g biomac article in press internationalare abbreviated as cpss, ecss, ehss, gkss, hsss, mass, mjss,...

G

B

St

1

2

KQ1

K3

4

a5b6c7

8

a9

10

A11

R12

R13

A14

A

15

K16

S17

P18

P19

H20

T21

122

23

a24

o25

n26

o27

a28

c29

[30

n31

p32

g33

T34

c35

a36

t37

h38

D39

e40

c41

p42

0h

ARTICLE IN PRESS Model

IOMAC 3437 1–13

International Journal of Biological Macromolecules xxx (2012) xxx– xxx

Contents lists available at SciVerse ScienceDirect

International Journal of Biological Macromolecules

jo u r n al hom epa ge: ww w.elsev ier .com/ locate / i jb iomac

tructure of SAICAR synthetase from Pyrococcus horikoshii OT3: Insights intohermal stability

avyashree Manjunatha, Shankar Prasad Kanaujiab, Surekha Kanagaraj c, Jeyaraman Jeyakanthanc,anagaraj Sekara,∗

Supercomputer Education and Research Centre, Indian Institute of Science, Bangalore 560 012, IndiaDepartment of Biotechnology, Indian Institute of Technology, Guwahati 781 039, IndiaDepartment of Bioinformatics, Alagappa University, Karaikudi 630 003, Tamilnadu, India

r t i c l e i n f o

rticle history:eceived 29 August 2012eceived in revised form 25 October 2012ccepted 26 October 2012vailable online xxx

a b s t r a c t

The first native crystal structure of Phosphoribosylaminoimidazole-succinocarboxamide synthetase(SAICAR synthetase) from a hyperthermophilic organism Pyrococcus horikoshii OT3 was determined intwo space groups H3 (Type-1: Resolution 2.35 A) and in C2221 (Type-2: Resolution 1.9 A). Both are dimericbut Type-1 structure exhibited hexameric arrangement due to the presence of cadmium ions. A compar-ison has been made on the sequence and structures of all SAICAR synthetases to better understand the

eywords:AICAR synthetaseurine de novo biosynthesisyrococcus horikoshii OT3yperthermophile

differences between mesophilic, thermophilic and hyperthermophilic SAICAR synthetases. These SAICARsynthetases are reasonably similar in sequence and three-dimensional structure; however, differenceswere visible only in the subtler details of percentage composition of the sequences, salt bridge interactionsand non-polar contact areas.

© 2012 Published by Elsevier B.V.

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hermostable proteins

. Introduction

Pyrococcus horikoshii OT3 is a hyperthermophilic anaerobicrchaeon which was isolated from the hydrothermal fluid samplesf Okinawa trough vents at a depth of 1395 m [1]. These orga-isms grow at an optimal temperature of 98 ◦C, but are capablef surviving at 105 ◦C over the pH range of 5–8 (optimal at pH 7)nd NaCl concentration of 1–5%, (optimal value of 2.4%) [1]. Theomplete genome sequence of this organism has been determined2]. These extremophiles have evolved using highly robust mecha-isms to adapt to the extreme conditions with exceptionally stableroteins [3,4]. A recent review [5] describes the different strate-ies adopted by them to survive at extremely high temperature.o mention a few, these organisms have histones to facilitate DNAompaction, high concentrations of linear polyamines (sperminesnd spermidines) to stabilize DNA and branched chain polyamineso stabilize tRNA. The reverse gyrase, which is present only inyperthermophiles, provides a positive superhelical structure toNA, stabilizing it further. In addition, they exhibit various differ-

Please cite this article in press as: K. Manjunath, et al., Int. J. Biol. Macromo

nces in protein sequences and structures. Many studies have beenarried out to reveal the possible evolutionary strategies of suchroteins [6–15].

∗ Corresponding author. Tel.: +91 80 22933059/22933060; fax: +91 80 23600683.E-mail addresses: [email protected], [email protected] (K. Sekar).

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141-8130/$ – see front matter © 2012 Published by Elsevier B.V.ttp://dx.doi.org/10.1016/j.ijbiomac.2012.10.028

Extensive experimental and theoretical studies have been car-

ried out exploring the sequence, structure and dynamic nature

discerning mesophilic, thermophilic and hyperthermophilic pro-

teins. Statistical studies on a large number of protein sequences

from mesophiles, thermophiles and hyperthermophiles have come

up with several observations unique to thermostable proteins.

Presence of higher Ala, preference for Lys to Arg, higher per-

centage of charged residues, lesser polar uncharged residues and

higher hydrophobic residues are some of the important observa-

tions [9]. Structural studies carried out on highly thermostable

proteins from hyperthermophilic bacteria Thermatoga maritima

[16] concluded that protein adopt different strategies for ther-

mostability mainly involving hydrophobic and ionic interactions.

Thermostable proteins exhibit various features like, enhanced

hydrophobic core [17] (with some exceptions [18]), increased salt-

bridges [13,19], higher aromatic and cation-pi interactions [9] and

shorter loop regions [20]. They also exhibit increased hydrogen

bonding interactions [21] (except in few cases [22]), disulfide bonds

[23], higher oligomerization states [24] and less number of cavities

[22]. Further, some evidences support that at ordinary temper-

atures, hyperthermophilic proteins are less flexible compared to

their mesophilic homologues [25], however, some studies disagree

l. (2012), http://dx.doi.org/10.1016/j.ijbiomac.2012.10.028

with this observation [26]. 65

The present work is based on an enzyme SAICAR synthetase (238 66

residues; 27,436 Da) from a hyperthermophilic organism, Pyrococ- 67

cus horikoshii OT3. This enzyme is involved in the de novo purine 68

dx.doi.org/10.1016/j.ijbiomac.2012.10.028


http://www.sciencedirect.com/science/journal/01418130

http://www.elsevier.com/locate/ijbiomac

mailto:[email protected]

mailto:[email protected]


ARTICLE IN PRESSG Model

BIOMAC 3437 1–13

2 K. Manjunath et al. / International Journal of Biological Macromolecules xxx (2012) xxx– xxx

esis pa

b69

s70

s71

b72

fi73

[74

i75

T76

r77

(78

s79

i80

b81

82

m83

c84

(85

(86

i87

o88

b89

t90

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(92

i93

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195

E96

M97

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l100

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Fig. 1. De novo purine biosynth

iosynthesis pathway. Nucleotides are bio-synthesized either inalvage pathway by joining already available bases with riboseugar units or de novo pathway where nucleotide bases are assem-led from simpler compounds. Purine nucleotide biosynthesis wasrst described by Buchanan [27]. De novo purine biosynthesis28] consists of 11 steps in bacteria, fungi and only ten stepsn some archaea and higher eukaryotes including humans [29].he difference arises during the conversion of 5-aminoimidazoleibonucleotide (AIR) to carboxyaminoimidazole ribonucleotideCAIR) [30,31]. The organism Pyrococcus horikoshii OT3 utilizes aingle step catalysis by AIR carboxylase (PurE class II) as illustratedn Fig. 1. Interestingly, the enzymes involved in the de novo purineiosynthesis pathway can be important drug targets [32,33].

