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Báo cáo Y học: Stepwise adaptations of citrate synthase to survival at life’s extremes From psychrophile to hyperthermophile pot

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Looking then at the trends towards inter/intrasubunit ion pairs, PfCS100 has the most interfacial ion pairs but both ArCS31 and pigCS37 have more intrasubunit interactions than the TaCS5

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Stepwise adaptations of citrate synthase to survival at

life’s extremes

From psychrophile to hyperthermophile

Graeme S Bell1, Rupert J M Russell2, Helen Connaris2, David W Hough1, Michael J Danson1

and Garry L Taylor1,2

1 Centre for Extremophile Research, Department of Biology and Biochemistry, University of Bath, UK; 2 Centre for Biomolecular Sciences, University of St Andrews, St Andrews, UK

The crystal structure of citrate synthase from the

thermo-philic Archaeon Sulfolobus solfataricus (optimum growth

temperature¼ 85 C) has been determined, extending the

number of crystal structures of citrate synthase from

differ-ent organisms to a total of five that span the temperature

range over which life exists (from psychrophile to

hyper-thermophile) Detailed structural analysis has revealed

possible molecular mechanisms that determine the different

stabilities of the five proteins The key to these mechanisms is

the precise structural location of the additional interactions

As one ascends the temperature ladder, the subunit interface

of this dimeric enzyme and loop regions are reinforced by complexelectrostatic interactions, and there is a reduced exposure of hydrophobic surface These observations reveal

a progressive pattern of stabilization through multiple additional interactions at solvent exposed, loop and inter-facial regions

Keywords: citrate synthase; Sulfolobus; citrate synthase; thermostability; crystal structure; ion networks

Comparative structural analysis of the same protein isolated

from mesophiles and thermophiles have highlighted many

structural adaptations that confer protein thermostability

[1–6] The importance of electrostatic interactions at specific

locations within the structure, and particularly the presence

of ion-pair networks, is a feature that is common to almost

all the hyperthermophilic proteins [7–10], although many

other additional differences such as improved hydrophobic

packing, compactness and additional hydrogen bonds have

been observed in other proteins

For our analysis we have chosen the enzyme citrate

synthase (CS) (EC 4.1.3.7), which catalyses the

condensa-tion of oxaloacetate and acetyl-CoA to form citrate and

CoA The enzyme from psychrophilic, mesophilic and

thermophilic sources has been intensively studied both

kinetically [11–13] and structurally [3,14] Crystal structures

exist for CS from a psychrophilic Antarctic bacterium

Arthrobacter strain DS2-3R (growth optimum ¼ 31 C)

[15], pig (37C) [16], and the Archaea Thermoplasma

acidophilum(55C) [17] and Pyrococcus furiosus (100 C)

[18] To extend our previous studies we have chosen the

organism Sulfolobus solfataricus, a thermophilic Archaeon

that optimally grows at 85C The gene for Sulfolobus

solfataricus CS has been cloned and sequenced [19], and

over-expressed in E coli The purified recombinant protein

exists as a homodimer of Mr¼ 81,000, with each monomer

comprising 379 amino acids The following abbreviations

will be used for the CSs, including their optimal growth

temperatures: Arthrobacter: ArCS(31), pig: PigCS(37),

T acidolphilum: TpCS(55), S solfataricus: ScCS(85), and

P furiosus: PfCS(100)

The structure of unliganded SsCS(85) reported in this paper can now be entered into the temperature ladder of CS structures, and fills in the gap between the 55C and 100 C enzymes SixCS crystal structures from five host organisms (Table 1) can now be used for comparative analysis in order

to identify some of the structural features that could confer (hyper)thermostability in this enzyme family As can be seen from Table 1, the organisms span the range of temperatures at which life is known to exist, and the inherent stability of each CS, from in vitro measured half-lives of thermal inactivation [19–21], increases with the optimum growth temperature of the host cells The structure

of the SsCS(85) is thus discussed in comparison with the other CS structures, and trends in structural changes are correlated with the increasing thermal stabilities across the homologous series of enzymes In terms of thermostability, the enzymes fall into two broad classes based on the temperature at which the half-life equals 8 min: the psychro-phile and pig enzymes at the lower end with temperatures of

45C and 58 C, and the archaeal enzymes at the upper end with temperatures of 87C, 95 C and 100 C

M A T E R I A L S A N D M E T H O D S

Crystallization and structure solution Recombinant SsCS(85) was purified as described previously [19] Crystallization trials were carried out using the hanging-drop vapour diffusion method using the Hampton Research Screens A single rod-like crystal of approximate dimensions 2· 0.1 · 0.1 mm grew in a 6-lL drop contain-ing 2 lL SsCS(85) (10 mgÆmL)1) with 10 m citrate and

Correspondence to G Taylor, Centre for Biomolecular Sciences,

University of St Andrews, St Andrews, KY16 9ST, UK.

