Starch-binding domains in the CBM45 family – low-affinity domains from glucan, water dikinase and a-amylase involved in plastidial starch metabolism Mikkel A. Glaring 1,2 , Martin J. Baumann 1 , Maher Abou Hachem 1 , Hiroyuki Nakai 1 , Natsuko Nakai 1 , Diana Santelia 3 , Bent W. Sigurskjold 4 , Samuel C. Zeeman 3 , Andreas Blennow 2 and Birte Svensson 1 1 Enzyme and Protein Chemistry, Department of Systems Biology, Technical University of Denmark, Kongens Lyngby, Denmark 2 VKR Research Centre Pro-Active Plants, Department of Plant Biology and Biotechnology, Faculty of Life Sciences, University of Copenhagen, Frederiksberg, Denmark 3 Department of Biology, ETH Zu ¨ rich, Switzerland 4 Department of Biology, University of Copenhagen, Denmark Introduction Starch is deposited as water-insoluble granules in the amyloplasts of tubers, roots and seeds for long-term storage, and in the chloroplasts of green tissues during the day as short-term storage for the following night (transitory starch). The granules are composed of two different polymers of glucose linked via a-(1,4)-glyco- sidic bonds: amylose, which is essentially linear, and amylopectin, which also contains a-(1,6)-glycosidic Keywords carbohydrate-binding module; starch metabolism; starch-binding domain; a-amylase; a-glucan, water dikinase Correspondence B. Svensson, Enzyme and Protein Chemistry, Department of Systems Biology, Technical University of Denmark, Søltofts Plads, Building 224, DK-2800 Kongens Lyngby, Denmark Fax: +45 45886307 Tel: +45 45252740 E-mail: bis@bio.dtu.dk (Received 17 November 2010, revised 5 January 2011, accepted 31 January 2011) doi:10.1111/j.1742-4658.2011.08043.x Starch-binding domains are noncatalytic carbohydrate-binding modules that mediate binding to granular starch. The starch-binding domains from the carbohydrate-binding module family 45 (CBM45, http://www.cazy.org) are found as N-terminal tandem repeats in a small number of enzymes, primarily from photosynthesizing organisms. Isolated domains from repre- sentatives of each of the two classes of enzyme carrying CBM45-type domains, the Solanum tuberosum a-glucan, water dikinase and the Arabid- opsis thaliana plastidial a-amylase 3, were expressed as recombinant pro- teins and characterized. Differential scanning calorimetry was used to verify the conformational integrity of an isolated CBM45 domain, reveal- ing a surprisingly high thermal stability (T m of 84.8 °C). The functionality of CBM45 was demonstrated in planta by yellow ⁄ green fluorescent protein fusions and transient expression in tobacco leaves. Affinities for starch and soluble cyclodextrin starch mimics were measured by adsorption assays, surface plasmon resonance and isothermal titration calorimetry analyses. The data indicate that CBM45 binds with an affinity of about two orders of magnitude lower than the classical starch-binding domains from extra- cellular microbial amylolytic enzymes. This suggests that low-affinity starch-binding domains are a recurring feature in plastidial starch metabo- lism, and supports the hypothesis that reversible binding, effectuated through low-affinity interaction with starch granules, facilitates dynamic regulation of enzyme activities and, hence, of starch metabolism. Abbreviations AMY3, a-amylase 3; AtAMY3, Arabidopsis thaliana a-amylase 3; CBM, carbohydrate-binding module; DSC, differential scanning calorimetry; GWD, a-glucan, water dikinase; IPTG, isopropyl thio-b- D-galactoside; ITC, isothermal titration calorimetry; PWD, phosphoglucan, water dikinase; SBD, starch-binding domain; SPR, surface plasmon resonance; StGWD, Solanum tuberosum a-glucan, water dikinase; TEV, tobacco etch virus; YFP ⁄ GFP, yellow ⁄ green fluorescent protein. FEBS Journal 278 (2011) 1175–1185 ª 2011 The Authors Journal compilation ª 2011 FEBS 1175 branch points. Together, these polymers are laid down as alternating semicrystalline and amorphous layers to form the supramolecular granule structure. The semi- crystalline layers are made up primarily of packed double helices formed by adjacent glucose chains in amylopectin. The amorphous layers are comprised mainly of amylopectin branch points and interspersed amylose [1]. This semicrystalline structure offers a sig- nificant challenge for degrading enzymes and, for the efficient amylolysis of raw starch, requires either sur- face substrate-binding sites on the catalytic module [2] or, more commonly, a specialized carbohydrate-bind- ing module (CBM) known as a starch-binding domain (SBD) [3]. CBMs are noncatalytic structural domains that mediate the binding of proteins to polysaccharides, thus bringing the appended catalytic modules into close contact with the substrate, enabling efficient hydrolysis of insoluble polysaccharides, such as starch and cellulose [4]. Based on their amino acid sequences, CBMs are currently grouped into 61 families. SBDs are found in CBM families 20, 21, 25, 26, 34, 41, 45, 48, 53 and 58 (http://www.cazy.org) [5,6]. The majority of characterized SBDs are found in extracellular microbial amylolytic enzymes, where they enhance binding to starch and related a-glucans. A significant and so far largely uncharacterized number of SBDs occur in nonamylolytic enzymes from all domains of life [3,6]. Among these is a recently established small family of SBDs, named CBM45 [7]. CBM45s are found primarily in photosynthesizing organisms in only two classes of