The enzyme SAICAR synthetase (E.C. 6.3.2.6) catalyzes the for-ation of N-succinyl-5-aminoimidazole-4-carboxamide ribonu-

leotide (SAICAR) from carboxyaminoimidazole ribonucleotideCAIR) [34] and aspartic acid in the presence of ATP. In archaeapurC), bacteria (purC), fungi (ADE1) and plants (pur7), this reactions catalyzed by a mono-functional enzyme but in higher eukary-tes, it is catalyzed by a bifunctional enzyme PAICS which hasoth AIR carboxylase and SAICAR synthetase activity. A total of 16hree-dimensional crystals structures of SAICAR synthetase fromifferent organisms have been deposited in the Protein Data BankPDB). The first crystal structure was solved from S. cerevisiae (PDB-d 1a48; [35]) at a resolution of 1.9 A. Subsequently, several crystaltructures of the native and its complex from S. cerevisiae (PDB-ids:obg, 1obd, 2cnu, 2cnv and 2cnq), T. maritima (PDB-id: 1kut [36]),. coli (PDB-ids: 2gqs and 2gqr [37]), G. kaustophilus (PDB-id: 2ywv),. jannaschii (PDB-ids: 2z02 and 2yzl), E. chaffeensis (PDB-id: 3kre),

. perfringens (PDB-id: 3nua), H. sapiens (PDB-id: 2h31 [38]) and M.


bscessus (PDB-id: 3r9r) have been reported. According to the pub-ished reports, the enzyme is a monomer in S. cerevisiae, a covalentimer in T. maritima and a non-covalent dimer in E. coli, while theifunctional enzyme PAICS in human is an octamer. In the present

thway in Pyrococcus horikoshii.

work, we report the crystal structure of the native SAICAR syn-

thetase from P. horikoshii OT3 in two different space groups. It is

noteworthy that this is the first uncomplexed SAICAR synthetase

structure from a hyperthermophilic organism. The sequence and

structures of all reported SAICAR synthetases have been examined

to distinguish between mesophilic, thermophilic and hyperther-

mophilic proteins. In the following text SAICAR synthetase from

C. perfringens, E. coli, E. chaffeensis, G. kaustophilus, H. sapiens, M.

abscessus, M. jannaschii, P. horikoshii, S. cerevisiae and T. maritima

are abbreviated as CpSS, EcSS, EhSS, GkSS, HsSS, MaSS, MjSS, PhSS,

ScSS and TmSS, respectively.

2. Materials and methods

2.1. Protein purification

The protein SAICAR synthetase was purified according to the

protocol mentioned in our previous work [39] with slight modifi-

cations. The clone of gene PH0239 in pET11a was transformed into

E. coli BL21-CodonPlus (DE3)-RIL cells. The transformed colonies

were grown at 37 ◦C in LB media containing 50 �g/ml of ampicillin

and 34 �g/ml chloramphenicol. After a post induction (0.05 mM

IPTG) growth of 4 hrs, cells were pelleted, re-suspended in lysis

solution and lysed by sonication. After heat treatment and centrifu-

gation of lysate, solution was desalted using Sephadex G-25 (GE

Healthcare) desalting column. The desalted protein solution was

loaded on to an anion exchange column, Sepharose Q (GE Health-

care), and eluted with a linear gradient of 0–0.5 M NaCl in buffer

A (20 mM Tris–HCl, pH 8.0). The fractions containing protein was

concentrated and loaded onto Sephacryl S200 (GE Healthcare) gel


filtration column, pre-equilibrated with buffer A containing 0.2 M 130

NaCl. Fractions containing pure protein were pooled and concen- 131

trated to 10–14 mg/ml as determined by measuring the absorbance 132

at 280 nm. The purity of the protein was confirmed by SDS–PAGE. 133


IN PRESSG Model

B

f Biological Macromolecules xxx (2012) xxx– xxx 3

U134

w135

2136

137

t138

o139

(140

o141

u142

o143

c144

s145

e146

w147

2148

c149

150

s151

s152

d153

s154

t155

a156

e157

w158

u159

a160

g161

d162

a163

r164

l165

S166

n167

a168

a169

t170

o171

s172

u173

c174

s175

t176

f177

P178

i179

t180

t181

f182

t183

u184

w185

g186

r187

o188

R189

f190

e191

r192

m193

3194

f195

Table 1Data collection and refinement statistics for Type-1 and Type-2 crystals. Q3

Type-1 Type-2

Data collection and processing statisticsWavelength (Å) 1.5418 1.5418Temperature (K) 100 100Crystal-to-detector-distance (mm) 200 150Space group H3 C2221

Unit cell parameters (Å) a = b = 95.42;c = 148.63

a = 44.10;b = 155.39;c = 78.35

Resolution range (Å) 55.26–2.35(2.48–2.35)

42.42–1.90(2.0–1.9)

Observed reflections 86,788 113,532Unique reflections 21,057 (3112) 21,450 (3041)Completeness (%) 100.0 (100.0) 98.9 (97.5)Rmerge (%) 8.2 (40.0) 4.2 (19.1)〈I/�(I)〉 13.2 (3.1) 24.1 (8.0)Multiplicity 4.1 (4.0) 5.3 (5.2)Matthews coefficient (Å Da−1) 2.37 2.45Solvent content 48.2 49.8Z 2 1

Refinement statisticsRwork (%) 23.5 18.4Rfree (%) 28.6 22.9

Protein modelProtein atoms 3529 1806Water oxygen atoms 165 211Metal ions (Cd2+) 12 –Others (BU1, SO4, ACT) 1BU1, 4 SO4, 1 ACT 2 SO4, 1ACT

RMS deviations from ideal geometriesBond lengths (Å) 0.012 0.007Bond angles (◦) 1.29 0.99

Average temperature factors (Å2)Protein atoms 25.38 22.36Water molecules 25.37 29.75Metals 52.69 -Others 43.98 35.01

Ramachandran statistics (%)Most favored 89.5 91.0Additionally allowed 10.0 8.5Generously allowed region 0.5 0.5

† Rmerge =∑

h k l

∑i|Ii(h k l) −

⟨I(h k l)

⟩|/∑

h k l

∑iIi(h k l), where Ii(h k l) is the ith

196

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212

ARTICLEIOMAC 3437 1–13

K. Manjunath et al. / International Journal o

nless otherwise mentioned, all the columns used in purificationere pre-equilibrated in buffer A.

.2. Crystallization

The purified protein sample was screened for crystalliza-ion conditions using Hampton crystal screen kits. Crystals werebtained in two different space groups, H3 (Type-1) and C2221Type-2). Crystallization condition for Type-1 is described previ-usly [39]. Type-2 crystals were obtained in another conditionsing the under oil method. Crystallization drop contained 1 �lf (∼10 mg/ml) protein and 1 �l of condition number 13 ofrystal screen kit II, containing 0.2 M ammonium sulfate, 0.1 Modium acetate trihydrate, pH 4.6 and 30% (w/v) PEG monomethylther 2000. Well-shaped good quality diffracting crystals appearedithin a week.

