This article was downloaded by: [] On: 19 July 2014, At: 02:26 Publisher: Taylor & Francis Informa Ltd Registered in England and Wales Registered Number: 1072954 Registered office: Mortimer House, 37-41 Mortimer Street, London W1T 3JH, UK

Journal of Biomolecular Structure and Dynamics Publication details, including instructions for authors and subscription information:

Single-stranded nucleic acid binding in Arabidopsis thaliana cold shock protein is cold shock domain dependent a


Ashutosh Mani & Dwijendra K. Gupta a

Center of Bioinformatics, Institute of Interdisciplinary Studies, University of Allahabad, Allahabad 211002, India Published online: 10 Apr 2014.

To cite this article: Ashutosh Mani & Dwijendra K. Gupta (2014): Single-stranded nucleic acid binding in Arabidopsis thaliana cold shock protein is cold shock domain dependent, Journal of Biomolecular Structure and Dynamics, DOI: 10.1080/07391102.2014.907747 To link to this article:

PLEASE SCROLL DOWN FOR ARTICLE Taylor & Francis makes every effort to ensure the accuracy of all the information (the “Content”) contained in the publications on our platform. However, Taylor & Francis, our agents, and our licensors make no representations or warranties whatsoever as to the accuracy, completeness, or suitability for any purpose of the Content. Any opinions and views expressed in this publication are the opinions and views of the authors, and are not the views of or endorsed by Taylor & Francis. The accuracy of the Content should not be relied upon and should be independently verified with primary sources of information. Taylor and Francis shall not be liable for any losses, actions, claims, proceedings, demands, costs, expenses, damages, and other liabilities whatsoever or howsoever caused arising directly or indirectly in connection with, in relation to or arising out of the use of the Content. This article may be used for research, teaching, and private study purposes. Any substantial or systematic reproduction, redistribution, reselling, loan, sub-licensing, systematic supply, or distribution in any form to anyone is expressly forbidden. Terms & Conditions of access and use can be found at http://

Journal of Biomolecular Structure and Dynamics, 2014

Single-stranded nucleic acid binding in Arabidopsis thaliana cold shock protein is cold shock domain dependent Ashutosh Mani1* and Dwijendra K. Gupta Center of Bioinformatics, Institute of Interdisciplinary Studies, University of Allahabad, Allahabad 211002, India Communicated by Ramaswamy H. Sarma (Received 7 January 2014; accepted 20 March 2014)

Downloaded by [] at 02:26 19 July 2014

Cold shock proteins (CSPs) are ancient nucleic acid-binding proteins and well conserved from bacteria to animals as well as plants. In prokaryotes, CSPs possess a single cold shock domain (CSD) while animal CSPs, flanked by N- and C-terminal domains, are commonly named Y-box proteins. Interestingly, the plants CSPs contain auxiliary C-terminal domains in addition to their N-terminal CSD. The CSPs have been shown to play important role in development and stress adaptation in various plant species. The objective of this study was to find out the possible nucleic acid-binding affinities of whole CSP as well as independent domains, so that role of each individual domain may be revealed in Arabidopsis thaliana, the model plant species. The structure of CSP 3 protein from A. thaliana was modeled by homology-based approach and docking was done with different nucleic acid types.

Introduction The response of prokaryotes to low-temperature stress has been studied comprehensively in Escherichia coli and well characterized by the accumulation of cold shock proteins (CSPs). Bacterial CSPs are small proteins that possess a single nucleic acid-binding domain, which is termed as the cold shock domain (CSD). The CSD is proposed to be an ancient molecule that was present even before the origin of single-cell life (Graumann & Marahiel, 1998) and is one of the most evolutionarily conserved nucleic acid-binding domains within prokaryotes and eukaryotes (Graumann & Marahiel, 1998; Wolffe, Tafuri, Ranjan, & Familiari, 1992; Wolffe, 1994). In eukaryotes, CSPs exist as multi-domain proteins with a nucleic acid-binding domain within them; they are also known as CSD proteins. It is supposed that RNA-binding proteins evolved from small molecules such as the CSD and contain combinations of various nucleic acid-binding components (Sommerville, 1999). Eukaryotic CSD proteins are characterized by a variable N-terminal domain (Kohno, Izumi, Uchiumi, Ashizuka, & Kuwano, 2003), a conserved CSD and diverse auxiliary C-terminal domains such as basic aromatic islands, RG repeats and retroviral-type Cysteine-Cysteine-HistidineCysteine (CCHC) zinc fingers (Graumann & Marahiel, 1998; Sommerville, 1999). This feature contrasts with bacterial CSPs, which are solely comprised of a single CSD. In plants, a distinctive class of CSD proteins has been discovered. Plant CSD homologs typically contain