E-mail: glt2@st-and.ac.uk

(Received 3 July 2002, revised 8 October 2002,

accepted 4 November 2002)

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CoA, 2 lL of 100 mM Tris/HCl, pH 7.2, containing 17%

(v/v) PEG 8K, and 2 lL of 0.1MCaCl2 The crystal grew in

a partially dried out drop after sixmonths X-ray data were

collected at room temperature on a 30-cm Mar image plate

detector Diffraction extended to 2.7 A˚ resolution The

crystal was translated stepwise perpendicular to the beam to

maximize the completeness of the data and to overcome

radiation damage of the crystal The data were reduced and

scaled using DENZO/SCALEPACK[22] (Table 2) The

asym-metric unit of the P21unit cell contains two dimers with a

solvent content of 51% The structure of SsCS(85) was

solved by molecular replacement using the programAMORE

[23] Because the crystallization solutions contained both

citrate and CoA, it was assumed that the closed form of

SsCS(85) had crystallized; therefore, initial attempts were

made to solve the structure using the closed structures of

PfCS(100) or ArCS(31) as the search model, but this did not

produce any clear solutions Attempts were subsequently

made using the open structure of the TaCS(55) dimer as the

search model Using data in the resolution range of 15–6 A˚

and a Patterson integration radius of 25 A˚, 50 solutions

from the rotation function were calculated Using the same

resolution range for the translation search, the top solution

(33rd highest from the rotation search) had a correlation

coefficient (CC) of 32.0 and R-factor of 53.4% (compared

with the next highest peak with a background CC of 23 and

R-factor of 56%) This solution was fixed, and a solution for the second dimer in the asymmetric unit was identified (CC of 37.7% and R-factor of 51.9%, compared to the next highest peak of 31 and 53%, respectively) After a rigid-body refinement in AMORE of the two dimers, the final solutions had a CC of 56.6 and R-factor of 41.3% The failure to find a solution using the closed form of the homologous enzyme, but a clear solution with the open forms, strongly suggested that the SsCS(85) had unexpect-edly crystallised in the open, unliganded form

Refinement and validation The restrained refinement of SsCS(85) was performed using REFMAC [24] The initial R-factor in REFMAC (after rigid body refinement) was 48.3% (Rfree ¼ 48.6%) and final R-factor of 20.8% (Rfree¼ 28.5%) for all data from 20.0 A˚

to 2.7 A˚

1,2 Tight non crystallographic symmetry (NCS) restraints for both main-chain and side-chain were used initially and sixcycles of refinement carried after which the R-factor was 36.3% (Rfree ¼ 40.5%) Keeping the tight NCS restraints, individual isotropic B-factor refinement was then carried out, bringing the R-factor down to 24.7% (Rfree 31.2%), after which the NCS restraints were gradually loosened and the four monomers were built independently NCS restraints were controlled in PROTINand, during the refinement procedure, side-chain followed by main-chain restraints were gradually loosened, with a final round removing the NCS restraints continuing to lower the Rfree value

The first two residues at the N-terminus and last seven residues of the C-terminal arm were not seen in the poorly defined electron density of these parts of the structure in all four monomers One conflict with the sequence data was residue 57, which had been assigned as arginine and was found from analysis of the electron density map to be a proline (this is a totally conserved proline in all the other known CSs) The position of the small domain with respect

to the large domain in SsCS(85) is the same as previously observed in TaCS(55) [17]; this together with the absence of density for substrates in the active site, supports the previous speculation that the structure is the open form of the enzyme After 24 rounds of refinement inREFMAC, the final R-factor was 20.8% (Rfree¼ 28.5%) (Table 2) The quality

of the final electron density is shown in Fig 1

Table 2 Table displaying native data collected and refinement statistics

for the SsCS Data in parentheses correspond to the high resolution

data shell (2.82–2.71 A˚).

Unit cell dimensions a ¼ 77.3 A˚, b ¼ 97.9 A˚,

c ¼ 119.3 A˚, b ¼ 107.6

Resolution limit 2.7 A˚

Data completeness 88.6% (91.2%)

Total No of reflections 148169

Unique reflections 46758

Free R-factor 28.5%

No protein atoms 11 742

Rmsd bond lengths (A˚) 0.009

Rmsd bond angles () 0.032

Table 1 CS structures used for analysis.