.3. Data collection, structure solution and refinement of Type-1rystal

For Type-1 crystal, the MAD data (collected previously usingynchrotron) had some problems during the refinement in P31pace group with a hexamer in the asymmetric unit. A secondata of SeMet protein crystal was collected at 100 K at the homeource using Cu K� radiation on a MAR345 image plate detec-or with a Bruker Microstar Ultra rotating anode X-ray generatorvailable at the Molecular Biophysics Unit, Indian Institute of Sci-nce, Bangalore. The crystal diffracted up to 2.35 A resolution whichas indexed and processed in the space group H3 (sg. no. 146)sing IMOSFLM [40]. The crystallization condition had cadmiumnd initial scaling indicated an overall RMS correlation ratio (RCR)reater than one (1.227), thus, the anomalous pairs were separateduring merging in SCALA. The data had a completeness of 100%nd anomalous completeness of 100%. The overall Rmerge for alleflections was 8.2% with an average mosaicity of 0.57◦. Data col-ection and processing statistics are given in Table 1. The programFCHECK [41] indicated the presence of a twinning, with a twin-ing fraction of 0.064, which was later supported by H-test, L-testnd Britton analysis, confirming a mild partial hemihedral twinninglong (k h −l). It also indicated the tentative presence of a pseudo-ranslation with 21.2% of peak at the origin, but more than otherff-origin peaks, along the vector (0.667, 0.333, 0.000). Preliminarytudies suggested the presence of two chains in the asymmetricnit with a total cell volume of 1172271.5 A3, Matthews’s coeffi-ient (Vm) [42] of 2.37 A3/Da and 48.2% solvent content. Structureolution was obtained by molecular replacement calculations usinghe three-dimensional atomic coordinates of SAICAR synthetaserom M. jannaschii (PDB-id: 2yzl; sequence identity of 49.6%) usingHASER [43]. Refinement was carried out using REFMAC [44] withntermediate rounds of model building using COOT [45]. Of theotal reflections, 5% were set aside for calculating Rfree value andhe refinement was carried out without twinning, as the twinningraction was found to be negligible. Non-crystallographic symme-ry restraints were applied to both the chains in the asymmetricnit and were maintained till the final refinement. Cadmium ionsere located using anomalous peak search using CAD and FFT pro-

rams available in CCP4 package. The Rwork and Rfree of the finalefined model was 23.5% and 28.6%, respectively. The validationf the structure was carried out using ADIT server available inCSB. The final structure had 89.5% of the residues in the most

avored, 10% in additionally allowed and remaining 0.5% in gen-rously allowed regions of the Ramachandran plot [46]. The final


efinement statistics are summarized in Table 1. The final refinedodel (Fig. 2a) consisted of two chains in the asymmetric unit with

529 protein atoms, 165 water oxygen atoms, 12 cadmium ions,our sulfate ions, one acetate ion and one 1,4-butanediol molecule.

observed intensity and 〈I(h k l)〉 is the weighted average intensity for multiple mea-surements. Values within the parenthesis correspond to the outermost resolutionshell.

The atomic coordinates and structure factors (PDB-id: 3U54) have

been deposited in PDB.

2.4. Data collection, structure solution and refinement of Type-2crystal

Type-2 crystal data were collected at 100 K using the home

source. The crystal diffracted up to a resolution of 1.9 A which was

indexed and processed in C2221 space group using IMOSFLM. The

data had an overall completeness of 98.9% with an overall Rmerge of

4.2% (mosaicity = 0.94◦). The data collection and processing statis-

tics are given in Table 1. The cell content analysis indicated a single

polypeptide chain in the asymmetric unit with a total cell vol-

ume 536909.3 A3 (Vm = 2.45 A3/Da and solvent content = 49.8%). The

Type-1 (chain A) structure was used as a search model to obtain the

solution for Type-2 data using the program PHASER. The refinement

and model building was carried out using REFMAC [44] and COOT

[45], respectively. A total of 5% of the reflections were set aside for

calculating Rfree value. At the end of refinement, after several rounds


of model building, the Rwork and Rfree were reduced to 18.4% and 213

22.9%, respectively. The validation of the structures was carried out 214

using ADIT server. The final refined model had 91% of the residues 215

in the most favored, 8.5% in the additionally allowed and remaining 216


ARTICLE ING Model

BIOMAC 3437 1–13

4 K. Manjunath et al. / International Journal of Biol

Fig. 2. The overall three-dimensional structure of (a) Type-1 crystal is showntogether with the secondary structural elements. The cadmium ions of the twochains are labeled and colored in violet and green, respectively. The sulfate ions,Q2acetate ion and butanediol are also labeled. (b) The overall three-dimensionalsit

0217

fi218

m219

n220

f221

f222

2223

224

C225

d226

d227

t228

i229

s230

[231

a232

g233

d234

t235

236

237

238

239

240

241

242

243

244

245

246

247

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249

250

251

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293

tructure of Type-2 crystal is shown along with the sulfate and acetate ions. (Fornterpretation of the references to color in this figure legend, the reader is referredo the web version of the article.)

.5% in generously allowed regions of the Ramachandran plot. Thenal refinement statistics are summarized in Table 1. The refinedodel (Fig. 2b) has one chain in the asymmetric unit with 1806

on-hydrogen protein atoms, 211 water oxygen atoms, two sul-ate ions and an acetate ion. The atomic coordinates and structureactors (PDB-id: 3U55) have been deposited in the PDB.

.5. Sequence and structure analysis

The sequences were retrieved from UNIPROT, the programlustalW [47] was used for multiple sequence alignment and ren-ered using ESPript [48]. The three-dimensional structures wereownloaded from PDB. Incomplete structures were modeled usinghe server SWISS-MODEL [49–51] and COOT was used for build-ng the missing residues. Energy minimization was done for thetructures (that were partially modeled) using GROMACS v4.5.352], with OPLS-AA (optimized potentials for liquid simulations all


tom) force field [53] and TIP4P water model [54] using conjugateradient method with convergence criteria of 1 kJ mol−1 nm−1. Aodecahedron box was chosen with a distance of 1.2 nm betweenhe protein and the box wall. The charges of the system were

PRESSogical Macromolecules xxx (2012) xxx– xxx

neutralized by replacing the water molecules with either Na+ or

Cl− ions. The long-range electrostatic interactions were treated

with Particle Mesh Ewald (PME) method [55] with a Fourier spac-

ing of 0.12 nm combined with the fourth order interpolation. The

short-range neighbor interactions, columbic and vdw cut-offs were

1.4 nm, 1.4 nm and 1.0 nm, respectively. A switch potential was

applied from 0.9 nm onwards for treating vdw forces. The bond

lengths were constrained with the LINCS algorithm [56]. Struc-

tural alignments were carried out using MUSTANG [57]. The PISA

(http://www.ebi.ac.uk/pdbe/prot int/pistart.html) web server [58]

was used to analyze the interfaces and quaternary structures. In

house server PDB Goodies [59] was used for some calculations

in the pdb file. The figures were generated using PyMOL (DeLano

Scientific; http://www.pymol.org). The plugin DSSP [60] was used

to generate secondary structures. Volume calculations were done

using the server 3V: Voss Volume Voxelator [61], radius of gyra-tion calculation of each protein structures was carried out usingHYDROPRO utility [62]. Secondary structure contents were calcu-lated using 2Struc server [63]. HBPLUS [64] was used for calculating

hydrogen bonds (with donor–acceptor cut-off distance of 3.5 A and

donor–hydrogen–acceptor angle to be at least 90◦) and salt-bridges

were detected using WHATIF server [65]. The atomic accessibility

was deduced using the program NACCESS [66]. NACCESS provides

the absolute accessibility RSA (residue surface accessibility) val-

ues of each residue, which is the sum of atomic accessibility of

the corresponding residues. The non-polar contact areas in pro-

tein were implemented using pdb np cont and clustering was done

using pdb np clus programs [67]. In addition, locally generated PERL

scripts were also used in the structure analysis.