two distinct nucleic acid-binding modules (a single N-terminal CSD and variable quantities of C-terminal retroviral-like CCHC zinc fingers), which are interspersed by glycine-rich regions, with a few exceptions (Karlson & Imai, 2003; Kingsley & Palis, 1994). Arabidopsis contains four unique types of CSD proteins and displays differential regulation activity in response to low-temperature stress (Karlson & Imai, 2003). In Arabidopsis thaliana as a model plant, its endogenous CSPs are demonstrated to be important during plant growth and development. When the expression of AtCSP2/AtGRP2/CSDP2 (At4g38680) was up or down regulated, many developmental abnormalities with respect to flowering time, apical dominance, and seed development were observed. Furthermore, the transcripts of this gene are abundant in meristematic areas in which rapid cell divisions occur suggesting this protein may function in mRNA storage (Fusaro et al., 2007). A subsequent extensive study characterized the expression of the entire Arabidopsis CSP family in relation to stages of floral and silique development (Nakaminami et al., 2009). A prominent effect of CSPs has also been noted on seed germination during abiotic stress conditions such as dehydration and salt stress (Park, Kwak, Oh, Kim, & Kang, 2009; Kim et al., 2013). AtCSP3, a nucleocytoplasmic protein (Yang & Karlson, 2012), has significant role in cellular functions like anti-freeze activity (Kim, Sasaki, & Imai, 2009) and leaf cell expansion (Yang & Karlson, 2012). The protein has also shown nucleic acid

*Corresponding author. Email: [email protected] 1 Present address: Department of Biotechnology, Motilal Nehru National Institute of Technology, Allahabad 211004, India © 2014 Taylor & Francis


A. Mani and D.K. Gupta

chaperone activity in recombinant AtCSP3 which induces the unwinding of double-stranded DNA and RNA (Kim et al., 2009; Park et al., 2009). Being an important protein for normal functioning under various conditions in A. thaliana, CSPs draw attention for their nucleic acid-binding property. The study becomes more interesting when domain-wise binding preferences may be revealed by molecular docking studies between the protein and nucleic acids. The results of the study contribute significant knowledge concerning amino acids that are involved in nucleic acid-binding activities in AtCSP3. Comparison of the amino acid sequences of several plants may indicate the amino acids that are functionally important since these should be conserved among the various species. Finally, docking studies have been performed to determine preferential bindings for AtCSP3.

Downloaded by [] at 02:26 19 July 2014

Experiments Multiple sequence alignment (MSA) and phylogeny Amino acid sequences of CSPs from seven plant species; A. thaliana CSD protein 3 (gi|330251603|gb| AEC06697.1|), Eutrema salsugineum CSD protein 3 (gi| 294470716|gb|ADE80750.1), Medicago truncatula Major cold-shock protein (gi|357518027|ref|XP_003629302.1|), Cucumis melo cold-shock DNA-binding family protein (gi|307136096|gb|ADN33944.1|), Ricinus communis CSP putative (gi|255545420|ref|XP_002513770.1|), Triticum aestivum CSP-1 (gi|21322752|dbj|BAB78536.2|), and Oryza sativa Japonica CSD protein 2-like protein (gi| 57900030|dbj|BAD88072.1|) were retrieved from NCBI Genbank (available at These sequences were analyzed by clustalW (Larkin et al., 2007) MSA tool. The phylogenetic tree was constructed for these sequences using Close-Neighbor-Interchange algorithm (Nei & Kumar, 2000) in MEGA 5.0 software (Tamura et al., 2011). The MP tree was obtained using the CloseNeighbor-Interchange algorithm (Nei & Kumar, 2000) with search level 0 in which the initial trees were obtained with the random addition of sequences (10 replicates). The analysis involved seven amino acid sequences. All positions containing gaps and missing data were eliminated. Prediction of 3-D structure and docking studies In order to identify a template for modeling, the threedimensional structure of AtCSD (Figure 4(a)) PDB (Protein Data Bank) was searched using NCBI BLAST program (Altschul et al., 1997). The search identified Salmonella CSP as the closest homolog of AtCSD. The structure of Salmonella CSP (Morgan et al., 2009) was taken as template for modeling of AtCSD protein using