Source

organism

Optimum growth temperature (C)

CS

Temperature (C) at which the half-life equals 8 min

Substrates in crystal structure

Data resolution (A˚)

Citrate and CoA

2.7 2.0

Pyrococcus furiosus 100 100 Citrate & CoA 1.9

a

It should be noted that, although Arthrobacter DS23R was isolated from a habitat temperature of approximately 0 C, this organism displays a relatively high optimum growth temperature; therefore, although it is described here as psychrophilic, it should perhaps more correctly be referred to as psychrotolerant.

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The Ramachandran plot shows that for the four

mono-mers in the asymmetric unit, 91.3% of residues lie in the

most favoured regions, with only 8.7% in the allowed

regions and no residues appearing in the generously allowed

or disallowed regions (excluding glycine and proline

residues) Atomic coordinates have been deposited in the

Protein Databank with accession code 107x

R E S U L T S

Overall structural comparisons

All the eukaryotic

4 , archaeal and Gram-positive bacteria CSs

are homo-dimeric structures with each monomer consisting

of a large and small domain In addition, they are almost

entirely a-helical; the pigCS contains 20 a-helices (A-T) with

the enzyme from ArCS(31), TaCS(55), SsCS(85) and

PfCS(100) containing 16 a-helices, all of which have an

equivalent in pigCS (helices A, B, H, and T are not present)

(Figs 2 and 3) Of the 16 equivalent helices, the large domain

comprises 11 helices (C-M and S) and the small domain five

helices (N-R) The small domain has been classed as

residues 217–321 inclusive for SsCS(85)

The active sites of CSs comprise residues from both

monomers and therefore CS is only active as a dimer,

stressing the importance of maintaining dimeric integrity as a

prerequisite for activity Binding of citrate and CoA to the

active site has been discussed in detail for PfCS(100) and

ArCS(31), and the differences with respect to the pig enzyme

noted [15,18] The SsCS(85) structure has no substrate

bound, but the location of active site residues can be

identified by comparison with the liganded PfCS(100)

structure The citrate-binding residues comprising three

arginine residues, R267 (helixP), R338 (helixS) and R358¢

(where the prime denotes the residue of the second

mono-mer), and three histidine residues, H183 (loop K-L), H218

(loop M-N) and H258 (loop O-P), are equivalent to those

found in PfCS(100) The binding residues for the three

Fig 2 Structurally based sequence alignment of the five CSs discussed Helices A to T are shaded, and the location of the small domain is indicated by lowercase sequence letters The three catalytic residues are indicated by fl The three arginines and histidines involved in binding citrate are marked with a C The residues involved in binding the three phosphates of CoA are marked with an A The sequence numbering is shown at the start of each line.

Fig 1 Stereo-diagram showing a typical region of the final 2Fo-Fc electron density map contoured at 1 r.

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phosphate groups of CoA are likely to be K250 (loop O-P)

and K306 (loop Q-R), R259 and K262 (both loop O-P), with

the third phosphate being co-ordinated by R355¢ from the

second monomer The catalytic residues H218, H258 and

D313 (loop Q-R) are also present in SsCS(85) and are in

a similar position to the PfCS(100) residues It is likely

therefore that SsCS(85) binds substrates in a similar man-ner to PfCS(100) and that the mechanism of catalysis is the same

The dimer interface of all the CSs is made up of two parts and comprises residues solely in the large domain; the main part is the eight a-helical sandwich of four antiparallel pairs

of helices (F, G, M and L), with the second being the additional interaction of N- and C-terminal regions (Fig 3) The pigCS is different from the other four CSs in terms of the topology of the C-terminal region In the other four, the C-terminal arm of one monomer wraps around the other monomer, clasping the two together [18], and results in more extensive interactions, including those with the N-terminus It is important to note that as the C-terminal arms of the TaCS(55) and SsCS(85) are not complete in the structures (see below), there may be additional interactions present that have not been observed This also suggests that the C-terminal arm seems to be ordered only in the presence

of substrates

Sequence and structural statistics Pairwise sequence alignments were carried out using the programBESTFITfrom the WisconsinGCGsequence analysis package, and superposition was carried out using the least squares fit in O [25] for fitting of alpha-carbon atoms (starting from three conserved atoms) These statistics are listed in Table 3

Sequence identities between the various CSs for which 3D-structures have been determined range from 20% [eukaryotic

5 vs bacterial or archaeal) to 60% (SsCS(85) and TaCS(55)] These identities are reflected in the root mean square (RMS) deviations between the alpha-carbons

of the structures, with the most similar structures being the TaCS(55) and SsCS(85), and with the PfCS(100) and ArCS(31) pair also showing a very low RMS deviation As some structures are in the open conformation and some have substrates bound, the large and small domains of each enzyme were compared separately (Table 3); in general, such an analysis shows the same trend as that for the whole dimer but the small domains tend to be more highly conserved As is suggested later, this may correlate with differences particularly relating to the dimer interface, to which the small domain does not contribute, and may reflect the fact that the majority of the substrate-binding and catalytic residues are from the small domain