3. Results and discussion

3.1. Structure description

3.1.1. OverviewAmong the nine known enzymes involved in the purine biosyn-

thetic pathway in P. horikoshii, six enzymes utilize ATP for the

catalysis [GAR synthetase (PurD), FGAR synthetase (PurT), FGAM

synthetase II (PurLQS), AIR synthetase (PurM), SAICAR synthetase

(PurC) and FAICAR synthetase (PurP)]. The two domains of the

enzyme SAICAR synthetase (PurC) correspond to the two domains

[‘C’ substrate-binding and ‘B’ ATP-binding domains] of ATP-grasp

family. However, it lacks the corresponding ‘A’ domain present in

the ATP-grasp family [68]. According to the SCOP classification,

the enzyme SAICAR synthetase from P. horikoshii OT3 belongs to

� + � architecture. The protein crystallized in two different space

groups, namely, H3 (Type-1, Fig. 2a) with two chains in the asym-

metric unit and C2221 (Type-2, Fig. 2b) with a single chain in the

asymmetric unit. The single chain has an approximate dimension

of 50 A × 50 A × 40 A with two domains (small and large). A small

domain ‘A’ (residues 12–81) includes six beta strands (�1–�6) and

a �-helix (�1). The large domain ‘B’ (residues 82–238) consist-

ing of eight �-strands (�7–�14), five helices (�2–�6) and a 310helix. The electron density is clearly visible only from the 12th

residue onwards. The mass spectrometric analysis on the protein

indicated (data not shown) that almost 90% of the species in the

sample have a mass corresponding to the full length protein. But,

the SeMet derivatized protein showed multiple peaks correspond-

ing to the full length and fragmented proteins. Thus, it is difficult

to say whether the missing region (first eleven residues) is due to

cleavage or because it is disordered.


The Type-1 refined model contains two chains in the asym- 294

metric unit (A and B) (Fig. 2a) with 3529 non-hydrogen protein 295

atoms, 165 water oxygen atoms, 12 cadmium (Cd2+) ions, four 296

sulfate ions, an acetate ion and a 1,4-butanediol molecule. The 297


http://www.ebi.ac.uk/pdbe/prot_int/pistart.html

http://www.pymol.org/

IN PRESSG Model

B

f Biological Macromolecules xxx (2012) xxx– xxx 5

o298

i299

c300

c301

C302

C303

t304

f305

fi306

d307

a308

n309

o310

G311

a312

A313

m314

f315

i316

d317

a318

m319

e320

a321

e322

c323

o324

t325

r326

d327

g328

S329

d330

b331

T332

a333

m334

(335

i336

a337

d338

o339

a340

s341

u342

o343

(344

f345

t346

L347

t348

i349

V350

t351

f352

G353

f354

i355

t356

(357

A358

r359

a360

361

i362

w363

Fig. 3. (a) Hexameric state of Type-1 crystal. Small domain is colored in red andthe large domain is colored in gold, cadmium ions are colored in violet, the truedimeric interface (interface-1) is colored blue and the pseudo-interface (interface-2) is colored green, (b) the identical true dimeric interface observed in Type-1 andType-2 forms. The interface is colored in blue and the small and large domains are

364

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373



verall RMSD (root mean square deviation) between the two chainss 0.13 A and no significant deviations were observed among thehains except near the N-terminal region. The cadmium sites wereonfirmed by the presence of anomalous peaks calculated usingCP4 tools. Among the 12 Cd2+ in the refined model, three ionsD400(A), CD401(A) and CD401(B) form a very strong coordina-ion with both the chains leading to a pseudo-interface (differentrom the true interface) as indicated (asymmetric unit) in thegure (Fig. 2a). The residues Glu158(A,B) and Asp162(A,B) coor-inate CD400(A), Glu111(B) and His63(A) coordinate CD401(A)nd Glu111(A), His63(B) and a 1,4-butanediol molecule coordi-ates CD401(B). The ions CD402(A,B), CD403(A,B) and CD404(A,B)ccupy identical positions in their respective chains. The residueslu89(A,B) and Asp128(A,B) coordinate CD402(A,B), His127(A,B)nd Asp35(A,B) coordinate CD403(A,B). Finally, Glu81(A,B) andsp181(A,B) coordinate CD404(A,B). In addition, there are waterolecules in the coordination shells of cadmium but are not uni-

ormly visible in both the chains. The remaining three cadmiumons are present in the positions unique to each chain. The coor-inating acidic residues Glu125 and Asp124 are 4.07 A and 4.31 Away, respectively, which is relatively far from CD405(A). The cad-ium ion CD405(B) is coordinated by Glu125(B), the symmetry

quivalent residues of chain A [Asp21(A), Asp19(A) and Lys22(A)]nd a water molecule HOH315(A). The above four residues aressential for the coordination but, the corresponding position inhain A does not have a cadmium ion which may be due the lackf similar combination of the four residues which are involved inhe coordination. The cadmium ion CD406(B) is coordinated by theesidues Glu226(B) and Glu229(B). The sulfate ions SO4420(A,B),esignated as the first sulfate ion, have ionic interaction with theuanidinium group of Arg93, Arg198 and backbone nitrogen ofer99 in their respective chains. Other sulfate ions SO4421(A,B),esignated as second sulfate ion, coordinate with Lys210, back-one nitrogen of Phe34 and Arg214 in their corresponding chains.he PISA server predicted that the interface between chain B and

symmetry equivalent (−y, x − y, z) molecule of chain A forms theost probable or true interface with an interfacial area of 1111.5 A2

with a predicted solvation free energy gain upon formation of thenterface, �iG of −18.4 kcal/mol). The dimeric orientation in thesymmetric unit does not represent the true orientation of theimer in solution, because the interfacial area is 241.5 A2 (�iGf −1.1 kcal/mol). The server predicted two types of quaternaryrrangements, the first being a hexamer (Fig. 3a) with a buriedurface area of 16,350 A2 (with the solvation free energy gainpon formation of assembly �intG −432.4 kcal/mol) and the sec-nd being a dimer (Fig. 3b) with a buried surface area of 4530 A2

�intG −132.2 kcal/mol). In the dimeric assembly, the true inter-ace consists of 32 residues (Leu100 to Leu106, Tyr110, Leu112o Leu119, Tyr121, Asn123, Leu126, Pro129 to His135, Lys137 toeu139, Lys147, Glu150 and Leu154) from each chain (A and B). Aotal of four residues from each chain form hydrogen bonds at thenterface [Asn132(B) with Val117(A), Tyr134(B) with Glu150(A),al117(B) with Asn132(A) and Glu150(B) with Tyr134(A)]. Further,

hree residues from each chain form salt bridges at the inter-ace [His135(B) with Glu118(A), Glu118(B) with His135(A) andlu118(B) with His135(A)]. In the hexameric assembly, one (inter-

ace 1) of the interfaces is same as the true interface observedn the dimeric assembly and the other (interface 2) interface ishe pseudo-interface having 12 residues from each chain, with tenHis63, Glu111, Pro113, Glu114, Lys155, Glu158, Lys161, Asp162,la165 and Lys166) common residues from chains A and B. Theemaining two residues are Glu62 and Ile159 (chain A) and Leu112


nd Ile170 (chain B).The Type-2 refined model has a single polypeptide chain (Fig. 2b)

n the asymmetric unit with 1806 non-hydrogen protein atoms, 211ater molecules, two sulfate ions and an acetate ion. The sulfate

colored in red and gold, respectively. (For interpretation of the references to colorin this figure legend, the reader is referred to the web version of the article.)

ions are coordinated to the same residues as found in Type-1 struc-

ture. The acetate ion is found interacting with the guanidinium

group of Arg103, Met130 and a water molecule. The quaternary

structure search using the server PISA predicted only a dimeric

assembly with an interface area of 1159 A2 (�iG = −17.7 kcal/mol)

which is similar to the true interface/interface-1 of Type-1 struc-ture. As opposed to Type-1 structure, in this case, a hexamer was not

predicted and all the identified residues at the true dimeric inter-

face of Type-1 (except Lys147) are also present in this structure.