Modeller 9v6, a homology modeling software for prediction of protein structures, which is based on satisfaction of spatial restraints (Šali & Blundell, 1993; Sánchez & Šali, 2000). Dual template-based modeling strategy was employed for prediction of the whole AtCSP structure as no single protein after PDB search was found to align along the full length of AtCSP protein sequence. However, the search identified Salmonella CSP and Mouse Lin 28 protein sequence as closest homologs that were aligned along the AtCSP sequence in two different regions of the length and covering almost whole AtCSP. Therefore, the structures of Salmonella CSP (Morgan, Mcnae, Wear, Gallagher, & Walkinshaw, 2009) and Mouse Lin 28 (Nam, Chen, Greogory, Chou, & Sliz, 2011) were used as template for homology modeling of AtCSP (Figure 3(a)) using Modeller. The quality of the predicted protein structure was checked by PROCHECK program (Laskowski, Macarthur, Moss, & Thornton, 1993) (Figures 3(b) and 4(b)). The structure of CSD of A. thaliana was used for docking with random single-stranded RNA, single-stranded (CCAAT) DNA and double-stranded (CCAAT) DNA (Figure 5). For docking studies, HADDOCK 2.0 software (Dominguez, Boelens, & Bonvin, 2003; de Vries et al., 2007) was used. During the rotational and translational rigid body minimization docking, 1000 structures were calculated with solvation. The best 200 solutions according to intermolecular energy were used for the semi-flexible annealing in torsion angle space. This was followed by a final refinement with explicit modeling of hydrating water molecules. The resulting 200 structures were clustered on the basis of the intermolecular van der Waals, electrostatic and restraint energy terms, buried surface area, desolvation energy, and binding energy as combined in the HADDOCK score vs. the backbone RMSD from the lowest HADDOCK score structure. The HADDOCK score is a weighted sum of various energy terms obtained in different phases of the docking. The energy was calculated in terms of van der Waals energy, electrostatic energy, ambiguous distance restraint energy, buried surface area, binding energy, and desolvation energy. The structure with the smallest weighted sum, which is with the smallest HADDOCK score, was ranked first. Results Sequence analysis The MSA of seven plants CSPs resulted into 372 aligned positions (Figure 1). The positions from 14 to 75 were found to be conserved and belong to CSD. Interspersed glycine-rich regions were found prevalently restricted to the C-terminal domain of the proteins. The CSPs from seven plant species were found to have four signature


Downloaded by [] at 02:26 19 July 2014

Single stranded nucleic acid binding in Arabidopsis thaliana cold shock protein

Figure 1. MSA of seven plants CSPs. Cold Shock domain has been shown in upper red quadrangle. The domain resulted into four conserved domain, CSD#1, CSD#2, CSD#3, and CSD#4, respectively. The lower quadrangle includes the C-terminal domain residues. The CTD is in prevailed with Glycine-rich regions (GRRs), shown in red circles. The secondary structures and motifs of the CSD are represented by cartoon above the domain, key is given at bottom.

motifs consisting of residues K/NGF/YG/SFI, E/ DLFVHQS/T, S/FD/EGFYRS/TL, and GK/RT/SAI/VE/ DVT/I. All these signature motifs belong mostly to the random coil as well as β strands. A comprehensive analysis of these motifs and their binding preferential has been shown in Table 1. The MSA of seven plants CSPs helped to identify conserved domains, glycine-rich regions, and signature motifs. In Figure 1, the CSD residues with their secondary structure type have been shown. Conserved domains

1 and 2 fall in β strand and β hairpin region. Conserved domain 3 specially belongs to γ turns while Conserved domain 3 includes the residues belonging to β hairpin flanked by β turn and β helix. Phylogenetic analysis A phylogenetic tree (Figure 2) was constructed using MEGA 5.0 for aligned amino acid sequences of the A. thaliana and the known plant CSD proteins.


A. Mani and D.K. Gupta

Table 1.

The four conserved domains, their secondary structure type and presence of RNA, ssDNA, and dsDNA binding residues.

Conserved domain (#) K/NGF/YG/SFI (1) E/DLFVHQS/T (2) S/FD/EGFYRS/TL (3) GK/RT/SAI/VE/DVT/I (4)

Helix Strand − + − −

+ + − +

β turn

γ turn

β hairpin

RNA binding residues

ssDNA binding residues

dsDNA binding residues

− − + +

− + + −

+ + − +

++ ++ − +

+ ++ ++ +

− + + +

Notes: (+) sign indicates that the domain falls in the category type of secondary structure/motif/nucleic acid binding residues. (−) sign indicates that the motif does not have any residue that fall in the category of the secondary structure/motif/nucleic acid binding residues.

