Chapter 4 Results - Shodhgangashodhganga.inflibnet.ac.in/bitstream/10603/39814/6/12...Chapter 4 Results Department of Biotechnology, Jamia Hamdard 54 4.2 Functional characterization

Chapter 4 Results

Department of Biotechnology, Jamia Hamdard 50

4. RESULTS

Plants respond to adverse environmental conditions by expression of specific genes and

synthesis of a large number of stress-related proteins that play crucial roles in stress adaptation

and/or plant defense (Reymond and Farmer 1998; Skriver and Mundy 1990). Transformation

with the osmotin gene has been reported to increase the tolerance to different biotic and abiotic

stresses, including desiccation in many plants (Goel et al. 2010; Grover et al. 2001; Husaini and

Abdin 2008). These plants were also reported to have increased levels of proline. These findings

suggest that osmotin may enhance osmotic potential of the cells by inducing synthesis and

accumulation of compatible osmolytes, namely proline.

During the course of this work, we studied the behaviour of osmotin protein. We worked on the

hypothesis that osmotin induces proline accumulation due to its involvement either as

transcription factor and/or cell signal pathway modulator in proline biosynthesis. We analyze

structure of osmotin protein for both, transcription factor and/or cell signal pathway modulator

for proline biosynthesis, using available bioinformatics tools. Rationalizing and classifying

information contained in the three dimensional structure of osmotin in terms of its functional

capabilities would ultimately help in understanding, at atomic level details, that in what ways

the biological organisms encode, make use of, and pass on information when exposed to

environmental stresses. Thus, besides helping in associating osmotin with a function, this study

would also provide ultimate insights into the mechanisms by which subsequent biological

events take place. The outcome of this study will help to elucidate and establish the role of

osmotin in proline biosynthesis and accumulation in plants and their tolerance against both

abiotic and biotic stresses.

4.1. Osmotin protein: Tobacco (Nicotiana tabacum)

Sequence and structural features of osmotin protein from tobacco (Nicotiana tabacum) were

downloaded from UniProtKB/Swiss-Prot database and RCSB, respectively (Table 1a, 1b, 1c

and Figure 5). The sequence annotation feature of osmotin showed the presence of signal

peptide and eight disulfide bond (Table 1b, 1c and Figure 6). Structure of osmotin is

predominantly a beta type with fifteen beta strands and only five helices (Table 1c). Amino acid

sequence of osmotin protein submitted to Compute pI/Mw at ExPASy Proteomics Server

showed that the osmotin protein is a cationic protein with theoretical pI of 8.13 and

corresponding theoretical molecular weight to be 26681.08 Da, which is in accordance with the

earlier published data (Singh et al. 1987; Singh et al. 1985).

Chapter 4 Results


10 20 30 40 50 60

MGNLRSSFVF FLLALVTYTY AATIEVRNNC PYTVWAASTP IGGGRRLDRG QTWVINAPRG

70 80 90 100 110 120

TKMARVWGRT NCNFNAAGRG TCQTGDCGGV LQCTGWGKPP NTLAEYALDQ FSGLDFWDIS

130 140 150 160 170 180

LVDGFNIPMT FAPTNPSGGK CHAIHCTANI NGECPRELRV PGGCNNPCTT FGGQQYCCTQ

190 200 210 220 230 240

GPCGPTFFSK FFKQRCPDAY SYPQDDPTST FTCPGGSTNY RVIFCPNGQA HPNFPLEMPG

SDEVAK

Figure 5: Amino acid sequence of osmotin from tobacco (Nicotiana tabacum).

Table 1a: Sequence annotation feature of osmotin from tobacco (UniProtKB/Swiss-ProtP14170).

Featurekey

Position(s)(aa)

Length(aa)

Description Graphical view Featureidentifier

Signalpeptide

1 – 21 21

Chain 22 – 246 225 Osmotin PRO_0000034043

Table 1b: Sequence annotation feature of osmotin from tobacco (UniProtKB/Swiss-ProtP14170).

Feature key Position(s) (aa) Graphical view

Disulfide bond 30 - 225



Disulfide bond 141- 213





Chapter 4 Results


Figure 6: Structural features of osmotin from tobacco (UniProtKB/Swiss-Prot P14170).

Table 1c: Sequence annotation feature of osmotin from tobacco (UniProtKB/Swiss-ProtP14170).

Feature key Position(s) (of aa) Length (in aa)

Beta strand 23 – 28 6



Turn 40 – 42 3







Turn 110 – 112 3





Helix 150 – 153 4

Turn 156 – 158 3

Helix 167 – 171 5

Helix 174 – 177 4

Helix 187 – 195 9

Helix 206 – 209 4



Chapter 4 Results


The sub-cellular location of osmotin protein was predicted using programs, TargetP 1.1 and

SecretomeP 1.0 Server, hosted at Technical University of Denmark, Denmark. In TargetP 1.1

program, the location assignment is based on the predicted presence of any of the N-terminal

pre-sequences: chloroplast transit peptide (cTP), mitochondrial ttransit peptide (mTP) or

secretory pathway signal peptide (SP). The score suggests the osmotin to be a secretary protein

(Table 2a and 2b) i.e it is not bound to any membrane. C-score (cleavage site score), Y-max(a

derivative of the C-score) combined with the S-scores of Signal Peptide-4.0 showed the presence

of 21 amino acid signal peptide at N-terminal sequence (Figure 7).

Table 2a: Targetp 1.1 prediction results for the osmotin protein.

Name Len cTP mTP SP other Loc Reliabilityclass

Predictedpresequencelength

Osmotin 246 0.014 0.052 0.956 0.011 S 1 21

Table 2b: SecretomeP 1 Server prediction results for the osmotin protein.

Name NN-score Odds Weighted by prior Warning

Osmotin 0.853 4.734 0.009 signal peptide predicted

Figure 7: Signal peptide prediction in osmotin using Signal Peptide-4.0.

Chapter 4 Results


4.2 Functional characterization of Osmotin as transcription factor: Prediction of DNA-

binding motifs

Several methods and web servers have been developed to predict DNA-binding residues from

the protein one dimensional (1D) sequence or three dimensional (3D) structures. The programs

that use the protein structure, if available, generally improve the DNA-binding site prediction, as

they replace the predicted solvent accessibility, hydrophobicity and secondary structure in

sequence-based methods with observed ones and can additionally employ energies or

frequencies, computed from the atomic coordinates, as well as experimental geometrical

features. Predictions of DNA-binding residues by structure-based methods employ mostly

electrostatic potentials in conjunction with other features such as surface/solvent accessibility,

the protein surface shape, amino acid conservation, propensity, hydrophobicity and hydrogen-

bonding potential, and structural motifs (Ferrer-Costa et al. 2005; Jones et al. 2003; Shanahan et

al. 2004; Stawiski et al. 2003; Tsuchiya et al. 2004; Wu et al. 2009). There are common types of

motifs that are responsible for binding to DNA found in different transcription factors. The most

common and best studied DNA-binding proteins are the zinc finger proteins, the helix-turn-helix

proteins, and the leucine zipper proteins. 2-zip server, DBD transcription factor prediction

database, GYM 2.0 and predictdnahth were used in the current study because of their user

friendly interface, speed and reliability. According to 2-zip server, DBD transcription factor

prediction database, GYM 2.0 and predictdnahth, osmotin lacked zinc fingers, leucine zippers

and HTH motif (Table 3). The results of predictdnahth showed the presence of HTH motif on

GT1 protein (the proteins know to have DNA binding domain) thus, implicating that it binds to

DNA molecule and may have a role in gene regulation.

Table 3: Results of DNA-binding motif prediction softwares.

Software Predicts Submission Results (Osmotin) Results (GT 1)

2-zip server Leucine zipper

domain

Amino acid

sequence

negative negative

DBD TF prediction

database

Transcription

factor

Amino acid

sequence

negative negative

GYM 2.0 HTH motif Amino acid

sequence

HTH motif was found but

with insignificant score

HTH motif was

found

predictdnahth HTH PDB file No DNA binding HTH

motif was found

Helix of HTH motif

was found

Chapter 4 Results


Osmotin protein was also compared with the transcription factors listed in Database of

Arabidopsis transcription factors (DATF) from Arabidopsis. 64 families of transcription factors

are listed in DATF. Protein sequences of randomly selected transcription factors from each of

the 64 families, listed in DATF (total 182 transcription factor sequences) were retrieved. For

realization of the information in terms of domains and motifs, the knowledge of the three-

dimensional structures of gene products is required. Therefore, protein sequences were submitted

to SWISS-MODEL for homology modelling of protein’s structure. The SWISS MODEL

workspace provided results for only 102 transcription factors. For the remaining 80 proteins it

failed to provide result due to unavailability of a corresponding template structure. BlastP with

default setting was performed to find templates for the remaining proteins, but template for only

10 more proteins could be ascertained and for others scores were very low (Appendix 1a). In

order to find unique domain, superimposition of osmotin and GT1 individually, was done with

modelled Arabidopsis transcription factors using Superpose. SuperPose provides a simple-to-use,

web accessible approach to performing a wide range of sophisticated structural superpositions. It

is unique in that it combines sequence alignment and difference distance matrix. GT1 was found

to be super-imposable on transcription factors belonging to Tri helix family, whereas no

significant superimposition was found in osmotin. Thus, the results indicated that probably there

are no DNA binding motifs present in osmotin, similar to those represented by our test set

(Appendix 1b).

4.3 Osmotin/ thaumatin like proteins in plants

Protein structures can be classified in a hierarchical fashion, in terms of folds, superfamilies and

families. Superfamilies comprise groups of proteins that share the same fold, and are probably

related by a common evolutionary origin, but have low levels of sequence identity. Existing

annotation of well-characterized tobacco osmotin protein was used for BlastP searches to

identify osmotin/ thaumatin like protein homologues among the completed genome of

Arabidopsis lyrata, Arabidopsis thaliana, Carica papaya, Medicago truncatula, Glycine max,

Cucumis sativus, Populus trichocarpa, Vitis vinifera, Oryza sativa, Zea mays and Sorghum

bicolor. The results showed that osmotin homologues were present in the genomes of all the 11

plants used for the study, either alone or in combination of 5 different partner domains; protein

kinase-like (PK-like), bifunctional inhibitor/lipid-transfer protein/seed storage 2S albumin,

mitochondrial carrier, Class II aaRS ABD-related and ARM repeats (Table 4).

Chapter 4 Results


Table 4: The domain architecture of Osmotin/thaumatin-like protein superfamily.

Genome

(complete)

No.of Proteins in the

superfamily

No. of Partner

Domains

Domain

Combination

Arabidopsis lyrata 30 2 1,2,3Arabidopsis thaliana 30 2 1,2,3Carica papaya 18 0 1Medicago truncatula 18 0 1Glycine max 57 1 1,4Cucumis sativus 29 0 1Populus trichocarpa 58 0 1Vitis vinifera 29 0 1Oryza sativasubsp. Japonica

44 1 1,2

Zea_mays subsp. mays 61 3 1,5,6Sorghum bicolor 45 1 1,2

1. Osmotin2. Protein kinase-like (PK-like)3. Bifunctional inhibitor/lipid-transfer protein/seed storage 2S albumin4. Mitochondrial carrier5. Class II aaRS ABD-related6. ARM repeat

Superfamily analyses showed that Osmotin/thaumatin-like protein family in A. thaliana contains

31 proteins (Table 5) occurring in 7 domain combinations with 2 different partner domains;

protein kinase-like (PK-like) and bifunctional inhibitor/lipid-transfer protein/seed storage 2S

albumin (Figure 8 and Table 5).

Table 5: Osmotin/thaumatin-like protein superfamily present in A. thaliana.

Gene SwissProt ID MW [Da] Structural Class

AT5G38280.1 Q9FF29 73951.0 segregated alpha/betaAT2G17860.1 Q4PSV2 26364.5 all betaAT4G38670.1 Q8GYY3 30095.2 all betaAT4G36010.1 O65638 30905.6 all betaAT1G75800.1 Q9LQT4 34883.8 all betaAT2G28790.1 Q8LDH6 27017.5 all betaAT1G18250.1 P50699 25906.5 all betaAT4G38670.2 A8MS42 29966.1 all betaAT4G38670.3 Q8GYY3 30095.2 all beta

Chapter 4 Results


AT1G18250.2 P50699 25977.5 all beta

AT4G36010.2 O65638 30905.6 all beta

AT1G75040.1 P28493 25252.1 all beta

AT1G20030.2 Q9LNT0 33494.2 all beta

AT4G36000.1 O65637 21979.2 all beta

AT4G24180.1 O22973 27450.3 all beta

AT4G38660.1 Q8VYN5 35436.8 all beta

AT4G18250.1 - 95264.9 all betaAT4G11650.1 P50700 26632.5 all betaAT4G38660.2 Q9SZP4 33126.2 all betaAT5G40020.1 Q9FLD4 28093.9 all betaAT5G24620.1 B3LFC8 44747.9 all beta

AT1G75050.1 Q1PFD2 25439.2 all beta

AT1G77700.1 Q9CA24 39535.5 all betaAT1G19320.1 Q9LN66 25430.2 all beta

AT5G02140.1 Q9LZL8 31173.5 all betaAT1G20030.1 Q9LNT0 31557.8 all betaAT5G24620.2 B3LFC8 32786.5 -

AT1G73620.1 Q9C9U9 28213.2 all beta

AT1G75030.1 Q9C9P9 25442.3 all betaAT2G24810.1 Q9SK51 21607.7 all beta

AT1G70250.1 - 87507.7 all alpha

Figure 8: The domain architecture of Osmotin/thaumatin-like protein superfamily present

in A. thaliana.