In addition to the hydrogen bonds and salt bridges at the interface


of Type-1 structure, two more hydrogen bonds [Arg103(A) with 374

Met130(A′) and Met130(A) with Arg103(A′)] and an additional salt 375

bridge are found [between His135(A) and Glu118(A′) (the symme- 376

try equivalent molecule is given as A′)]. 377


ARTICLE ING Model

BIOMAC 3437 1–13

6 K. Manjunath et al. / International Journal of Biol

Fig. 4. The superposition of Type-1 and Type-2 structures. Secondary structures � –helices (purple), � – sheets (orange), 310 helices (green) and turns (cyan) are assignedbased on DSSP and colored accordingly in both the structures. Type-1 structure iscolored in light shade while Type-2 structure is colored in dark shade. It can beobserved that only Type-1 structure has a second 310 helix while it is a turn in Type-2ct

3378

379

T380

�381

h382

o383

T384

a385

r386

(387

o388

T389

s390

n391

t392

t393

2394

L395

t396

t397

d398

i399

d400

b401

c402

b403

n404

s405

3406

407

E408

t409

w410

L411

g412

t413

414

415

416

417

418

419

420

421

422

423

424

425

426

427

428

429

430

431

432

433

434

435

436

437

438

439

440

441

442

443

444

445

446

447

448

449

450

451

452

453

454

455

456

457

458

459

460

461

462

463

464

465

466

467

468

469

470

471

472

473

structure. All the deviating regions (>0.5 A) are labeled and the cadmium ions areolored yellow. (For interpretation of the references to color in this figure legend,he reader is referred to the web version of the article.)

.1.2. Comparison of Type-1 and Type-2 structuresMinor differences are found when the topologies of Type-1 and

ype-2 structures are examined. A short 310 helix between �3 and4 is found in both Type-1 and Type-2 structures, but, a second 310elix between the strands �9 and �10 is present only in the chainsf Type-1 structure while it is a turn in the Type-2 structure. Theype-1 (chain B) and Type-2 structures superpose with an over-ll RMSD of 0.62 A. The residues deviating more than 0.5 A (nineegions) are labeled in the Fig. 4. Two of the most deviating regionsAsp29 to Gly44 and Lys122 to Asp128) are present near the vicinityf the cadmium ions CD402(B), CD403(B) and CD405(B) (Type-1).he other two regions (Val96 to Ile116 and Arg214 to Lys217) areubstantially deviated (especially the former) even though they areot coordinated to any cadmium ions. However, the region (Val96o Ile116) is very crucial as it contributes to the true interface ofhe dimer. Upon comparing the two structures (Type-1 and Type-), it is quite clear that when the two regions, Asp29 to Gly44 andys122 to Asp128, are moved toward each other as seen in Type-1,here will be a drastic conformational change in the region, Val96o Ile116, located at the true dimeric interface. These structuraleviations may be correlated to a situation similar to the bind-

ng of the ligands ATP or CAIR at the active site influencing theeviations at the dimeric interface. The Type-1 structure resem-les more, although not completely, to other SAICAR synthetaseomplex structures. Thus, it may be concluded that the cadmiuminding induces the structural deviation which is significant onlyear the active site and provides a possible clue to the allostericignal between the active site and the true interface.

.1.3. Substrate binding sites in SAICAR synthetaseThe PhSS structures (Type-1 and Type-2) are compared with

cSS and ScSS to identify a probable substrate binding region andhe residues in the active site of PhSS. The structure superposition


ith EcSS shows an RMSD of 1.35 A for 223 residues. The residuesys11, Ala12 and Lys13 which stabilizes the � and � phosphateroups of ADP in EcSS are conserved in PhSS, although the elec-ron density of Lys11 is not visible in PhSS. The residues Leu24


and Met86 in EcSS interacting with the adenine ring of ADP corre-

spond to Leu23 and Met85 in PhSS. From the above, it is clear that

these residues (Lys11, Ala12, Lys13, Leu23 and Met85) are essential

for the binding of ATP. Thus, the N-terminal residues along with

the seven beta strands (�12, �13, �1, �2, �6, �5 and �7) and a

loop connecting �13 and �14 form the binding pocket for ATP. The

conserved residues Arg93, Ser99 and Arg198 of PhSS are probably

the CAIR binding residues which correspond to Arg94, Ser100 and

Arg199 in EcSS. The position of the first sulfate ion, in both Type-1

and Type-2, corresponds to the position of the phosphate group of

CAIR in EcSS structure. Further, the position of the second sulfate

ion corresponds to the carboxylic group of SAICAR moiety in ScSS

or the hetero-atom position in the aspartic acid (Asp1308) bound

structure of ScSS (PDB-id: 2cnu). The platform of the cleft for the

binding of CAIR is formed by the beta strands �8, �9, �11 and �14

while 310, �4, loops between �9–�10 and �3–�4 form a supporting

ridge like structure for the binding of CAIR. When compared to thestructure of ScSS (PDB-id: 2cnu), it is believed that the most proba-ble binding site of aspartic acid is near the turn between �3 and �4

where the second sulfate ion was found in the present structures.

3.1.4. Dimeric interfaceType-1 structure was predicted as a hexamer (Fig. 3a) by PISA

server. This hexamer is a dimer of trimers placed one above the

other with a 60◦ rotation with respect to each other. In this arrange-

ment, two different dimeric interfaces are observed and one of

them is the crystallographic interface (pseudo-interface/interface-

2) held by strong coordinating interaction with the cadmium ions.

The other is the true dimeric interface/interface-1 as predicted in

Type-2 structure. The absence of a hexameric orientation in Type-

2 structure indicates that it may be a crystallographic artifact. As

described earlier, the interfacial residues of the true dimeric assem-

bly of both Type-1 and Type-2 structures (Fig. 3b) are formed by the

residues from a 310 helix (which is common in Type-1 and Type-

2), �9, �10 and �2. Examining the oligomeric assembly of SAICAR

synthetases from different organisms shows that the structures of

EcSS, EhSS, GkSS, CpSS, MjSS, and TmSS are dimeric while the struc-

tures of ScSS and MaSS exist as monomer and the bifunctional HsSS

is an octamer. It was previously reported [69] that the structure of

EcSS exists as a trimer in solution, however, the crystal structure

revealed that it is a dimer [37]. The presence of an additional �-turn

between a 310 helix and the strand �9 (according to PhSS) probably

prevents the dimerization in the structures of MaSS and ScSS. In case

of octameric assembly of HsSS, it is clear that the SAICAR synthetase

domain from one chain has a weak interaction with the corre-

sponding domain of the other chain as observed in the dimers of

other structures. The dimeric interfacial area of SAICAR synthetase

from different organisms, EcSS (996.5 A2), EhSS (942.1 A2), GkSS

(858.9 A2), HsSS (451.1 A2), CpSS (1118.2 A2), MjSS (1053 A2), TmSS

(943.9 A2) and PhSS (1111.5 A2 for Type-1 and 1159 A2 for Type-2)

are found to be similar except for HsSS, which is significantly less

compared to all other dimers.