Arabidopsis thaliana* Eutrema salsugineum Medicago truncatula Cucumis melo subsp. Melo Ricinus communis Triticum aestivum Oryza sativa Japonica

Downloaded by [] at 02:26 19 July 2014

Figure 2. Most Parsimonious tree inferred for seven plant species with reference to their cold shock domain proteins.

The evolutionary history was inferred using the Maximum Parsimony method. The most parsimonious tree with length = 263 is shown. The consistency index is .935361 (.876812), the retention index is .734375 (.734375), and the composite index is .686906 (.643909) for all sites and parsimony-informative sites (in parentheses). There were a total of 145 positions in the final data-set. Evolutionary analyses were conducted in MEGA5 (14). A close analysis of the tree confirms that plant CSD proteins have been highly conserved during the course of evolution. The A. thaliana CSD is evolutionarily closest to Eutrema while it is distantly related to Oryza species. Structure prediction of whole CSP and CSD only The Ramachandran plot (Figure 3(b)) for the predicted structure of AtCSP, which has a total of 301 residues, reported 84.5% residues in most favored regions and 14.5% residues in additionally allowed regions. No residues were reported in generously allowed regions and only 1% (two) residues in disallowed regions. This model, having approximately 99% residues in allowed region with no residues in generously allowed region, was finally selected for nucleic acid docking studies. The Ramachandran plot (Figure 4(b)) for the predicted structure of AtCSD reported 94.4% residues in most favored regions and 5.6% residues in additionally allowed regions. No residues were reported in generously allowed regions and disallowed regions. This model, having approximately 95% residues in most favored regions with no residues in disallowed region, was finally selected for nucleic acid docking studies.

The structure has five β strands which form a barrel. In the AtCSD, the first β strand (Blue color) which has residues RSIGKVSWF is a nine-residue long strand. Second β strand (sea blue color) is a run of six amino acids comprising of residues GYGFIT. The third β strand (green color) which has residues LFVHQSS is a sevenresidue long strand. The Fourth β strand (orange color) comprises GESVEYEIA, nine residues. Fifth β strand (Red color) is the smallest strand having only four amino acids TKAI. The first and second β strands are connected by SDGK, while the second and third β strands are connected by PDDGGEEL. The third and fourth β strands are connected by IVSDGFRSLTL residues. The fourth and fifth strands are connected by ALGSDGK residues. All the four random coils connecting the five β strands belong to the most accessible regions of the domain while all the five β strands fall in buried regions. Random coils interconnecting these five β strands belong to the most exposed regions in the structure. The nucleic acid binding results for the CSD show that, for RNA and ssDNA, the binding affinity is considerably better and the complex is more stabilized when the length of nucleic acid is increased from 5mer to 10mer, as evident from scores shown in Table 2. But the docking scores for AtCSD and dsDNA complex decrease considerably, when the length of dsDNA is increased from 5 to 10 residues. In silico nucleic acid docking results The results of docking (Table 2) demonstrate that, for 10 most stable complexes, AtCSP3 forms the docking structure complex with 5mer ssRNA in the score range of 37.6 ± 10.1; and when the length of ssRNA was increased to 10mer, the score was improved to the range of −20.5 ± 8.2. Similarly the score of AtCSP3 and 5mer ssDNA complex changed from 11.0 ± 11.4 to −90.6 ± 6.9 after increasing the length of ssDNA from 5mer to 10mer. (Supplementary material: Athalfull_5merRNA, Athalfull_10merRNA, Athalfull_5merssDNA,Athalfull_ 10merssDNA.) On docking the AtCSP3with 5 bp dsDNA, the stable structures showed scores in the range of 74.1 ± 7.2. Thus,

Downloaded by [] at 02:26 19 July 2014

Single stranded nucleic acid binding in Arabidopsis thaliana cold shock protein


Figure 3. (a) Structure of cold shock protein of A.thaliana predicted using structure of Mouse Lin 28 and Salmonella CSP as template, (b) Ramachandran plot for the predicted structure of A.thaliana CSP.

