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Structural study of LESV from Arabidopsis thaliana, a new protein involved in starch metabolism
Emaelle Rimbaut, N Szydlowski, F. Wattebled, Christophe d’Hulst, Xavier Roussel, Coralie Bompard
To cite this version:
Emaelle Rimbaut, N Szydlowski, F. Wattebled, Christophe d’Hulst, Xavier Roussel, et al.. Structural study of LESV from Arabidopsis thaliana, a new protein involved in starch metabolism. Congrès de Biologie Structurale Intégrative (BSI 2019), Oct 2019, Toulouse, France. 2019. �hal-02402236�
Structural study of LESV from Arabidopsis thaliana, a new protein involved in starch metabolism
Emaelle Rimbaut, Nicolas Szydlowski, Fabrice Wattebled, Christophe d’Hulst, Xavier Roussel & Coralie Bompard
CNRS, Univ. Lille, INRA, Unité de Glycobiologie Structurale et Fonctionnelle, 59000 Lille, France
Starch is vital in human nutrition and its unique physical properties are widely used in industrial applications. It accumulates in plants as water-insoluble, semi-crystalline granules. Plants produce and consume their starch over time scales from hours to years. Leaf starch is metabolized in chloroplasts during the diel cycle. The synthesis and degradation rates are under photoperiodic control. Starch is comprised of two glucan polymers (amylose and amylopectin) and its physicochemical properties are defined by their organization within the granule. It has been assumed that the polymers self-organize to form the granule matrix. Recently, however, two conserved but uncharacterized proteins, Early Starvation 1 (ESV1) and Like Early Starvation 1 (LESV), were proposed to organize the polysaccharides within the granules. The mechanism by which this occurs is unknown. We report here preliminary results on the structural organization of LESV from Arabidopsis thaliana.
Starch granule organization
ESV1 & LESV are involved in starch metabolism in plants
conserved in plant kingdom
important for normal starch turnover in leaves
chloroplastic, soluble and bound to starch granules
have no CBM but Trp-rich region for carbohydrate binding
altered levels of ESV1 and LESV
-affect starch granule morphology, number, & composition -have minor effects on amylopectin structure
ESV1 & LESV may play a role in determining the final organization of glucans in the starch granule matrix
ESV1 may promote a high level of organization of the granule matrix
whereas LESV may reduce this level of organization.
• glucose polymer
• 2 types of O-glycosidic bonds
• 2 polyglucanes
α(1→4) α(1→6)
Amylose (20-30%)
Semi-cristalline starch granule
Amylopectin (70-80%)
Branch Points
linear α-helical glucans Crystalline lamella mainly
linear
Branching points Amorphous lamella
Amylose and amylopectin biosynthesis (SSs, Bes, DBEs…)
Amylose and amylopectin packing in starch granule
(ESV1 & LESV?)
ESV1 and LESV : two related new proteins with unknown function
Feike et al (2016) the plant Cell, 28,p1472-89 [6]
Structural study of LESV shows an original sickle-shaped modular structure
Molecular model of AtLESV computed by I-Tasser [7]
AtLESV is organized in five modules (I to V from N to C-ter)
Modules folded into nine-stranded immunoglobulin-like 𝛽 sandwich
Exclusively composed by -strands .
The modules form an elongated folded sickle-shaped structure.
Conserved W are exposed on the surface of module IV and V and can constitute SBSs
Far UV Circular Dichroïsm (CD) spectrum of purified AtLESV
LESV is composed exclusively by -strands I
II
III IV
V NH2
COOH
Starch accumulates in plants as water-insoluble granules composed of amylose and amylopectin. These polymers are made of glucose residues linked together by α-(1→4) more or less ramified by α-(1→6) O-glycosidic bonds also called “branch points”. Amylose is essentially linear while amylopectin is moderately branched with 5 to 6 % of α-1,6 linkages [1]. Amylopectin, the major component of starch, is synthesized by the concerted activities of soluble starch synthases (SSs), starch branching enzymes (BEs) and starch debranching enzymes (DBEs) [2]. Branch points are introduced by BEs. DBEs hydrolyse the α-1,6 bonds of a developing amorphous lamellae to ordinate the branch points and allow the amylopectin to crystallize [3, 4]. Crystalline lamellae formed by amylopectin linear chains organized in double helices consisting of 2 poly-glucosyl chains forming A or B crystal forms. Amylose molecules are interspaced among amylopectin molecules. Most proteins involved in starch metabolism are present in starch granules. They contain Carbohydrate Binding Modules (CBMs), or Surface Binding Sites (SBSs) [5] located at the surface of the proteins. While CBMs can be identified by sequence analysis, SBSs requires the atomic structure determination to be localized.
