Báo cáo khoa học: Cys126 is a completely conserved residue in triosephosphate isomerase that docx

A previous study of the C126S and C126A mutants of yeast TIM reported substantial catalytic activity for the mutant enzymes, leading to the suggestion that this residue is implicated in

triosephosphate isomerase by site-specific

mutagenesis – distal effects on dimer stability

Moumita Samanta1, Mousumi Banerjee1, Mathur R N Murthy1, Hemalatha Balaram2

and Padmanabhan Balaram1

1 Molecular Biophysics Unit, Indian Institute of Science, Bangalore, India

2 Molecular Biology and Genetics Unit, Jawaharlal Nehru Centre for Advanced Scientific Research, Bangalore, India

Introduction

The conserved amino acids in enzymes are, most often,

associated with the key steps of substrate recognition

and catalysis The availability of rapidly expanding

databases of enzyme sequences may be effectively used

to identify key residues Triosephosphate isomerase (TIM) is an extremely well-studied enzyme [1–4], and

Keywords

dimer interface; dimer stability;

Plasmodium falciparum; thermal stability;

triosephosphate isomerase

Correspondence

P Balaram, Molecular Biophysics Unit,

Indian Institute of Science,

Bangalore-560012, India

Fax: +91 80 2360 0535

Tel: +91 80 2293 3000

E-mail: pb@mbu.iisc.ernet.in

(Received 2 February 2011, revised 22

March 2011, accepted 28 March 2011)

doi:10.1111/j.1742-4658.2011.08110.x

Cys126 is a completely conserved residue in triosephosphate isomerase that

is proximal to the active site but has been ascribed no specific role in catal-ysis A previous study of the C126S and C126A mutants of yeast TIM reported substantial catalytic activity for the mutant enzymes, leading to the suggestion that this residue is implicated in folding and stability [Gonz-alez-Mondragon E et al (2004) Biochemistry 43, 3255–3263] We re-exam-ined the role of Cys126 with the Plasmodium falciparum enzyme as a model Five mutants, C126S, C126A, C126V, C126M, and C126T, were characterized Crystal structures of the 3-phosphoglycolate-bound C126S mutant and the unliganded forms of the C126S and C126A mutants were determined at a resolution of 1.7–2.1 A˚ Kinetic studies revealed an approximately five-fold drop in kcat for the C126S and C126A mutants, whereas an approximately 10-fold drop was observed for the other three mutants At ambient temperature, the wild-type enzyme and all five mutants showed no concentration dependence of activity At higher tem-peratures (> 40C), the mutants showed a significant concentration dependence, with a dramatic loss in activity below 15 lM The mutants also had diminished thermal stability at low concentration, as monitored by

far-UV CD These results suggest that Cys126 contributes to the stability of the dimer interface through a network of interactions involving His95, Glu97, and Arg98, which form direct contacts across the dimer interface

Database Structural data are available in the Protein Data Bank under the accession numbers 3PVF, 3PY2, and 3PWA.

Structured digital abstract

l Tim binds to Tim by x-ray crystallography (View interaction)

Abbreviations

GAP, glyceraldehyde 3-phosphate; DHAP, dihydroxyacetone phosphate; PDB, Protein Data Bank; Pf TIM, Plasmodium falciparum

triosephosphate isomerase; PGA, phosphoglycolate; TIM, triosephosphate isomerase; Tm, melting temperature.

provides a good model system for exploring the role of

residues that are completely conserved or minimally

replaced during evolution Examination of a dataset of

503 sequences of TIM from different organisms reveals

only nine fully conserved residues: Lys12, Thr75,

His95, Glu97, Cys126, Glu165, Pro166, Gly209, and

Gly228 [the numbering scheme used here corresponds

to that for Plasmodium falciparum TIM (Pf TIM),

and, for all of the fully conserved residues, this is

iden-tical to that of yeast TIM] Of these, Lys12, His95,

Glu97 and Glu165 surround the substrate, with the

carboxylate of Glu165 acting as the base for

abstrac-tion of a proton from the C2 posiabstrac-tion of

glycer-aldehyde 3-phosphate (GAP) and dihydroxyacetone

phosphate (DHAP) [5–8] Lys12 and His95 are

involved in substrate⁄ transition state binding and

pro-ton transfer, respectively [6,9,10] Pro166 is a hinge

res-idue located in loop 6, which undergoes dynamic

interconversion between open and closed states, with

the latter corresponding to the catalytically competent

form [11–15] Gly209 is located near the active site in

the highly conserved 208–212 segment Gly228 adopts

a backbone conformation accessible only for Gly

resi-dues, enabling appropriate positioning of the facing

208–209 segment by backbone–backbone hydrogen

bonds Thr75 is a critical residue at the dimer interface

[16]; the side chain of this residue from one subunit

makes key hydrogen bonding contacts with Asn10 and

Glu97 of the other subunit, which are proximal to

the active site Cys126 is a completely conserved

resi-due that is spatially proximal to the active site resiresi-due

Glu165 (Fig S1)