The molecular mechanisms underlying protein thermal stability

In our comparison of CS atomic structures from organisms spanning a wide-range of growth temperatures, the deter-mination of the SsCS(85) structure fills an important gap between the enzyme from Thermoplasma (55C) and Pyrococcus (100C) With the structure reported in this paper, we can now look for trends in the structures that might correlate with the increasing thermostabilities of these enzymes However, the complexnature of the stabilization

of a protein structure lends itself to many types of comparative analysis, and the results presented below are those where significant differences exist between the struc-tures Other types of analysis (e.g of hydrogen bonds and helixcapping) have been performed but are not included

Fig 3 Schematic drawings of CS From top to bottom: ArCS(31),

PigCS(37), TaCS(55), SsCS(85) and PfCS(100) The right hand

col-umn represent views obtained by rotating the images in the left hand

column by 90 about a horizontal axis N- and C-terminii are denoted

by blue and red spheres, respectively The small domains (helices N to

R) are coloured in a lighted shade Catalytic residues, and citrate and

Co-A where appropriate, are shown in ball-and-stick representation.

All figures were created using BOBSCRIPT [51] and GL _ RENDER (L Esser

and J Deisenhofer, unpublished results).

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due to there being no significant differences between the

different structures

Compactness and surface characteristics

The accessible surface area was calculated using the program

GRASP [26] and the volume and cavity detection were

determined using the program VOIDOO [27] with a probe

radius 1.4 A˚ and grid spacing of 0.75 A˚ All calculations for

closed structures were carried out in the absence of substrate,

and the results are summarized in Table 4

ArCS(31), TaCS(55) and PfCS(100) have very similar

surface areas, with that of SsCS(85) being slightly higher;

however, all four enzymes have a considerably smaller

surface area and volume than the pigCS(37), even when

deleting the first 35 residues from the pig enzyme (these 35

amino acids comprise helices A and B, which are absent in

the other CSs being considered) A similar pattern to the

total accessible surface area (ASA)

compar-ing the overall volume, with pigCS(37) havcompar-ing a

consider-ably larger volume than the other CSs (again, even when

calculated with the N-terminally deleted structure)

However, it is also notable that the smallest volume is

exhibited by the psychrophilic CS (8.36· 104A˚3) All the CSs have a similar percentage of atoms buried, although the hyperthermophilic Pf CS(100) exhibits the highest with 54.5%

Examination of the exposed hydrophobic area shows a more obvious trend Despite all the archaeal and bacterial CSs having a similar overall ASA, there is quite a difference

in hydrophobic exposure when comparing ArCS(31) with the other CSs; the closed conformation of the ArCS(31) has

7854 A˚2 overall exposed hydrophobic area (representing 29% of the total ASA) compared with the closed PfCS(100) with only 4942 A˚2(18% of total ASA) Thus, on average, ArCS(31) exposes 23 A˚2per hydrophobic residue compared with 16 A˚2 per hydrophobic residue in PfCS(100) The total amount (A˚2) of hydrophobic surface area shows a decrease as the thermostability of the protein increases, a trend observed in other structural comparisons [4] SsCS(85) also follows the trend observed in PfCS(100), with the elimination of all cavities capable of accommodating a solvent molecule, indicating that this is a prerequisite for maintaining integrity at high temperatures The number of internal cavities (and their total volumes in A˚2calculated by ) are 1 (104), 6 (476), 3 (218), 3 (184), 0 (0) and 0 (0)

Table 3 Overall comparison of primary and 3D structures of CSs In the top half of the table the RMS deviations between Ca atoms (in A˚) are given for complete dimers, the large domain and the small domain, with the number of contributing pairs of Ca atoms in parantheses In the bottom half

of the table, the percentage sequence identities and similarities are shown, the latter in parentheses.

Enzyme

(open)

ArCS(31) (closed) PigCS(37) PigCS(37) TaCS(55) SsCS(85) PfCS(100)

ArCS(31) – 2.27 (560) 2.12 (630) 1.97 (604) 1.94 (610) 1.32 (719)

1.74 (242) 1.53 (252) 1.57 (259) 1.07 (262) 1.79 (96) 1.59 (90) 1.51 (90) 1.27 (92) PigCS(37) (open) – – 1.19 (730) 1.95 (651) 1.88 (646) 2.15 (550)

2.16 (533) 2.08 (519) 2.04 (631) PigCS(37) (closed) 27% (50%) – – 1.81 (233) 1.79 (232) 1.84 (245)

1.60 (82) 1.55 (82) 1.49 (90)

0.87 (719) 2.03 (581) TaCS(55) 32% (54%) – 22% (48%) – 0.76 (256) 1.66 (247)

0.76 (104) 1.02 (95) SsCS(85) 34% (55%) – 27% (50%) 59% (76%) – 1.94 (597)

1.53 (245) 1.03 (96) PfCS(100) 40% (58%) – 31% (53%) 42% (62%) 46% (67%) –

Table 4 Accessible surface area (ASA) and volume statistics of CSs.