Figure 4. (a) Structure of cold shock domain of A. thaliana predicted using structure of and Salmonella CSP as template, (b) Ramachandran plot for the predicted structure of A. thaliana CSD.

less-stable structure complex was formed with 5mer dsDNA. However, a small increase in stability was observed when 10 bp ds DNA was allowed to form structure complex with CSP as the score came in the range of 60.1 ± 11.3. (Supplementary material: Athalfull_5merdsDNA,Athalfull_10merdsDNA.) When the study was repeated by docking only the AtCSD, it was observed that the score of −13.8 ± 3.0 for 5mer ssRNA changed to −67.7 ± 4.0 for 10merRNA. Similar trend was followed by ssDNA also. (Supplementary material: Athalcsd_5merRNA,Athalcsd_10merRNA, Athalcsd_5merssDNA,Athalcsd_10merssDNA.)

Interestingly, when A. thaliana CSD was used for docking with a dsCCAAT DNA, docking structure complexes were formed in the range of 49.0 ± 6.0 and with the increased length of the dsDNA up to 10mer the complexes with lower stability scoring 121.8 ± 2.4 were formed (Table 2). (Supplementary material: Athalcsd_5merdsDNA,Athalcsd_10merdsDNA.) These results show that the whole CSP has good ssDNA and ssRNA binding activity, but it has modest dsDNA binding activity comparatively. When compared with whole CSP, the CSD preferentially binds to single-stranded nucleic acid entities and shows better

Downloaded by [] at 02:26 19 July 2014


A. Mani and D.K. Gupta

Figure 5. Structures of (A) ssRNA CGAAU, (B) single-stranded 10mer ssRNA, (C) ssCCAAT, (D); dsCCAAT, (E) 10mer ssCCAAT repeat, (F) 10mer dsCCAAT repeat (see supplementary material for coordinate files).

binding, but does not show any considerable binding activity with double-stranded DNA. The docking of the CTD of A. thaliana CSP did not show preferred binding with any type of nucleic acid. (Data shown in Supplementary files). It supports the view that in plants the single-stranded nucleic acid binding is CSD dependent, the whole CSP is needed for a double-stranded DNA binding activity and CTD does not play any major role in any nucleic acid-binding activity. From the studies, it has become clear that conserved domains 1,2, including a variable site consisting of 4–5 residues, preceding conserved domain 1 (Figure 1) are preferred binding sites for 5mer RNA while 5mer ssDNA and 5mer dsDNA bind at conserved domain numbers 3 and 4, including a variable site consisting of 4–5 residues, preceding the conserved domain number 4. For 10mer RNA, an additional site for binding was identified as conserved domain No. 4. Binding of 10mer ds DNA was observed at different sites from the binding sites of 5mer ds DNA. From these observations, it is clear that in CSD RNA and ssDNA binds on definite sites while dsDNA binding is random. In case of whole CSP majority of binding sites for ssDNA and RNA are restricted to the CSD but binding of dsDNA is dependent on 4–5 residues that are not predefined (see supplementary material). Discussion CSPs are known to be involved in various cellular functions. Wheat CSP (WCSP1), identified a protein that is highly inducible by cold but not by other stresses such as heat, salt, and drought, was the first plant CSD protein

to be functionally characterized (Karlson, Nakaminami, Toyomasu, & Imai, 2002). Since then, two rice CSPs and four Arabidopsis CSPs have been confirmed as functional nucleic acid-binding proteins. Several studies on plants CSPs have suggested their putative functional role as RNA chaperones. (Chaikam & Karlson, 2008; Fusaro et al., 2007; Kim et al., 2007, 2009; Nakaminami, Karlson, & Imai, 2006; Nakaminami et al., 2009; Park et al., 2009; Sasaki, Kim, & Imai, 2007; Yang & Karlson, 2011). All the four AtCSPs are highly similar at the amino acid sequence level (Karlson & Imai, 2003) and have been confirmed as functional nucleic acid-binding proteins. The AtCSP3 melts nucleic acids and has been reported to complement a bacterial mutant lacking four endogenous CSPs, supporting the hypothesis that AtCSP3 functions as an RNA chaperone in plants (Kim et al., 2009; Park et al., 2009). In our previous study (Mani, Yadava, & Gupta, 2012), it has been shown that CSD from Philosamia ricini YBP, an insect CSD protein, shows preferential binding for single-stranded nucleic acids. This trend is followed by AtCSD too, a plant CSP, adding that this binding preference is exclusive to CSD only, as the whole CSP did not follow the same pattern of nucleic acid binding. However, whole CSP has shown some improvement in binding for dsDNA in comparison to the CSD only. In our previous study, we observed that the bacterial CSP, where there is not any NTD or CTD to flank the CSD, did not show any binding preferential for dsDNA and upon increasing the length of dsDNA, the stability of Protein–DNA complex decreased. It is also important to mention here that the plants CSPs show more homology with bacterial CSPs rather than with