Of 31 proteins, only three proteins (AT4G18250.1, AT5G38280.1, AT1G70250.1) has PK-like

and only one (AT1G70250.1) has bifunctional inhibitor/lipid-transfer protein/seed storage 2S

albumin domain (Figure 9).

Table cont.

Chapter 4 Results


Protein kinase ATP-binding region signatureIGKGGFGTVYKGKLPDASGRDIALK

AT4G18250.1 RYSFEKVKKMTNSFDHVIGKGGFGTVYKGKLPDASGRDIALKILKESKGNGEEFINELVS 567AT5G38280.1 RYSYTRVKKMTNSFAHVLGKGGFGTVYKGKLADS-GRDVAVKILKVSEGNGEEFINEVAS 378AT1G70250.1 RFSYVQVKKMTKSFENVLGKGGFGTVYKGKLPDG-SRDVAVKILKESNEDGEDFINEIAS 506

Serine/Threonine protein kinases active-site signature:IVHFDIKPQNILI

AT4G18250.1 GLEYLHNSCVSKIVHFDIKPQNILIDEDLCPKISDFGLAKLCKKKESIISMLDARGTVGY 687AT5G38280.1 GLEYLHNRCVTRIVHFDIKPQNILMDENLCPKISDFGLAKLCKNKESIISMLHMRGTFGY 498AT1G70250.1 GLEYLHSHCVSRIVHFDIKPQNILIDGDLCPKISDFGLAKLCKNNESIISMLHARGTIGY 626

Figure 9: The Protein kinases domains present in Osmotin/thaumatin-like protein

superfamily.

Although, the osmotin protein was isolated from the cells of tobacco var. Wisconsin 38 that were

cultured for several generations in the culture medium with high NaCl concentration, till

recently, the genome of tobacco has not been published with complete details. A BAC library

with 9.7-fold genome coverage of N. tabacum genome has been constructed, BAC end

sequencing and construction of a physical map, however, is in progress (Opperman,

http://www.pngg.org/tgi/index.html), (SGN,http://solgenomics.net/). However, to study the

behaviour of osmotin through in silico tools, knowledge about the sequence and structural details

of probable receptor(s) and subsequent signaling molecule, through which it exerts its effect, is

required. Arabidopsis thaliana, a small flowering plant, has been used for over fifty years to

study plant mutations and for classical genetic analysis. Its genome became the first plant

genome to be fully sequenced (The Arabidopsis Genome Initiative 2000). Since the genome of

A. thaliana is completely sequenced, therefore, it was considered for further studies.

The sequence alignment of N. tabacum osmotin with members of Arabidopsis

Osmotin/thaumatin-like protein superfamily showed AT4G 11650 (ATOSM 34) to be most

similar (71.71%) to the N. tabacum osmotin, while AT4G 36000.1 is least (29.27%) (Table 6

and Figure 10). Target P server results for 31 members of A. thaliana Osmotin/ thaumatin like

protein superfamily suggests that all, except proteins encoded by these ten genes; AT4G18250.1,

AT1G20030.1, AT1G73620.1, AT1G77700.1, AT4G36000.1, AT4G38660.1, AT4G38660.2,

AT4G38670.1, AT4G38670.2, AT4G38670.3, are secretory in nature (Table 7). The secretory

nature and multiple locations targeting of osmotin are in agreement to their multi functional role

in plants exposed to biotic and abiotic stresses.

Chapter 4 Results


Table 6: Sequence alignment scores using Clustal W of members of A. thaliana Osmotin/

thaumatin like protein superfamily.

Osmotin Length(aa)

OLPs(Arabidopsis)

Length(aa)

Score

1pcv 205 AT1G18250.1 243 44.88

1pcv 205 AT1G18250.2 244 44.88

1pcv 205 AT1G19320.1 247 42.44

1pcv 205 AT1G20030.1 299 46.34

1pcv 205 AT1G20030.2 316 46.34

1pcv 205 AT1G70250.1 799 36.59

1pcv 205 AT1G73620.1 264 44.88

1pcv 205 AT1G75030.1 246 47.32

1pcv 205 AT1G75040.1 239 46.34

1pcv 205 AT1G75050.1 246 48.29

1pcv 205 AT1G75800.1 330 47.8

1pcv 205 AT1G77700.1 356 46.83

1pcv 205 AT2G17860.1 253 46.34

1pcv 205 AT2G24810.1 193 24.87

1pcv 205 AT2G28790.1 249 36.1

1pcv 205 AT4G11650.1 244 71.71

1pcv 205 AT4G18250.1 853 34.151pcv 205 AT4G24180.1 260 44.39

1pcv 205 AT4G36000.1 208 29.27

1pcv 205 AT4G36010.1 301 48.29

1pcv 205 AT4G36010.2 301 48.29

1pcv 205 AT4G38660.1 345 43.41

1pcv 205 AT4G38660.2 323 43.41

1pcv 205 AT4G38670.1 281 43.9

1pcv 205 AT4G38670.2 280 43.9

1pcv 205 AT4G38670.3 281 43.9

1pcv 205 AT5G02140.1 294 41.46

1pcv 205 AT5G38280.1 665 41.46

1pcv 205 AT5G24620.1 420 48.29

1pcv 205 AT5G24620.2 303 25.85

1pcv 205 AT5G40020.1 256 36.59

Chapter 4 Results


Figure 10: Phylogenetic tree of Osmotin/thaumatin-like protein superfamily with 1PCV.

Table 7: Location prediction results by TargetP v1.1 using PLANT networks.

Name Length Chloroplasttransitpeptide

MitochondrialSignalpeptide

Signalpeptide

other Location Reliabilityclass

PredictedPreSequencelength

AT1G18250.1 243 0.108 0.008 0.919 0.027 S 1 20

AT1G18250.2 244 0.075 0.009 0.947 0.026 S 1 20

AT1G19320.1 247 0.108 0.006 0.977 0.018 - 5 25

AT1G20030.1 299 0.336 0.085 0.101 0.380 S 3 -

AT1G20030.2 316 0.203 0.041 0.698 0.010 S 2 19

AT1G70250.1 799 0.059 0.013 0.869 0.194 - 4 19

AT1G73620.1 264 0.128 0.079 0.143 0.532 S 1 -

AT1G75030.1 246 0.055 0.006 0.987 0.025 S 1 22

AT1G75040.1 239 0.087 0.005 0.982 0.040 S 1 23

AT1G75050.1 246 0.038 0.010 0.989 0.020 S 1 23

AT1G75800.1 330 0.103 0.012 0.916 0.015 S 1 22

AT1G77700.1 356 0.243 0.071 0.057 0.819 - 3 -

AT2G17860.1 253 0.059 0.005 0.939 0.023 S 1 22

AT2G24810.1 193 0.026 0.013 0.671 0.016 S 2 26

AT2G28790.1 249 0.060 0.009 0.990 0.009 S 1 21

AT4G11650.1 244 0.022 0.007 0.988 0.021 S 1 22

AT4G18250.1 853 0.460 0.024 0.111 0.386 C 5 48

Chapter 4 Results


AT4G24180.1 260 0.003 0.037 0.970 0.035 S 1 30

AT4G36000.1 208 0.082 0.108 0.148 0.847 _ 2 -

AT4G36010.1 301 0.021 0.011 0.960 0.016 S 1 22

AT4G36010.2 301 0.021 0.011 0.960 0.016 S 1 22

AT4G38660.1 345 0.011 0.392 0.253 0.037 M 5 9

AT4G38660.2 323 0.155 0.420 0.013 0.194 M 4 55

AT4G38670.1 281 0.482 0.003 0.305 0.038 C 5 78

AT4G38670.2 280 0.405 0.006 0.339 0.015 C 5 77

AT4G38670.3 281 0.482 0.003 0.305 0.038 C 5 78

AT5G02140.1 294 0.016 0.014 0.968 0.077 S 1 20

AT5G38280.1 665 0.030 0.013 0.869 0.052 S 1 24

AT5G24620.1 420 0.020 0.021 0.945 0.016 S 1 26

AT5G24620.2 303 0.003 0.761 0.189 0.340 M 3 117

AT5G40020.1 256 0.025 0.022 0.917 0.092 S 1 21

4.4 Sequence analysis, homology modelling, model optimization, quality assessment and

visualization of Arabidopsis osmotin (ATOSM34)

The amino acid sequences of Arabidopsis osmotin (ATOSM34) and Nicotiana osmotin were

aligned with the program ClustalW with protein weight matrix as Gonnet, gap opening penalty

as 10 and gap extension penality as 0.1. Over all similarity of ATOSM34 and osmotin is 63%.

Signal peptide of ATOSM34 and osmotin has 28 % similarity, and the protein without signal

peptide has 71.71% similarity. The ATOSM34 and osmotin share 10 out of 14 residues in the

acidic cleft (Figure 11). 1PCV, X-ray diffraction model of osmotin from Nicotiana at a

resolution of 2.3 Å, hence, was used as template for homology modelling. The query sequence

and template structure was submitted to Discovery studio 2.0 for modelling.

Figure 11: Alignment of 1PCV and ATOSM34 by alignment using Discovery studio 2.0.

Table cont.

Chapter 4 Results


Ramachandran plot statistics and G- factor statistics for ATOSM34 showed 83% amino acid in

most favorable region (Figure 12). The main chain conformation within the favored or allowed

region of Ramachandran plot and G-factor indicated the accuracy of generated models (Table 8

and 9).

Figure 12: Ramachandran plot for ATOSM34.

Table 8: Ramachandran Plot statistics of ATOSM34.

No. of residues %

Most favoured regions[A,B,L] 155 83.3%

Additional allowedregions[a,b,l,p]

26 14.0%

Generously allowedregions[~a,~b,~l,~p]

1 0.5%

Disallowed regions[XX] 4 2.2%

Non-glycine and non-prolineresidues

186 100.0%

End-residues (excl. Gly andPro)

2 -

Glycine residues 21 -

Proline residues 13 -

Total number of residues 222 -

Chapter 4 Results


Table 9: G factor statistics of ATOSM34.

Parameter Score Average Score

Dihedral angles:Phi-psi distributionChi1-chi2 distributionChi1 onlyChi3 and chi4Omega

-0.45-0.130.180.63-0.07 -0.11

Main-chain covalent forces:-Main-chain bond lengthsMain-chain bond angles

-0.18-0.46 -0.34

OVERALL AVERAGE -0.19

The positioning of secondary structural elements was generated from PDBsum. The predicted

model of ATOSM34 showed 3 sheets, 5 beta hairpins, 3 beta bulges, 14 strands, 5 helices, 5

helix-helix interactions, 27 beta turns, 2 gamma turns and 8 disulphide linkages (Figure 13). The

superimposition of main-chain atoms of ATOSM34, by Discovery studio, showed 0.462 Å

RMSD and overlap among 199 amino acids residues with respect to 1PCV (Figure 14).

Figure 13: Secondary structural elements of ATOSM34 generated from PDBsum.

(Secondary structure: helices, sheets, turn, turn, hairpin,

disulphide bond).

Chapter 4 Results


a

b

Figure 14: a: Model of ATOSM34 (pink) and 1PCV (blue), b: Superimposition of

modelled ATOSM34 (pink) and 1PCV (blue) using Discovery Studio 2.0.

4.5 Osmotin as cell signaling molecule

4.5.1 Osmotin as homologue of adiponectin, a cell signaling molecule in oxidative stress

tolerance

The literature mining showed that osmotin protein has similar structural folds as that of

adiponectin (Yamauchi et al. 2003). Adiponectin, a 28–30 kDa protein, is an important

adipocyte-derived hormone that regulates metabolism of lipids and glucose. Adiponectin and

osmotin are shown to transduce their effects by activating numerous signaling molecules,

including adenosine monophosphate-activated protein kinase (AMPK), p38-MAPK, JNK,

PPARa transcription factor and NF-kB via the human receptors of adiponectin, ADIPORs

(Tang et al. 2005; Yamauchi et al. 2003). Therefore, a homologue of ADIPORs in plant might

be a transducer of signals mediated by osmotin in the cell. The BlastP carried out in this study

showed that heptahelical proteins (HHPs) viz. HHP1 (At2g24150), HHP2 (At4g30850), HHP3

Chapter 4 Results


(At4g37680), HHP4 (At4g38320), and HHP5 (At5g20270) from Arabidopsis were homologue

of human adiponectin receptors, ADIPOR1 and ADIPOR2 (Table 10).

