gb-2002-3-10-research0056 GBJ Research <p>Analysis of EF-hand-containing proteins in <it>Arabidopsis</it></p> Day S Irene Reddy S Vaka Shad Ali Gul Reddy ASN reddy@lamar.colostate.edu

Department of Biology and Program in Cell and Molecular Biology, Colorado State University, Fort Collins, CO 80523, USA

 Genome Biology 1465-6906 2002 3 10 research0056.1 research0056.24 http://genomebiology.com/2002/3/10/research/0056 10.1186/gb-2002-3-10-research0056 12372144 8 4 2002 14 6 2002 19 8 2002 23 9 2002 2002 Day et al., licensee BioMed Central Ltd

Analysis of EF-hand-containing proteins in Arabidopsis

Three out of the four classes of Ca2+-binding proteins in plants contain Ca 2+ -binding EF-hand motif(s). A maximum of 250 proteins possibly having EF-hands were identified. Diverse proteins, including enzymes, proteins involved in transcription and translation, protein- and nucleic-acid-binding proteins and a large number of unknown proteins, have one or more putative EF-hands.

Abstract

Background

In plants, calcium (Ca2+) has emerged as an important messenger mediating the action of many hormonal and environmental signals, including biotic and abiotic stresses. Many different signals raise cytosolic calcium concentration ([Ca2+]cyt), which in turn is thought to regulate cellular and developmental processes via Ca2+-binding proteins. Three out of the four classes of Ca2+-binding proteins in plants contain Ca2+-binding EF-hand motif(s). This motif is a conserved helix-loop-helix structure that can bind a single Ca2+ ion. To identify all EF-hand-containing proteins in Arabidopsis, we analyzed its completed genome sequence for genes encoding EF-hand-containing proteins.

Results

A maximum of 250 proteins possibly having EF-hands were identified. Diverse proteins, including enzymes, proteins involved in transcription and translation, protein- and nucleic-acid-binding proteins and a large number of unknown proteins, have one or more putative EF-hands. Phylogenetic analysis identified six major groups that contain some families of proteins.

Conclusions

The presence of EF-hand motif(s) in a diversity of proteins is consistent with the involvement of Ca2+ in regulating many cellular and developmental processes. Thus far, only 47 of the possible 250 EF-hand proteins have been reported in the literature. Various domains that we identified in many of the uncharacterized EF-hand-containing proteins should help in elucidating their cellular role(s). Our analyses suggest that the Ca2+ messenger system is widely used in plants and that EF-hand-containing proteins are likely to be the key transducers mediating Ca2+ action.

 Biochemistry and structural biology Bioinformatics Plant biology Genome studies Physiology

Background

Organisms must be able to respond to their environment to survive. In plants, mechanisms have evolved for sensing and responding to hormonal and environmental signals, both biotic (for example, pathogens) and abiotic (for example, heat, cold, light and salt/drought stresses). To elicit a response, the perceived signal must be conveyed to the cellular machinery. Messengers such as Ca2+, cyclic nucleotides (cAMP and cGMP), hydrogen peroxide (H2O2) and nitric oxide transduce the perceived stimulus to proteins that initiate a response. Ca2+ is one of the important messengers that mediate plant responses to hormones, developmental cues and external stimuli [1,2,3]. It is implicated in regulating such diverse and fundamental cellular process as cytoplasmic streaming, thigmotropism, gravitropism, cell division, cell elongation, cell differentiation, cell polarity, photomorphogenesis and plant defense and stress responses [1,3,4]. The Ca2+ concentration in the cytoplasm ([Ca2+]cyt) is maintained in the nanomolar range (approximately 100-200 nM), whereas the concentration in organelles and cell wall is in the millimolar range [1,3,5,6]. Several signals (hormonal, abiotic and biotic) have been shown to cause transient elevation of [Ca2+]cyt [1,2,5,6,7]. This transient increase in [Ca2+]cyt is sensed by Ca2+-binding proteins [4,8,9]. The conformation of the Ca2+-binding protein changes on binding Ca2+, resulting in modulation of its activity or its ability to interact with other proteins or nucleic acids and modulate their function or activity. The Ca2+ sensors in plants can be broadly divided into four major classes [2,9]: calmodulin (CAM) (class A), CAM-like and other EF-hand-containing Ca2+-binding proteins (class B), Ca2+-regulated protein kinases (class C) and Ca2+-binding proteins without EF-hand motifs (class D).