intracellular enzyme: the a-glucan, water dikinases (GWDs, EC 2.7.9.4), which phosphor- ylate starch, and the plastidial a-amylases (AMYs, EC 3.2.1.1). Two types of CBM45-containing GWD have been identified in plants, one of which is plastid- ial and essential for normal starch metabolism (GWD1 ⁄ R1 ⁄ SEX1) [8,9]. The second GWD, called GWD2 in Arabidopsis, is extraplastidial and has no apparent role in starch degradation [10]. The plastidial a-amylase AMY3 is not required for normal transitory starch metabolism in Arabidopsis [11], but a functional role in planta has been inferred from knock-out studies in phosphoglucan phosphatase (SEX4) and quadruple debranching enzyme (ISA1 ⁄ ISA2 ⁄ ISA3 ⁄ LDA) mutant backgrounds [12,13]. In both enzyme classes, the CBM45s are present as N-terminal tandem domains, separated by a linker domain of varying length. No three-dimensional structure is available for CBM45, but a recent bioinformatic analysis produced a rough model and identified two tryptophan residues as puta- tive binding sites in the N-terminal CBM45 from Ara- bidopsis GWD1 [14]. These two tryptophans have previously been experimentally confirmed as pivotal for the starch-binding ability of potato GWD [7]. The metabolism of plastidial starch in leaves of plants is a tightly regulated process. The available pho- tosynthate has to be balanced with the energy and car- bohydrate needs of the plant during the subsequent dark period. Perturbations in this process lead to severe phenotypes and retardation of growth [15,16]. Plastidial starch metabolism has been well character- ized in the model plant Arabidopsis thaliana, and numerous enzymes are involved in the process of building and degrading the insoluble granule (for recent reviews, see refs. [17–19]). Many of these enzymes contain SBDs representing several different CBM families, and starch and ⁄ or a-glucan binding has been demonstrated in a number of cases [20–26]. Starch binding in the plastidial system is influenced by potential regulatory mechanisms that can reversibly affect the interaction with the granule [24,27]. Binding of potato GWD to starch in planta has been shown to be diurnally regulated [23] and potentially influenced by the redox status of the enzyme [23,27]. The redox status also influences the binding of the CBM48-con- taining glucan phosphatase SEX4 to starch in vitro [24]. Phosphoglucan, water dikinase (PWD ⁄ GWD3, EC 2.7.9.5), a second type of plastidial GWD carrying an N-terminal CBM20 SBD, shows a relatively low affinity towards the starch mimics, cyclodextrins [20], and it has been proposed that this could facilitate more dynamic interactions with starch, allowing the modulation of affinity necessary for metabolic regula- tion in the plastid [6,20,28]. In this article, we report the characterization of SBDs from representatives of the two classes of enzyme carrying CBM45-type domains, the Sola- num tuberosum a-glucan, water dikinase (StGWD) and the A. thaliana plastidial a-amylase 3 (AtAMY3), sug- gesting that the evolution of low-affinity domains is a recurring and functionally important theme in plastid- ial starch metabolism. Results and Discussion Identification and bioinformatic analysis of CBM45s from GWD and AMY3 Family CBM45 sequences were obtained from the car- bohydrate-active enzymes database (CAZY, http:// www.cazy.org). Furthermore, a search of the translated nucleotide database at the National Center for Bio- technology Information (http://www.ncbi.nlm.nih.gov) uncovered several additional sequences with homology to StGWD and AtAMY3, which were included in the Starch-binding domains in the CBM45 family M. A. Glaring et al. 1176 FEBS Journal 278 (2011) 1175–1185 ª 2011 The Authors Journal compilation ª 2011 FEBS analysis. Alignment of these sequences showed not only extensive conservation in the catalytic domains, suggesting a preservation of function, but also the exis- tence of the N-terminally appended CBM45 domains (data not shown). The sequences used in the subse- quent analysis included CBM45s from 45 proteins from primarily photosynthetic eukaryotes (plants, green algae and red algae), as well as from apicom- plexan parasites (Doc. S1). A common characteristic of these organisms is the presence of starch or starch- like crystalline polysaccharides. The starch synthesis ability of the apicomplexan parasites is believed to be derived from an endosymbiosis of a red alga, with following loss of photosynthetic capacity [29]. The CBM45s are present as tandem domains in the N-terminal part of StGWD and AtAMY3 and sepa- rated by a linker of approximately 200 and 50 amino acids, respectively (Fig. 1A). The alignment of all iden- tified CBM45s revealed that each contains five aro- matic amino acids that are widely conserved across all species (Fig. S1). These residues are also present in StGWD and AtAMY3 (Fig. 1B). The ability to bind to starch has been associated with certain consensus aromatic residues in other CBM families [6] and, although there is evidence that two of the aromatic residues (W139 and W194) are necessary for the starch-binding ability of StGWD [7], the lack of struc- tural information on CBM45 precludes the assignment of residues to specific binding sites as has been per- formed for CBM20 from Arabidopsis PWD ⁄ GWD3 (AtPWD) [20]. It was clear from the collected sequences that the tandem organization of two domains is a common