Báo cáo khoa học: Structural adaptation to low temperatures ) analysis of the subunit interface of oligomeric psychrophilic enzymes pdf

The following structural parameters were calculated: overall and core interface area, characteriza-tion of polar⁄ apolar contributions to the interface, hydrophobic contact area, quantit

the subunit interface of oligomeric psychrophilic enzymes Daniele Tronelli1, Elisa Maugini1, Francesco Bossa1and Stefano Pascarella1,2

1 Dipartimento di Scienze Biochimiche ‘A Rossi Fanelli’, Universita` degli Studi di Roma ‘La Sapienza’, Rome, Italy

2 Centro Interdipartimentale di Ricerca per la Analisi dei Modelli e dell’Informazione nei Sistemi Biomedici (CISB), Universita` degli Studi di Roma ‘La Sapienza’, Rome, Italy

Many terrestrial environments present physical and

chemical conditions that can be defined as extreme

from a human point of view Among these, permanent

cold environments are the most common In fact,

about 70% of the earth’s surface is covered by the

oceans, whose temperature is constantly at 4–5C

below a depth of 1000 m, regardless of the latitude

Moreover, polar regions constitute a further 15% of

the earth, and there are also alpine regions and gla-ciers Ectothermic organisms that have colonized such environments are called psychrophiles, and, consider-ing their spread, represent a considerable component

of the biosphere, in terms of species diversity and biomass Psychrophilic organisms include eubacteria, archaea, protozoa, fungi and multicellular eukaryotes such as algae, invertebrates and fish [1,2]

Keywords

cold-adapted enzymes; electrostatic and

hydrophobic interactions; interface; protein

quaternary structure; psychrophiles

Correspondence

S Pascarella, Dipartimento di Scienze

Biochimiche, Universita` ‘La Sapienza’,

P le A Moro 5, 00185 Rome, Italy

Fax: +39 06 49917566

Tel: +39 06 49917574

E-mail: Stefano.Pascarella@uniroma1.it

Website: http://schubert.bio.uniroma1.it/

(Received 8 June 2007, revised 12 July

2007, accepted 13 July 2007)

doi:10.1111/j.1742-4658.2007.05988.x

Enzymes from psychrophiles show higher catalytic efficiency in the 0–20C temperature range and often lower thermostability in comparison with meso⁄ thermophilic homologs Physical and chemical characterization of these enzymes is currently underway in order to understand the molecular basis of cold adaptation Psychrophilic enzymes are often characterized by higher flexibility, which allows for better interaction with substrates, and

by a lower activation energy requirement in comparison with meso⁄ ther-mophilic counterparts In their tertiary structure, psychrophilic enzymes present fewer stabilizing interactions, longer and more hydrophilic loops, higher glycine content, and lower proline and arginine content In this study, a comparative analysis of the structural characteristics of the inter-faces between oligomeric psychrophilic enzyme subunits was carried out Crystallographic structures of oligomeric psychrophilic enzymes, and their meso⁄ thermophilic homologs belonging to five different protein families, were retrieved from the Protein Data Bank The following structural parameters were calculated: overall and core interface area, characteriza-tion of polar⁄ apolar contributions to the interface, hydrophobic contact area, quantity of ion pairs and hydrogen bonds between monomers, inter-nal area and total volume of non-solvent-exposed cavities at the interface, and average packing of interface residues These properties were compared

to those of meso⁄ thermophilic enzymes The results were analyzed using Student’s t-test The most significant differences between psychrophilic and mesophilic proteins were found in the number of ion pairs and hydrogen bonds, and in the apolarity of their subunit interface Interestingly, the number of ion pairs at the interface shows an opposite adaptation to those occurring at the monomer core and surface

Abbreviations

CS, citrate synthase; MDH, malate dehydrogenase; TIM, triose phosphate isomerase.