Interestingly, a preliminary analysis of a dataset of

over 800 putative TIM sequences extracted from a

dataset of bacterial sequences of marine origin [17]

also revealed the occurrence of Cys at position 126

Inspection of several 3D structures of TIM from

diverse organisms available in the Protein Data Bank

(PDB) does not immediately suggest a structural

expla-nation for the complete conservation of this residue

Indeed, an earlier investigation of the C126S and

C126A mutants of yeast TIM revealed that their

activ-ity remained undiminished, with the mutants

display-ing a significantly lower degree of thermal stability

This study suggested that Cys126 may be required for

efficient folding and stability rather than being

involved in maintaining catalytic activity [18] A recent

treatise on enzymology highlights Cys126 in a

discus-sion of TIM [19] As part of a program directed

towards understanding the role of conserved residues,

we describe the characterization of five Cys126

mutants of Pf TIM The mutants studied were C126S,

C126A, C126V, C126M, and C126T We describe

crystal structures of unliganded forms of the C126S and C126A mutants, and the liganded form of the C126S mutant Temperature-dependent activity mea-surements and spectroscopic studies suggest that Cys126 may be involved in maintaining the structural integrity of the active site in the temperature range 40–

50C Furthermore, the residue also contributes to the thermal stability of the dimer interface through an extended interaction network involving His95, Glu97 and Thr75 of the neighboring subunit, all of which are fully conserved residues

Results

Analysis of crystal structures Diffraction-quality crystals were obtained for the C126S mutant complexed with phosphoglycolate (PGA) and the unliganded C126S mutant For the C126A mutant, a structure could be determined only for the unliganded form PGA was bound to the active site of the C126S mutant structure in a manner similar

to that for wild-type Pf TIM, whereas the C126A mutant structure had no ligand bound to the active site after cocrystallization The difference in electron density at the ligand position is shown in Fig 1A Figures were generated with pymol (http://www pymol.org) The active site loop 6 was in the ‘closed’ form in the structure of the C126S–PGA complex In the unliganded forms of the C126S and C126A mutants, both of which contained a dimer in the asym-metric unit, the active site loop 6 was in the ‘open’ conformation In the C126S mutant unliganded struc-ture, the active site was occupied by an ethylene glycol molecule and a single water molecule in one subunit

In addition, a proximal sulfate ion, derived from the lithium sulfate in the crystallization medium, could also be identified near the active site The other sub-unit in the C126S mutant and both subsub-units in the C126A mutant contained two water molecules in the active site, along with a distal sulfate ion The electron density maps (2Fo) Fc, contoured at 1.0r) surround-ing the residues at position 126 for the mutants are shown in Fig 1

Figure S2 compares the relationships between the active site residues and Cys⁄ Ser126 in the unliganded and liganded forms of the wild-type enzyme and the C126S mutant The most notable difference is in the orientation of the Ser side chain, with the hydroxyl group forming a hydrogen bond with the carboxylate

of Glu165 in the liganded form A change of v1 from )62.5 in the unliganded form to )170.8 in the ligan-ded form is observed In contrast, the Cys126 side

chain remains unchanged in orientation upon ligand

binding Interestingly, both the unliganded forms

contain two invariant water molecules, which form

hydrogen bonds with one another Water 512 in the

wild-type enzyme (PDB ID: 1LYX) and water 349 in

the C126S mutant also form hydrogen bonds with the

fully conserved His95 and highly conserved Asn10

(Asn in 465 out of 470 sequences) side chains

Water 558 in wild-type TIM (PDB ID:1LYX) and

water 36 in the C126S mutant form hydrogen bonds with the side chain of the active site Glu165 and the backbone CO of the fully conserved Gly209 These two invariant water molecules form a similar network

of interactions in the C126A unliganded structure and also in the previously reported, unliganded yeast struc-ture (PDB ID:1YPI) [20] Ligand binding and loop closure result in the expulsion of these water molecules and a change in the backbone conformational angles for the highly conserved Gly209-Gly210-Ser211 seg-ment This results in a change in orientation of the Gly209 backbone CO group

Kinetic parameters The kinetic parameters determined for Pf TIM and the five mutants at position 126 are listed in Table 1 The parameters determined for the wild-type yeast enzyme and the C126S and C126A mutants by Gonzalez-Mondragon et al [18] are also shown for comparison