CS

ArCS(31) (closed)

PigCS(37) (open)

PigCS(37) (closed)

PigCS(37) (closed, N-terminally deleted)

TaCS(55) (open)

SsCS(85) (open)

PfCS(100) (closed)

ASA ( · 10 4 A˚ 2 ) 2.72 3.34 3.20 2.99 2.72 2.82 2.72

No of atoms calculated for 5784 6888 6884 6344 5722 5879 5961

No of atoms buried 3044 3469 3601 3307 2955 3014 3248 Atoms buried (%) 52.6 50.4 52.3 52.1 51.6 51.3 54.5 Volume ( · 10 4

Total area hydrophobic exposed (A˚2) 7854 6654 6246 – 6001 5513 4942

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for ArCS(31), PigCS(37) open, PigCS(37) closed, TaCS(55),

SsCS(85) and PfCS(100), respectively

The subunit interface: ion pairs, hydrophobicity

and complementarity

Ion pairs were classed as residues of opposite charge

situated 4.0 A˚ or less apart [28] Looking simply at the total

numbers of ion pairs, it can be seen that all the thermophilic

CSs have a greater total number of ion pairs than the pig

enzyme, but that the psychrophilic enzyme actually has the

greatest number of all (Table 5) Looking then at the trends

towards inter/intrasubunit ion pairs, PfCS(100) has the

most interfacial ion pairs but both ArCS(31) and pigCS(37)

have more intrasubunit interactions than the TaCS(55) and

SsCS(85), and therefore the location of these ionic

inter-actions may be particularly relevant

With respect to the ionic interactions at the dimer

interface (Fig 4), the five CSs show considerable variation

Interactions in the pig enzyme are all unique with respect

to the other CSs and involve residues near the termini and

outer helices (F and L) of the eight-helical sandwich; the

central helices of the dimer interface have no ionic

interactions associated with them In marked contrast,

the other four CSs all have ionic interactions associated

with the two internal helices of the interface (G and M) as

well as those involved with the N-terminus and the

C-terminal arm Firstly, with respect to the interfacial helices,

one completely conserved ionic interaction in ArCS(31),

TaCS(55), SsCS(85) and PfCS(100) is that between a

conserved aspartate at the N-terminal end of helixM with

a lysine at the C-terminal end of helixM in the other

subunit (D205 and K218 in TaCS) The lysine residue is

not conserved in the pig enzyme This leads to a single ion

pair at each end of helixM in TaCS(55), whereas in

SsCS(85) there is an additional salt bridge in close

proximity with the first one, between E89 (loop F-G) of

one monomer and K108 at the C-terminal end of helixG

in the other; thus in SsCS(85) both central helices have ion

pairs at either end In PfCS(100), the G-M interhelical

electrostatic interactions are even more pronounced than

in either TaCS(55) or SsCS(85), with a five-residue ionic

network comprising H93 and R99 (helixG) and D113 in

loop G-I, in addition to the above-mentioned Asp-Lys pair

Interestingly, in the psychrophilic ArCS(31) the first Asp-Lys ion pair is part of a four-residue network (in conjunction with D95 and R98, both in helixG) The four residue network in ArCS(31) only comprises two single interactions directly across the interface, whereas the PfCS(100) five-residue network has four such interactions This would suggest that the PfCS(100) networks contri-bute considerably more to the intermolecular interactions than they do in the psychrophilic enzyme Even so, the psychrophilic enzyme does have an intersubunit ionic network, which may be related to cold-stability in the face

of diminished hydrophobic interactions at very low temperatures [15] The ionic interactions at the central helices G and M of the five CS structures are shown in Fig 5

The nature of the dimer interface of SsCS(85) differs from PfCS(100) in that there is a higher degree of hydrophobic interactions (Table 6) Similar levels to those in SsCS(85) are also observed in TaCS(55) All the thermophilic CSs show a low value for the gap volume index, the ratio of the gap volume to the accessible surface area of the interface, an indication that these proteins exhibit greater surface com-plementarity at the interface compared with ArCS(31) and pigCS(37)

Finally, examining the part of the dimer interface near the active site, all the archaeal CSs and the ArCS(31) have ionic interactions that also tend to stabilize the N-terminus; however, PfCS(100) certainly has the most extensive ionic interactions with two four-residue networks (E189,D12, R356¢ and R358¢) and two single ion pairs (E11-R353¢) A cluster of sixisoleucines, three from each monomer, was observed in this region for PfCS(100) [18], indicative of a strong hydrophobic interaction in this region Two of the three isoleucines are conserved in TaCS(55) and SsCS(85), residues 15 and 357, suggesting a similar role in these enzymes

Ionic interactions at the termini Ionic interactions may also be important for prevention of fraying of the N- and C-termini; several of these interactions

Table 5 Total number of ion pairs and ion pair networks in CS A and B refer to the two subunits of the dimer.