To survive at temperatures close to the freezing

point of water, psychrophiles have evolved some

important cellular adaptations, including mechanisms

to maintain membrane fluidity [3,4], synthesis of

cold-acclimation proteins [5], freeze tolerance strategies [6],

and cold-active enzymes Psychrophilic enzymes are of

great interest in the scientific community, and are

cur-rently under study to characterize their physical and

chemical properties in an attempt to understand the

molecular basis of cold adaptation

Low temperatures have a negative effect on enzyme

kinetics: any decrease in temperature results in an

exponential decrease in reaction rate For example,

lowering the temperature by 10 C causes a two-fold

to four-fold decrease in enzyme activity [1,7]

There-fore, enzymes from psychrophiles show high catalytic

efficiency in the 0–20C temperature range,

tempera-tures at which counterparts from mesophilic or

ther-mophilic organisms do not allow adequate metabolic

rates to support life or cellular growth Such high

activity balances the cold-induced inhibition of

reac-tion rates However, the structure of cold-adapted

enzymes is also heat-labile Indeed, low stability at

moderate temperatures (usually > 40C) is the other

peculiar characteristic of psychrophilic enzymes [8,9]

This trend was revealed by calorimetric analysis of

residual enzyme activities after incubation at increasing

temperatures (it should be pointed out, however, that

the loss of activity at moderate temperatures might not

be always directly related to the loss of enzyme

struc-ture) It is generally believed that cold adaptation

results from a combination of lack of selective pressure

for thermostability and strong selection for high

activ-ity at low temperatures [1]

Psychrophilic enzymes are often characterized by high

flexibility [10], which allows better interaction with

sub-strates, and by lower activation energy requirements in

comparison with their mesophilic and thermophilic

counterparts Hence, the presence of high flexibility

could explain both thermolability and high catalytic

effi-ciency at low temperatures [11] The higher structural

flexibility of psychrophilic enzymes, as compared to

their mesophilic and thermophilic counterparts, could

be the result of a combination of several features:

weak-ening of intramolecular bonds (fewer hydrogen bonds

and salt bridges as compared to mesophilic and

thermo-philic homologs have been shown); a decrease in

com-pactness of the hydrophobic core; an increase in the

number of hydrophobic side chains that are exposed to

the solvent; longer and more hydrophilic loops; a

reduced number of proline and arginine residues; and a

higher number of glycine residues [12–15] However,

each protein family adopts its own strategy to increase

its overall or local structural flexibility by using one or a combination of these structural modifications

Earlier studies on the structural adaptation of extremophilic enzymes [16–19] were based on compara-tive analysis, also using homology modeling in cases where no experimental three-dimensional structures were available [20,21] These approaches could give valuable information on rules to be followed by pro-tein engineers to produce modified enzymes with suit-able features for biotechnological applications [22] In fact, because of their high catalytic efficiency at low temperatures, psychrophilic enzymes are investigated for their high potential economic benefit: in particular, they could be utilized in industrial processes as energy savers, and in the detergent industry as additives [23,24] Also, the possibility of selecting and rapidly inactivating these enzymes, due to their high thermola-bility, makes psychrophilic enzymes extremely useful in biomolecular applications [25]

Previous comparative studies investigated factors governing cold adaptation occurring in the protein structure core, in the enzyme active site, and in the overall protein structure However, although the molecular adaptation of enzymes to extreme conditions has been intensively studied, not very much is known about the adaptations that have occurred at the inter-face of oligomeric enzymes Even less is known regard-ing the adaptation of the psychrophilic interface of oligomeric enzymes Intersubunit interactions have spe-cial importance in the stability of oligomeric psychro-philic enzymes and their function Indeed, interface regions between protein monomers are mainly respon-sible for the maintenance of the quaternary structure

in oligomeric enzymes The hydrophobic interaction is

at the base of the process of folding and the stabiliza-tion of protein associastabiliza-tion [26,27] The hydrophobic interaction occurs when apolar residues aggregate in

an aqueous environment, achieving a loss in free energy that stabilizes the protein structure During association of monomers, hydrophobic residues are buried in the interface region, minimizing the number

of thermodynamically unfavorable solute–solvent inter-actions Other important features, such as interface extension, residue packing, hydrogen bonds, salt bridges and internal cavities, can play a significant role

in the stability of the quaternary structure in cold-adapted enzymes

Our work is aimed at detecting the structural varia-tion related to the cold adaptavaria-tion of the subunit inter-face of oligomeric psychrophilic enzymes To our knowledge, this is the first study entirely focused on the analysis of the molecular adaptations that have occurred at the level of subunit interfaces of