In the earlier study of the yeast enzyme, the wild-type and the Cys126 mutant enzymes had comparable kinetic parameters, with a small reduction in kcat (approxi-mately four-fold) Temperature-dependent activity mea-surements were not reported in that study In the present study of Pf TIM, an approximately 5.8-fold drop in kcat was observed for both the C126S and C126A mutants The other three mutants, C126V, C126M, and C126T, showed significantly lower kcat values, corresponding to a reduction of approximately 10-fold in catalytic activity These results suggest that all five Cys126 mutants show a high degree of catalytic activity, despite the fact that a completely conserved residue, proximal to the active site Glu165 and His95 side chains, has been replaced by residues of varying size and hydrogen-bonding ability.Figure 2A compares the temperature dependence of the specific activity of wild-type Pf TIM and the five Cys126 mutants, at a protein concentration of 3.7 nm For the wild-type enzyme, there was the expected increase in activity over the temperature range 25–40C, with a leveling off between 40C and 50 C In sharp contrast, all five mutants showed a dramatic reduction in activity in the temperature range 40–50C, with essentially com-plete absence of activity at 50 C The activities of the wild-type enzyme and the five Cys126 mutants were also measured as a function of protein concentration at

50C The results summarized in Fig 2B establish that all of the Cys126 mutants exhibited a pronounced fall in activity upon lowering of the protein concentration to below 20 lm Indeed, a fall in activity of approximately 10–1000-fold was observed on the change from 30 lm

to 1 lm The pronounced concentration dependence of

A

B

C

Glu165

Ser126

His95

Lys12

Glu165

Ser126

His95

Lys12

Glu165

Ala126

His95

Lys12

Fig 1 The electron density maps (2F o ) F c contoured at 1.0r) at

position 126 and the active site residues in: (A) the Pf TIM C126S

PGA-bound structure; (B) the Pf TIM C126S-unliganded structure

with the molecule ethylene glycol (cryoprotectant) at the active

site; and (C) the Pf TIM C126A-unliganded structure.

activity in the Cys126 mutants is suggestive of

diminished stability of the dimeric protein at high

temperature

Structural stability

Figure 3A,B show the far-UV CD and fluorescence

emission spectra of Pf TIM and the five Cys126

mutants, determined at a protein concentration of

3 lm The near identity of the observed spectra

estab-lished that there were no dramatic structural

conse-quences of the mutations at position 126 The far-UV

CD spectra also remained unchanged over the

concen-tration range 0.5–15 lm at 25C, suggesting the

absence of any concentration-dependent structural effects at ambient temperature Figure 3C shows a comparison of the thermal melting profiles for wild-type Pf TIM and the five mutants, obtained by moni-toring the CD ellipticity at 222 nm as a function of temperature, at a protein concentration of 15 lm The sharp reduction in CD ellipticity at temperatures greater than 60C corresponds to unfolding, aggrega-tion, and precipitation The wild-type and the mutant enzymes behaved in a very similar way under these conditions These results suggest that replacement of Cys at position 126 does not significantly perturb the overall folded structure of the protein or its thermal stability, at this relatively high protein concentration However, when the protein concentration was reduced

to 0.5 lm, the melting curves determined using the fall

in ellipticity at 222 nm (shown in Fig 3D) were dra-matically different for the wild-type enzyme and the mutants The melting temperature (Tm) for wild-type

PfTIM was unaffected by lowering the concentration, whereas the mutants melted at a significantly lower temperature (midpoint of transition, 50C) This con-centration dependence of protein thermal stability is consistent with the fall in enzyme activity of the mutants at low concentration and high temperature The reversibility of the thermal unfolding transition was investigated by measurements of ellipticity at

222 nm upon cooling from a temperature of 55C for the mutants and 60C for the wild-type enzyme, at a protein concentration of 0.5 lm Under these condi-tions, aggregation and irreversible precipitation of the thermally unfolded protein structure was minimized

Figure 4 summarizes the results obtained for the heat-ing and coolheat-ing cycles for the wild-type enzyme and the five Cys126 mutants Wild-type Pf TIM recovered almost 90% of the original ellipticity upon cooling to

20C The observed hysteresis in the cooling cycle has also been previously noted for the wild-type enzyme from Saccharomyces cerevisiae [18,21] In the case of all five mutants, only 60% of the CD ellipticity was recovered upon cooling These results correspond well with those reported in the previous study of yeast TIM C126S and C126A mutants Gonzalez-Mondragon

et al have noted that the reduction in Tmobserved for the C126S and C126A mutants of the yeast enzyme