ArCS(31)

PigCS(37) (closed) TaCS(55) SsCS(85) PfCS(100)

Intra A networks

Inter AB networks

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interlink the two terminal regions, and therefore they may have additional relevance to the strength of the subunit interactions The lengths of the C-terminal arms vary, with ArCS(31) being sixresidues shorter than those of PfCS(100) and TaCS(55), and five shorter than SsCS(85) ArCS(31) has fewer interactions of the C-terminal arm with the other monomer than the three thermophilic CSs (including one ion pair that appears to anchor the end of the arm: R375-E48¢ in PfCS(100)) R375 and E48 are conserved in TaCS(55) and SsCS(85) suggesting the likelihood of this ion pair being present at their C-termini ArCS(31) also has

an arginine residue (R375) which interacts with E56¢ but, as this residue is four residues from the end of the C-terminus, there may be more chance of fraying of this terminal arm in the psychrophile Both N-termini in PfCS(100) also have an interconnecting ion pair (K8-D16¢) but this is a three residue interaction in ArCS(31) (K7, D15¢ and D359¢) SsCS(85) also has several terminal interactions (E9,R259 and R355¢) but TaCS(55) does not

Loop regions: length and ionic interactions

It has previously been suggested that loop regions tend to be the most flexible regions within a protein, and are therefore often the first areas to be subject to proteolytic cleavage or heat denaturation [29] It is possible therefore that increased thermostability may be achieved by shortening loops or by additional interactions stabilizing these regions

The equivalent loop regions of the five CSs have therefore been compared (several extra loops are present in the pig enzyme) Although some of the differences in loop conformations (particularly near the active site) may be due to the open or closed nature of the structures, it is immediately obvious on comparison of the ionic interac-tions that there is considerable difference between the five enzymes There is a distinct absence of ionic interactions in the loops of the pig enzyme, with the thermophilic and psychrophilic CSs showing more extensive interactions Many of these ionic interactions are involved with the dimer interface as well as those that interconnect one loop region with another, thus possibly pinning loops together The total number of ionic interactions present within the loop and dimer interface regions of the five CSs is: ArCS(31) 12, pigCS(37) 8, TpCS(55) 16, SsCS(85) 24 and PfCS(100) 18 As in evident, the thermophilic CSs have the highest number of these interactions, with SsCS(85) having the most However, the interactions in PfCS(100) tend to

be more complex, perhaps affording a greater degree of stabilization in particular areas of the protein structure; also, as detailed below, the PfCS(100) often has the shortest loops, reducing the need for a large number of ionic interactions in those specific regions The results for individual loops are:

Loop E-F The loop in pigCS(37) is one residue shorter than in ArCS(31), SsCS(85), and PfCS(100), and two residues shorter than TaCS(55), but the loops superimpose

Fig 4 Residues involved in ion-pair interactions at the dimer interface Spheres are drawn for at the Ca atoms of acidic (red) and basic (blue) side-chains involved in intersubunit ionic interactions From top to bottom: ArCS(31), PigCS(37), TaCS(55), SsCS(85) and PfCS(100).

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well and there are no ionic interactions in pigCS(37)

compared with a single ion pair in ArCS(31) TaCS(55)

has a four residue intramolecular network and SsCS(85) has

a five residue network that involves a residue at the

N-terminal end of the loop

Loop I-J All the enzymes have similarly large loops but

there are no ion pairs in pigCS(37) with one ion pair in

TaCS(55) ArCS(31), SsCS(85) and PfCS(100) all have

multiple ionic interactions linking loops I-J and J-K

Loop J-K The loop in pigCS(37) is slightly shorter than the

others and contains no interactions, whilst the TaCS(55)

loop has an ion pair ArCS(31), SsCS(85) and PfCS(100)

have interactions linking this loop with the previous loop I-J

Loop N-O This appears to be a long and flexible loop in

ArCS(31) and contains sixcharged residues, but no ion

pairs PigCS(37) also has a longer and more extended loop

than SsCS(85) and TaCS(55) (which both contain ion pairs)

and PfCS(100) has the shortest loop

Loop O-P Although not obvious from the length of loops

as designated byPROMOTIF[30], the loop in the pig enzyme

is considerably more extended than the others ArCS(31),

pigCS(37) and PfCS(100) all have single ion pairs

stabilizing this loop (the interaction in the pigCS(37) loop

links it to loop B-C), with TaCS(55) and SsCS(85) loops

having multiple ionic interactions that link loops O-P and

K-L

Loop P-Q TaCS(55) and SsCS(85) loops both contain ionic interactions In the case of SsCS(85), this is in the form

of a three residue network linking it with loop J-K This loop is absent in PfCS(100)