1867.7 ± 108.6

−.9 ± 2.6 427.2 ± 10.92

17.1 ± 1.9 1888.3 ± 28.76 1060.0 ± 18.5

−41.5 ± 4.0 −213.0 ± 25.4

−51.4 ± 9.2 −199.5 ± 20.2 9.1 ± 1.5 1311.2 ± 55.89 1205.0 ± 62.3 −2.2 ± 11.7 2546.7 ± 142.40 2734.4 ± 64.5 17.0 ± 8.9 779.7 ± 134.56 2572.0 ± 161.5 1428.3 ± 113.2 Buried Surface Area

Desolvation energy Restraints violation energy

−63.4 ± 1.9 −265.1 ± 35.2 30.5 ± 5.2 968.2 ± 81.60

−84.8 ± 8.6 −336.0 ± 38.4 18.2 ± 8.6 1133.3 ± 24.60 2280.9 ± 110.1 −56.2 ± 1.2 −176.0 ± 21.1 17.5 ± 12.2 1116.2 ± 43.07 1211.7 ± 123.6

−102.4 ± 8.4 −416.0 ± 16.8

−71.5 ± 5.5 −288.1 ± 32.5 16.3 ± 5.5 1869.9 ± 54.51 1865.9 ± 126.9

−101.3 ± 4.0 −455.5 ± 32.4

−42.6 ± 3.1 −136.8 ± 15.9 .3 ± 2.3 558.2 ± 31.81 921.5 ± 51.0

−77.5 ± 3.1 −207.5 ± 34.9 3.2 ± 3.7 481.1 ± 47.64 1637.8 ± 56.8

−50.6 ± 5.5 −180.6 ± 12.1 2.7 ± 2.7 581.0 ± 33.01 1105.0 ± 26.3

−85.4 ± 4.8 −262.6 ± 13.5

121.8 ± 2.4 15 11.8 ± .2 49.0 ± 6.0 7 6.4 ± .3 11.0 ± 11.4 31 4.0 ± .2 −20.5 ± 8.2 14 1.1 ± .9

HADDOCK score Cluster size RMSD from the overall lowestenergy structure Van der Waals energy Electrostatic energy

37.6 ± 10.1 22 3.9 ± 0.1

−90.6 ± 6.9 22 5.6 ± .1

74.1 ± 7.2 43 2.6 ± .1

60.1 ± 11.3 14 1.9 ± 1.2

−13.8 ± 3.0 41 6.3 ± .6

−67.7 ± 4.0 64 6.9 ± .2

−25.9 ± 2.6 19 5.0 ± .2

−96.2 ± 5.1 36 1.1 ± .7

csd_10mer dsDNA csd_5mer dsDNA csd_10mer ssDNA csd_5mer ssDNA csd_10mer RNA csd_5mer RNA Full_10mer dsDNA Full_5mer dsDNA Full_10mer ssDNA Full_5mer ssDNA Full_10mer RNA Full_5mer RNA

Table showing docking results between A.thaliana CSD/CSP and ssRNA/ssDNA/dsDNA. (see supplementary material for coordinate files of docking complexes.) Table 2.

Downloaded by [] at 02:26 19 July 2014

Single stranded nucleic acid binding in Arabidopsis thaliana cold shock protein


animal YBPs. Here it becomes clear that bacterial CSPs, as whole protein itself, exhibit preferential binding for single-stranded nucleic acids whereas in case of plants CSPs binding with nucleic acid, the role of each domain is different and depends on nucleic acid type.