‘should be taken as an indication of diminished kinetic, rather than thermodynamic, stability of the native dimer’ [18] They have also presented evidence for the dependence of refolding rates of the C126S yeast mutant at 30C and enzyme concentrations of 1.1 lm and 1.9 lm More rapid refolding is observed at higher protein concentrations [18] Our present study points

to a greater tendency of the Cys126 mutants than of

Fig 2 (A) Temperature dependence of specific activity of wild-type

Pf TIM (TWT) and the Cys126 mutants (B) Concentration

depen-dence of specific activity of the five Cys126 mutants at 50 C.

the wild-type enzyme to dissociate at low

concentra-tions and high temperatures

The relative stability of the wild-type enzyme and

the five mutants with respect to guanidinium

chloride-induced and urea-chloride-induced perturbation was probed by

measuring the position of fluorescence maxima Unfolding results in a shift in the emission maximum from 328 to 355 nm It is evident from the data in

Fig 5 that all five mutants were significantly less stable

to urea-induced and guanidinium chloride-induced

Fig 3 CD and fluorescence spectra of wild-type Pf TIM (TWT) and the five Cys126 mutants (A) Far-UV CD, protein concentra-tion 15 l M , 25 C (B) Fluorescence spectra, protein concentration 3 l M , 25 C (C) Thermal melting profile monitored at

222 nm, pathlength 1 mm, and protein concentration 15 l M (D) Thermal melting profile monitored at 222 nm, pathlength

1 cm, and protein concentration 0.5 l M All spectra were recorded in 20 m M Tris ⁄ HCl (pH 8.0).

TWT

Fig 4 Thermal unfolding and refolding study on wild-type Pf TIM and the five Cys126 mutants A protein concentration of 0.5 l M was used

in 20 m M Tris ⁄ HCl (pH 8.0) Ellipticity changes at 222 nm were monitored with heating and cooling rates of 0.5 CÆmin)1 The cooling cycle was started immediately after completion of the denaturation transition Black line: unfolding Gray line: refolding.

denaturation The observed Cm values (midpoint of

transition) for guanidinium chloride-induced

denatur-ation were 1.7 m for wild-type Pf TIM and  1.0 m

for all five Cys126 mutants; in the case of urea-induced

denaturation, the Cm for wild-type Pf TIM was

> 8 m, and that for all five Cys126 mutants was

 4 m The precise nature of the side chain at

posi-tion 126 did not appear to have a significant influence,

with all of the mutants exhibiting very similar

unfold-ing transitions, suggestunfold-ing that the Cys side chain is

unique in imparting local stability

Discussion

We began this study with the intention of establishing

the role of the completely conserved Cys126 in the

structure and function of TIM In a previously

reported study of S cerevisiae TIM,

Gonzalez-Mondragon et al had concluded that Cys126 ‘is

required not for enzymatic activity but for folding and

stability’ [18] Their studies of the C126S and C126A

mutants of the yeast enzyme established that these

mutations had little effect on enzymatic activity, but

resulted in greater susceptibility to thermal

denatur-ation In addition, the mutations slowed down the

folding rate by a factor of 10 We have now

re-exam-ined the C126S and C126A mutants of Pf TIM, and

determined their 3D structures by X-ray diffraction, in

order to gain further insights into the structural

conse-quences of mutations at position 126 We have also

compared the kinetic and biophysical properties of

three additional mutants: C126V, C126M, and C126T

The C126S and C126A mutants show a five-fold drop

in kcat, whereas the other three mutants show a 10-fold

drop The observation of significantly high catalytic

rates in all five mutants suggests that the conservation

of Cys126 cannot be directly attributed to the

impera-tives of catalysis

Our results clearly establish that the temperature dependence of enzyme activity is strongly concen-tration-dependent At a temperature of 50C, the measured activity of all of the mutants show a concen-tration dependence over the range 1–20 lm At low concentrations (3.7 nm), whereas the wild-type enzyme does not show marked temperature dependence over the range 40–50C, all of the mutants show a sharp loss in activity beyond 40C Biophysical studies also confirm a concentration dependence of thermal stability, as probed with CD ellipticities at 222 nm The mutants are significantly less stable with respect to thermal unfolding at low protein concentrations Fur-thermore, the mutants are also much more structurally labile at appreciably lower concentrations of the dena-turants urea and guanidinium chloride than the wild-type enzyme These results lead to the conclusion that mutation at position 126 must cause a destabilization

of subunit interactions, despite the apparent nonin-volvement of this residue in any direct contacts across the dimer interface We therefore turned to a re-exami-nation of the structures of wild-type Pf TIM and the C126S and C126A mutants and the yeast enzyme DHAP complex reported by McDermott et al (PDB ID: 1NEY) [22]