characteristic of most CBM45-containing enzymes, suggesting that this is essential for the functionality of the appended catalytic modules. Isoforms of GWD and AMY3 from the green algae Chlamydomonas reinhardtii (CreGWD) and Micromonas (MiGWDb, MpGWDb and MpAMY3; Doc. S1) contained only one identifiable CBM45 domain. Whether this repre- sents a simple misannotation or a distinct function of single-CBM45 SBDs is unknown. A previous analysis of a recombinant truncated StGWD lacking CBM45-1 showed an altered specificity on soluble substrates with a preference for the phosphorylation of shorter glucan chains [7]. A phylogenetic tree based on the complete amino acid alignment of all CBM45s showed obvious groupings of the individual domains from plant sequences (Fig. S2). Most of the nonplant sequences formed a separate, mixed group, reflecting the evolu- tionary distance and low homology between these sequences. Overall, it appears as though CBM45s and the tandem structure of these domains arose early in evolution, perhaps in an ancestor of the current photo- synthetic eukaryotes. It has been proposed that GWD sequences were a prerequisite for the appearance of semicrystalline starch-like polymers [29]. If this is indeed the case, the appended CBM45 domains could represent a truly ancient SBD and, perhaps, be one of the first CBMs dedicated to starch binding. Expression and purification of CBM45s from potato GWD and Arabidopsis AMY3 In order to characterize the CBM45s from StGWD, several expression constructs were produced and tested. Because there is no structural information on CBM45, putative domain borders were assigned on the basis of the predicted secondary structure and homol- ogy to other CBMs. Thirteen constructs with an N-ter- minal tobacco etch virus (TEV) protease-cleavable Histidine (His)-tag, containing both the single and A B Fig. 1. Overview of the CBM45 domains. (A) Domain structure of StGWD and AtAMY3 showing the chloroplast transit peptide (TP, black), tandem CBM45s (grey) and catalytic domain (light grey). The size of the proteins is given in amino acids (aa). (B) Sequence alignment of CBM45 domains 1 and 2 from StGWD and AtAMY3 created using C LUSTALW2 (http://www.ebi.ac.uk/ tools/clustalw2). Identical (*), conserved (:) and semiconserved (.) residues are indicated below the alignment. The arrows indicate the five conserved aromatic amino acid residues. M. A. Glaring et al. Starch-binding domains in the CBM45 family FEBS Journal 278 (2011) 1175–1185 ª 2011 The Authors Journal compilation ª 2011 FEBS 1177 double modules with or without the intervening sequence, were produced and tested for soluble expres- sion (Fig. S3). Two of each of the recombinant single modules, as well as the double module (CBM45-1B, amino acids 77–217; CBM45-1E, amino acids 109–217; CBM45-2A, amino acids 405–545; CBM45-2B, amino acids 405–551; CBM45-1,2, amino acids 77–551; Fig. S3), were further purified. Although the CBM45- 1s and double module could be expressed with reason- able yields, they precipitated rapidly after His-tag puri- fication and were not suitable for detailed analyses. The CBM45-2s were stable at pH 8.0 and could be subjected to TEV protease cleavage and dialysis with- out significant loss of protein. The more stable of the two resulting proteins (CBM45-2A) was chosen for detailed characterization. This protein precipitated slowly when stored at 4 °C. The general problem of aggregation observed with the recombinant CBM45s suggests that the isolated SBD is destabilized as a result of the exposure of hydrophobic surface, which would otherwise be packed on other domains in the native full-length GWD. Initial differential scanning calorimetry (DSC) screening of the His-tagged StGWD CBM45-2A indi- cated a high unfolding temperature. This protein gave rise to a broad asymmetric thermogram with T m = 78.1 °C (data not shown). Proteolytic removal of the N-terminal His-tag yielded a symmetric single peak thermogram with T m = 84.8 °C at pH 8.0. Inter- estingly, the unfolding was partially reversible, as dem- onstrated by the 83% and 74% area recovery of the second and third scans, respectively (Fig. 2). Fitting a two-state model to the reference- and baseline-corrected calorimetric trace resulted in an excellent fit, yielding DH = 414.1 ± 0.7 kJÆmol )1 , attesting to the high ther- mal stability and conformational integrity of CBM45- 2A from StGWD. This CBM displayed similarly high conformational stability at pH 7.0 (T m = 87.1 °C), but the reversibility was decreased significantly and the pro- tein was prone to aggregation (data not shown). The reason for this extraordinarily high stability is unknown, but it strongly suggests that CBM45-2A is correctly folded and justifies the use of the isolated domain to investigate the binding properties of CBM45. Thermostability is often associated with lower structural flexibility, which may influence ligand inter- actions, but how this affects the binding properties of the domain is unclear. In the case of the CBM20 domain from Aspergillus niger glucoamylase, binding site 2, which is characterized by high conformational flexibility and large rearrangements on binding, dis- plays higher affinity towards b-cyclodextrin when com- pared with the structurally rigid site 1 [30,31]. AtAMY3 was expressed as either the full-length pro- tein, excluding the chloroplast transit peptide (amino acids 68–887), or