Loop Q-R This loop is shortest in ArCS(31) and it has already been suggested that the reason for this is that it seems to allow greater accessibility to the active site [15] However, recent site-directed mutagenesis studies to increase the length of this loop to mimic the situation in the PfCS(100), reveal that the cold activity of the ArCS(31)

is not significantly compromised by the mutations [31]

D I S C U S S I O N

The determination of the crystal structure of SsCS(85), and its comparison with four other CSs from organisms that essentially span the temperature range over which life exists, have allowed a detailed structural analysis to be performed

to investigate the structural mechanisms underlying protein thermal stability in this enzyme This has been possible because, in general, the 3D structures of the CSs are highly similar and we have therefore not only been able to identify specific differences, but have also succeeded in finding trends in structural changes that correlate with increasing thermostability of the individual proteins These identified structural differences are mainly concerned with the protein’s compactness, both in general and in the loop regions, and with the nature of the interactions at the dimer interface, possibly indicating that the respective protein thermostabilities are largely determined by these parts of the protein

General compactness The compactness of heat-stable proteins has often been found to be synonymous with their thermostability, and can be described in a number of ways There is a tendency towards a smaller accessible surface area and volume when comparing the thermophilic archaeal CSs with the pig-CS(37), and the tendency towards fewer cavities should also correlate with the improved hydrophobic packing of these proteins The increased complementarity of the dimer interface, as measured by the gap volume index, in the thermophilic enzymes may also be a significant feature Although the total percentage of atoms buried is similar for all the CSs, the decreased burial of hydrophobic groups

of ArCS(31) compared with the other CSs probably reflects the decreased entropic penalty of exposure of hydrophobic side-chains at psychrophilic temperatures (reviewed by [32,33])

Loop regions There is a tendency towards shorter (even absent) loop regions in the thermophilic CSs, correlating with the compactness of these proteins when compared with pig-CS(37) This trend has also been highlighted by analysis of mesophilic and thermophilic genome sequences, and was suggested to be a general strategy for thermostabilization [34] However, many of the shorter loops in the thermophilic

CS are similarly short in ArCS(31) (apart from loop N-O)

A more dramatic difference in the loops is seen in the

Fig 5 Diagram showing ionic interactions in the central helices (G and

M) of the dimer interface of ArCS, PigCS, TaCS, SsCS and PfCS.

Helices from different monomers are coloured blue and orange.

Table 6 The dimer interface of CS Statistics are calculated using the

protein–protein interactions server (Jones and Thornton, 1995) for the

CS crystal structures with the C-terminal arm removed.

ArCS

PigCS (closed) TaCS SsCS PfCS

Interface ASA ( A˚ 2 ) 3403 4934 3154 3363 3698

% of total ASA 21.0 24.0 19.0 19.8 22.6

% polar atoms 34.8 37.8 32.6 32.3 39.2

% nonpolar atoms 65.2 62.2 67.4 67.7 60.8

Hydrogen bonds 44 42 24 28 54

Gap volume ( A˚3) 10591 17164 6474 8474 9605

Gap volume index1.52 1.74 1.03 1.26 1.29

Trang 9

comparison of the ionic interactions in these regions: very

few are present in pigCS(37), but a large number occur in

the archaeal and bacterial proteins, with the thermophilic

CSs, particularly SsCS(85), having the most extensive

networks that cross-link loop regions A significant increase

in the number of long-range (in sequence terms) electrostatic

interactions is also observed in SsCs(85) and PfCS(100),

where they serve to tether different parts of the structure

together This compares with the observations of

b-glyco-sidase from S solfataricus [35], which was shown to have

ionic interactions (specifically networks) over the surface

of the protein such that they cross-linked areas of

surface structure Similarly, a mutational analysis of the

hyperthermostable indoleglycerol-phosphate synthase from

T maritima, in which an ion-pair linking two a-helices was

disrupted, resulted in a less stable protein [36]

Ionic interactions and hydrophobicity

at the subunit interface

The archaeal and bacterial CSs have a higher total number

of ionic interactions than the pigCS(37), which in fact

exhibits the lowest percentage participation of charged

residues in ion pairs or networks of the five enzymes in the

comparison The psychrophilic ArCS(31) actually has the

most ionic interactions, which we have suggested may be

related to cold stability [15], but with respect to subunit

association, PfCS(100) has the most extensive interactions

across the dimer interface whilst ArCS(31) has more than

either TaCS(55) or SsCS(85)