From Fig S2, it can be seen that Cys126 closely approaches two active site residues, His95 and Glu165 The shortest contact distances lie between 4.0 and 4.5 A˚ in the case of the Pf TIM–PGA complex In the ligand-bound C126S mutant structure, the serine OH group swings away from His95, in order to form a hydrogen bond with the carboxylate of Glu165

Figure 6 provides a view of the environment of Cys126, illustrating a network of interactions that con-nect this site to key residues at the subunit interface The Cys126 backbone CO and NH groups are held by

a pair of hydrogen bonds to the Arg99 guanidine side chain and the backbone CO of Ile93, respectively

Fig 5 Unfolding study on wild-type Pf TIM

(TWT) and the five Cys126 mutants in the

presence of urea and guanidinium chloride,

by fluorescence Protein at a concentration

of 3 l M was incubated with different

con-centrations of urea and guanidinium chloride

in 20 m M Tris ⁄ HCl (pH 8.0) for 45 min.

The data for unfolding were normalized by

taking the spectroscopic parameter to be

100% in the absence of any denaturant.

The CO group of the fully conserved Gly94 is also

held by a second guanidine group on the side chain of

Arg99 The Cb methylene group of Cys126 is in close

proximity to Gly94 (3.71 A˚) Arg 99 is also a very

highly conserved residue, and is found in as many as

464 of 470 bacterial and eukaryotic sequences Crucial hydrogen bond interactions across the subunit inter-face are made between the carboxylate of the fully conserved Glu97 and the Cb hydroxyl of the fully con-served Thr75 from the other subunit The guanidine group of Arg98 of one subunit also forms hydrogen bonds with the backbone CO of Thr75 and the side chain carboxylate of Glu77 The residues at posi-tions 98 and 77 are also strongly conserved Arg98 occurs in 441 of 470 sequences in our dataset, whereas,

at position 77, Glu is observed in 409 examples and Asp in 51 examples from 470 sequences

Figure 7 shows a view of the environment of the Cys126 side chain The thiol group of Cys126 does not appear to be involved in any significant hydrogen-bonding interaction The closest potential hydrogen bond acceptors are the backbone carbonyl oxygen atoms of Ile93 (S–O=C: 4.12 A˚) and Ile124 (S–O=C: 4.39 A˚) A similar observation has been made in the atomic resolution structure of Leishmania mexicana TIM (0.83 A˚), where the distances are as follows: 3.91 A˚ for S(Cys126)–O=C(Leu93); and 4.17 A˚ for S(Cys 126)–O=C(Ile124) [23] No evidence for the involvement of the Cys126 thiol group in strongly

Arg99

2.87

2.81

Gly94

2.88

Cys126

Ile93

His95

3.57 Glu97 2.72

Thr75

2.85 2.88

Glu77 Arg98

Fig 6 Environment of Cys126 in Pf TIM (PDB ID: 1O5X), showing

the important network of hydrogen bond interactions involving

sub-unit interface residues Thr75 and Glu77 are from the other

subunit.

Val91

Gly94 3.71

Cys126 4.03

4.10 4.68 4.13

4.68 Ile92

His95

Glu165

Glu97

Ser126

Gly94

Glu165

2.80 4.29

6.23 4.56

4.26

4.66 His95 5.60

Glu97 Ile92

Val91

Val91

Ala126

5.49 4.71 4.23

4.68

His95 4.06

Glu97

Glu165 Gly94

Ile92

Gly94

Ser126

4.75 4.58

4.10 4.69 His95

Glu97

3.04 Glu165

4.48

Ile92 Val91

Fig 7 View of the Cys ⁄ Ser ⁄ Ala126 side chain with 92, 94, 95 and 165 residues (A) Wild-type Pf TIM PGA-bound structure (PDB ID: 1LYX) (B) C126S PGA-bound structure (C) C126S-unliganded structure (D) C126A-unliganded structure.