as the tandem CBM45s (amino acids 68–388). Two versions of the double module carrying a His-tag at either end gave rise to some soluble pro- tein but, as these proteins were prone to aggregation and rapid degradation, they could not be characterized any further. In contrast, full-length AtAMY3 carrying a C-terminal His-tag was soluble and was produced in satisfactory yields (2–3 mgÆL )1 ) in a fermentor. This recombinant AtAMY3 was capable of releasing reduc- ing sugars from amylopectin, glycogen and b-limit dex- trin at both pH 6.2 and pH 8.0 and 30–37 °C (data not shown), indicating that the recombinant protein was correctly folded. Affinity measurements using surface plasmon resonance (SPR) and isothermal titration calorim- etry (ITC) It has been shown previously that the isolated CBM20 domain from AtPWD has relatively low affinity towards cyclodextrins [20]. SPR was employed to mea- sure the affinity of StGWD CBM45-2A for selected soluble oligosaccharides. The domain was biotinylated, immobilized on a streptavidin-coated chip and probed for carbohydrate-binding ability at pH 8.0 (Fig. 3). The resulting dissociation constants (K d ) towards both a- and b-cyclodextrin, as well as 6-O-a-maltosyl-b- Fig. 2. Differential scanning calorimetry (DSC) analysis of StGWD CBM45-2A. Reference subtracted thermograms of 50 l M StGWD CBM45-2A in 25 mM Hepes, pH 8.0. The full black line, grey line and broken black line are the thermograms of the first, second and third scans, respectively, at a rate of 1 °CÆmin )1 . Starch-binding domains in the CBM45 family M. A. Glaring et al. 1178 FEBS Journal 278 (2011) 1175–1185 ª 2011 The Authors Journal compilation ª 2011 FEBS cyclodextrin, were in the submillimolar range and com- parable with the values obtained for the AtPWD CBM20 (Table 1) [20]. The related function of the cat- alytic modules in At PWD and StGWD is thus matched by a similar range of affinity of their SBDs, despite the fact that they have been assigned to two different CBM families. This property was confirmed by SPR analysis of the binding of b-cyclodextrin to AtAMY3 in a similar experimental set-up, using a dif- ferent immobilization chemistry. The K d value obtained was in the same range as for StGWD CBM45-2A, suggesting that the weak binding of the starch mimic b-cyclodextrin is a general feature of CBM45s (Table 1). For both proteins, an approximate two-fold variation was observed in the calculated K d value between identical experiments. This was most probably a consequence of the low affinity manifested in low signal-to-noise ratios, particularly at high cyclo- dextrin concentrations, resulting in elevated back- ground levels. For this reason, cyclodextrin concentrations above 1 mm were excluded from the subsequent data analysis. The data in Table 1 were obtained from a representative experiment giving the best fit to the binding curve (lowest v 2 ). The StGWD CBM45-2A domain showed no detectable affinity towards maltoheptaose. This is not surprising, as the affinity of SBDs for linear oligosaccharides is generally much lower than for cyclodextrins, because of the additional entropic penalty associated with the stabil- ization of the conformation of the linear ligand upon binding [32]. To corroborate the affinity range acquired in the SPR experiment, StGWD CBM45-2A was analysed by ITC with b-cyclodextrin at pH 7.0 and pH 8.0. Although binding was evident in both cases, the heat responses were small. The data obtained at pH 7.0 were noisy, suggesting that the protein was more prone to aggregation at this pH. A one-site binding model was fitted to the integrated ITC data, giving a K d value of 0.68 ± 0.02 mm for the binding of b-cyclodextrin to StGWD CBM45-2A at pH 8.0 (Fig. 4), in good agreement with the value obtained using SPR (Table 1). The measured heat of dilution was negligible and was disregarded in the integrations. The binding was driven by a favourable enthalpy change, which compensated for an unfavourable entropy change (DH = )42.1 ± 0.9 kJÆmol )1 ; TDS = )24.1 kJÆmol )1 ). The binding affinity at pH 7.0 (K d = 0.44 mm) was similar to that at pH 8.0. This thermodynamic finger- print is consistent with the binding of b-cyclodextrin to other SBDs [33]. The observed binding affinity of CBM45 for cyclo- dextrins is considerably lower than that of other char- acterized SBDs from microbial amylolytic enzymes. Analysis of CBM20 and CBM21 SBDs from two glu- coamylases gave K d values of 14.4 and 5.1 lm, respec- tively, for the interaction with b-cyclodextrin [30,34]. 0 5 10 15 20 25 30 35 0 200 400 600 800 1000 Response (RU) -cyclodextrin (µM) Fig. 3. Surface plasmon resonance (SPR) analysis of b-cyclodextrin binding to CBM45. The instrument response level (RU, response units) is plotted (±SE) as a function of b-cyclodextrin concentration for StGWD CBM45-2A (squares) and full-length AtAMY3 (triangles). The full lines represent the fit to a one-site binding model. The experiments were carried out in triplicate at 25 °C and pH 8.0. Table 1. Dissociation constants for CBM45 determined using SPR. Data are from representative experiments (±SE) giving the best fit (low- est v 2 ) to the binding curves. Each experiment was run in triplicate. K d , dissociation constant; R max , maximum binding response; RU, response units; v 2 , chi-squared test value for the fitted curve. Protein Ligand K d (mM) R max (RU) v 2 (RU 2 ) StGWD CBM45-2A