Three classes (A, B, and C) contain proteins with EF-hand motifs. This motif is a helix-loop-helix structure that binds a single Ca2+ ion [10]. The loop consists of 12 residues with the pattern X*Y*Z*-Y*-X**-Z. The residues X, Y, Z, -Y, -X, -Z participate in binding Ca2+and the intervening residues are represented by asterisks (*). Asp or Asn is usually found at X and Y; Asp, Asn, or Ser at Z; a variety of residues at -Y; usually Asp, Asn, or Ser at -X, but this position is more variable; and usually Glu at -Z [11]. The helix-loop-helix is only 29 residues long, the E α-helix being residues 1-10, the loop 10-21, and the F α-helix 19-29 [11]. Residue 1 is often Glu (E), and a Gly at residue 15 is highly conserved, as is Ile at residue 17. It has been reported that some of the EF-hand domains do not bind Ca2+ [11].

In quiescent cells, proteins with EF-hands are in an apoprotein form; when [Ca2+]cyt increases they bind Ca2+ and change their conformation. Some EF-hands can also bind Mg2+ (for example, the third and fourth EF-hands of troponin C bind Ca2+/Mg2+, whereas the first and second EF-hands are Ca2+ specific [12]). Ca2+/Mg2+ discrimination relies on the affinities of the EF-hands for these cations, which is dependent on the types of amino-acid residues in the binding loop [11,13].

EF-hands can be present in proteins with no other known domains, as is the case for CAM, or in proteins with other domains such as a protein kinase. In most cases EF-hand motifs are found in pairs, and proteins with four EF-hands usually have two domains with a pair of EF-hands in each. Calpain is an exception to the pairing rule. It comprises a large subunit with five EF-hands at the carboxyl terminus and a small subunit that also has five EF-hands. The two unpaired hands in these subunits pair to form a heterodimer [14]. The large superfamily of EF-hand proteins has been divided into 66 subfamilies on the basis of differences in number and organization of EF-hand pairs, amino-acid sequences within or outside the motifs, affinity for Ca2+ and/or selectivity and affinity for target proteins [11]. Of the subfamilies, 28 consist of a unique single member. The EF-hand proteins used in the classification by Nakayama et al. [11] include proteins from animals, plants, fungi and protists, with plants represented in only nine of the 66 subfamilies.

Several EF-hand proteins have been identified in the model plant Arabidopsis thaliana, including several CAMs [15,16,17,18], a Ca2+-binding protein (CaBP-22)[19], touch-induced proteins TCH2 and TCH3 [20], centrin [21], Ca2+-dependent protein kinases (CPKs) [22], calcineurin B-like proteins/salt-overly-sensitive3 family (CBLs/SOS3) [23,24], fimbrins [25], respiratory burst oxidase homologs (Rbohs) [26,27], a phospholipase [28,29], channel proteins [30,31], a NAD(H)-dependent glutamate dehydrogenase [32], a protein phosphatase [33,34], a NaCl-inducible protein [35], and a Ca2+-binding protein in pollen [36]. Some have been identified by screening with animal homologs whereas others have been identified by sequencing the genes induced in response to biotic and abiotic signals. The first method would miss plant-specific EF-hand proteins and the second method relies on comprehensive analysis of all genes that might be induced or activated by various signals. With the recent completion of the sequencing of the A. thaliana genome, the first plant genome to be sequenced, new methods of identifying genes encoding proteins with specific domains have become possible [37]. Insight into the function of the proteins can be gained by identifying and characterizing EF-hand proteins encoded in the Arabidopsis genome. Classification of the EF-hand-containing proteins can be a starting point in identification of Ca2+-binding proteins that might be involved in a particular cell process. With this in mind, we searched the Arabidopsis genome for genes encoding proteins with EF-hand motifs. We used three approaches to identify EF-hand-containing proteins in Arabidopsis. First, we analyzed data from the Munich Information Center for Protein Sequences (MIPS) A. thaliana database (MAtDB) [38,39]; second, we carried out BLAST searches using different known EF-hand sequences (nucleotide and amino acid) against the Arabidopsis genome database and third, we searched the literature. We identified 250 EF-hand or putative EF-hand proteins. This estimate represents a maximum number of possible EF-hand-containing proteins in the Arabidopsis genome as our analysis was very inclusive. Of these 250 proteins, only 47 have been reported in the literature. Of the 250, 73 were identified by only one prediction program as having an EF-hand and the rest were identified by two or more programs. Further study is needed to verify Ca2+-binding activity of many of these proteins. Each protein sequence was analyzed for domains other than EF-hands. Several have a variety of domains, which may be useful in determining protein function.