Table 10: Comparison of receptors for osmotin in humans and plants.

GeneName

ADIPOR1 ADIPOR2 HHP1 HHP2 HHP3 HHP4 HHP5

Proteinname

Adiponectinreceptor

Adiponectinreceptor

HeptahelicalTM protein1

HeptahelicalTM protein2

Heptahelicalprotein3



Organism Homosapiens

Homosapiens

Arabidopsis Arabidopsis Arabidopsis Arabidopsis Arabidopsis

Locus tag Adipor2 Adipor1 At5g20270 At4g30850 At2g24150 At4g37680 At4g38320

Location Integral tomembrane

Integral tomembrane

Integral tomembrane

Integral tomembrane

Integral tomembrane

Integral tomembrane

Integral tomembrane

Biologicalfunction

Hormonebinding,

Hormonebinding,

Receptoractivity

Receptoractivity

Receptoractivity

Receptoractivity

Receptoractivity

Cellularcomponent

Integral toPlasmamembrane

Integral toPlasmamembrane

Integral tomembrane

Integral tomembrane

Integral tomembrane

Integral tomembrane

Integral tomembrane

No. ofamino acid

375aa 386aa 332aa 358aa 344aa 385aa 374aa

Topology prediction program (TMHMM) showed that HHP family membrane proteins

contained seven transmembrane (7TM) domains, which is in concordance with the published

results (Hsieh and Goodman 2005). The topological predictions of Arabidopsis HHPs are

shown in Figure 15 and Table 11. For Arabidopsis HHP1, HHP2 and HHP3, the TMHMM

program predicted an intracellular N-terminal domain, 7TM domains and a short extracellular

C-terminal segment; and for HHP4 and HHP5 it predicted a similar topology, but with 6TM

domains.

Table 11: Comparison of TMHMM results for HHP proteins from Arabodopsis.

Gene name HHP1 HHP2 HHP3 HHP4 HHP5

Organism Arabidopsis Arabidopsis Arabidopsis Arabidopsis Arabidopsis

Locus tag At5g20270 At4g30850 At2g24150 At4g37680 At4g38320

Length 332aa 358aa 344aa 385aa 374aa

Number 7TMH 7TMH 7TMH 6TMH 6TMH

Chapter 4 Results


of TMHs

Location Amino acid positions

Start End Start End Start End Start End Start End

inside 1 96 1 70 1 73 1 78 1 78

TMhelix 97 119 71 90 74 93 79 101 79 101

outside 120 138 91 161 94 147 102 191 102 191

TMhelix 139 161 162 184 148 170 192 214 192 214

inside 162 167 185 190 171 176 215 225 215 220

TMhelix 168 190 191 213 177 199 226 248 221 243

outside 191 199 214 222 200 208 249 251 244 257

TMhelix 200 222 223 245 209 231 252 274 258 280

inside 223 234 246 251 232 242 275 286 281 286

TMhelix 235 254 252 274 243 265 287 309 287 309

outside 255 263 275 283 266 269 310 313 310 313

TMhelix 264 283 284 306 270 292 314 336 314 336

inside 284 303 307 326 293 312 337 385 337 374

TMhelix 304 326 327 349 313 335 - -

outside 327 332 350 358 336 344

a.

Table cont.

Chapter 4 Results


b.

c.

d.

Chapter 4 Results


e.

Figure 15: Topological predictions of Arabidopsis HHPs: a; HHP1 (At2g24150),b; HHP2

(At4g30850),c; HHP3 (At4g37680),d; HHP4 (At4g38320), and e; HHP5 (At5g20270)

Amino acid sequence alignment of the Arabidopsis HHP1 and human ADIPOR1 revealed that

they share 33% amino acid sequence similarities. The TMHMM and Jpred results, however,

showed high conservation in their predicted 7TM topology and secondary structures (Figure 16

and Table 12).

ADIPOR1 TGNIWTHLLGFVLFLFLGILTMLRPNMYFMAPLQ-----EKVVFGMFFLGAVLCLSFSWL 55HHP1 -LNVWTHLIGFIFFVALTVANIIHHDGFFPVDAKSPGNVTRWPFFVFLGGSMFCLLASSI 59

*:****:**::*: * : .::: : :* . : : * :*: *:::** * :

ADIPOR1 FHTVYCHSEKVSRTFSKLDYSGIALLIMGSFVPWLYYSFYCSPQPRLIYLSIVCVLGISA 115HHP1 CHLFCCHSKELNVFLLRIDYAGITAMIITSFFPPIFYIFQCTPRWYFIYLAGITSMGIFT 119

* . ***:::. : ::**:**: :*: **.* ::* * *:*: :***: : :** :

ADIPOR1 IIVAQWDRFATPKHRQTRAGVFLGLGLSGVVPTMHFTIAEGFVKATTVGQMGWFFLMAVM 175HHP1 IITLFTPSLSAPKYRAFRALLFASMGLFGIVPAAHALVVN-WGNPQRNVTLVYELLMAVF 178

**. :::**:* ** :* .:** *:**: * :.: : :. : : :****:

ADIPOR1 YITGAGLYAARIPERFFPGKFDIWFQSHQIFHVLVVAAAFVHFYGVSNLQEFRYGLEGGC 235HHP1 YLVGTGFYVGRVPERLKPGWFDRVGHSHQIFHVFVLLGALSHYAAALLFLDWRD--HVGC 236

*:.*:*:*..*:***: ** ** :*******:*: .*: *: .. : ::* . **

ADIPOR1 TDDTLL 241HHP1 ------

Figure 16: Amino acid sequence alignment of ADIPOR1and HHP1 using Clustal W.

Chapter 4 Results


Table 12: Comparison of TMHMM results for HHP1 and ADIPOR1.

HHP1 ADIPOR1

OrganismLocus tagLengthNo. of TMHs

ArabidopsisAt5g20270332aa7TMH

HumanADIPOR1375aa7TMH

Location Amino acidpositions

No. of aminoacids presentin helix

Aminoacidpositions

No. of amino acidspresent in helix

Start End Start Endinside 1 96 1 134TMhelix 97 119 23 135 157 23outside 120 138 19 158 171 14TMhelix 139 161 23 172 194 23inside 162 167 195 206TMhelix 168 190 23 207 226 20outside 191 199 227 235TMhelix 200 222 23 236 255 20inside 223 234 256 267TMhelix 235 254 20 268 287 20outside 255 263 288 296TMhelix 264 283 20 297 319 23inside 284 303 320 331TMhelix 304 326 23 332 354 23outside 327 332 355 375

Chen et al (2009) have shown that HHP1 is induced significantly by salt. To avoid the

complexity because of the diversity within the HHP family and to narrow down our target, the

HHP1 was chosen for further studies. TMHMM, TMpred, PRED-TMR, HmmTop and Phobius

were used to predict the transmembrane domains in HHP1 (Table 13).

Table 13: Prediction of trans-membraneous helices in HHP1 from Arabodopsis.

TMHelix TMHMM TMpred PRED-TMR

HmmTop Phobius Consensus

Amino acid positionsStart End Start End Start End Start End Start End Start End

TMhelix1 97 119 99 118 99 118 97 118 99 119 99 118TMhelix2 139 161 137 159 137 159 139 160 139 161 139 160

TMhelix3 168 190 173 194 175 194 175 194 173 194 173 194TMhelix4 200 222 201 227 200 220 203 222 200 220 200 222

Chapter 4 Results


TMhelix5 235 254 231 254 234 254 235 254 233 253 233 254

TMhelix6 264 283 263 284 263 283 263 284 265 283 263 283

TMhelix7 304 326 301 322 304 324 305 324 304 324 304 324

4.5.2. Structure prediction of receptor molecules (ADIPOR1 and HHP1)

4.5.2.1 Secondary structure prediction

In the absence of clear structural homologue, profile representations such as presence of an

amino acid exposed on the surface or buried in the core of the protein, local secondary structure

(amino acid as a part of an α helix) and/or magnitude of conservation among the amino acid

(evolutionary information) is generally taken into account for determination of the template for

modelling of the proteins. Here, we first determined the secondary structure of the

transmembrane protein (ADIPOR1 and HHP1) and then compared their secondary structure

with the other members of 7 helix trans-membrane proteins to find the closest homologue for

3D structure modelling.

Secondary structure of ADIPOR1 was predicted using Jpred (Jnet,Jpssm,Jhmm) nnPredict,

GOR4, HNN, SOPMA, PSIPRED, Prepro, Sspro webservers. All these prediction servers use

different algorithms and were used so as to minimize the error in the secondary structure

prediction. The consensus secondary structure prediction showed that ADIPOR1 had 57.3 %

helix, 34.4 % coil and 8.2% extended sheets (Figure 17).ADIPOR1 MSSHKGSVVAQGNGAPASNREADTVELAELGPLLEEKGKRVIANPPKAEEEQTCPVPQEEEEEVRVLTLPJnet -----------------------------------HH--------HHHHHHHH-------------HHHHGor4 MSSHKGSVVAQGNGAPASNREADTVELAELGPLLEEKGKRVIANPPKAEEEQTCPVPQEEEEEVRVLTLP

CccccceeeeeccccccccchhhhhhhhhhhhhhhhhcceeecccccccccccccccccchhhhhhhhhhHNN MSSHKGSVVAQGNGAPASNREADTVELAELGPLLEEKGKRVIANPPKAEEEQTCPVPQEEEEEVRVLTLP

cccccceeeeecccccccccchchhhhhhhccchhhccceeeeccccccccccccccccchhhhehhcccSOPMA MSSHKGSVVAQGNGAPASNREADTVELAELGPLLEEKGKRVIANPPKAEEEQTCPVPQEEEEEVRVLTLP

ettttceeeetttccccccchhhhhhhhhhhhhhhhttceeeeccccccccccccccccchhhheeeeccPSIPRED MSSHKGSVVAQGNGAPASNREADTVELAELGPLLEEKGKRVIANPPKAEEEQTCPVPQEEEEEVRVLTLP

CCCCCCCCCCCCCCCCCCCCCCCHHHHHHCCCCCCCCCCCCCCCCCCHHHCCCCCCCCCHHHHHHHCCCCPrepro MSSHKGSVVAQGNGAPASNREADTVELAELGPLLEEKGKRVIANPPKAEEEQTCPVPQEEEEEVRVLTLP

EE HHHHHHHHHH HHHHH HHHSspro MSSHKGSVVAQGNGAPASNREADTVELAELGPLLEEKGKRVIANPPKAEEEQTCPVPQEEEEEVRVLTLP

CCCCCCCCCCCCCCCCCCCCCCCCHHHHHCCCHHHHCCCEEECCCCCCHHHHCCCCCCHHHHHCCECCCHCSS CCCCCCCEECCCCCCCCCCCCCCCHHHHHHHHHHHHCCCEEECCCCCHHHHHHCCCCCCHHHHHHHHHHH

140ADIPOR1 LQAHHAMEKMEEFVYKVWEGRWRVIPYDVLPDWLKDNDYLLHGHRPPMPSFRACFKSIFRIHTETGNIWTJnet HHHHHHHHHHHHHHHHH------EEE-----------------------HHHHHHHHHHHH---HHHHHHGor4 LQAHHAMEKMEEFVYKVWEGRWRVIPYDVLPDWLKDNDYLLHGHRPPMPSFRACFKSIFRIHTETGNIWT

hhhhhhhhhhhhhhhhhhcccceeeccccccccccccceeecccccccccccccccceeeeeeeccccchHNN LQAHHAMEKMEEFVYKVWEGRWRVIPYDVLPDWLKDNDYLLHGHRPPMPSFRACFKSIFRIHTETGNIWT

cchhhhhhhhhhhhhhhhccceeeeccccchhhccccceeecccccchhhhhhhhhhhhheccccchhhhSOPMA LQAHHAMEKMEEFVYKVWEGRWRVIPYDVLPDWLKDNDYLLHGHRPPMPSFRACFKSIFRIHTETGNIWT

cchhhhhhhhhhhhhhhhhttceeechtccchhccttceeeetccccccchhhhhhhheeecttthhhhhPSIPRED LQAHHAMEKMEEFVYKVWEGRWRVIPYDVLPDWLKDNDYLLHGHRPPMPSFRACFKSIFRIHTETGNIWT

HHHHHHHHHHHHHHHHHHCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCHHHHHHHHHHHCCCCHHHHHPrepro LQAHHAMEKMEEFVYKVWEGRWRVIPYDVLPDWLKDNDYLLHGHRPPMPSFRACFKSIFRIHTETGNIWT

HHHHHHHHHHHHHHHHHHHH EEEE EEE HHHHHHHHHHHHHHHHHHHHHSspro LQAHHAMEKMEEFVYKVWEGRWRVIPYDVLPDWLKDNDYLLHGHRPPMPSFRACFKSIFRIHTETGNIWT

HHHHHHHHHHHHHHHHHHHHHHHHCCHCCCCCHCCCCCEEECCCCCCCHHHHHHHHHHHHHHHHHHHHHHCSS HHHHHHHHHHHHHHHHHHCCCCCEEECCCCCCCCCCCCEEECCCCCCCHHHHHHHHHHHHHHHHHHHHHH

Table cont.