3.1.5. Comparison with other structures

Multiple sequence alignment of all SAICAR synthetase

sequences (Fig. 5a) shows the conservation of the two signa-

ture motifs and other additional residues (Gly10, Lys11, Lys122,

Asp190, Arg198, Arg214, Asp209, Lys210, Ala32, Asp195, Lys45

and Leu60 of PhSS). A careful examination of the PhSS structure

shows that, among these conserved residues (other than the two

signature motifs), the first four residues are near the ATP binding

region while next four residues are present near the CAIR and


aspartate binding sites. The next three residues (Ala32, Asp195 474

and Lys45) are present between the ATP and CAIR binding sites 475

and the residue Leu60 lies in the position of the helix �1. In case 476

of ScSS sequence, there are three major insertions consist of 21 477


Please cite this article in press as: K. Manjunath, et al., Int. J. Biol. Macromol. (2012), http://dx.doi.org/10.1016/j.ijbiomac.2012.10.028


BIOMAC 3437 1–13

K. Manjunath et al. / International Journal of Biological Macromolecules xxx (2012) xxx– xxx 7

Fig. 5. (a) Multiple sequence alignment of PhSS, MjSS, GkSS, CpSS, EhSS, TmSS, HsSS, EcSS, ScSS and MaSS using the program ClustalW. The signature motifs 1 and 2 arehighlighted in green box. The residues highlighted in red box are conserved in all the sequences and those outlined in blue box are semi conserved. (b) A structure basedsequence alignment obtained from Mustang (numbering is according to the residues aligned). (c) Superposition of the structures of MjSS (brown), GkSS (brown), CpSS (brown),EhSS (brown), EcSS (brown), TmSS (red), HsSS (green), ScSS (cyan), MaSS (cyan) with PhSS (dark blue). It clearly shows that the structures in brown are very similar to PhSS,while TmSS and HsSS are slightly deviated. Monomeric SAICARs (ScSS and MaSS) are significantly deviated. (For interpretation of the references to color in this figure legend,the reader is referred to the web version of the article.)



BIOMAC 3437 1–13


Fig. 5. (a) Multiple sequence alignment of PhSS, MjSS, GkSS, CpSS, EhSS, TmSS, HsSS, EcSS, ScSS and MaSS using the program ClustalW. The signature motifs 1 and 2 arehighlighted in green box. The residues highlighted in red box are conserved in all the sequences and those outlined in blue box are semi conserved. (b) A structure basedsequence alignment obtained from Mustang (numbering is according to the residues aligned). (c) Superposition of the structures of MjSS (brown), GkSS (brown), CpSS (brown),EhSS (brown), EcSS (brown), TmSS (red), HsSS (green), ScSS (cyan), MaSS (cyan) with PhSS (dark blue). It clearly shows that the structures in brown are very similar to PhSS,while TmSS and HsSS are slightly deviated. Monomeric SAICARs (ScSS and MaSS) are significantly deviated. (For interpretation of the references to color in this figure legend,t

(478

r479

S480

T481

c482

A483

s484

o485

E486

H487

P488

d489

S490

a491

s492

s493

a494

c495

s496

t497

3498

3499

500

t501

m502

S503

a504

(505

T506

507

508

509

510

511

512

513

514

515

516

517

518

519

520

521

522

523

524

525

526

527

528

529

530

531

532

533

534

he reader is referred to the web version of the article.)

Asp71 to Ala91), 13 (Tyr133 to Pro145), 18 (Asp265 to Gln282)esidues and one minor insertion of six residues (Ala247 to Gly252).imilar insertions are also observed in MaSS SAICAR synthetase.hus, it is concluded that the two signature motifs and additionalonserved residues lay the platform for the binding of CAIR andTP. In order to see any changes in the structure, a pairwisetructural alignment of all SAICAR synthetases has been carriedut against PhSS. It shows an RMSD of less than 1.36 A with CpSS,cSS, EhSS, GkSS, MjSS and an RMSD of more than 1.86 A with TmSS,sSS, MaSS and ScSS. The first five structures are more similar tohSS compared to the last four. In all the superposed structures,eviations are more pronounced at the N- and C-terminal regions.tructure based sequence alignment of all SAICAR structuresgainst PhSS (Fig. 5b) shows the conservation of all the activeite residues near the ATP or CAIR binding sites. Fig. 5c shows thetructural superposition of all SAICAR synthetases against PhSSnd it is clear that the monomeric SAICAR’s are somewhat differentompared to the dimeric SAICAR’s. From the sequence and thetructural alignments, it is difficult to delineate the mesophilic,hermophilic and hyperthermophilic SAICAR synthetases.

.2. Thermal stability analysis

.2.1. Amino acid compositionThe amino acid composition is examined to look for features

hat could delineate mesophilic, thermophilic and hyperther-ophilic SAICAR synthetases. Among them (EcSS, EhSS, CpSS, GkSS,


cSS, MaSS, HsSS, MjSS, TmSS and PhSS), two (ScSS and MaSS)re monomeric, one (HsSS) is bifunctional octamer and anotherTmSS) is a dimer with a disulfide bond between the monomers.he remaining six are non-covalent dimers. The bifunctional

enzyme (HsSS) is excluded from the composition analysis. Due

to lack of experimental results, the value of the melting temper-

ature Tm is assumed to correlate with the optimal temperature

To of the organisms. The percentages mentioned in the rest of

this section are calculated by averaging the composition of the

amino acid residues separately for mesophilic, thermophilic and

hyperthermophilic proteins. The percentage composition of each

residue (Table 2, higher percentage compositions are in bold

and lower percentage compositions are shaded) highlights that

only two (Lys and Gln) residues delineate hyperthermophiles

from mesophiles. For instance, the residue Lys is ∼4% more

in hyperthermophiles while Gln is marginally less by ∼1.6% in

hyperthermophilic structures. On examining further, in hyperther-

mophiles, the charged residues (D + E + H + R + K) together are ∼6%

more and polar uncharged residues (S + T + N + Q) are ∼7% less than

mesophiles. The value (D + E + H + R + K)/Q is observed to be extraor-

dinarily high in hyperthermophiles than the corresponding values

of thermophiles and mesophiles. This is the first report to distin-

guish hyperthermophiles from mesophiles based on the value of

the (D + E + H + R + K)/Q ratio. This could be further confirmed with

a larger data set of hyperthermophilic proteins. However, no con-

clusions could be drawn about the uniqueness of the hydrophobic

residues in hyperthermophiles. Further, the thermophilic protein

(GkSS) has less Lys residues but more Arg than hyperthermophiles,

while Ala and Leu content are more and the residue Met is less

than in mesophiles and hyperthermophiles. Surprisingly, in GkSS

(being a thermophile with To of 60 ◦C), the composition of the

charged residues (D + E + H + R + K) and polar uncharged residues


(S + T + N + Q) are comparable to mesophiles which contradicts the 535

general observation that the polar residues are less and the charged 536

residues are higher in thermophiles than mesophiles. However, 537

GkSS has a high composition of aliphatic residues (A + I + L + V) 538



BIOMAC 3437 1–13


Table 2Percentage composition of amino acids of SAICAR synthetase structures.

a

w539

t540

541

c542

o543

544

TR

(M) Mesophile; (T) Thermophile; (H) Hyperthermophile.

hich is ∼6.1% higher than mesophiles and 4.7% higher than hyper-


hermophilic proteins.Monomeric enzymes have some unique features. The per-

entage composition of Trp is ∼2.24 times greater than the restf SAICARs, while Pro is ∼3.5% greater than mesophilic and

able 3adius of gyration and secondary structures.