b-Cyclodextrin 0.38 ± 0.03 33 ± 0.61 0.29 a-Cyclodextrin 0.40 ± 0.06 36 ± 1.4 1.4 6-O-a-Maltosyl-b-cyclodextrin 0.44 ± 0.04 35 ± 0.97 0.51 AtAMY3 b-Cyclodextrin 0.19 ± 0.20 38 ± 0.88 1.3 AtPWD CBM20 a b-Cyclodextrin, pH 7.0 1.07 ± 0.19 b-Cyclodextrin, pH 9.0 0.56 ± 0.12 a Previously published data [20]. M. A. Glaring et al. Starch-binding domains in the CBM45 family FEBS Journal 278 (2011) 1175–1185 ª 2011 The Authors Journal compilation ª 2011 FEBS 1179 Similarly, a CBM41 SBD from Thermotoga maritima pullulanase showed a K d value of 42 lm for the inter- action with b-cyclodextrin [35]. An amylase from Bacillus halodurans carrying both a CBM25 and a CBM26 SBD gave K d values in the range 0.01–1 mm for the binding of various linear maltooligosaccharides to the individual SBDs [36]. As mentioned above, the affinity for linear ligands is generally lower than for cyclic ligands, and StGWD CBM45-2A showed no binding to maltoheptaose. Taken together, the data presented here show that the binding affinity of the CBM45 SBD is one to two orders of magnitude lower than the SBDs typically appended to microbial amylo- lytic enzymes. This clearly distinguishes the CBM45s from these more thoroughly studied SBDs and, together with the previous report on the CBM20 domain from AtPWD [20], suggests that low-affinity interactions are a recurring characteristic of plastidial starch metabolism. This would permit a more dynamic interaction with the starch granule, which may be nec- essary for the accurate control of the rate of degrada- tion [6,20,28]. The glucan phosphorylation carried out by GWD and PWD is an essential initial step in starch degradation in both tubers and leaves, and it has been suggested that the plant controls the release of energy from starch at this crucial step [18,19]. It is possible that the low binding affinity of the single domain is masked by avidity effects of the tandem arrangement of CBMs in the native enzyme. This has recently been observed for the triple-CBM53-containing chloroplas- tic starch synthase III from Arabidopsis [25]. The low binding affinity of native AtAMY3 towards b-cyclo- dextrin would suggest that this is not the case for the tandem CBM45s but, based on the current data, a small avidity effect cannot be entirely ruled out. AtAMY3 binding to starch in vitro The full-length AtAMY3 offered an advantage when examining the binding affinity of CBM45 SBDs to starch, as it displayed catalytic activity and, being a full-length enzyme, misinterpretation of binding data as a result of instability or aggregation would most likely be minimal compared with the isolated CBM45s. Hence, the starch-binding ability of purified recombi- nant AtAMY3 was demonstrated by incubation with starch isolated from leaves of tobacco plants. Binding was carried out at 4 °C and the a-amylase activity of the unbound fraction was subsequently measured. A one-site binding model was fitted to the binding iso- therm (Fig. 5), resulting in a K d value of 36 ± 6.8 mgÆmL )1 and maximum binding capacity (B max )of 93 ± 5.6%. This affinity is up to two orders of magni- tude lower than that reported previously for the bind- ing of various CBM20 domains to starch [6]. A similar experiment using maize starch resulted in comparable affinity, but substantially lower binding capacity (K d = 21 ± 9.5 mgÆmL )1 , B max = 42 ± 4.2%). A pre- vious binding analysis of a construct encompassing the isolated recombinant StGWD CBM45-1 to granular potato starch yielded a dissociation constant in the same range ( K d = 7.2 mgÆmL )1 , B max = 53%) [7]. This construct, however, contained approximately 70 amino acids of the C-terminal intervening sequence of Fig. 4. Isothermal titration calorimetry (ITC) analysis of StGWD CBM45-2A interaction with b-cyclodextrin. The top panel depicts the raw heat response for each b-cyclodextrin injection, and the bottom panel depicts the binding isotherm; open circles represent the integrated binding heat of the data in the top panel, and the full line is the fit of a one-site binding model to the integrated binding data. The experiment was carried out at 25 °C by titrating 50 l M protein in 25 mM Hepes, pH 8.0 with 4 mM b-cyclodextrin in the same buffer. Starch-binding domains in the CBM45 family M. A. Glaring et al. 1180 FEBS Journal 278 (2011) 1175–1185 ª 2011 The Authors Journal compilation ª 2011 FEBS unknown function (amino acids 68–286) and the struc- tural integrity of the protein was not verified. The data obtained not only support the affinity range measured using SPR and ITC with the cyclodex- trin starch mimics, but also verify the starch-binding ability of AtAMY3 in vitro. It cannot be precluded that some binding may involve secondary binding sites in the catalytic domain, but the demonstrated starch- binding ability of different isolated CBM45s used in both the present and other studies [7,10] suggests that AtAMY3 does indeed interact with starch granules through the tandem CBM45 domains. CBM45 interaction with starch granules in planta It has been shown by immunoblotting that StGWD binds to starch in planta in its active full-length form [23]. To investigate whether the isolated CBM45s from StGWD function as SBDs in planta, they were C-termi- nally fused to yellow fluorescent protein (YFP), either singly or as the double module, and transiently expressed in tobacco leaves. The constructs were tar- geted to the