The eight-helical sandwich part of the dimer interface

shows a definite trend towards increasing hydrophobicity

going from ArCS(31) and pigCS(37) to TaCS(55) and

SsCS(85), and this may be indicative of the increasing

strength of the hydrophobic interaction with temperature,

at least to temperatures approaching 100C [37] PfCS(100)

also has a greater degree of hydrophobicity in this region

than ArCS(31) and pigCS(37) but lower than the other two

thermophilic CSs, and this may be compensated by the

more extensive ionic interactions in the hyperthermophilic

protein That is, the ionic interactions in the central helices

(G and M) of the eight-helical sandwich also show an

increase from none in pigCS(37), two single ion-pairs in

TaCS(55), four single ion-pairs in SsCS(85) and the two

five-residue networks in PfCS(100) ArCS(31) also has two

four-residue networks here, but these seem to be less

extensive than those in PfCS(100) (with fewer interactions

actually across the interface) PfCS(100) also has the

additional two four-residue networks near the active site

region (with the other archaeal and bacterial enzymes

displaying interactions to a lesser degree) as well as the

isoleucine cluster [18] which is partly conserved in TaCS(55)

and SsCS(85), again suggesting a better hydrophobic

packing than with either the mesophilic or psychrophilic

enzyme

Finally, the parts of the dimer interface associated with

both termini seem to be stabilized by ionic interactions

particularly in the PfCS(100), but also to some degree in

ArCS(31) and SsCS(85)

These conclusions with respect to the ionic interactions at

the subunit interface and termini are supported by

muta-genesis studies [38] Analysis of chimeric mutants between

the TaCS(55) and PfCS(100), where the large and small

domains were swapped, demonstrated that the determinants

of thermostability lie mainly with the large (subunit contact) domain, possibly correlating with the trend of increasing ionic interactions that are seen at the subunit interface as the thermostability of the enzyme increases Additionally, mutagenesis of the PfCS(100) where we have disrupted the ionic network at the subunit interface, and have removed the C-terminal ionic interaction, support the role

of these electrostatic bonds in the stability of the enzyme In nearly all cases, the catalytic parameters of the mutants were not significantly different from the wild-type enzyme, supporting the contention that we have not grossly altered the structure of the enzyme but have merely disrupted stabilizing ionic interactions

The importance of electrostatic interactions and their precise location to stabilizing proteins has been shown in other crystal structures of (hyper)thermostable proteins, as discussed in the recent review by Karshikoff and Ladenstein [10] The most striking examples include glutamate dehy-drogenase [39–41], glyceraldehyde 3-phosphate dehydro-genase [42,43] and lumazine synthase [44] Again, the electrostatic strengthening of the intersubunit contacts is a common theme in these proteins Finally, computational analyses [7,45,46] and genomic comparisons [8,9,47] add further support to these findings

Concluding remarks The importance of the determination of the structure of the SsCS(85) is principally that it completes a series of CS structures from which we are now able to identify trends in the structures of CSs that appear to be correlated with the different degrees of thermostability Our findings correlate well with the growing number of studies that conclude that ionic interactions stabilizing crucial areas of structure are perhaps the most common

proteins at high temperatures, particularly for oligomeric proteins Recent thermodynamic studies on a mesophilic and thermophilic pair of CheY proteins, have suggested that

a reduced change in heat capacity upon unfolding is a possible indicator of thermostability [48], also supported by studies on a mesophilic and thermophilic pair of Rnase H proteins [49] These studies suggest that it may be difficult to dissect the contributions of individual interactions to thermostability This may be true for small monomeric proteins such as CheY and RnaseH, but for oligomeric proteins that make up > 85% of intracellular proteins, the nature of the oligomer interface is key Ionic networks at interfaces, however, are not the exclusive means of gaining thermostability, as the tetrameric triosephosphate isomerase structure from P furiosus has shown [50] Nevertheless, for the family of CSs presented here, increased ionic interac-tions either between loops or at the dimer interface do appear to correlate with increasing thermostability The results presented here lay the foundation for a suite of site-directed mutagenesis experiments to investigate the precise role of each of the sets of individual interactions in the five dimeric CSs Preliminary experiments that remove interac-tions and destabilize the enzyme without affecting catalytic activity have already been successfully carried out, but the real proof of their importance is now to introduce ionic bonds and networks into the less stable CSs to increase their thermostability

Trang 10

A C K N O W L E D G E M E N T S

This work was supported by the Biotechnology and Biological Sciences

Research Council.

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