Results and discussion

Identification of EF-hand-containing proteins

To identify EF-hand-containing proteins in Arabidopsis, the protein sequences listed as having EF-hands in the InterPro Domain Table at MAtDB [39] were retrieved. Each protein sequence was then analyzed for the presence of an EF-hand motif and other domain(s) using InterProScan [40]. There are many databases for analyzing proteins using different approaches to search for patterns, profiles and hidden Markov models [41]. InterProScan was chosen because it integrates SWISS-PROT, PROSITE, PRINTS, Pfam, ProDom, SMART and TIGRFAMs programs into a single comprehensive format. Therefore, scanning one site is the equivalent of scanning seven databases that use different approaches [40]. The Inter-Pro Domain Table at MAtDB listed 219 proteins as having EF-hands. Eighteen sequences did not have EF-hands identifiable by InterProScan and so were eliminated from our analysis. We also did sequence-similarity searches using three different EF-hand proteins that have been characterized in Arabidopsis. The nucleotide and protein sequences of Arabidopsis CAM4, a protein containing four EF-hands, were used to do BLAST searches (TbIastN, BlastP) against the Arabidopsis genome at MAtDB [39]. We also used the protein sequences of a Ca2+-dependent protein kinase (CPK1) and a small protein with one EF-hand domain (At2g46600). Proteins showing similarity to these proteins were checked for the presence of EF-hands using InterProScan as above. Additional EF-hand proteins were found that had not been included in the MAtDB Inter-Pro domain table. We also searched the literature for reports of EF-hand-containing proteins in Arabidopsis that had been identified by various experimental approaches. Additional EF-hand proteins were identified from this search. Together, these searches resulted in identification of a possible total of 250 EF-hand-containing proteins (Tables 1,2,3). Seventy-three of the EF-hands were identified by only one of the seven prediction programs included in InterProScan. These proteins, which are indicated in bold in Table 1, could be false positives. Further studies are needed to verify the Ca2+-binding ability of these putative EF-hands. It is, however, worth noting that the activity of two of the proteins in this category (AtPLC1 and KCO1) has been shown to be dependent on Ca2+ [31,42]. All proteins are listed by their protein ID number except CAM6, which has not been assigned an ID number. CPKs and closely related CRKs (CPK-related protein kinases) are listed in Table 2, as this is a large family of proteins that has been relatively well studied in Arabidopsis.

<p>Table 1</p>

EF-hand-containing proteins (excluding CPKs and CRKs) in Arabidopsis

ID number§

Size (amino acids)

Number of EF hands

Domains*/remarks†

Targeting‡

Published name

References

Group I

At1g02150

638

1

PPR repeats, NLS

sc (.616)

At1g06220

987

2

Elongation factor aEF-2

At5g25230

973

1

ATP_GTP_A, EFG_C, GTP_EFTU(_D2)

At1g17090

93

1

-

sc (.944)

At1g55040

849

1

Zn-finger in Ranbp and others

ch (.823)

At1g74430

271

1

Myb DNA-binding domain

At2g03150

1276

1

-

mt (.870)

At5g40690

210

1

-

mt (.656)

At3g13500

110

1

-

At1g69030

320

1

-

At3g59820

755

1

-

mt (.666)

At1g73440

254

2

Josephin, UIM

ch (.941)

At3g07740

548

1

Zinc finger, Myb DNA-binding, GatC

At5g06260

424

1

-

At4g00140

257

1

-

ch (.792)

At1g20760

1019

2

EPS15 repeat, pfkB

At1g21630

1181

4

EPS15 repeat

At3g20290

485

2

ATP_GTP_A, EPS15, NLS_BP

At4g05520

514

1

EPS15 repeat, dynamin family

At1g47550

861

1

NLS_BP

At1g47560

1564

1

NLS_BP

At3g59270

335

1

-

At5g41840

540

1

F-box domain

Group II

At1g02270

484

1

-

At5g54130

232

1

-

At5g46830

511

1

bHLH

At1g05150

808

1

Zinc finger, TPR/put O-GlcNAc transferase

At2g32450

802

1

Zinc finger, TPR/put O-GlcNAc transferase

At2g35380

336

1

Plant peroxidase

sc (.960)

At1g20490

530

1

AMP-dependent synthetase and ligase

At1g20510

546

1

-

ch (.747)

At1g21540

550

1

AMP-dependent synthetase and ligase

At1g23160

578

1

-

At3g04860

289

1

-

ch (.666)

At5g28150

289

1

-

ch (.651)

At1g44780

471

1

NLS_BP

At3g01780

1192

1

-

At3g02270

676

1

eIF4-gamma/eIF

At5g64060

356

1

No apical meristem protein

At4g05110

394

1

Nucleoside_tran

At4g05120

418

1

Nucleoside_tran

At4g05140

419

1

Nucleoside_tran

At5g22840

538

1

Eukaryotic protein kinase

At5g12120

619

1

Ubiquitin-associated domain

ch (.811)

At1g64850

162

1

-

At3g46220

804

1

Biotin operon repressor

At3g44330

565

1

-

sc (.697)