chloroplasts by an N-terminal fusion to the transit peptide of Arabidopsis GWD1. In a similar experimental set-up, fusions between green fluorescent protein (GFP) and either full-length AtAMY3 or the tandem CBM45s were analysed. Investigation of locali- zation using confocal laser scanning microscopy showed clear targeting to the chloroplasts of mesophyll cells and binding to disc-shaped transient starch granules for StGWD CBM45-2, CBM45-1,2 and full-length AtAMY3 (Fig. 6). The StGWD CBM45-1 fusion, in contrast, gave rise to numerous highly fluorescent inclu- sion body-like structures with no clear targeting (data not shown). The double module from AtAMY3 did not yield any visible signal. The behaviour of these proteins is likely to be affected by their instability and observed tendency to aggregate in isolated form (see above), sug- gesting that the CBM45s depend on packing contacts with other domains in the native enzyme. Further support for the binding of isolated CBM45s to starch comes from a previous report showing binding to starch in vitro of an StGWD construct encompassing CBM45- 1 [7] and an in planta localization analysis of GFP- tagged CBM45-1 from Arabidopsis GWD2 [10]. In the present study, it has been demonstrated that both iso- lated single and double CBM45 domains from StGWD are capable of binding to starch, and that full-length AtAMY3 binds to starch both in vitro and in planta. Conclusion In the present study, the carbohydrate-binding proper- ties of two representative plastidial enzymes containing CBM45-type SBDs were characterized. The CBM45s A B C Fig. 6. Transient expression of CBM45-YFP ⁄ GFP fusions in tobacco leaf mesophyll cells. Single and double CBM45 domains from StGWD and the full-length AtAMY3 were fused to YFP or GFP, respectively, and transiently expressed in Nicotiana benthami- ana leaves by infiltration with Agrobacterium tumefaciens. Expres- sion and localization were investigated by confocal laser scanning microscopy. YFP ⁄ GFP fluorescence (green), chlorophyll autofluores- cence (red) and a merged image of the two channels are shown. (A) StGWD CBM45-2 fused to YFP. (B) StGWD CBM45-1,2 fused to YFP. (C) Full-length AtAMY3 fused to GFP. Scale bar, 20 lm. Starch (mg·mL –1 ) 0 50 100 150 200 AtAMY3 bound (%) 0 20 40 60 80 100 Fig. 5. Binding of AtAMY3 to tobacco leaf starch in vitro. Recombi- nant AtAMY3 protein was incubated with starch isolated from leaves of Nicotiana benthamiana for 45 min at 4 °C. Unbound pro- tein was assayed for activity by measuring the release of reducing- end sugars from amylopectin. Each data point (±SE) is the average of four independent experiments. M. A. Glaring et al. Starch-binding domains in the CBM45 family FEBS Journal 278 (2011) 1175–1185 ª 2011 The Authors Journal compilation ª 2011 FEBS 1181 were demonstrated to exhibit up to two orders of mag- nitude lower affinity towards both cyclodextrins and granular starch, compared with typical SBDs encoun- tered in microbial amylolytic enzymes. This behaviour is analogous to that of the CBM20-type SBD from AtPWD [20] and supports the hypothesis that low- affinity SBDs are important for dynamic and reversible interactions in starch metabolism [28]. It remains unclear how the functionality of these low-affinity SBDs is integrated with other levels of regulation, such as the observed diurnal effects of light and redox conditions [23,27]. Further studies will be required to elucidate these details of starch metabolism and the structural elements responsible for the lower affinity of CBM45. The outcome of the present study demon- strates the large functional diversity of SBDs that has only started to be addressed, and the investigation of SBDs occurring in nonhydrolytic, starch- and glyco- gen-active enzymes will be essential to understand the contribution of such atypical SBDs to this group of important enzymes. Materials and methods Cloning, expression and purification of CBM45 domains from potato GWD DNA fragments of S. tuberosum GWD (accession number AY027522) were amplified as outlined in Fig. S3 using the primers given in Table S1. The PCR products were cloned using Gateway technology (Invitrogen, Carlsbad, CA, USA) via the entry vector pENTR ⁄ TEV ⁄ D-TOPO, and subsequently moved into the expression vector pDEST17 (Invitrogen) with an N-terminal TEV protease-cleavable His-tag. The expression vectors were transformed into Escherichia coli BL21 cells. Cultures were grown in 6 · 1L scale in Tunair flasks (Sigma-Aldrich, St. Louis, MO, USA) at 37 °C, cooled to 16 °C before induction with 0.5 mm iso- propyl thio-b-d-galactoside (IPTG), and harvested 16 h after induction. For protein purification, cell pellets were lysed in 15 mL Bugbuster (Novagen, Merck4Biosciences, Nottingham, UK) with 5 lL of Benzonase Nuclease (Sigma-Aldrich). Following centrifugation, the supernatant was loaded onto a HisTrap HP, 5 mL column (GE Health- care, Uppsala, Sweden) and eluted by a 40–400 mm imidaz- ole gradient in 20 mm Tris ⁄ HCl pH 8.0, 500 mm NaCl, 10% v ⁄ v glycerol and 0.5 m betaine according to the manu- facturer’s instructions. TEV protease cleavage of the His-tag was performed with AcTEV protease according to the manufacturer’s instructions (Invitrogen). For large-scale production, incu- bation was performed overnight at room temperate using 25% of the recommended amount of protease. The cleaved untagged protein was purified by anion