Chapter 4 Results


210ADIPOR1 HLLGFVLFLFLGILTMLRPNMYFMAPLQEKVVFGMFFLGAVLCLSFSWLFHTVYCHSEKVSRTFSKLDYSJnet HHHHHHHHHHHHHHHH-------------HHHHHHHHHHHHHHHHHHHHHHHHH---HHHHHHHHHHHHHGor4 HLLGFVLFLFLGILTMLRPNMYFMAPLQEKVVFGMFFLGAVLCLSFSWLFHTVYCHSEKVSRTFSKLDYS

hhhhhhhhhhhhhhhhcccchhhhcccchhhhhhhhhcceeeeeccceeeceeeccccccccccccccccHNN HLLGFVLFLFLGILTMLRPNMYFMAPLQEKVVFGMFFLGAVLCLSFSWLFHTVYCHSEKVSRTFSKLDYS

hhhhhhhhhhhhhhhhcccchhhhhhhhhhhhhhhhhhhhhhhhhhhhheeeeeccccchhhhhhhccccSOPMA HLLGFVLFLFLGILTMLRPNMYFMAPLQEKVVFGMFFLGAVLCLSFSWLFHTVYCHSEKVSRTFSKLDYS

hhhhhhhhhhhhhhhhccccccccccchhhheeehhhhhhhhhhhhhhhhhhhcccchhhhhhhhhhhhtPSIPRED HLLGFVLFLFLGILTMLRPNMYFMAPLQEKVVFGMFFLGAVLCLSFSWLFHTVYCHSEKVSRTFSKLDYS

HHHHHHHHHHHHHHHCCCCCCCCCCCCHHHHHHHHHHHHHHHHHHHHHHHHHCCCCCHHHHHHHHHCCCCPrepro HLLGFVLFLFLGILTMLRPNMYFMAPLQEKVVFGMFFLGAVLCLSFSWLFHTVYCHSEKVSRTFSKLDYS

HHHHHHHHHHHHHHHHHH HHHEEEHHHHHHH HHHHHHHHHHHH HHHHHHHHHHSspro HLLGFVLFLFLGILTMLRPNMYFMAPLQEKVVFGMFFLGAVLCLSFSWLFHTVYCHSEKVSRTFSKLDYS

HHHHHHHHHHHHHHHCCCCCCCCCCCHHCHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHCSS HHHHHHHHHHHHHHHHCCCCCCCCCCCCHHHHHHHHHHHHHHHHHHHHHHHHHCCCCHHHHHHHHHHHHH

280ADIPOR1 GIALLIMGSFVPWLYYSFYCSPQPRLIYLSIVCVLGISAIIVAQWDRFATPKHRQTRAGVFLGLGLSGVVJnet HHHHHHHHHHHHHHHHH----HHHHHHHHHHHHHHHHHHHHHHH-----------HHHHHHHHHHHHHHHGor4 GIALLIMGSFVPWLYYSFYCSPQPRLIYLSIVCVLGISAIIVAQWDRFATPKHRQTRAGVFLGLGLSGVV

ceeeeeeccccceeeeccccccccceeeeeeeccccceeeehhhhhccccccchhhhheeeeecccccccHNN GIALLIMGSFVPWLYYSFYCSPQPRLIYLSIVCVLGISAIIVAQWDRFATPKHRQTRAGVFLGLGLSGVV

cheeeehccchhheeheecccccccheeeehhhhhchhhhhhhhhhhccccccccccceeeeeeccccccSOPMA GIALLIMGSFVPWLYYSFYCSPQPRLIYLSIVCVLGISAIIVAQWDRFATPKHRQTRAGVFLGLGLSGVV

teeeeeehccccteeeeeecccchhheehhhhhhhhhhheeeehccccccccccchhheeeeeeccccccPSIPRED GIALLIMGSFVPWLYYSFYCSPQPRLIYLSIVCVLGISAIIVAQWDRFATPKHRQTRAGVFLGLGLSGVV

HHHHHHHHCCCCEEEEEECCCCHHHHHHHHHHHHHHHHHHHHHHCCCCCCCCCCCCCHHHHHHHHHHCCHPrepro GIALLIMGSFVPWLYYSFYCSPQPRLIYLSIVCVLGISAIIVAQWDRFATPKHRQTRAGVFLGLGLSGVV

EEEEEE HHHEEEEEEE HHHHHHHHHHHHHHHHHHHHHHHH EEHHHHHHHHHHHHHSspro GIALLIMGSFVPWLYYSFYCSPQPRLIYLSIVCVLGISAIIVAQWDRFATPKHRQTRAGVFLGLGLSGVV

HHHHHHHHCCCHHEEEEEECCCCHHHHEEEHHHHEHHHHHHHHHHHHHCCCCCCCHEEEEEECHHHHHHHCSS HHHHHHHHCCCEEEEEEEECCCCHHHHHHHHHHHHHHHHHHHHHHHCCCCCCCCCCEEEEEEEHHHHHHH

350ADIPOR1 PTMHFTIAEGFVKATTVGQMGWFFLMAVMYITGAGLYAARIPERFFPGKFDIWFQSHQIFHVLVVAAAFVJnet HHHHHHHH-----------HHHHHHHHHHHHHHHHHHHHH--------EEEEEE---HHHHHHHHHHHHHGor4 PTMHFTIAEGFVKATTVGQMGWFFLMAVMYITGAGLYAARIPERFFPGKFDIWFQSHQIFHVLVVAAAFV

ceeeeeeecccceeeeecccceeeeeeeeeecccchhhhcccccccccccceeecccchhhhhhhhhhhhHNN PTMHFTIAEGFVKATTVGQMGWFFLMAVMYITGAGLYAARIPERFFPGKFDIWFQSHQIFHVLVVAAAFV

hchhhhhhhhhhccccccchhhhhhhhhhhhhccchhhhhcchcccccccheeehhhhhhhhhhhhhhhhSOPMA PTMHFTIAEGFVKATTVGQMGWFFLMAVMYITGAGLYAARIPERFFPGKFDIWFQSHQIFHVLVVAAAFV

chhhheeettcchhhhhhhhhhhhhhhhhhhhhhheehtccccccccccceeeecccchheeeehhhhhhPSIPRED PTMHFTIAEGFVKATTVGQMGWFFLMAVMYITGAGLYAARIPERFFPGKFDIWFQSHQIFHVLVVAAAFV

HHHHHHEEECCCCCCCCHHHHHHHHHHHHHHHHHHHHCCCCCCCCCCCCCCEECCCHHHHHHHHHHHHHHPrepro PTMHFTIAEGFVKATTVGQMGWFFLMAVMYITGAGLYAARIPERFFPGKFDIWFQSHQIFHVLVVAAAFV

HHHHHHHHHHHHHH HHHHHHHHHHHHHHHEEEEEEEEE HHHHHHHHHHHHHSspro PTMHFTIAEGFVKATTVGQMGWFFLMAVMYITGAGLYAARIPERFFPGKFDIWFQSHQIFHVLVVAAAFV

CEEEEEEEECCCCCCCCCCCHHHHHHHHHHHHCCCEEEECCCCHCCCCCEEEECCCHHHHHHHHHHHHHHCSS HHHHHHHEECCCCCCCCCHHHHHHHHHHHHHHHHHHHHHCCCCCCCCCCCCEEECCCHHHHHHHHHHHHH

375ADIPOR1 HFYGVSNLQEFRYGLEGGCTDDTLLJnet HHHHHHHHHHHHHHH----------Gor4 HFYGVSNLQEFRYGLEGGCTDDTLL

cccccccceeeeeeeccccccceecHNN HFYGVSNLQEFRYGLEGGCTDDTLL

hhhcccchheeeeccccccccccccSOPMA HFYGVSNLQEFRYGLEGGCTDDTLL

hhhhhhhhhhhhhhtccccchhhhhPSIPRED HFYGVSNLQEFRYGLEGGCTDDTLL

HHHHHHHHHHHHHHCCCCCCCCCCCPrepro HFYGVSNLQEFRYGLEGGCTDDTLL

HHHHHHHHHHHHHHHSspro HFYGVSNLQEFRYGLEGGCTDDTLL

HHHHHHHHHHHHHHCCCCCCCCCCCCSS HHHHHHHHHHHHHCCCCCCCCCCCC

Figure 17: Secondary structure prediction for ADIPOR1.

Chapter 4 Results


Similarly, a consensus secondary structure prediction was drawn from the predictions of Jnet,

GOR4, HNN, SOPMA, PSIPRED, Prepro webservers for HHP1. The consensus secondary

structure prediction showed that HHP1 had 59% helix, 32.4% coil and 8.6% extended sheets

(Figure 18). Further, secondary structure alignment of HHP1 and ADIPOR1 showed similar

lengths of helices and turns (Figure 19).

HHP1 MDQNGHNDEAETVSCGNGNCKSKIVPGDDHGGDESSGTKRRKKRKTQQKTMKRRELMSYCELPEYMKDNEJnet -------HHHHEEE--------EEE------------------------------EEE------------GOR4 ccccccccceeeeeccccccceeeeccccccccccccchhhhhhhhhhhhhhhhhhhhhhhhccccccceHNN ccccccccccceeecccccccceeeccccccccccccchhhhccccchhhhhhhhhhhhhcccccccchhSOPMA ecccccccceeeeeccccccceeeeccccccccccttcccccccchhhhhhhhhhhechhccchhctttcPred: CCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCHHHHHHHHHHHHCCCCCCEECCCCCCCCCCCCPSIPRED -----------EEEE----------------------------HHHHHHH----EEEEHHH--HHH----CSS cccccccccccccccccccccceeeccccccccccccchhhhhhhhhhhhhhhhhhhhhhhhhccccccc

HHP1 YILNYYRADWSIRDAFFSVFSFHNESLNVWTHLIGFIFFVALTVANIIHHDGFFPVDAKSPGNVTRWPFFJnet EE---------HHHHHHHHHHHH--HHHHHHHHHHHHHHHHHHHHHH-----EEE---------HHHHHHGOR4 eeeeeccccccccccccceeeecccccccccceeceeeehhhhhhceecccccccccccccccccccceeHNN hhhhhcccccchhhhhhhhhhhchhhhhhhhhhhhhhhhhhhhhhhhheccccccccccccccccccceeSOPMA eeeetccccccchhhhhhhhhhctthhhhhhhhhhhhheehhhhhhhhcccccccheeecccccccceeePred: CCCCCCCCCCCHHHHHHHHHCCCCCCHHHHHHHHHHHHHHHHHHHHHCCCCCCCCCCCCCCCCCCHHHHHPSIPRED EE---------HHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHH-----HHHHHHHCSS EEEEEECCCCCHHHHHHHHHHHCCHHHHHHHHHHHHHHHHHHHHHHHHCCCCCCCCCCCCCCCCCCHHHH

HHP1 VFLGGSMFCLLASSICHLFCCHSKELNVFLLRIDYAGITAMIITSFFPPIFYIFQCTPRWYFIYLAGITSJnet HHHHHHHHHHHHHHHHHHHH---HHHHHHHHHHHHHHHHHHHHHHHHHHHHHH-----HHHHHHHHHHHHGOR4 eeecccceeeeeccccceeccccccccceeeeecccccceeeeeccccceeeeecccccceeeeecccccHNN eeechchhhhhhhhhhhhhhhcchhhhhhheehccccceeeeeecccchhhhhhccccchhhehhhhhccSOPMA eeehhhhhhhhhhhhhhhhhccchhhhhhhhhhhhtteeeeeecccccteeeeecccccchheeehhhhhPred: HHHHHHHHHHHHHHHHHHHCCCCHHHHHHHHHCCCHHHHHHHHHCCCCCEEEEECCCHHHHHHHHHHHHHPSIPRED HHHHHHHHHHHHHHHHHH-----HHHHHHHHHHHHHHHHHHHHHHHHHHHHHHH----HHHHHHHHHHHHCSS HHHHHHHHHHHHHHHHHHHCCCCHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHCCCHHHHHHHHHHHHH

HHP1 MGIFTIITLFTPSLSAPKYRAFRALLFASMGLFGIVPAAHALVVNWGNPQRNVTLVYELLMAVFYLVGTGJnet HHHHHHHHHHH---------HHHHHHHHHHHHHHHHHHHHHHHH------HHHHHHHHHHHHHHHHHHHHGOR4 cceeeeeeccccccccchhhhhhhhhhhhccccccchhhheeeecccccccchhhhhhhhhhhhhhccccHNN cceeeeeeeecccccchhhhhhhhhhhhhhchhchhhhhhhheeeccccccchhhhhhhhhhhhhhhcccSOPMA hhhhhheeehcccccccccchhhhhheehhhhttccchhhheeecccccchhhhhhhhhhhhhhhhhhhePred: HHHHHHHHHHCCCCCCCCHHHHHHHHHHHHHHHHHHHHHHHHHHCCCCCCCCCHHHHHHHHHHHHHHHHHPSIPRED HHHHHHHHHHHHH-------HHHHHHHHHHHHHHHHHHHHHHHHHHH---HHHHHHHHHHHHHHHHHHHHCSS HHHHHHHHHHHHHHHHHHHCCCCHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHCCCHHHHHHHHHHHHH

HHP1 FYVGRVPERLKPGWFDRVGHSHQIFHVFVLLGALSHYAAALLFLDWRDHVGCJnet HHHH---------EEEEEE---HHHHHHHHHHHHHHHHHHHHHHHHHHH---GOR4 eeecccccccccccccccccccchhhhhhhhhhhhhhhhhhhhhcccceeecHNN eeeeccccccccccccccccchhhhhhhhhhhhhhhhhhhhhhhhhhhccccSOPMA eeeccccccccccceeeecccchhheeeeehhhhhhhhhhhhhhhhccccccPred: HHCCCCCCCCCCCCCCCCCCCHHHHHHHHHHHHHHHHHHHHHHHHHHHCCCCPSIPRED HHH-----------------HHHHHHHHHHHHHHHHHHHHHHHHHHHHH---CSS HHCCCCCCCCCCCCCCCCCCCCHHHHHHHHHHHHHHHHHHHHHHHHHHH---

Figure 18: Secondary structure prediction for HHP1.