At5g21130

281

1

ATP synthase alpha and beta subunit

At3g14270

1791

1

FYVE, PIP5K

At3g32010

603

1

ATHILA

At3g32880

503

1

ATHILA

At3g32970

289

1

ATHILA

At4g08080

561

1

-

At4g08050

1428

1

-

At4g03790

1064

1

ATHILA

At1g80680

1032

1

Aminoacyl-transfer RNA syn class II

At2g30110

1080

1

UBACT, ThiF_family

At5g46360

260

1

Potassium channel/KCO1-like

At5g46370

443

1

Potassium channel/KCO1-like

At5g55630

363

1

Potassium channel

ch (.772)

KCO1

[31]

At2g35800

844

1

Mitoch_carrier

At5g07320

479

4

Mitoch_carrier, Graves disease

At5g51050

487

2

Mitoch_carrier, Adptrnslcase

ch (.621)

At5g61810

476

4

Mitoch_carrier

At1g33790

745

1

Jacalin-related lectin domain

At5g28340

390

2

PPR repeats

At5g28380

283

2

PPR repeats

At5g58670

559

1

PIPLC

AtPLC1

[29,42]

At4g26700

687

1

Calponin homology

ATFIM1

[25]

At5g55400

714

1

Calponin homology

Group III

At1g03960

534

1

/protein phosphatase 2A group

At5g44090

538

2

/protein phosphatase 2A group

[58]

At1g54450

535

2

/protein phosphatase 2A group

At5g28850

536

3

/protein phosphatase 2A group

At5g28900

536

3

/protein phosphatase 2A group

At5g18580

464

3

NLS_BP

At5g49480

160

3

/NaCl-inducible Ca2+ binding protein-like

AtCP1

[35]

At1g32410

1048

1

PPR repeats

sc (.813)

At3g18430

175

1

-

At1g64480

214

3

Recoverin

CBL8

[13]

At5g24270

222

3

-

CBL4

[51]

At4g33000

246

3

Recoverin/CBL-like

sc (.931)

At4g16350

227¥

3#

-

CBL6

[13]

At5g55990

226

3

-

CBL3

[23]

At4g26560

214

3

Recoverin

CBL7

[13]

At4g26570

226

3

-

CBL2

[23]

At4g17615

252

3

Recoverin

CBL1

[23]

At5g47100

213

2

Recoverin

CBL9

[13]

At4g01420

192

3

-

CBL5

[13]

At1g54530

127

1

-

At2g44310

142

2

-

At5g28830

324

2

-

At5g22760

1516

1

PHD-finger, DDT domain

At4g38810

375

2

-

At4g13440

154

2

-

At4g32060

498

4

-

At4g39420

781

1

-

At1g29020

1062

4

-

sc (.939)

At2g34020

462

2

sc (.965)

At2g34030

423

2

mt (.648)

At1g53210

574

2

-

sc (.907)

At4g25970

628

2

PS_Dcrbxylase, Porpobil_deam

ch (.849)

At5g57190

615

4

PS_Dcrbxylase, Porpobil_deam

At2g46600

135f

1

-

KIC

Unpublished

At4g27280

130

1

-

At5g54490

127

1

-

At3g17470

570

2

-

At5g62250

549

1

-

At4g27790

345

6

ER_TARGET

sc (.941)

At5g08580

391

4

-

sc (.631)

Group IV

At1g05990

150

4

-

At4g03290

154

4

-

At2g43290

169

4

-

At3g59440

195

4

-

sc (.844)

At3g59450

148

1

-

At3g07490

153

4

-

At4g12860

152

4

-

At1g18210

170

4

-

At1g73630

163

4

-

At1g66400

157

4

Calflagin

At5g37770

161

4

-

TCH2

[20]

At2g36180

146

4

-

At5g17470

146

4

Calflagin

At3g03400

137

3

-

At3g03410

131

4

-

At2g15680

187

4

Recoverin

ch (.725)

At1g21550

155

3

-

At4g20780

191

3

-

At5g44460

181

3

-

At1g12310

148

3

-

At1g62820

148

3

-

At1g66410

149

4

EPS15 repeat

CaM4

[17]

At5g37780

149

4

-

CaM1

[16]

At2g27030

149

4

EPS15 repeat

CaM5

[17]

At2g41110

149

4

EPS15 repeat

CaM2

[16]

At3g56800

149

4

EPS15 repeat

CaM3

[15]

At3g43810

149

4

EPS15 repeat

CaM7

[18]

N/A

149

4

EPS15 repeat

CaM6

[17]

At2g41090

191

4

-

CaBP-22

[17]

At3g22930

173

4

-

mt (.702)

At4g14640

151

4

-

CaM8

[18]

At2g41100

324

6

-

TCH-3

[20]

At3g51920

151

4

-

CaM9

[18]

At3g50360

169

4

-

mt (.609)

Centrin

[21]

At4g37010