directional hydrogen bond interactions is obtained

from the crystal structures of TIMs from diverse

organisms The three proximal side chains are those of

Glu165, His95, and Ile92 The closest distances of

approach involving the thiol sulfur atom are 3.85 A˚

for S(Cys126)–OOC(Glu165), 4.21 A˚ for S(Cys126)–

Cd2(His95), (Fig S1) and 4.10 A˚ for S(Cys126)–Cc2H3

(Ile 92) (Fig 7) The corresponding residues are shown

in the same orientation in the C126S–PGA complex

structure It is evident that the only difference is with

respect to the orientation of the Ser126 hydroxyl

group The absence of any significant change in the

relative orientations of His95 and Glu165 is consistent

with the relatively high kcatvalues determined for the

mutants at ambient temperature However, creation of

a cavity at position 126 in the case of the mutants (as

shown in Fig 7) may be expected to result in enhanced

flexibility of the fully conserved Gly94-His95 segment,

with the possibility of greater variability of the His95

side chain conformations upon heating

The structural data provide a possible explanation

for the observed instability of the dimeric structure in

the Cys126 mutants at elevated temperature

Perturba-tion of dimer interface contacts may be mediated by

altered interactions between His95 and Glu97, and also

through the Arg98-Arg99 segment (Fig 6) The

space-filling interactions involving the side chain of Cys126

(Fig 7) appear to be critical in maintaining the

observed network of hydrogen-bonding interactions,

which must contribute to the stability of both active

site residue orientation and subunit interface structure

Complete conservation of Cys126 suggests that

selec-tive pressures for optimal dimer stability at low

con-centrations and physiological temperatures may have

been operative during the evolution of TIM sequences

Experimental procedures

Mutagenesis

The Pf TIM gene was cloned into the pTrc99A vector

pARC1008 [24] The protein was overexpressed in

Escheri-chia coli strain AA200, which has a null mutation for the

host TIM gene [25] For the present study, the five single

mutants at position 126 were constructed by site-directed

mutagenesis with the single primer method [26] A single

primer was sufficient to generate mutant ssDNA, which

was subsequently transformed into E coli DH5a cells to

finally obtain the plasmid DNA with the desired mutation

As only one primer was used to achieve the mutation, the

mutation site lies in the middle of a stretch of

oligonucleo-tides, with sufficient flanking residues to obtain a high Tm,

close to 78C A primer length of 35-mer to 40-mer was

successfully used to obtain the required mutations The thermostable proofreading polymerase enzyme Pfu was used The PCR mixture contained, in a total volume of

25 lL: template DNA, 150 ng; mutagenic primer, 20 pmol; thermostable polymerase buffer (· 10), 2.5 lL; dNTPs,

6 lL of a solution containing 2.5 mm each dNTP; and polymerase, 2.5 U The cycling conditions for the PCR were as follows The PCR tube was initially taken to 95C for 5 min, and then 40 cycles consisting of 1 min at 95C, annealing at 45C for 1 min and extension at 72 C for

10 min were applied Following this, a final extension at

72C for 20 min was applied One microliter of DpnI (equivalent to 10 U) was directly added to the reaction mix-ture and incubated for 6–8 h at 37C, to digest the methy-lated template (parent) DNA Ten microliters of the reaction mix was directly transformed into chemically com-petent DH5a cells, after which the presence of mutations was confirmed by restriction digestion and sequencing In this study, five mutations were constructed at the same position Because of the absence of a restriction site at the desired mutation position, a two-step process was followed: step 1, generating an intermediate clone, C126int, with the introduction of EcoRV restriction site at the desired muta-tion posimuta-tion; and step 2, taking C126int as the template and generating the mutant clones C126S, C126A, C126V, C126M, and C126T, with the subsequent removal of the EcoRV restriction site at the desired mutation position The primer used for generating the C126int clone, with the introduction of the EcoRV restriction site, was 5¢-TAAT TTAAAAGCCGTGATATCTTTTGGTGAATCTT-3¢, and the primers used for generating the five mutants were: C126S, 5¢-TAATTTAAAAGCCGTTGTATCCTTTGGT GAATCTT-3¢; C126A, 5¢-TAATTTAAAAGCCGTTGT AGCTTTTGGTGAATCTT-3¢; C126V, 5¢-TAATTTAAAA GCCGTTGTAGTTTTTGGTGAATCTT-3¢; C126M, 5¢-T AATTTAAAAGCCGTTGTAATGTTTGGTGAATCTT-5¢; and C126T, 5¢-TAATTTAAAAGCCGTTGTAACTTTT

GG TGAATCTT-3¢

Protein expression and purification The TIM gene carrying the mutation was expressed in

E coliAA200 (a null mutant for the inherent TIM gene) cells carrying the pTrc99A recombinant vector Cells were grown at 37C in Terrific broth, containing 100 lgÆmL)1 ampicillin Cells were induced with 300 lm isopropyl thio-b-d-galactoside at a D600 nmof 0.6–0.8, harvested by centri-fugation at 4C, resuspended in lysis buffer containing