Chapter 4 Results


-LNVWTHLIGFIFFVALTVANIIHHDGFFPVDAKSPGNVTRWPFFVFLGGSMFCLLASSICHLFCCHSKELNVFLLRI---HHHHHHHHHHHHHHHHHHHH----EEE---------HHHHHHHHHHHHHHHHHHHHHHHHHH---HHHHHHHHHH---HHHHHHHHHHHHHHHHHHHH-----------------HHHHHHHHHHHHHHHHHHHHHHHHH---HHHHHHHHHHTGNIWTHLLGFVLFLFLGILTMLRPNMYFMAPLQ-----EKVVFGMFFLGAVLCLSFSWLFHTVYCHSEKVSRTFSKL

DYAGITAMIITSFFPPIFYIFQCTPRWYFIYLAGITSMGIFTIITLFTPSLSAPKYRAFRALLFASMGLFGIVPAAHAHHHHHHHHHHHHHHHHHHHHH----HHHHHHHHHHHHHHHHHHHHHHH----------HHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHH-----HHHHHHHHHHHHHHHHHHHHHHH----------HHHHHHHHHHHHHHHHHHHHDYSGIALLIMGSFVPWLYYSFYCSPQPRLIYLSIVCVLGISAIIVAQWDRFATPKHRQTRAGVFLGLGLSGVVPTMHF

LVVN-WGNPQRNVTLVYELLMAVFYLVGTGFYVGRVPERLKPGWFDRVGHSHQIFHVFVLLGALSHYAAALLFLDWRDHHH---------HHHHHHHHHHHHHHHHHHHHHH---------EEEEEH---HHHHHHHHHHHHHHHHHHHHHHHHHHHHH-----------HHHHHHHHHHHHHHHHHHHH---------EEEEEE---HHHHHHHHHHHHHHHHHHHHHHHHHHTIAEGFVKATTVGQMGWFFLMAVMYITGAGLYAARIPERFFPGKFDIWFQSHQIFHVLVVAAAFVHFYGVSNLQEFRY

--HVGCH-----H-----------GLEGGCTDDTLL

Figure 19: Secondary structure alignment of HHP1 (Grey, amino acid sequence; Red,

secondary structure) and ADIPOR1 (Blue, amino acid sequence; Green, secondary

structure) by Jpred.

4.5.2.2 Three dimension structure modelling of ADIPOR1 and HHP1

In absence of clear structural homologue, template for modelling the 3D structure of ADIPOR1

was searched using secondary structure alignment of ADIPOR1 with single subunit of proteins

containing the 7 helix trans-membrane topology from OPM database. The alignment was done

manually as well as by online software (SSEA program), for lengths of helices and loops

between the helices of ADIPOR1 with proteins from OPM database. The SSEA server uses the

fold recognition program MANIFOLD (Bindewald et al. 2003) and is able to compute

either local or global alignments of secondary structures in analogy to regular sequence

alignment methods. It computes the compatibility in terms of secondary structure of one

protein against either a representative library of known protein folds (database alignment

mode) or a single secondary structure (one vs. one alignment mode). The alignment scores

were generated using one vs. one alignment mode. Although the scores of 1VGO, 3DDL,

1UAZ and 1PY6 were more however, on manual alignment of helices and the loops between

the helices of these proteins showed difference in the secondary structure. In 1VGO, first helix

was absent. In 3DDL, helices were much longer. In 1UAZ, first helix was very short and fifth

and six were quite long. In 1PY6, first was absent, fifth was long and an addition helix was

present in it which correspond to the loop region between the six and seventh helices of

Chapter 4 Results


ADIPOR1. The SSEA scores along with the secondary structure alignment results therefore,

suggested that the structure of eubacteria (Anabena nostoc sp.) sensory rhodopsin (PDB code:

1XIO) had similar lengths of helices, and loops between the helices with respect to ADIPOR1

(Table 14 and Figure 20). Therefore, this protein was used as the template for predicting the

three-dimensional structure of ADIPOR1 by comparative modelling strategy. Modelled

structure of ADIPOR1 was obtained from Discover studio 2.0.

Table 14: Alignment scores for Secondary Structure alignment for ADIPOR1 and other

members of the 7 helix members using SSEA program.

PDB ID Name OrganismAlignmentScore Region

1VGO Archaerhodopsin-2 Archaebacteria 69.230823 - 241 length 2411 - 253 length 253

3DDL Xanthorodopsin Eubacterium 68.09341 - 241 length 24112 - 273 length 273

1UAZ Archaerhodopsin-1Halorubrumchaoviator 67.8788

1 - 241 length 2414 - 254 length 254

1PY6Bacteriorhodopsin Halobacterium

salinarum 66.530623 - 241 length 2415 - 249 length 249

1XIOAnabena sensoryrhodopsin Anabena sp. 64.1434

1 - 241 length 2414 - 261 length 261

1GU8 Sensory rhodopsinNatronomonaspharaonis 61.7954

1 - 241 length 2413 - 238 length 238

2Y02Beta 1 Adrenergicreceptor

Meleagrisgallopavo 62.0504

1 - 241 length 2419 - 315 length 315

2ZIY:A RodopsinOdarodespacificus 55.9543

1 - 241 length 24130 - 349 length 372

1F88 Rodopsin Bos taurus 55.44221 - 241 length 24134 - 347 length 347

3PBL

Dopamine Gprotein coupledreceptor Homo sapiens 51.595

1 - 241 length 24172 - 372 length 480

3EML:AA2A Adenosinereceptor Homo sapiens 46.7764

1 - 241 length 24118 - 308 length 488

3NY8:ABeta 2 Adrenergicreceptor Homo sapiens 46.238

1 - 241 length 24138 - 380 length 490

3P0GBeta 2 Adrenoreceptor Homo sapiens 45.1482

1 - 241 length 24175 - 440 length 501

Chapter 4 Results


ADIPOR1 HHHHHHHHHHHHHHHHHHHHHH---------CCCCCCCCCCCC-HHHHHHHHHHHHHHHHHHHHHHHHH---------------1VGO -------------------------------CCCCCCCCCCCCCHHHHHHHHHHHHHHHHHHHHHH------------------3DDL HHHHHHHHHHHHHHHHHHHHHHHHHHHHH--CCCCCC-------HHHHHHHHHHHHHHHHHHHHHHHHHHHHHH----------1UAZ HHHHHHH------------------------CCCC---------HHHHHHHHHHHHHHHHHHHHHHH-----------------1PY6 -------------------------------CCCCC--------HHHHHHHHHHHHHHHHHHHHHH------------------1XIO HHHHHHHHHHHHHHHHHHHHHHHHH------CCC----------HHHHHHHHHHHHHHHHHHHHHHHH----------------1GU8 HHHHHHHHHHHHHHHHHHHHHHH--------CCCC---------HH--------------------------------------2YO2 HHHHHHHHHHHHHHHHHHHHHHHHHHHHHH-CCCCCC-------HHHHHHHHHHHHHHHHHHHHHHHHHHHHHH----------2ZIY HHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHCCCCCC-------HHHHHHHHHHHHHHHHHHHHHHHHHHHHHHH---------1F88 HHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHCCCCCC-------HHHHHHHHHHHHHHHHHHHHHHHHHHHHHH----------3PBL HHHHHHHHHHHHHHHHHHHHHHHHHHHHHH-CCCCCC-------HHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHH3EML HHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHCCCCCC-------HHHHHHHHHHHHHHHHHHHHHHHHHHHH------------3NY8 HHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHCCCCCC-------HHHHHHHHHHHHHHHHHHHHHHHHHHHHHH----------3POG HHHHHHHHHHHHHHHHHHHHHHHHHHHHHH-CCCCC--------HHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHH------

ADIPOR1 ---CCCC---------------HHHHHHHHHHHHHHHHHHHHH-------------------CCC-------EEEEEEEE----1VGO ---CCCCC--------------HHHHHHHHHHHHHHHHHHHHHHHHHH--------------CCCC------EEEEE-------3DDL ---CCCCCCCCCCCC-------HHHHHHHHHHHHHHHHHHHHHHHHHH------------------------------------1UAZ ---CCCCCC-------------HHHHHHHHHHHHHHHHHHHHHHHH----------------CCCC------EEEEEE------1PY6 --- CCCCCC------------HHHHHHHHHHHHHHHHHHHHHHHHH---------------CCCC------EEEE--------1XIO ---CCCCCCCCCCEEE------HHHHHHHHHHHHHHHHHHHHHHH-----------------CCCCCC----HHHHHHHHHHHH1GU8 ---CCEEEE-------------HHHHHHHHHHHHHHHHHH----------------------CCC—------EEE---------2YO2 ---CCCCC--------------HHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHH----------------------2ZIY ---CCCCC--------------HHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHH--------------------------1F88 ---CCCC---------------HHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHH------------------------3PBL HHHCCCCC--------------HHHHHHHHHHHHHHHHHHHHHHHHHHHH------------CCCC------EEEEEE—-----3EML ---CCCC---------------HHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHH-----CCCCCCC---EEE---------3NY8 ---CCCCC--------------HHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHH------CCCCCCCCCCHH----------3POG ---CCCCCCCCCCCCCCCCEEEHHHHHHHHHHHHHHHH------------------------CCCCCCCCCCCCEEEE------

ADIPOR1 -------------CCCC----HHHHHHHHHHHHHHHHHHHHHHH---------------------------------------C1VGO -------------CCCCCCCCHHHHHHHHHHHHHHHHHHHHHHH---------------------------------------C3DDL -------------CCC-----HHHHHHHHHHHHHHHHHHHH------------------------------------------C1UAZ -------------CCCCEEEEHHHHHHHHHHHHHHHHHHHHHH----------------------------------------C1PY6 -------------CCCCCCC-HHHHHHHHHHHHHHHHHHHHHHHH--------------------------------------C1XIO HHHHHHHHHHHHHCCCC----HHHHHHHHHHHHHHHHHHHHHHHHHHHHHH--------------------------------C1GU8 CCC------EEEE--------HHHHHHHHHHHHHHHHHHHHH-----------------------------------------C2YO2 CCCCC----------------HHHHHHHHHHHHHHHHHHHHHHHHH-------------------------------------C2ZIY CCCCCCCCC------------HHHHHHHHHHHHHHHHHHHHHHHH--------------------------------------C1F88 CCCCCCC--------------HHHHHHHHHHHHHHHHHHHHHHHH--------------------------------------C3PBL CC-------------------HHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHH----------------------C3EML C--------------------HHHHHHHHHHHHHHHHHHHHHHHHHHH-----------------------------------C3NY8 ---------EEE---------HHHHHHHHHHHHHHHH----------------------------------------------C3POG CCCCCCCCC------------HHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHCCCHHHHHHHHHCCCCCCHHHHC

