity among plant glycoside hydrolase family 32 enzymes , 2009, The FEBS journal.

[6]  Y. Ruan,et al.  Posttranslational Elevation of Cell Wall Invertase Activity by Silencing Its Inhibitor in Tomato Delays Leaf Senescence and Increases Seed Weight and Fruit Hexose Level , 2022 .

[7]  M. Zanor,et al.  RNA Interference of LIN5 in Tomato Confirms Its Role in Controlling Brix Content, Uncovers the Influence of Sugars on the Levels of Fruit Hormones, and Demonstrates the Importance of Sucrose Cleavage for Normal Fruit Development and Fertility1[W][OA] , 2009, Plant Physiology.

[8]  J. Janin,et al.  Protein–protein interaction and quaternary structure , 2008, Quarterly Reviews of Biophysics.

[9]  L. Camardella,et al.  The peculiar structural features of kiwi fruit pectin methylesterase: Amino acid sequence, oligosaccharides structure, and modeling of the interaction with its natural proteinaceous inhibitor , 2008, Proteins.

[10]  W. Van den Ende,et al.  Crystal structures of Arabidopsis thaliana cell-wall invertase mutants in complex with sucrose. , 2008, Journal of molecular biology.

[11]  B. De Coninck,et al.  Unraveling the Difference between Invertases and Fructan Exohydrolases: A Single Amino Acid (Asp-239) Substitution Transforms Arabidopsis Cell Wall Invertase1 into a Fructan 1-Exohydrolase1[C] , 2007, Plant Physiology.

[12]  R. Pickersgill,et al.  Molecular basis of the activity of the phytopathogen pectin methylesterase , 2007, The EMBO journal.

[13]  K. Koch,et al.  Regulation of invertase: a 'suite' of transcriptional and post-transcriptional mechanisms. , 2007, Functional plant biology : FPB.

[14]  Airlie J. McCoy,et al.  Solving structures of protein complexes by molecular replacement with Phaser , 2006, Acta crystallographica. Section D, Biological crystallography.

[15]  C. D. De Ranter,et al.  X-ray diffraction structure of a cell-wall invertase from Arabidopsis thaliana. , 2006, Acta crystallographica. Section D, Biological crystallography.

[16]  K. Scheffzek,et al.  Multiple crystal forms of the cell-wall invertase inhibitor from tobacco support high conformational rigidity over a broad pH range. , 2006, Acta crystallographica. Section D, Biological crystallography.

[17]  Steven M. L. Smith,et al.  Arabidopsis AtcwINV3 and 6 are not invertases but are fructan exohydrolases (FEHs) with different substrate specificities , 2005 .

[18]  D. Tsernoglou,et al.  Structural Basis for the Interaction between Pectin Methylesterase and a Specific Inhibitor Protein , 2005, The Plant Cell Online.

[19]  P. Aloy,et al.  Structural Insights into the Target Specificity of Plant Invertase and Pectin Methylesterase Inhibitory Proteins , 2004, The Plant Cell Online.

[20]  T. Roitsch,et al.  Function and regulation of plant invertases: sweet sensations. , 2004, Trends in plant science.

[21]  Kevin Cowtan,et al.  research papers Acta Crystallographica Section D Biological , 2005 .

[22]  F. Carrari,et al.  Zooming In on a Quantitative Trait for Tomato Yield Using Interspecific Introgressions , 2004, Science.

[23]  T. Rausch,et al.  In Arabidopsis thaliana, the invertase inhibitors AtC/VIF1 and 2 exhibit distinct target enzyme specificities and expression profiles , 2004, FEBS letters.

[24]  T. Roitsch,et al.  Extracellular Invertase Is an Essential Component of Cytokinin-Mediated Delay of Senescence , 2004, The Plant Cell Online.

[25]  B. Henrissat,et al.  The Three-dimensional Structure of Invertase (β-Fructosidase) from Thermotoga maritima Reveals a Bimodular Arrangement and an Evolutionary Relationship between Retaining and Inverting Glycosidases* , 2004, Journal of Biological Chemistry.

[26]  T. Rausch,et al.  Plant protein inhibitors of invertases. , 2004, Biochimica et biophysica acta.

[27]  I. D'Angelo,et al.  The invertase inhibitor Nt-CIF from tobacco: a highly thermostable four-helix bundle with an unusual N-terminal extension. , 2004, Journal of molecular biology.

[28]  T. Rausch,et al.  Identification of pollen‐expressed pectin methylesterase inhibitors in Arabidopsis , 2003, FEBS letters.

[29]  G. Stier,et al.  Bacterial expression, purification and preliminary X-ray crystallographic characterization of the invertase inhibitor Nt-CIF from tobacco. , 2003, Acta crystallographica. Section D, Biological crystallography.

[30]  W. Van den Ende,et al.  Properties of Fructan:Fructan 1-Fructosyltransferases from Chicory and Globe Thistle, Two Asteracean Plants Storing Greatly Different Types of Inulin1 , 2003, Plant Physiology.

[31]  Dagmar Ringe,et al.  POVScript+: a program for model and data visualization using persistence of vision ray-tracing , 2003 .

[32]  Alain Roussel,et al.  Structural analysis of xylanase inhibitor protein I (XIP-I), a proteinaceous xylanase inhibitor from wheat (Triticum aestivum, var. Soisson). , 2003, The Biochemical journal.

[33]  Ian W. Davis,et al.  Structure validation by Cα geometry: ϕ,ψ and Cβ deviation , 2003, Proteins.

[34]  T. Roitsch,et al.  Induction of male sterility in plants by metabolic engineering of the carbohydrate supply , 2001, Proceedings of the National Academy of Sciences of the United States of America.

[35]  D. Higgins,et al.  T-Coffee: A novel method for fast and accurate multiple sequence alignment. , 2000, Journal of molecular biology.

[36]  A Sturm,et al.  Invertases. Primary structures, functions, and roles in plant development and sucrose partitioning. , 1999, Plant physiology.

[37]  C. Chothia,et al.  The atomic structure of protein-protein recognition sites. , 1999, Journal of molecular biology.

[38]  B. Svensson,et al.  Barley alpha-amylase bound to its endogenous protein inhibitor BASI: crystal structure of the complex at 1.9 A resolution. , 1998, Structure.

[39]  T. Rausch,et al.  Sucrose protects cell wall invertase but not vacuolar invertase against proteinaceous inhibitors , 1996, FEBS letters.

[40]  M. Karplus,et al.  Evaluation of comparative protein modeling by MODELLER , 1995, Proteins.

[41]  T. Rausch,et al.  Acid invertase in Nicotiana tabacum crown-gall cells: Molecular properties of the cell-wall isoform , 1994, Planta.

[42]  Wolfgang Kabsch,et al.  Automatic processing of rotation diffraction data from crystals of initially unknown symmetry and cell constants , 1993 .

[43]  Mukesh Jain,et al.  Genetic control of cell wall invertases in developing endosperm of maize , 2005, Planta.

[44]  T. Rausch,et al.  A 17-kDa Nicotiana tabacum cell-wall peptide acts as an in-vitro inhibitor of the cell-wall isoform of acid invertase , 2004, Planta.

[45]  G N Murshudov,et al.  Use of TLS parameters to model anisotropic displacements in macromolecular refinement. , 2001, Acta crystallographica. Section D, Biological crystallography.