Source (group)a Rgb (nm) Reff

c (Å) Percentage of secondary struc

B E G

CpSS (M) 1.81 10.29 0.8 35.3 1.3

EcSS (M) 1.81 10.51 0.8 31.6 1.3

EhSS (M) 1.86 10.25 0.8 33.1 1.2

GkSS (T) 1.85 10.12 0.8 33.1 1.2

MjSS (H) 1.89 10.16 1.2 30.2 1.2

PhSS (H) 1.86 9.98 1.3 31.1 2.5

TmSS (H) 1.85 9.33 0.4 30.9 0

a (M) Mesophilic; (T) Thermophilic; (H) Hyperthermophilic.b Radius of gyration.c Effective radius – radius of a sphere whose surface area to volume ratio is same as thed Secondary structures assigned by DSSP-B, beta-bridge; E, strand; G, 310 helix; I, � hel

∼2.24% greater than thermophilic and hyperthermophilic SAICARs


contributing to the conformational rigidity. The composition 545

of polar uncharged residues (N + Q) is closer to hyperther- 546

mophilic or thermophilic than mesophilic enzymes, specifically 547

the residue Asn. These monomeric enzymes notably have more 548

turesd % Loop (S + B + I)

I H S T Coil

0 29.4 6.3 13.0 13.9 7.10 30.0 6.3 11.8 18.1 7.10 25.2 7.4 16.5 15.7 8.20 30.2 8.7 9.1 16.9 9.50 29.8 7.9 14.0 15.7 9.10 27.7 7.6 14.3 15.5 8.90 33.0 6.1 11.3 18.3 6.5

object in question. Reff in thermophiles and hyperthermophiles are highlighted.ix; H, alpha helix; S, bend; T, turn; Coil, random coil.



BIOMAC 3437 1–13


Table 4Total number of hydrogen bonds and number of buried hydrogen bonds.

Source No. of HBa MMb SSc MS/SMd Buried MM Buried SS Buried SM/MS

CpSSe 238 148 44 46 102 2 16EcSSe 224 148 40 36 98 4 11EhSSe 220 133 42 45 93 2 10GkSSf 232 134 55 43 94 6 12MjSSg 232 155 37 40 116 2 10PhSSg 220 140 45 35 94 2 10TmSSg 204 127 44 33 93 5 11

a Hydrogen bond.b Main chain – main chain hydrogen bond.c Side chain – side chain hydrogen bonds.d Main chain – side chain/side chain – main chain hydrogen bonds.

s549

(550

h551

S552

r553

554

TC

a

b

Sc

e Mesophiles.f Thermophiles.g Hyperthermophiles.

hort chain polar residues (S + T), less number of charged residues


D + E + H + K + R) compared to other SAICARs. Finally, highlyydrophobic residues (L + I + F + W + V + M) are less in monomericAICARs. Thus, it may be concluded that the monomeric SAICARsesembles mesophilic dimeric SAICARs in terms of charged and

555

556

able 5lassification of salt-bridges based on RASB values.

Total number of salt bridges. Greater number of SBs in hyperthermophiles is in bold.RASB = (Sum of absolute accessibility)/(Sum of standard accessibility) × 100. This value is

Bs in hyperthermophiles compared to mesophiles (in each RASB bins) are indicated in bolompared to mesophiles (in each RASB bins) is indicated to bold with a darker backgroun

polar (S + T) residues content. On contrary, it also resembles

thermophilic and hyperthermophilic SAICARs in terms of N + Q con-

tent. It is concluded that in the case of SAICAR synthetase, the


monomers and dimers have to be separately analyzed for thermal 557

stability. 558

calculated for the residues involved in SB formation. Relatively higher percentage ofd with lighter background. Relatively lower percentage of SBs in hyperthermophilesd.



BIOMAC 3437 1–13


Table 6Clustering of non-polar contact areas in the SAICAR synthetase structures.

Cut-offa CpSS EcSS EhSS GkSS MjSS PhSS TmSS

Nil 7072.05 7255.60 7675.05 7291.50 7820.65 7849.25 7110.205 219

7037.552247236.10

2257626.05

2277243.90

2287775.75

225-37810.25

2167076.15

10 1996873.70

2067080.55

2147541.65

2097061.15

2117599.85

206-47647.12

2046993.95

15 184-3-46733.65

185-46853.33

1937238.25

188-46857.46

193-47182.85

192-37461.39

182-36706.11

20 1736455.30

4-1756678.63

1746882.40

175-36590.46

1787051.75

179-37231.79

3-5-1506114.05

25 8-6-121-35423.40

8-6-121-105745.11

12-4-7-1306180.67

123-6-155649.36

140-96250.78

17-6-130-36505.61

11-6-113-45449.87

30 3-6-673371.91

87-33997.37

5-8-80-5-64555.05

3-6-12-613494.58

10-733776.12

91-94701.32

76-6-43867.34

31 3-3-6335.17

3-763589.88

8-60-9-5-4-64078.53

3-6-9-613395.00

7-733671.44

90-94670.93

6-75-43836.54

32 3-3145.78

23-3-41171.23

8-57-8-63675.21

3-562742.75

72-73640.22

884250.69

74-43615.63

33 3-3145.78

377.03

7-191161.27

385.60

72-63607.90

703513.83

713410.17

34 376.21

377.03

7–191161.27

385.60

– 703513.83

592924.83

35 376.21

377.03

7260.11

385.60

– 696957.72

–

36 376.21

377.03

7260.11

385.60

– 19875.20

–

37 376.21

– 7260.11

385.60

– – –

38 – – – 385.60

– – –

41 – – – 385.60

– – –

a Cut-off for the non-polar interactions. First data line is the non-polar contact area with no cut-off. Subsequent data represent the number of residues in the cluster ate ow th

3559

560

(561

l562

r563

t564

G565

y566

f567

t568

n569

m570

r571

f572

b573

t574

c575

t576

(577

‘578

t579

9580

m581

e582

t583

y584

3585

586

b587

d588

r589

590

591

592

593

594

595

596

597

598

599

600

601

602

603

604

605

606

607

608

609

610

611

612

613

614

615

616

617

sum of the standard accessibility of these residues. The resulting 618

ach cut-off. The non-polar contact area of each cluster is indicated in bold font bel

.2.2. Overall structural featuresThe structures of CpSS (3nua), EcSS (2gqr), EhSS (3kre), GkSS

2ywv), MjSS (2z02), TmSS (1kut) and PhSS (Type-2) are ana-yzed. The structures of SAICAR synthetases having the missingesidues are modeled and the missing side chains are built. Finally,he structures are energy minimized using OPLS-AA force field inROMACS, before subjecting the structures for interaction anal-sis. It is reported that the compactness of the protein increasesrom mesophiles to hyperthermophiles [14]. The radius of gyra-ion (Rg), which is a measure of the compactness of protein, didot show any distinction between the mesophilic and hyperther-ophilic/thermophilic proteins (Table 3). However, the effective

adius (Reff), which is the radius of a sphere that has the same sur-ace area to volume ratio as the protein in question, is observed toe marginally smaller in thermophilic and hyperthermophilic pro-eins (Table 3). It has been studied that the contents of the loop areomparatively less in thermostable proteins [20]. A close examina-ion on the content of (Table 3) the secondary structural elementscalculated using 2Struc according to DSSP and considering theloop’ as the sum of bend, �-bridge and �-helix) shows that thehermophilic (GkSS, 9.5%) and hyperthermophilic proteins (MjSS,.5%; PhSS, 8.9%) in fact have a higher percentage of loops thanesophilic (CpSS, 7.1%; EcSS, 7.1%; EhSS, 8.2%) proteins with an

xception of TmSS (6.5%). The atomic packing, packing density inhe protein have been investigated (data not shown), but it did notield any correlation with temperature.