20 mm Tris⁄ HCl (pH 8.0), 1 mm EDTA, 0.01 mm phen-ylmethanesulfonyl fluoride, 2 mm dithiothreitol, and 10% glycerol, and disrupted by sonication After centrifugation (7245 g, 15 min, 4C) and removal of cell debris, the super-natant was fractionated with ammonium sulfate The pro-tein fraction containing TIM was precipitated between 60%

and 80% ammonium sulfate saturation The precipitate was

obtained by centrifugation (19 320 g, 45 mins, 4C), and

after resuspension in buffer A (20 mm Tris⁄ HCl (pH 8.0),

2mm dithiothreitol, and 10 % glycerol), the following steps

were followed Firstly, it was subjected to gel filtration

chromatography (Sephacryl-200), equilibrated with the

same buffer A The fractions containing the protein were

pooled and further purified by anion exchange

(Q-Sephar-ose) chromatography, with a linear gradient of 0-1 m NaCl

The purified protein obtained was then extensively dialyzed

overnight against buffer A at 4C Protein purity was

checked by 12% SDS⁄ PAGE Mutations were confirmed

by ESI MS: mobs (mcalc): wild-type TIM, 27 831 Da

(27 831 Da); C126S, 27 815.7 Da (27 815 Da); C126A,

27 799.8 Da (27 799 Da); C126V, 27 827.2 Da (27 827 Da);

C126M, 27 859.6 Da (27 859 Da); and C126T, 27 829 Da

(27 829 Da) (Fig S3) The protein concentration was

determined with the Bradford method [27], using BSA as a

standard

Enzyme activity

Enzyme activity was measured by a coupled assay

method The conversion of GAP to DHAP by TIM was

monitored in the presence of the coupling enzyme,

a-glyc-erol phosphate dehydrogenase [28] Enzymes were freshly

prepared in 100 mm triethanolamine-HCl (pH 7.6) The

reaction mixture contained (final volume, 1 mL) 100 mm

triethanolamine-HCl, 5 mm EDTA, 0.5 mm NADH and

20 lgÆmL)1 a-glycerol phosphate dehydrogenase and GAP,

to which TIM was added to initiate the reaction In the

case of the wild-type enzyme, the assay was started by

addition of 10 ng of protein, and in the case of the

Cys126 mutants 100 ng was used Substrate concentrations

varied from 0.25 mm to 4.0 mm The progress of the

reac-tion was monitored by the decrease in absorbance of

NADH at 340 nm The extinction coefficient of NADH

was taken to be 6220 m)1Æcm)1 at 340 nm [29] The initial

rates showed a linear dependence on the enzyme

concen-tration in the range studied This ensures the validity of

the assay [28] The values for the kinetic parameters (Km,

kcat) were determined by fitting to the Michaelis–Menten

equation with graphpad prism (Version 5 for windows;

graphpad Software, San Diego, CA, USA; http://www

graphpad.com)

Fluorescence spectroscopy

Fluorescence emission spectra were recorded on a

HIT-ACHI-250 spectroflorimeter The protein samples were

excited at 295 nm, and the emission spectra were recorded

from 300 nm to 400 nm Excitation and emission bandpasses

were kept as 5 nm and 10 nm, respectively Denaturation

studies were performed by incubating 3 lm protein with

different concentrations of urea and guanidinium chloride

for 45 min Spectra were acquired from 300 nm to 400 nm, after excitation at 295 nm

CD Far-UV CD measurements were carried out on a

JASCO-715 spectropolarimeter equipped with a thermostatted cell holder The temperature of the sample solution in the cuv-ette was controlled with a Peltier device For thermal melting studies, ellipticity changes at 222 nm were monitored The temperature was varied at a rate of 0.5CÆmin)1 to follow the unfolding and refolding transitions Spectra were aver-aged over four scans at a scanning speed of 10 nmÆmin)1 The change of ellipticity was measured as a function of temperature for thermal melting Individual spectra (250–200 nm) were averaged over four scans

Table 1 Kinetic parameters of Pf TIM and its five Cys126 mutants.

k cat ⁄ K m

(m M )1Æs)1)

Wild type a (4.3 ± 0.3) · 10 3 0.35 ± 0.05 1.2 · 10 4

C126S a (7.5 ± 0.1) · 10 2 1.4 ± 0.20 5.4 · 10 2

C126A a (7.7 ± 0.2) · 10 2 1.5 ± 0.20 5.2 · 10 2

C126V a (1.6 ± 0.8) · 10 2 1.0 ± 0.10 1.6 · 10 2

C126Ma (1.9 ± 0.3) · 10 2

1.5 ± 0.10 1.2 · 10 2

C126T a (3.3 ± 0.2) · 10 2 1.2 ± 0.20 2.8 · 10 2

Wild type b (4.7 ± 0.7) · 10 3 1.1 ± 0.4 4.3 · 10 3

C126Sb (1.1 ± 0.2) · 10 3

0.3 ± 0.1 3.7 · 10 3

C126A b (3.1 ± 0.2) · 10 3 0.8 ± 0.3 3.9 · 10 3

a P falciparum (present study) b S cerevisiae [18].