ADIPOR1 CCCCCCCCCEEEEEEE---HHHHHHHHHHHHHH----------------------------------EE--------CCCCCCC1VGO CC-----------------HHHHHHHHHHHHHHHHHHHHHHH-----------------------------------CCCC---3DDL CCCCCCCCCC---------HHHHHHHHHHHHHHHHHHHHHHHHHHHHHH----------------------------CCC----1UAZ CC-----------------HHHHHHHHHHHHHHHHHHHHHHH-----------------------------------CCCCC--1PY6 CC-----------------HHHHHHHHHHHHHHHHHHHHHHH-----------------------------------CCCC---1XIO C------------------HHHHHHHHHHHHHHHHHHHHHHHHHHH-------------------------------CCCCCCC1GU8 CCC----------------HHHHHHHHHHHHHHHHHHHHHHH-----------------------------------CCC----2YO2 CC-------EE--------CCCCC-------------------------------------------EEEEE-----CCCCCCC2ZIY CCC------EEE-------CCC---------------------------------------------EEEEEE----CCCCCCC1F88 CC-------EEE-------CCCC--------------------------------------------EEEEEE----CCCCCC-3PBL CCCC---------------HHHHHHHHHHHHH-----------------------------------EEEEEEEEE-CCCCCCC3EML CCCCCCCCCCCCCCCCCCCHHHCHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHCCCCCCCHHHCCC----3NY8 CCCCCCCCCCCCEEECCCCHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHCCCCCCC3POG CCCCCCCCCCCCCCCCCCCHHHHHH----------------------------------------CCCCHHHCCCCCCCCCCCC

ADIPOR1 CC------------------HHHHHHHHHHHHHHHHHHHHH-------------------------------------------1VGO --------------------HHHHHHHHHHHHHHHHHHHHHHHHHHHHHHH---------------------------------3DDL --------------------HHHHHHHHHHHHHHHHHHHHHHHHHH--------------------------------------1UAZ --------------------HHHHHHHHHHHHHHHHHHHHHHHHHHHHHH----------------------------------1PY6 --------------------HHHHHHHHHHHHHHHHHHHHHHHHHHHHHHH---------------------------------1XIO CC------------------HHHHHHHHHHHHHHHHHHHHHHHHHHHHH-----------------------------------1GU8 --------------------HHHHHHHHHHHHHHHHHHHHHHHHHHHHHH----------------------------------2YO2 C-------------------HHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHH--------------------------2ZIY C-------------------HHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHH1F88 --------------------HHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHH3PBL CCCCCCCCCCCCCCCCCCC--HHHHHH----------------------------------------------------------3EML ---------------------HHHHH-----------------------------------------------------------3NY8 HHHHCCCCCCCCCCCCCCCCCHHHHHH----------------------------------------------------------3POG CCCCCCCCCCCCCCCCCCCCCCCCCHHHHHHHHHHHHHHHHHHHH----------------------------------------

Chapter 4 Results


ADIPOR1 ----------------------------CCCCCCCCCCCC-------------EEE----------------------------1VGO ---------------------------CC------------------------HHHHHHHHHHHHHHHHHHHHHHHHHHHH---3DDL ---------------------------CCCCCCCCCCCCCCCC-----------------------------------------1UAZ ---------------------------CC------------------------HHHHHHHHHHHHHHHHHHHHHHHHHHHH---1PY6 ---------------------------CC------------------------HHHHHHHHHHHHHHHHHHHHHHHHHHHH---1XIO ---------------------------CCCCCCCCCCCHH-------------CCCC---------------------------1GU8 ---------------------------CCC-----------------------HHHHHHHHHHHHHHHHHHHHHHHHHHHHH--2YO2 ---------------------------CCCCC---------------------HHHHHHHHHHHHHHHHHHHHHHHHHHHHHHH2ZIY HHHHHHHHHHHHHHHHHHHHHHHHHHHCCCCCC--------------------HHHHHHHHHHHHHHHHHHHHHHHHH------1F88 HHHHHHHHHHHHHHHHH----------CCCCC---------------------HHHHHHHHHHHHHHHHHHHHHHHHHH-----3PBL ---------------------------CCCCCCCCCCCCCCCCCCCCCCCCCCCCCCHHHHH----------------------3EML ---EEEEEEEEE---------------CCCCCCCCCCCHHH------------EEE----------------------------3NY8 --------------------CCCCHHHCCCCCCCCCCCCCCCCCCCCCCCCCCCHH----------------------------3POG ---------------------------CCCCCCCC------------------HHHHHHHHHH---------------------

ADIPOR1 ----------CCC----------HHHHHHHHHHHHHHHHHHHHHHHHHH--------------CCCCCCCCCCCC---------1VGO ----------CCCCCC-------HHHHHHHHHHHHHHHHHHHHHHHHHHHHH-----------CCCCCCCCCCCCCCCCCCC--3DDL -----------------------HHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHH---CCCCCCCCCCCC---------1UAZ ----------CCCCCCCC-----HHHHHHHHHHHHHHHHHHHHHHHHHHHHH-----------CCCCCCCCCCCCCCCCCCC--1PY6 ----------CCCCCCCC-----HHHHHHHHHHHHHHHHHHHHHHHHHHHHH-----------CCCCCCCCCCCCCCCCCCCC-1XIO ----------EEEEEEEE-----HHHHHHHHHH------------------------------CCC------------------1GU8 ----------CCCCCCC------HHHHHHHHHHHHHHHHHHHHHHHHHHHHHHH---------CCCCCCCCCCCCCCCCCCC--2YO2 HHHHHHHHHHCC-----------HHHHHHHHHHHHHHHHHHHHHHHHH-CHHHHHHHHHHH—CCCCCCCCCCCCCCCC------2ZIY ----------CC-----------HHHHHHHHHHHHHHH-------------------------CCCCCCCCCCCCCCCC-----1F88 ----------CC-----------HHHHHHHHHH-----------------------CCCCCCCCCCCCCCCCCCCCCCCCCCCC3PBL ----------CCCCCCCCCCCCCHHHHHHHHHHHHHHHHHHHHHHHH----------------CCCC-----------------3EML ----------CCCCCCCC-----HHHHHHHHHHHHHHHHHHHHH-------------------CCCCCC---------------3NY8 ----------CCCCCCCCCC---HHHHHHHHH HHHHHHHHHHH-------------------CCCCCCCC-------------3POG ----------CCCCC--------HHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHCCCCC----------------

Figure 20: Alignment of secondary structure of members of 7 helix protein family with

ADIPOR1.

The Ramachandran plot for the ADIPOR1 model showed 93.45% residues in most favored

regions and other parameters for PROCHECK was in the allowed range (Figure 21 and Table

15). Ramachandran plot and G-factor values, compared with those of the template structure,

indicate that our model has a good energetic and stereo-chemical quality (Table 15 and 16).

Figure 21: Ramachandran plot of ADIPOR1.

Chapter 4 Results


Table 15: Ramachandran Plot statistics of ADIPOR1.

Ramachandran Plotstatistics

No. ofresidues

% residue


Additional allowedregions[a,b,l,p]

11 5.6%


2 1.0%



196 100.0%

End-residues (excl. Gly andPro)

2 -




Table 16: G-Factors statistics of ADIPOR1.

Parameter Score AverageScore

Dihedral angles:Phi-psi distributionChi1-chi2 distributionChi1 onlyChi3 and chi4Omega

-0.49-0.71-0.280.71-0.35

-0.06


-0.16-1.10

-0.71


The positioning of secondary structural elements was generated from PDBsum and was in

agreement with the predicted consensus secondary structure. The predicted model of

ADIPOR1 contained seven helices with five continuous and two with breaks (Figure 22).

Chapter 4 Results


a. b.

Figure 22: a; Super-imposition of modelled 3D structure of ADIPOR1 onto 1XIO usingPymol. b; Secondary structural elements of ADIPOR1 generated from PDBsum(Secondary structure: helices, sheets , turn, turn, hairpin,disulphide bond).

Since the structure of ADIPOR1 and HHP1 were quite similar, same protocol was followed

for determining the suitable template for 3D structure modelling of HHP1. Although the

SSEA program score for 3DDL was more than the XIO1 however, upon manual alignment,

the helices and the loops between the helices of 3DDL and HHP1 showed difference in

lengths of helices. Thus the manual alignment of the secondary structure and alignment

scores for secondary structure alignment from SSEA program suggested that the structure

of HHP1 and eubacteria (Anabena nostoc sp.) sensory rhodopsin (PDB code: 1XIO) had

similar lengths of helices and loops between the helices (Table 17 and Figure 23).

Therefore, this protein was used as the template for predicting the three-dimensional

structure of HHP1 by comparative modelling strategy. Modelled structure of HHP1 was

obtained from Discover studio 2.0.

HHP1 CC-----------HHHHHHHHHHHHHHHHHHHHH------CCCCEE-------------------------CCCCCCCC-----HHH3DDL CCCCCCCCCCC--HHHHHHHHHHHHHHHHHHHHHHHHHHHHH-----------------------------CCCCCC-------HHH1PY6 CCCCCC-------HHHHHHHHHHHHHHHHHHHHHHHHH—-CCCCEE-EE----------------------CCCCCCC------HHH1XIO CCC----------HHHHHHHHHHHHHHHHHHHHHHHHH--CCC—HHHHHHHHHHHHHHHHHHHHHHHH---CCCCCCCCCCEEEHHH1VGO CCCCC--------HHHHHHHHHHHHHHHHHHHHHHHHHH-CCCCEE-EEE---------------------CCCCCCCC-----HHH1GU8 CCCCCCCC-----HHHHHHHHHHHHHHHHHHHHHH-----CCCCEE-EE----------------------CCEEEE-------HHH1UAZ CCCCCC-------HHHHHHHHHHHHHHHHHHHHHHHH---CCCCEEEEEE---------------------CCCC-----EEEEHHH2YO2 CCCCCCCC-----HHHHHHHHHHHHHHHHHHHHHHHHHHHHHH----------------------------CCCCCC-------HHH1F88 ----------------------------------------CCCCCCCCCEE--CCCCCCCCCCCCCCCCCCCCCC---------HHH2ZIY ----------------------------------------------------CCCCCCCCCCCCCCCCCCCCCCCCCCCC----HHH3PBL CCCCCCCCCCCCCHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHCCCCCCHHHHHHHHHHHHHHHHHHHHHHHHHHCC-------HHH3NY8 -------------CCCCCCCCCCCCCCC---------------HHH-------------------CCCCCCCCCCCCCCCCCCCHHH3EML -------------------------------------------------------------------CCCCCCCCCCCCCCCCCHHH3POG -------------CCCCCCCCCCCCCCC---------------HHH-------------------CCCCCCCCCCCCCCCCCCCHHH

Chapter 4 Results


HHP1 HHHHHHHHHHHHHHHHHHHHHHH-----------------CCCC-------------HHHHHHHHHHHHHHHHHHHHHHHHHHHHHH3DDL HHHHHHHHHHHHHHHHHHHHHHHHHHH-------------CCCCCCCCCCCCCCCCCHHHHHHHHHHHHHHHHHHHHHHHHHH----1PY6 HHHHHHHHHHHHHHHHHHHHH-------------------CCC--------------HHHHHHHHHHHHHHHHHHHHHHH-------1XIO HHHHHHHHHHHHHHHHHHHH--------------------CCCCCC-----------HHHHHHHHHHHHHHHHHHHHHHHHH-----1VGO HHHHHHHHHHHHHHHHHHHH--------------------CCC--------------HHHHHHHHHHHHHHHHHHHHHHH-------1GU8 HHHHHHHHHHHHHHHHHHH---------------------CCC--------------HHHHHHHHHHHHHHHHHHHHHHH-------1UAZ HHHHHHHHHHHHHHHHHHH---------------------CCC--------------HHHHHHHHHHHHHHHHHHHHHHH-------2YO2 HHHHHHHHHHHHHHHHHHHHHHHHHHH-------------CCCC-------------HHHHHHHHHHHHHHHHHHHHHHHHHHHHHH1F88 HHHHHHHHHHHHHHHHHHHHHHHHHHHH------------CCCCCC-----------HHHHHHHHHHHHHHHHHHHHHHHHHHHHHH2ZIY HHHHHHHHHHHHHHHHHHHHHHHHHHHH------------CCCCCC-----------HHHHHHHHHHHHHHHHHHHHHHHHHHHHHH3PBL HHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHCCCCC------------HHHHHHHHHHHHHHHHHHHHHHHHHHHH--3NY8 HHHHHHHHHHHHHHHHHHHHHHHHHHHH------------CCCCCC-----------HHHHHHHHHHHHHHHHHHHHHHHHHHHHHH3EML HHHHHHHHHHHHHHHHHHHHHHHHHHHH------------CCCCCC-----------HHHHHHHHHHHHHHHHHHHHHHHHHHHH--3POG HHHHHHHHHHHHHHHHHHHHHHHHHHHH------------CCCCCC-----------HHHHHHHHHHHHHHHHHHHHHHHHHHHHHH