Conclusions All the four AtCSPs are quite similar at amino acid sequence level. They are reported to be involved in different functions at cellular level and known to exhibit nucleic acid-binding activity. This investigation reveals nucleic acid-binding preferences of AtCSP3 that is supposed to work as RNA chaperone besides possessing nucleic acid melting properties (Kim et al., 2009; Park et al., 2009). Therefore, in this protein, it is interesting to inspect the binding preferences of different domains for various nucleic acid types. The present study appreciates the role of AtCSP3 protein from A. thaliana as well as the CSD domain independently in binding with nucleic acids. The results, which comply with the previous studies in other organisms, confirm that the CSD plays a major role in singlestranded nucleic acid binding. Whereas the CTD did not show any considerable nucleic acid binding. Importantly, the whole CSP forms stable complex with dsDNA in comparison to the CSD underlining the role of NTD in dsDNA binding. Here, we report that A. thaliana CSP prefers dsDNA binding while the CSD of the protein has greater affinity for RNA and ssDNA. These results may accelerate the studies on domaindependent nucleic acids binding in CSPs and help in increasing focus on the residues involved in different types of nucleic acids binding in plants as well as other organisms. These findings will promote study on the multilevel role of plants CSPs in transcription and translational machinery, focusing on their domain-specific binding for nucleic acids.

Abbreviations AtCSP Arabidopsis thaliana cold shock protein AtCSD Arabidopsis thaliana cold shock domain CSD cold shock domain YBP Y-box protein CSP Cold shock protein CDS Coding DNA sequence MSA multiple sequence alignment ss single stranded ds double stranded bp base pairs NTD N-terminal domain CTD C-terminal domain


A. Mani and D.K. Gupta

Supplementary material The Supplementary material for this paper is available online at http://dx.doi/10.1080/07391102.2014.907747. Acknowledgments The work was supported by funds from Department of Science Technology –Nanomission Grant and Department of Biotechnology – BIF Grant under its BTISNet Scheme to DKG.

Downloaded by [] at 02:26 19 July 2014

References Altschul, S. F., Madden, T. L., Schaffer, A. A., Zhang, J., Zhang, Z., Miller, W., & Lipman, D. J. (1997). Gapped BLAST and PSI-BLAST: A new generation of protein database search programs. Nucleic Acids Research, 25, 3389–3402. Chaikam, V., & Karlson, D. (2008). Functional characterization of two cold shock domain proteins from Oryza sativa. Plant, Cell and Environment, 31, 995–1006. Dominguez, C., Boelens, R., & Bonvin, A. M. J. J. (2003). HADDOCK: A protein-protein docking approach based on biochemical and/or biophysical information. Journal of American Chemical Society, 125, 1731–1737. Fusaro, A. F., Bocca, S. N., Ramos, R. L., Barroco, R. M., Magioli, C., Jorge, V. C., … Sachetto-Martins, G. (2007). AtGRP2, a cold-induced nucleo-cytoplasmic RNA-binding protein, has a role in flower and seed development. Planta, 225, 1339–1351. Graumann, P. L., & Marahiel, M. A. (1998). Superfamily of proteins that contain the cold-shock domain. Trends in Biochemical Sciences, 23, 286–290. Karlson, D., & Imai, R. (2003). Conservation of the cold shock domain protein family in plants. Plant Physiology, 131, 12–15. Karlson, D., Nakaminami, K., Toyomasu, T., & Imai, R. (2002). A cold-regulated nucleic acid-binding protein of winter wheat shares a domain with bacterial cold shock proteins. Journal of Biological Chemistry, 277, 35248–35256. Kim, J. S., Park, S. J., Kwak, K. J., Kim, Y. O., Kim, J. Y., Song, J., … Kang, H. (2007). Cold shock domain proteins and glycine-rich RNA-binding proteins from Arabidopsis thaliana can promote the cold adaptation process in Escherichia coli. Nucleic Acids Research, 35, 506–516. Kim, M. H., Sasaki, K., & Imai, R. (2009). Cold shock domain protein 3 regulates freezing tolerance in Arabidopsis thaliana. Journal of Biological Chemistry, 284, 23454–23460. Kim, M. H., Satoa, S., Sasakia, K., Saburic, W., Matsuic, H., & Imaia, R. (2013). Cold shock domain protein 3 is involved in salt and drought stress tolerance in Arabidopsis. FEBS Open Biology, 3, 438–442. Kingsley, D. P., & Palis, J. (1994). GRP2 protein contain both CCHC fingers and a cold shock domain. Plant Cell, 6, 1522–1523. Kohno, K., Izumi, H., Uchiumi, T., Ashizuka, M., & Kuwano, M. (2003). The pleiotropic functions of the Y-box-binding protein, YB-1. BioEssays, 25, 691–698. Larkin, M. A., Blackshieds, G., Brown, N. P., Chenna, R., McGettigan, P. A., McWilliam, H., … Higgins, D. G. (2007). ClustalW and ClustalX version 2. Bioinformatics, 23, 2947–2948. Laskowski, R. A., Macarthur, M. W., Moss, D. S., & Thornton, J. M. (1993). PROCHECK: A program to check the stereochemical quality of protein structures. Journal of Applied Crystallography, 26, 283–291.