exchange on a Mono Q 10 ⁄ 100 GL column (GE Healthcare) in 20 mm Tris ⁄ HCl pH 8.0 and eluted with 20 column volumes of a 0–0.5 m NaCl gradient. After dialysis to remove NaCl, the protein was stored at 4 °C. Cloning, expression and purification of A. thaliana AMY3 An AtAMY3 cDNA clone (At1g69830, accession number AY050398) was obtained from the RIKEN Arabidopsis Genome Encyclopedia (RARGE, http://rarge.psc.riken.jp). Full-length AtAMY3 excluding the chloroplast transit pep- tide and stop codon (amino acids 68–887) was amplified (primers AtCBM1-NcoI and AtpAMY- NotI, Table S1) and cloned into the NcoI and NotI sites of the expression vector pET-28a containing a C-terminal 6 · His-tag. The con- struct was transformed into E. coli BL21 Rosetta (DE3) cells (Novagen). Protein expression was carried out in either a 5 L bioreactor (Biostat B, B. Braun Biotech International, Melsungen, Germany) on defined medium [37] by induction at an absorbance at 600 nm (A 600 ) of 5 with 0.1 mm IPTG at 16 °C and harvesting after 22 h, or in shake-flasks by induction with 0.2 mm IPTG at 20 ° C and harvesting after 16–18 h. The cell pellet was resuspended in buffer A (20 mm Hepes pH 7.5, 500 mm NaCl, 40 mm imidazole, 40% v ⁄ v glycerol, 0.1% v ⁄ v Triton X-100, 0.5 mm CaCl 2 ) containing 2 mm dithithreitol, 0.1 lLÆmL )1 Benzonase Nuclease (Sigma-Aldrich) and one Complete Mini protease inhibitor tablet (Roche, Basle, Switzerland), and lysed using a French press. The lysate was incubated on ice for 30 min, clarified by centrifugation and filtered through a 0.22 lm filter. The filtrate was applied to a HisTrap HP, 1 mL col- umn (GE Healthcare), washed with a 40–70 mm imidazole gradient for 10 column volumes, and eluted with 20 column volumes of a 70–400 mm imidazole gradient at 0.5 mLÆ min )1 . Concentrated, partially pure AtAMY3 was applied to a HiLoad Superdex 200 16 ⁄ 60 gel filtration column (GE Healthcare) and eluted in 20 mm Hepes pH 7.5, 150 mm NaCl, 25% v ⁄ v glycerol and 0.5 mm CaCl 2 . The fractions containing AtAMY3 were pooled and concentrated to approximately 1 mgÆmL )1 and stored at 4 °C. DSC analysis DSC analysis was performed using a VP-DSC calorimeter (MicroCal, Northampton, MA, USA) with a cell volume of 0.52061 mL at a scan rate of 1 °CÆmin )1 . Samples were dialysed in at least 500 volumes of 25 mm Hepes–NaOH, pH 7.0 or pH 8.0, and degassed for 10 min at 20 °C. Base- line scans collected with buffer in the reference and sample cells were subtracted from sample scans. The reversibility of the thermal transitions was evaluated by checking the reproducibility of the scan on immediate cooling and rescanning. The initial screening of the conformational sta- bility of purified StGWD constructs was performed using a Starch-binding domains in the CBM45 family M. A. Glaring et al. 1182 FEBS Journal 278 (2011) 1175–1185 ª 2011 The Authors Journal compilation ª 2011 FEBS protein concentration of 0.5 mgÆmL )1 in 25 mm Hepes– NaOH, pH 8.0. The DSC analysis of the form with the highest T m value (StGWD CBM45-2A) was performed fol- lowing cleavage of the His-tag with TEV protease (see above), and subsequent repurification and dialysis of 50 lm protein as mentioned above. Origin v7.038 software with a DSC add-on module was used for data analysis, T m (unfolding temperature, defined as the temperature of maxi- mum apparent heat capacity) assignment and unfolding enthalpy calculations. SPR analysis Measurements of interactions with soluble ligands using SPR were carried out on a Biacore T100 (GE Healthcare). Domains of StGWD were biotinylated using EZ-Link Sul- fo-NHS-LC-Biotin (Pierce, Thermo Scientific, Rockford, IL, USA) in 10 mm Mes pH 6.8, 5 mm CaCl 2 and 8 mm b-cyclodextrin, and immobilized on a streptavidin-coated chip (sensor chip SA, GE Healthcare) using a standard pro- gram, aiming for a density of 1250 response units (RU). AtAMY3 was immobilized on a carboxymethylated dextran chip (sensor chip CM5, GE Healthcare) in 10 mm sodium acetate pH 4.6, 20% v ⁄ v glycerol, 1 mm CaCl 2 and 2 mm b-cyclodextrin, using a standard program, aiming for a den- sity of 7500 RU. Sensograms were collected at 25 °Cin 25 mm Hepes pH 8.0, 150 mm NaCl, 0.5 mm CaCl 2 and 0.005% v ⁄ v P20 surfactant (GE Healthcare) at a flow rate of 30 lLÆmin )1 , contact time of 90–180 s and dissociation time of 100–240 s. Experiments were run in triplicate in the range 0–2000 lm for each carbohydrate dissolved in the same buffer. All data evaluation was carried out using the Biacore T100 evaluation software. ITC analysis Experiments were performed using an MCS isothermal titration calorimeter (MicroCal). Titrations were performed by injecting 5 lL b-cyclodextrin in 25 mm Hepes–NaOH pH 7.0 or 8.0 into a stirred (400 rpm) 1.3187 mL cell con- taining 50 lm StGWD CBM45-2A in the same buffer. For each titration of enzyme, the dialysis buffer of the sample was titrated as a control using the same b-cyclodextrin stock to measure the heat of dilution. The control titration consisted of 10 injections of 1 lL in 2.5 s for the first injec- tion and 5 lL for the rest, and with 180 s of equilibration between injections. Titrations of the protein were carried out similarly, but were continued until no significant response was observed on ligand injections. Origin software supplied with the instrument was used to analyse the data. Starch-binding assays Tobacco leaf starch was isolated from 5-week-old