Table 2 Data collection statistics.

C126S-liganded

C126S-unliganded

C126A-unliganded

Unit cell

Resolution range (A ˚ ) 26.2–1.7 43.4–1.9 51.8–2.0

No of unique reflections 25 341 31 243 29 675 Completion (%)a 94.2 (79.7) 85.6 (78.0) 93.2 (75.3) Overall R merge(%) a 3.3 (22.5) 8.2 (25.2) 8.2 (26) Multiplicity a 2.1 (2.1) 6.67(6.67) 10 (10.3)

<I> ⁄ <rI> a

20.2 (5.3) 17.1 (8.3) 20.2 (8.9)

a Values in parentheses correspond to the last resolution shell.

Crystallization of Pf TIM Cys126 mutants

The Cys126 mutants were purified as described, and

con-centrated to approximately 10 mgÆmL)1 Crystals were

allowed to grow by the hanging drop method, at 23C

[30] The C126S–PGA crystal was obtained under the

fol-lowing conditions: 20% poly(ethylene glycol), 1 m Hepes

buffer (pH 7.5), and 10 mm lithium sulfate The unliganded

C126S crystal was obtained under the following conditions:

24% poly(ethylene glycol), 1 m Hepes buffer (pH 7.0), and

10 mm lithium sulfate The unliganded C126A crystal was

obtained under the following conditions: 24% poly(ethylene

glycol), 1 m Hepes buffer (pH 7.0), and 10 mm lithium

sul-fate The crystals appeared within 2 days, and grew to the

required sizes within 4–5 days

Data collection and processing

Ethylene glycol (20%) was used as the cryoprotectant

before flash-freezing of the crystals X-ray diffraction data

were collected with a Rigaku rotating anode generator and

a MAR Research image plate detector system The data

were processed with mosflm and scala [31] of the ccp4

suite of programs [32] The details of the datasets collected

and the data collection statistics are shown inTable 2

Structure solution and refinement The mutant structures were solved with the molecular replacement program phaser of the ccp4 package [33] The native Pf TIM crystal structure (PDB ID: lLYX) was used

as the starting model for structure determination for the datasets of C126S-liganded The structure with the PDB ID

of 1O5X was used as the starting model in the case of the datasets for C126S-unliganded and C126A The coordinates

of 1LYX and of 1O5X were modified by removing the loop 6 residues, ligand, water molecules, and alternative conformations Refinements of all the structures were car-ried out with refmac [34], with an initial 20 cycles of rigid body refinement followed by 50 cycles of restrained refine-ment The loop 6 residues, ligand and water molecules were added on the basis of 2Fo) Fc and Fo) Fc maps con-toured at 1r and 3r, respectively Model building was per-formed with coot [35] One subunit in the case of the C126S-liganded structure and two subunits in the case of the C126S-unliganded and C126A structures were present

in the asymmetric unit The existence of the C126S and C126A mutations was confirmed from difference Fourier maps Water molecules were first located automatically by coot, and validated if a peak was observed above 3r on a difference map and above 1.5r on a double difference map The B-factors of all atoms were also refined, and alternative conformations were included wherever necessary All of the structures were refined to reasonable Rwork and Rfree values and good geometry, and then validated with pro-check[36] in the ccp4 package The electron density maps (2Fo) Fc contoured at 1.0r) surrounding the residues at position 126 for the mutants are shown in Fig 1 The refinement statistics for the mutant structures are shown in

Table 3

Acknowledgements

One of us (P Balaram) is deeply indebted to N V Joshi for his analysis of TIM sequences and helpful discussions M Samanta was supported by a Senior Research Fellowship from the Council of Scientific and Industrial Research (India) X-ray diffraction and

MS facilities are supported by program grants from the Department of Biotechnology (India)

References

1 Knowles JR (1991) Enzyme catalysis: not different, just better Nature 350, 121–124

2 Wierenga RK (2001) The TIM-barrel fold: a versatile framework for efficient enzymes FEBS Lett 492, 193–198

3 Cui Q & Karplus M (2003) Catalysis and specificity in enzymes: a study of triosephosphate isomerase and comparison with methyl glyoxal synthase Adv Protein Chem 66, 315–372

Table 3 Refinement statistics.

C126S-liganded

C126S-unliganded

C126A-unliganded Resolution range (A ˚ ) 26.2–1.7 43.4–1.9 51.8–2.0

Number of

subunits ⁄ asymmetric unit

Number of used reflections 25 341 31 297 29 723

Model quality

Number of ligand

atoms (PGA)

Average B-factor (A ˚ 2 )

rmsd from ideal

Ramachandran statistics

Generously allowed

region (%)