Mani, A., Yadava, P. K., & Gupta, D. K. (2012). Cold shock domain protein from Philosamia ricini prefers single starnded nucleic acid binding. Journal of Biomolecular Structure and Dynamics, 30, 532–541. Morgan, H. P., Mcnae, I., Wear, M. A., Gallagher, M., & Walkinshaw, M. D. (2009). Crystallization and X-ray structure of cold-shock protein E from Salmonella typhimurium. Acta Crystallographica Section F Structural Biology and Crystallization Communications, 65, 1240–1245. Nakaminami, K., Karlson, D. T., & Imai, R. (2006). Functional conservation of cold shock domains in bacteria and higher plants. Proceedings of the National Academy of Sciences, USA, 103, 10122–10127. Nakaminami, K., Hill, K., Perry, S. E., Sentoku, N., Long, J. A., & Karlson, D. T. (2009). Arabidopsis cold shock domain proteins: relationships to floral and silique development. Journal Experimental Botany, 60, 1047–1062. Nam, Y., Chen, C., Greogory, R. I., Chou, J. J., & Sliz, P. (2011). Molecular basis for interaction of Let-7 molecular basis for interaction of let-7 microRNAs with Lin28. Cell, 147, 1080–1091. Nei, M., & Kumar, S. (2000). Molecular evolution and phylogenetics. New York, NY: Oxford University Press. Park, S. J., Kwak, K. J., Oh, T. R., Kim, Y. O., & Kang, H. (2009). Cold shock domain proteins affect seed germination and growth of Arabidopsis thaliana under abiotic stress conditions. Plant Cell and Physiology, 50, 869–878. Šali, A., & Blundell, T. L. (1993). Comparative protein modelling by satisfaction of spatial restraints. Journal of Molecular Biology, 234, 779–815. Sánchez, R., & Šali, A. (2000). Comparative protein structure modeling: Introduction and practical examples with MODELLER. In: D. M. Webster (Ed.), Protein structure prediction: Methods and protocols (pp. 97–129). New Jersey, NJ: Humana Press. Sasaki, K., Kim, M. H., & Imai, R. (2007). Arabidopsis COLD SHOCK DOMAIN PROTEIN2 is a RNA chaperone that is regulated by cold and developmental signals. Biochemical and Biophysical Research Communications, 364, 633–638. Sommerville, J. (1999). Activities of cold-shock domain proteins in translational control. BioEssays, 21, 319–325. Tamura, K., Peterson, D., Peterson, N., Stecher, G., Nei, M., & Kumar, S. (2011). MEGA5: Molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods. Molecular Biology and Evolution, 28, 2731–2739. de Vries, S. J., van Dijk, A. D. J., Krzeminski, M., van Dijk, M., Thureau, A., Hsu, V., … Bonvin, A. M. J. J. (2007). HADDOCK versus HADDOCK: New features and performance of HADDOCK2.0 on the CAPRI targets. Proteins: Structure, Function & Bioinformatics, 69, 726–733. Wolffe, A. P. (1994). Structural and functional properties of the evolutionarily ancient Y-box family of nucleic acid binding proteins. BioEssays, 16, 245–251. Wolffe, A. P., Tafuri, S., Ranjan, M., & Familiari, M. (1992). The Y-box factors: A family of nucleic acid binding proteins conserved from Escherichia coli to man. The New Biologist, 4, 290–298. Yang, Y., & Karlson, D. T. (2011). Over-expression of AtCSP4 affects late stages of embryo development in Arabidopsis. Journal of Experimental Botany, 62, 2079–2091. Yang, Y., & Karlson, D. (2012). Effects of mutations in the Arabidopsis cold shock domain protein 3 (AtCSP3) gene on leaf cell expansion. Journal Experimental Botany, 63, 4861–4873.

Single-stranded nucleic acid binding in Arabidopsis thaliana cold shock protein is cold shock domain dependent.

Cold shock proteins (CSPs) are ancient nucleic acid-binding proteins and well conserved from bacteria to animals as well as plants. In prokaryotes, CS...
1018KB Sizes 0 Downloads 3 Views