-1,7 -1,2 -0,7 -0,2 0,3 0,8 1,3
-0,15 0,05 0,25 0,45
2,68 2,88
4,50E-046,50E-048,50E-04
0,00 0,01 0,02 0,03 0,04
0,00 50,00 100,00
Rg=36.8Å
Dmax=122Å
SAXS model of AtLESV
LESV molecular model superposed with the average SAXS envelope computed with DAMAVER (from 10 ab initio LESV protein models computed by GASBOR, in white) and with the most probable model
computed by DAMFILT (Red) [8]
-20 -10 0 10 20
190 210 230 250
SAXS data of purified AtLESV
A) experimental data plotted as a function of the scattering vector q. The Guinier plot is represented in inset
B) Distance distribution function
Data were collected on the SWING beamline of synchrotron SOLEIL
A B
amylose
amylopectin 1 2 3
*
* *
Determination of LESV affinity for amylose & amylopectin
LESV (1µg) was loaded on native PAGE containing increasing amounts (from 0 to 0.3%) of amylose (top panel) or amylopectin (bottom panel).
For each gel the first lane is loaded with a reference protein with no affinity for starch, the second lane with LESV and the third with both proteins. The black arrow indicates the migration of the reference protein the red star indicate the position of the shifted LESV
LESV binds specifically to amylopectin molecules but has no affinity for amylose
Introduction
Results
Manual fitting of the atomic model of LESV with B form crystalline amylopectin double helices
A form
B form
AtLESV is structured in five modules folded into nine-stranded immunoglobulin-like 𝛽 sandwich and exclusively composed by -strands.
The five modules sharing important surface contact are arranged one after the other to form an elongated folded sickle-shaped structure.
This original shape is in agreement with the global envelope of the protein, measured by SAXS and the secondary structure composition in -strands has been confirmed by CD.
In the LESV model, the Trp conserved region is present in modules IV, V and part of domain III and many conserved W side chains are directed toward the semicircular region delimited by modules II to V suggesting that this region could constitute the glucan binding site of LESV through several SBSs.
LESV is specific to amylopectin, suggesting a structural recognition of the 3D structure of the polyglucan. Furthermore, the diameter of the semicircular region (~50 Å) of LESV is compatible with the diameter of a hexameric helix organization of the amylopectin cluster (B-allomorph).
C-score of -0.86
Conclusion
Bibliographie
1. Buleon A, Colonna P, Planchot V, Ball S. Int J Biol Macromol. 1998;23(2):85-112.
2. D'Hulst C, Wattebled F, Szydlowski N. In: Nakamura Y, editor. Starch. Japan: Springer; 2015. p. 211-37 3. Wattebled F, Dong Y, Dumez S, Delvalle D, Planchot V, Berbezy P, et al. Plant Physiol. 2005;138(1):184-95.
4. Wattebled F, Planchot V, Dong Y, Szydlowski N, Pontoire B, Devin A, et al. Plant Physiol. 2008;148(3):1309-23.
5. Baroroh U, Yusuf M, Rachman SD, Ishmayana S, Syamsunarno M, Levita J, et al. Enzyme Res. 2017;2017:4086845.
6. Feike D, Seung D, Graf A, Bischof S, Ellick T, Coiro M, et al. Plant Cell. 2016;28(6):1472-89.
7. Yang J, Yan R, Roy A, Xu D, Poisson J, Zhang Y. Nat Methods. 2015;12(1):7-8.
8. Franke, D., Petoukhov, M.V., Konarev, P.V., Panjkovich, A., Tuukkanen, A., Mertens, et. al(2017)J. Appl. Cryst.50, 1212-1225