HHP1 H---------CCC---------HHHHHHHHHHHHHHHHHHHHHHHHH---------------CCCCCCCC------HHHHHHHHHHH3DDL ----------CCC---------HHHHHHHHHHHHHHHHHHHH--------------------CCCCCCCCCCC---HHHHHHHHHHH1PY6 ----------CCCC--------HHHHHHHHHHHHHHHHHHHHHHHHHHHHHHH---------CC------------HHHHHHHHHHH1XIO ----------CCCC--------HHHHHHHHHHHHHHHHHHHHHHHHHHHHHH----------CC------------HHHHHHHHHHH1VGO ----------CCCC--------HHHHHHHHHHHHHHHHHHHHHHHHHHHHHHH---------CC------------HHHHHHHHHHH1GU8 ----------CCCC--------HHHHHHHHHHHHHHHHHHHH--------------------C-------------HHHHHH-----1UAZ ----------CCCCC-------HHHHHHHHHHHHHHHHHHHHHHHHHHHHHH----------CC------------HHHHHHHHHHH2YO2 HHHHHHHHHHCCCCC-------HHHHHHHHHHHHHHHHHHHHHHHHHCCCEECCCCCEEEEECCCCCCCC------HHHHHHHHHHH1F88 ----------CCCC--------HHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHH--CCCCCCC-------HHHHHHHHHHH2ZIY H---------CCCCC-------HHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHH----CCCCCCCCC-----HHHHHHHHHHH3PBL ----------CCCCEEEEEECCHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHCCCCC---------HHHHHHHHHHH3NY8 ----------CCCCC-------HHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHH------CCCCCCCCCCCCCCHH---------3EML ----------CCCC--------HHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHH-----CCCCCCC-------EEE--------3POG ----------CCCCC-------HHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHH--------------------CCCCCCCCCCC

HHP1 HHHHHHHHHHHHH--------------CCCC-----HHHHHHHHHHHHHHHHHHHHHHHHHH---------------CCCCCCCCCC3DDL HHHHHHHHHHHHHHHHHH---------CCC------HHHHHHHHHHHHHHHHHHHHHHHHHH---------------CCCCCCCCCC1PY6 HHHHHHHHHHHHHHHHHH---------CCCCCCCC-HHHHHHHHHHHHHHHHHHHHHHHHHHHHH------------CCCCCCCCCC1XIO HHHHHHHHHHHHHHHH-----------CCCCCCCCCHHHHHHHHHHHHHHHHHHHHHHHHHHHHH------------CCCCCCCCCC1VGO HHHHHHHHHHHHHHHHH----------CCCCCCCC-HHHHHHHHHHHHHHHHHHHHHHHHHHHHH------------CCCCCCCCCC1GU8 ---------------------------CCCC-----HHHHHHHHHHHHHHHHHHHHHHHHHHH--------------CCCCCCCCC-1UAZ HHHHHHHHHHHHHHHHH----------CCCCCCCC-HHHHHHHHHHHHHHHHHHHHHHHHHHHHH------------CCCCCCCCCC2YO2 HHHHHHHHHHHHHHHHHHHHHHHHHHHCCCCC----HHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHCCCCCC----1F88 HHHHHHHHHHHHH--------------CCC------EEE--------------------------------------CCC-------2ZIY HHHHHHHHHHHHH--------------CCCC-----EEE--------------------------------------CCC-------3PBL HH-------------------------EEEEEEEEECCCCCCCCCCCCCCCCCCCCCCCCCCC--------HHHHHHCCCCCCCCCC3NY8 ---------------------------EEE------HHHHHHHHHHHHHHHH-------------------------CCCCCCCCCC3EML ---------------------------C--------HHHHHHHHHHHHHHHHHHHHHHHHHHH--------------CCCCCCCCCC3POG CCCCC----------------------EEE------HHHHHHHHHHHHHHHH-------------------------CCCCCCCCCC

HHP1 CC--------EEE---CC----------------HHHHHHHHHHHHHHHHHHHHHHHHHHHH-------------------------3DDL CCCCCC----------------------------HHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHH----------------1PY6 CCCCCCCCCC-----------------------------------------------------------------------------1XIO C-------HHCCCCEEEEEEEE------------HHHHHHHHHH-------------------------------------------1VGO CCCCCCCCC------------------------------------------------------------------------------1GU8 ----------------------------------HHHHHHHHHHHHHHHHHHHHHHHHHHHHH------------------------1UAZ CCCCCCCCC------------------------------------------------------------------------------2YO2 ----------------------------------HHHHHHHHHHHHHHHHHHHHHHHHH----------------------------1F88 ---------EEEEEECCCCCC-------------HHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHH2ZIY ---------EEEEEECCCCCCCC-----------HHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHH3PBL CCCCCCCCCCCCCCCCHHHHHCCCCCCC CCCCCCHHHHHHHHHHHHHHHHHHHHHHHH-----------------------------3NY8 CCC------EEECCCCCCCCCC------------HHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHH3EML CCCCCCCCCHHHCCC-------------------HHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHH---------3POG CC-------EEEECCCCCCCCC------------HHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHH-------------

HHP1 -----------------------CCC---------3DDL -----------------------CCCCCCCCCCCC1PY6 -----------------------------------1XIO -----------------------CCC---------1VGO -----------------------------------1GU8 -----------------------C-----------1UAZ -----------------------------------2YO2 -----------------------C—----------

Chapter 4 Results


1F88 HHHHHHHHHHHHHHHHHHHHHHHCCCCC-------2ZIY HHHHHHHHHHHHHHHHHHHHHHHHHHHHHCCCCCC3PBL -----------------------CCCC--------3NY8 HHHHHHHHH--------------CCCCCCC-----3EML -----------------------CCCCCCC-----3POG -----------------------CCC---------

Figure 23: Secondary structure alignment of HHP1 with 7 helical trans-membrane

proteins.

Table 17: Alignment scores for Secondary Structure alignment for HHP1 and othermembers of the 7 helix members using SSEA program.

PDB ID Name OrganismAlignmentScore Region

3DDL Xanthorodopsin Eubacterium 77.01381 - 236 length 2361 - 273 length 273

1XIOAnabena sensoryrhodopsin Anabena sp. 75.8551

1 - 236 length 2361 - 261 length 261

1PY6 BacteriorhodopsinHalobacteriumsalinarum 75.0515

1 - 200 length 23632 - 249 length 249

1VGO Archaerhodopsin-2 Archaebacteria 74.43761 - 200 length 23636 - 253 length 253

1GU8 sensory rhodopsin iiNatronomonaspharaonis 70.8861

1 - 200 length 23634 - 238 length 238

1UAZ Archaerhodopsin-1Halorubrumchaoviator 70.6122

1 - 200 length 23638 - 254 length 254

2Y02Beta 1 Adrenergicreceptor

Meleagrisgallopavo 65.3358

1 - 236 length 2361 - 288 length 315

1F88 RodopsinOdarodespacificus 60.3774

24 - 236 length 2361 - 281 length 347

2ZIY:A Rodopsin Bos taurus 56.578930 - 236 length 2361 - 291 length 372

3PBLDopamine G proteincoupled receptor Homo sapiens 50.4881

1 - 236 length 2361 - 372 length 481

3NY8:ABeta 2 Adrenergicreceptor Homo sapiens 48.6226

3 - 236 length 2361 - 273 length 490

3EML:AA2A Adenosinereceptor Homo sapiens 47.7901

30 - 236 length 2361 - 236 length 488

3P0GBeta 2 Adrenoreceptor Homo sapiens 46.2687

3 - 236 length 2361 - 246 length 501

The Ramachandran plot for the HHP1 model showed 86.3% residues in most favored regions

and other parameters for PROCHECK was in the allowed range (Figure 24 and Table 18). The

theoretical model was hence, a good quality model. The values from Ramachandran plot and

G-factor statistics, when compared with those of the template structure, indicated that our

Chapter 4 Results


model had a good energetic and stereo-chemical quality (Table 18and 19). Secondary structure

predictions made by above also agree with the obtained structure by comparative modelling

(Figure 25).

Figure 24: Ramachandran plot of HHP1.

Table 18 : Ramachandran Plot statistics for HHP1.

Ramachandran Plot statistics No. ofresidues

% residues


Additional allowed regions[a,b,l,p] 22 10.8%


4 2.0%



204 100.0%

End-residues (excl. Gly and Pro) 2 -




Chapter 4 Results


Table 19 : G factor statistics for HHP1.

Parameter Score AverageScore

Dihedral angles:Phi-psi distributionChi1-chi2 distributionChi1 onlyChi3 & chi4Omega

0.28-0.82-0.270.62-0.30

-0.15


-0.17-1.21

-0.77


The positioning of secondary structural elements was generated from PDBsum and was in

agreement with the predicted consensus secondary structure.. The predicted model of HHP1

contained seven helices, five continuous and two with breaks (Figure 25).

Figure 25: a; Super-imposition of modelled 3D structure of HHP1 and 1XIO usingPymol. b; Secondary structural elements of HHP1 generated from PDBsum.(Secondary structure: helices, sheets , turn, turn, hairpin,disulphide bond)

Although, Arabidopsis HHP1 and human ADIPOR1 shared very less amino acid sequence

similarities, the modelled and superimposed structures, however, showed high conservation

in their predicted 3D structures (Figure 26).

Chapter 4 Results


Figure 26: Super-imposition of modelled 3D structures of ADIPOR1 and HHP1.

4.5.3 Molecular docking of osmotin - HHP1 and ATOSM34 - HHP1.

The binding of a ligand to the extracellular domain of its cognate receptor is the starting point

of the signaling process. Therefore, we modelled the complex between osmotin-HHP1 and

ATOSM34-HHP1. The output of Discovery Studio 2.0 is a list of candidate complexes

between receptor and ligand molecules identified as poses. Out of the list of 2000 poses for

osmotin-HHP1 complex, eleven top scoring poses around the loops extending towards the

extracellular spaces (Loop I; Phe28-Trp41, Loop II; Ile 94-Trp104, LoopIII; 162-Val167, and

free C-Terminal end) were selected. Upon manual checking and correlation of available data, a

single poses was selected.

In this complex, osmotin exposes Arg 38, Asp 88, Asp 185, Met 42, Ser 91, Lys 119, Tyr 179

and Phe 90, whereas the HHP1 exposes Arg 165, Ser 34, Gln 99, Cys 236, Thr 39 and Trp 41

(Table 20 and Figure 27a and b). The complex structure is also stabilized by the pi and pi-pi

interactions. This data suggest that predominant interaction between the osmotin and HHP1 are

on electrostatic basis.

Table 20: Interacting amino acids between the osmotin and HHP1 in osmotin-HHP1

HHP1 osmotinInteractingamino acid

Positionof aminoacid

Interactingamino acid


Gln 99 Arg 38Arg 165 Met 42Thr 39 Ser 91Ser 34 Lys 119Cys 236 Tyr 179Cys 236 Asp 185

Chapter 4 Results


Thr 39 Asp 88Thr 39 Phe 90Trp 41 Phe 90

a. b.

Figure 27: a; 3D model of HHP1 and osmotin complex. b; Interacting residues of

HHP1 and osmotin complex.

Similarly, Discovery studio output of the list of 2000 poses for ATOSM34-HHP1 complex, the

fifteen top scoring poses around the loops extending towards the extracellular spaces (Loop I;

Phe28-Trp41, Loop II; Ile 94-Trp104, LoopIII; 162-Val167, and free C-Terminal end) were

selected. Upon manual checking and correlation of available data, only one poses was selected

(Table 21 and Figure 28 a and b). In this complex, ATOSM34 exposes Arg 172, Asp 175, Asn

115, Met 42, Thr 185, Leu 120 and Thr 189, whereas the HHP1 exposes Arg 165, Asn 37, Cys

100, Thr101, Gln 99 and Cys 236. The complex structure is also stabilized by the pi and pi-pi

interactions. This data suggest that predominant interaction between the ATOSM34 and HHP1

are also on electrostatic basis.

Table 21: Interacting amino acids between the ATOSM34 and HHP1 in ATOSM34-

HHP1

HHP1 ATOSM34Interactingamino acid


Interactingamino acid


Asn 37 Asp 175Asn 37 Asp 175

Table cont.

Chapter 4 Results


Cys 100 Asn 115

Arg 165 Thr 185

Arg 165 Thr 185

Thr 101 Asn 115

Asn 37 Leu 120

Gln 99 Arg 172

Cys 236 Thr 189

a. b.

Figure 28: a; 3D model of HHP1 and ATOSM34 complex, b; Interacting residues ofHHP1 and ATOSM34 complex.

4.5.4 Signaling through the N terminal domain of HHP1

Once the ligand is bound to the extracellular domain of its cognate receptor, it induces changes

in structure of the receptor. These structural changes in the receptor initiates cascade of

reaction mediated by its cytoplasmic domain. Receptors at their cytoplasmic domain have

specialized enzyme/s site that becomes activated whenever the extracellular domain of the

receptor encounters a ligand. The C terminus of ADIPOR1 is suggested to bind to adiponectin,

whereas the adaptor protein containing the pleckstrin homology domain (APPL1), RACK,

CKII kinase and ERp46 interacts with the cytoplasmic N terminus of the receptor (Charlton et

al. 2010; Deepa and Dong 2009; Heiker et al. 2009; Xu et al. 2009; Yamauchi et al. 2003).