Nicoti- ana benthamiana. The harvested leaves were homogenized in 0.2% SDS in a polytron PT3000 blender (Kinematica AG, Lucerne, Switzerland) and filtered sequentially through 2 · 100 lm and 2 · 20 lm filtration cloth. Following cen- trifugation, the starch pellet was washed twice in 0.2% SDS, three times in water, twice in 96% ethanol and air dried. Recombinant AtAMY3 (3 lg) was incubated with tobacco leaf starch granules in a 350 lL mixture containing 20 mm Hepes pH 7.5, 0.5 mm CaCl 2 , 0.05 mgÆmL )1 BSA and 0–200 mgÆmL )1 starch. The suspension was incubated with gentle mixing at 4 °C for 45 min. The supernatant (198 lL) containing unbound AtAMY3 was treated with 10 mm dithiothreitol for 20 min at 25 °C, and a-amylase activity was measured by adding 50 lL of a 25 mgÆmL )1 amylopectin solution (Fluka 10118, dissolved in 20 mm Hepes pH 7.5, 0.5 mm CaCl 2 ) and incubating for 45 min at 37 °C. Reactions were stopped by mixing with an equal volume of 0.5 m NaOH, and liberated reducing ends were determined by the 3-methyl-2-benzothiazolinone hydrazone method, as described previously [38]. The activity (expressed as the percentage of bound At AMY3 when com- pared with a no-starch control) was plotted against the starch concentration, and the data were fitted to a one-site binding model. Transient expression of CBM45s in tobacco The CBM45s from StGWD were C-terminally fused to YFP, either singly (CBM45-1, amino acids 109–217; CBM45-2, amino acids 405–545) or as the entire double module (CBM45-1,2, amino acids 77–551). Fragments were PCR amplified using uracil-containing primers (Table S1) and cloned into the vector pPS48uYFP using an improved USERÔ (uracil-specific excision reagent; New England Biolabs, Ipswich, MA, USA) cloning procedure [39]. The chloroplast transit peptide of Arabidopsis GWD1 (amino acids 1–77) was fused to each construct by simultaneous cloning of both fragments as described previously [10]. The full-length ORF of AtAMY3 (primers AMY3-F and AMY3-R, Table S1), as well as an N-terminal fragment (amino acids 1–391) covering both CBM45s (primers AMY3-F and AMY3SBD-R, Table S1), were fused to enhanced GFP in the binary vector pK7FWG2 [40] using GATEWAYÔ cloning technology (Invitrogen). Constructs were transformed into Agrobacterium tumefaciens and tran- siently expressed by infiltration in Nicotiana benthamiana as described previously [41]. Expression and localization were analysed by a confocal laser scanning microscope (TCS SP2, Leica Microsystems, Wetzlar, Germany) equipped with a 20 ·⁄0.70 or 63 ·⁄1.20 PL APO water immersion objective. A 488 nm laser line was used for excitation, and emission was detected between 520 and 550 nm for YFP fluorescence, between 510 and 535 nm for GFP fluores- cence, and between 600 and 750 nm for chlorophyll auto- fluorescence. M. A. Glaring et al. Starch-binding domains in the CBM45 family FEBS Journal 278 (2011) 1175–1185 ª 2011 The Authors Journal compilation ª 2011 FEBS 1183 Acknowledgements MAG was supported by a grant from the Danish Research Council for Technology and Production Sci- ences (grant no. 274-06-0312) and MJB by a Hans Christian Ørsted postdoctoral fellowship from the Technical University of Denmark. The financial sup- port from the Carlsberg Foundation (to BS), the Vil- lum Kann Rasmussen Foundation (to the VKR Research Centre Pro-Active Plants) and ETH Zu ¨ rich and the Swiss–South African Joint Research Pro- gramme (grant no. 08 IZ LS Z3122916, to SCZ and DS) is gratefully acknowledged. References 1 Tester RF, Karkalas J & Qi X (2004) Starch – composi- tion, fine structure and architecture. J Cereal Sci 39, 151–165. 2 Nielsen MM, Bozonnet S, Seo ES, Motyan JA, Ander- sen JM, Dilokpimol A, Abou Hachem M, Gyemant G, Naested H, Kandra L et al. (2009) Two secondary carbohydrate binding sites on the surface of barley a-amylase 1 have distinct functions and display synergy in hydrolysis of starch granules. Biochemistry 48, 7686–7697. 3 Machovic M & Janecek S (2006) Starch-binding domains in the post-genome era. Cell Mol Life Sci 63, 2710–2724. 4 Guillen D, Sanchez S & Rodriguez-Sanoja R (2010) Carbohydrate-binding domains: multiplicity of biologi- cal roles. 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Overview of the StGWD constructs produced and tested in the present study Doc S1 Complete list of species and accession numbers Table S1 Table of oligonucleotide primers This supplementary material can be found in the online version of this article Please note: As a service to our authors and readers, this journal provides supporting information supplied by the authors Such materials are peer-reviewed and. .. & Law V (2006) A structural and functional analysis of a-glucan recognition by family 25 and 26 carbohydrate-binding modules reveals a conserved mode of starch recognition J Biol Chem 281, 58 7–5 98 Ramchuran SO, Karlsson EN, Velut S, de Mare L, Hagander P & Holst O (2002) Production of heterologous thermostable glycoside hydrolases and the presence of host-cell proteases in substrate limited fed-batch . Starch- binding domains in the CBM45 family – low-affinity domains from glucan, water dikinase and a-amylase involved in plastidial starch metabolism Mikkel. 2011) doi:10.1111/j.1742-4658.2011.08043.x Starch- binding domains are noncatalytic carbohydrate-binding modules that mediate binding to granular starch. The starch- binding domains from the carbohydrate-binding