.2.3. Interaction analysisIntra-molecular interactions such as hydrogen bonds and salt


ridges, in the protein structures are analyzed to decipher theifferences in the thermostability. Hydrogen bonding interactionsevealed that (Table 4), the number of hydrogen bonds is similar

e clusters.

in mesophilic, thermophilic and hyperthermophilic SAICARs. The

percentage of buried (the sum of accessibility of both the atomsinvolved in the hydrogen bonding is zero) hydrogen bonds is higher

in the case of MjSS and TmSS but not in the case of GkSS or

PhSS. Thus, among the set of proteins considered in the study,

an increased number of hydrogen bonds, compared to mesophilic

proteins, is not observed in thermophilic or hyperthermophilic pro-

teins as opposed to a general opinion that number of hydrogen

bonds increase as the thermophilicity of the protein increases. The

salt-bridge (SB) is a long-range interaction compared to the hydro-

gen bonding interaction. Salt-bridge interaction distances (distance

between the positively charged residues Arg, Lys, His and the neg-

atively charged residues Asp, Glu) which are less than or equal to

4 A are considered as strong [70], 4–6 A are weak and 6 A or more

are considered as weaker. The thermophilic/hyperthermophilic did

not have more number of strong SBs (within a cut-off distance 4 A

and 5 A) compared to mesophiles except for PhSS. However, higher

cut-off distances (6 A and 7 A) revealed a positive correlation in

the number of SBs with Tm of the protein. Thus, hyperthermophilic

proteins exhibited higher number of weaker SBs than mesophilic

proteins. In case of PhSS, the SBs are high in number (in all distance

cut-offs) compared to all other proteins, contributing a dominant

feature for stability. In GkSS, as mentioned above, the percentage

composition of the charged residues is comparable to mesophilic

proteins as a result; the number of its SBs is also closer to mesophilic

proteins. In order to calculate the relative accessibility of the salt-

bridges, the absolute accessibility value (RSA) of the residue pairs

involved in the SB formation is added and the sum is divided by the


ratio is multiplied by 100. This value is designated as RASB (rel- 619

ative accessibility of SBs). Within each distance cutoff (4–7 A) of 620

SBs, the percentage of SBs with certain value of RASBs is clustered 621


ING Model

B

1 of Biol

t622

t623

S624

b625

i626

s627

c628

t629

S630

a631

s632

t633

t634

635

k636

i637

s638

i639

t640

s641

e642

c643

c644

r645

c646

e647

o648

G649

a650

t651

T652

t653

(654

a655

a656

H657

e658

p659

4660

661

h662

t663

b664

a665

a666

h667

d668

S669

h670

D671

o672

t673

e674

w675

a676

e677

T678

b679

p680

a681

t682

T683

h684

685

686

687

688

689

690

691

692

693

694

695

696

697

698

699

700

701

702

703

704

705

706

707

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712

713

714

715

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717

[ 718

719

[ 720

[ 721

[ 722

723

724

[ 725

726

[ 727

728

[ 729

730

[ 731

732

[ 733

[ 734

[ 735

[ 736

[ 737

[ 738

739

[ 740

[ 741

742

[ 743

744

745

[ 746

[ 747

748

[ 749

750

[ 751

752

[ 753

754

[32] R.I. Christopherson, S.D. Lyons, P.K. Wilson, Accounts of Chemical Research 35 755


2 K. Manjunath et al. / International Journal

ogether (Table 5). Table 5 shows the number of SBs in each struc-ure at various cut-offs (4 A, 5 A, 6 A and 7 A) and the percentage ofBs in the corresponding RASB bins. The values in bold font (lightackground) indicate higher percentage (relatively exposed) of SBs

n hyperthermophiles compared to mesophiles. Further, the valueshown in bold font with dark background indicate a lower per-entage of (relatively buried) SBs in hyperthermophiles comparedo mesophiles. It can be inferred from Table 5 that the percentage ofBs having higher RASBs are more in hyperthermophiles especiallyt higher cut-offs (6 A and 7 A). However, the enzyme GkSS does nothow such a trend. To conclude, most of the SBs (weak or strong)end to reside on the surface of the hyperthermophilic comparedo mesophilic proteins.

Hydrophobic interactions are long-range [71] interactionsnown to play a significant role in the protein folding and stabil-ty. The hydrophobic contribution to the thermal stability in SAICARynthetase are investigated by studying the non-polar contact areasn protein structures using the tool pdb np cont and these interac-ions are clustered using the tool pdb np clus. The total non-polarurface area is calculated for the whole protein and for differ-nt contact area cut-offs (5, 10, 15, etc.) (Table 6). Later, they arelustered with a minimum of three members in each cluster. Theut-off indicates the minimum pairwise contact area between theesidues for clustering. The first row data shows the total non-polarontact area and the subsequent rows show the total residues inach clusters separated by a hyphen (‘-’). The total contact areaf all the clusters in a particular cut-off is in bold. The enzymekSS has a cluster of three residues even at a cut-off of 41 A2

nd among the mesophilic proteins, the enzyme EhSS appearso have relatively higher stability in terms of non-polar contacts.he hyperthermophilic proteins have higher total non-polar con-act area than mesophilic proteins. At higher contact area cut-off≥33 A2), the total non-polar contact area of hyperthermophilesre greater than mesophilic proteins by at least 1000 A2 and prob-bly provides the stability for the protein at higher temperature.owever, these theoretical observations need to be ascertainedxperimentally by measuring the melting temperatures of theseroteins.

. Conclusion

The first native crystal structure of SAICAR synthetase from ayperthermophilic organism has been reported. The Type-1 struc-ure of PhSS resembles the complex bound form of EcSS due to theound cadmium ions near the active site, inducing significant devi-tion at the dimeric interface. These cadmium ions also give rise to

pseudo-interface leading to a hexameric form. The PhSS being ayperthermophilic protein has very similar sequence and three-imensional structure compared to all other mesophilic dimericAICARs. The amino acid composition analysis revealed that theyperthermophilic SAICARs, in general, has higher percentage of

+ E + H + R + K and lesser percentage of S + T + N + Q compared tothers. Further, the ratio of (D + E + H + R + K)/Q is found to be excep-ionally high in hyperthermophiles. The thermophilic enzyme GkSSxhibited comparable percentage of D + E + H + R + K and S + T + N + Qith the mesophilic SAICARs. However, a very high percentage of

liphatic residues (A + L + I + V) are found. The monomeric SAICARsxhibited a unique composition with a large number of Pro andrp residues. Hyperthermophiles have more number of weak saltridges than mesophiles. Hyperthermophilic SAICARs have higherercentage of SBs with higher RASB values. It means higher percent-ge of SBs (both weak and strong) tend to reside on the surface of


he hyperthermophilic SAICARs compared to mesophilic SAICARs.he total non-polar contact area is observed to be the highest inyperthermophiles at higher contact area cut-offs.

[


Author’s contribution

KM purified, crystallized, collected the data, solved, refined and

analyzed the structures. SPK and SK assisted in the purification

process. JJ provided the plasmid. KS supervised the project and

critically read the manuscript.

Acknowledgements

The authors gratefully acknowledge the facilities offered by the

Interactive graphics facility and the Supercomputer Education and

Research Centre. The authors acknowledge the X-ray data collec-

tion facility at the Molecular Biophysics Unit. One of the authors(KM) thanks Eleanor Dodson for her valuable suggestions while

solving the structure. The authors thank the Department of Science

and Technology (DST) for financial support. The authors thank the

Spring-8 beam line BL44XU (proposal number 2011B6653).

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ING Model

B

f Biol

[760

761

[762

763

[764

765

[766

767

[768

769

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773

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g biomac article in press internationalare abbreviated as cpss, ecss, ehss, gkss, hsss, mass, mjss,...

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