Clustal W analysis of ADIPOR1 and HHP1 N-terminal showed 17 % similarity (Figure 29).

The HHP1 N-terminal amino acid sequence showed no site for CKII kinase and ERp46. The

Table cont.

Chapter 4 Results


APPL1 interacts with ADIPOR1’s cytoplasmic tail by PTB domain and RACK with WD

domain, but the molecular details of the interactions are yet to be determined. Since the

sequence homology is very less in N-terminus of ADIPOR1 and HHP1, therefore, it is difficult

to say that there is binding site for APPL1 and RACK. However, the possibility of their

interaction cannot be ruled out and, hence, experimental process is required to prove the same.

Figure 29: Clustal W analysis of ADIPOR1 and HHP1 N-terminal amino acid sequence.

Recently, evidence are coming up to indicate a previously undescribed role for β-arrestins

as signal transducers and scaffolding molecules that can function independent of G proteins

in addition to the classically described functions as terminators of G protein-dependent

seven TM receptor signaling (McDonald et al. 2000). Upcoming reports have demonstrated

that, upon activation of angiotensin II (AngII) type 1A (AT1A) (Luttrell et al. 2001),

neurokinin 1 (DeFea et al. 2000), and protease-activated receptors, β-arrestin scaffolds the

components of the extracellular signal-regulated kinase (ERK) cascade, Raf-1, MEK1, and

ERK1 2, into complex with the receptors leading to activation of ERK1.

Interestingly, recent reports suggest that adiponectin mount antiapoptotic responses mediated

by extracellular signal-regulated kinase (ERK) (Wijesekara et al. 2010). Therefore, to explore

the possibility of involvement of this signaling pathway in osmotin induced proline

biosynthesis and accumulation, submission of N-terminal amino acid sequence of HHP1 to

Group-based Phosphorylation Scoring Method (GPS) was done. Results showed the presence

of six G-protein-coupled receptor kinase (GRKs) phosphorylation sites on the N-terminus

HHP1 protein (Table 22). The phophorylation of activated GPCRs by GRKs, that are

constitutively located on the membrane, initiates the binding of β-arrestins to phosphorylated

GPCRs/7TM proteins. Published reports indicate that, for a variety of receptors, β-arrestins

functions as molecular mediators of G protein-independent signaling by acting as scaffold to a

variety of signaling proteins including ERK-MAP kinase, apart from its earlier established role

Chapter 4 Results


in the termination of G-protein signaling (Pierce and Lefkowitz, 2001; Kim and Shenoy, 2005).

Therefore, we suggest that GRKs present on the membrane would phosphorylate either one or

multiple GRKs phosphorylation sites on the N-terminus of HHP1 protein leading to β-arrestins

mediated activation of ERK. Activated ERK would mediate its effect through phosphorylation

of substrates.

There are many cytosolic substrates and several transcription factors that translocate to the

nucleus following phosphorylation and activation by ERK in the cytoplasm (Ebisuya et al.

2005). To dissect further the signaling pathway, we determined the enzymes and transcription

factors involved in proline biosynthesis and accumulation and subsequently determine the ERK

phosphorylation sites on them. These phosphorylation sites may serve as switches to regulate

their activities by ERK.

Table 22: GRKs phosphorylation site on the N-terminus of HHP1 protein predicted by

Group-based Phosphorylation Scoring Method (GPS).

Position Code Kinase Peptide Score Cutoff

35 S AGC DDHGGDESSGTKRRK 1.234 0.949

36 S AGC DHGGDESSGTKRRKK 1.343 0.949

38 T AGC GGDESSGTKRRKKRK 2.048 0.949

46 T AGC KRRKKRKTQQKTMKR 3.167 0.949

50 T AGC KRKTQQKTMKRRELM 1.067 0.949

12 T AGC/GRK GHNDEAETVSCGNGN 0.75 0.583

4.5.5 Nuclear (Transcription) factors and Cytosolic enzymes targets for ERK-MAP

kinase in proline biosynthesis.

Phosphorylation is the key regulatory step in signaling pathways responsible for cellular

development and differentiation, cell cycle control, metabolism and the immune response. It

acts not only as a veritable on and off switch for protein activity, but also as a precise

mechanism of redirecting a protein's cellular localization and, thus, its function in the cell.

Therefore, we first identified the nuclear (transcription) factor/s those modulate the expression

of genes of proline biosynthesis and accumulation and may serve as suitable targets for ERK

phosphorylation.

Chapter 4 Results


4.5.5.1 Identification of Nuclear (Transcription) factors in Proline biosynthesis

The cell- and tissue-specific gene expression is modulated by the binding of specialized

transcription factors (activators or repressors) to the promoters and/or enhancers of the gene.

The discovery of regulatory motifs and their organization in promoter sequences is an

important first step to improve understanding of gene expression and regulation. Since co-

expressed genes are likely to be regulated by the same transcription factor, the identification of

shared, and thus, overrepresented motifs insets of potentially co-regulated genes may provide

an insight to the regulation of expression of whole metabolic pathway.

Table 23: Genes involved in proline metabolism and transport.

Group Gene symbol Description Accession TSS

I

P5CS1Delta1-pyrroline-5-

carboxylate synthytase 1At2g39800.1 Chr.2

16610040 (-)

P5CR Pyrroline-5-carboxylatereductase

At5g14800 Chr.54787842 (-)

II

At5g38710Proline oxidase, putative /osmotic stress-responsiveproline dehydrogenase

At5g38710 Chr.515518475 (+)

ALDH12A1Mitochondrial Delta-pyrroline-5- carboxylatedehydrogenase

At5g62530 Chr.525120524 (-)

III

ProT1Proline transporter withaffinity for gly betaine,proline and GABA

At2g39890 Chr.216662731 (+)

ProT2 Proline transporter 2 At3g55740 Chr.320706491 (+)

ProT3 Proline transport3 At2g36590 Chr.215352416 (-)

Based on the literature and genome information, Arabidopsis genome was parsed for the

proline biosynthetic genes by using mapviewer. The proline metabolism genes were divided

into three sub-groups according to the metabolic functions. Group 1 consisting of Pyrroline-5-

Chapter 4 Results


carboxylate synthetase (P5CS) and Pyrroline-5-carboxylate reductase (P5CR). Group 2

consisting of Osmotic stress-responsive proline dehydrogenase (PO/PDH) and Pyrroline-5-

carboxylate dehydrogenase (P5CDH). Group 3 consisting of Proline transporter with affinity

for glycine betaine, proline and GABA (AtProT1), Proline transporter 2 (AtProT2) and Proline

transporter 3 (AtProT3) (Table 23).

F-Match analysis compares the number of sites found in a query sequence set against the

background set and provides as results the Position Weight Matrices (PWMs), whose

frequencies are higher in the query sequence set compared to the background set. For analysis,

background set or no-set comprised of housekeeping genes as they are ubiquitously present and

constitutively expressed in most cells and under most conditions, and so are not likely to

exhibit the same regulatory pattern as proline biosynthesis gene set. The F-match analysis

results showed over represented TFBS in the proline biosynthetic gene set (Figure 30). The

composite module analysis was then used to determine the combination of binding sites most

commonly found within this set of genes. Upon analysis, the binding sites matching matrices

for Opaque2 (O2_Q2; O2) and Ocs element binding factor (OCSBF-1) appeared most

commonly in differing combinations within the promoters of proline biosynthesis genes

(Figure 31 and Table 24).

Table 24: Composite Module Analysis of proline biosynthesis pathway genes.

Genesymbol

TRANSProID P$OCSBF1_01 P$O2_Q2

P5CS1 ATH_3213 -286 to -282 -250 to -240

P5CR ATH_14248 -854 to -850 -834 to -774

AT5G38710 ATH_16278 -825 to -821 -792 to -780

ALDH12A1 ATH_18736 -471 to -467 -533 to -465

ProT1 ATH_3223 -636 to -632 -756 to -712

ProT2 ATH_8478 -914 to -910 -951 to -941

ProT3 ATH_2891 -351 to -347 -353 to -308

Chapter 4 Results


Figure 30: Over represented Transcription factor binding sites (TFBS) in Prolinebiosynthesis promoter set using F- MATCH Analysis.

Figure 31: Composite Module Analysis of proline biosynthesis pathway genes.

Chapter 4 Results


The presence of O2 and OCSBF-1 consensus binding sites occurring in combination within the

promoters of proline biosynthetic genes suggests a role for bzip family proteins in the

regulation of expression of this gene set. The presence of O2 and OCSBF-1 consensus binding

sites in the members of proline metabolism genes set suggests common sensor for the

concerted regulatory control of proline metabolism. The BlastP analysis showed O2 and

OCSBF1 show homology with members of C and S groups of AtbZIP transcription factors,

respectively. The results showed transcription factors AtbZIP10 and AtbZIP25 closest to

Opaque2 and AtbZIP53 and AtbZIP2 to OCSBF1.

Composite module analysis also showed consensus binding sites of MYBAS1 in P5CS1,

P5CR, ProT1 and ProT2, SBF-1 in PDH, and GT-1 in P5CDH (Table 25) promoters

suggesting that the proline metabolism gene cluster is likely to be under the combinatorial

control of more than one class of transcription factors.

Table 25: Composite module analysis for exclusive transcription factor binding sites

present on the promoters of proline metabolism gene cluster.

Matrixidentifier

Position(strand) Corematch

Matrixmatch

Sequence (alwaysthe(+)-strand isshown

Factorname

P5CS1 (delta1-pyrroline-5-carboxylate synthase 1)

P$MYBAS1_01 413 (+) 1.000 0.999 ccccaaccgcc MYBAS1

P5CR (pyrroline-5- carboxylate (p5c) reductase)

P$MYBAS1_01 574 (+) 0.999 0.995 acgcaacagcc MYBAS1

Proline oxidase, putative / osmotic stress-responsive proline dehydrogenase, putative

P$SBF1_01 692 (-) 1.000 0.967 tatattaacaaata SBF-1

1-pyrroline-5-carboxylate dehydrogenase/ 3-chloroallyl aldehyde dehydrogenase

P$GT1_Q6 961 (+) 1.000 1.000 gtaaata GT-1

Proline transporter 1

P$MYBAS1_01 1012 (+) 0.997 0.997 cctcaacggct MYBAS1

Proline transporter 2

P$MYBAS1_01 565 (-) 0.999 0.996 agcagttacgt MYBAS1

Chapter 4 Results


4.5.5.2 Determination of ERK-MAP kinase phosphorylation sites on the identified

nuclear (transcription) factors and cytosolic (enzymes) targets of proline metabolism

The available programs such as GPS, Kinophos, etc. were able to find the sites for MAP

kinases on amino acid sequence of proteins. No program however, specifically determined

ERK-MAP (ERK) kinase phosphorylation sites. Therefore, amino acid sequences of all the

enzymes of proline biosynthesis (Table 23) and identified transcription factors AtbZIP10,

AtbZIP25, AtbZIP53, AtbZIP2, MYBAS1 and GT-1 were manually searched for ERK

phosphorylation sites. Apart from ERK Phosphorylation site, docking motifs on the substrate

were also searched. D domain is a distal docking site that is ubiquitous in mitogen activated

protein kinase substrates. The sequence of the D domain usually conforms to an (R/K)1–2-

(X)2–6-Φ-X-Φ pattern, where Φ is a hydrophobic residue. Many substrates also have a second

ERK-binding region, the DEF motif which possesses a consensus sequence of F-X-F-P.

P5CS1 showed two sites for ERK phosphorylation both followed by well defined D domain,

whereas P5CSR showed none (Table 26). PROD and P5CDH showed two sites for ERK

phosphorylation (Table 26). In PROD, however, both ERK phosphorylation sites were

followed by well defined D domain. The P5CDH had one ERK phosphorylation site followed

by D domain and the other by FXFP domain. Similar analyses were performed for the

transcription factors AtbZIP10, AtbZIP25, AtbZIP53 and AtbZIP2. Among these, only

AtbZIP25 showed one single site as phosphoacceptor motif for ERK kinase. The presence of

ERK phosphorylation site and the docking site suggests an important role of ERK kinase in

modulating the signals in the proline biosynthetic pathway. The effect of phosphorylation

however, has to be determined by experimental methods.

Table 26: ERK phosphorylation site and the docking site on enzymes and transcriptionfactors involved in proline metabolism.Protein ERK Phosphorylation site ERK Docking site

Amino

acid No.

Amino acid

composition

Amino

acid No.

Amino acid

composition

P5CS1 362

403

TP

SP

388

471

KKTEVADGLVL

REEIPDLLKL

PRODH 18

217

SP

SP

37

300

KPEVDLDL

KDAGERLHL

P5CDH 111

351

SP

TP

206

453

FNFP

KKDQLPLVL

AtbZIP25 71 SP 141 KSKLEL

Chapter 4 Results


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