psychro-philic enzymes Some of the key questions are as

fol-lows Are these interfaces significantly different from

the interfaces of mesophilic enzymes? Which structural

features are mostly variable? Are the interface

adapta-tions different from those occurring at the level of

monomer hydrophobic core and surface? The answers

to these questions can give indications about aspects

of protein–protein interaction at low temperatures and

suggest rules for interface engineering

Results

The main dataset utilized for the analysis (Table 1)

contained five psychrophilic, 20 mesophilic, four

thermophilic and six hyperthermophilic structures, accounting for a total of 35 oligomeric enzymes corre-sponding to a total of 30 pairwise comparisons The proteins belong to five families: citrate synthase (CS) [17], triose phosphate isomerase (TIM) [28], malate dehydrogenase (MDH) [18], alkaline phosphatase [29] and glyceraldehyde-3-phosphate dehydrogenase [30] In total, 21 incomplete side chains distributed in six pro-teins (1cer, 1a59, 1ixe, 1ew2, 4gpd, 1bmd) were rebuilt

as described in Experimental procedures The careful reconstruction of the 21 incomplete side chains was nec-essary to include in the working dataset as much infor-mation as possible Ideally, only complete coordinate sets should be used, to avoid any artefact However, the

Table 1 List of enzymes used in the work (main dataset) AP, alkaline phosphatase; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; PDB, Protein Data Bank.

Growth temperature (C)

Structure resolution (A ˚ )

Identitya (%)

PDB ID

Sequence length (monomer) No of subunits

a Percentage residue identity to the psychrophilic reference (Ref.) homolog sequence.

21 side chains represent in this case only 0.3% of all the

interface residues contained in the working dataset

Consequently, even if, in the worst case, the

reconstruc-tion was not correct, the potential effect on the final

statistics would be negligible The average sequence

identity was calculated between enzyme pairs of the

same family, and gave a value of 49.0% over all protein

families

The differences in structural features observed

between the psychrophilic and mesophilic enzymes

were compared with the differences between the same properties calculated from an oligomeric mesophilic reference dataset (Table 2) This nonredundant refer-ence dataset contained 148 protein structures belonging

to 43 oligomeric enzyme families, with an average sequence identity between enzyme pairs of the same family of 52.4% The dataset generated a total of 514 pairwise comparisons The whole dataset included 10 mainly-a domains, two mainly-b domains, and 53 a–b domains The taxonomic composition of the

Table 2 List of mesophilic enzymes used in the reference dataset.

20 Superoxide dismutase (iron ⁄ manganese) 1isa, 3sdp, 2awp, 2a03, 1uer, 1y67

36 Hypoxanthine phosphoribosyltransferase (dimeric) 1tc1, 1pzm, 1hgx

37 Hypoxanthine phosphoribosyltransferase (tetrameric) 1grv, 1z7g, 1cjb

dataset included species belonging to the prokaryotes

and eukaryotes (comprising protozoa and multicellular

organisms such as invertebrates, fish, and mammals)

The significance of the observed differences in

structural properties, calculated as described in

Experimental procedures, was measured by a t-value

t-values¼ +1.96 or t-values ¼ )1.96 with a number

of degrees of freedom > 500 correspond to a

P-value¼ 5% that the null hypothesis is true

This value represents the significance threshold

adopted in our analyses

The structural properties tested at the subunit

interface were: interface and core interface extension;

number of ion pairs and hydrogen bonds; fraction of

apolar contact surface; atomic packing; volume and

internal surface area of interface cavities; and fraction

of apolar surface in the interface and in the core

inter-face

Whenever applicable, the properties were normalized

by interface extension and number of interface

resi-dues However, as there was no difference between the

two normalizations, only the former is considered

here

Table 3 shows the t-values and the percentage

prob-abilities relative to the structural differences in the

number of strong, weak and total ion pairs and

hydro-gen bonds between psychrophilic and meso⁄

thermo-philic homologs The t-value relative to the strong ion

pairs at the interface indicates a significant increase in

these electrostatic interactions in the psychrophilic

enzymes as compared to the mesophilic enzymes The

same results were found for weak and total ion pairs

The significance of this trend decreased in the

compari-son of psychrophilic with both mesophilic and

thermo-philic enzymes Figure 1 shows the normalized mean number of total ionic interactions at the interface, calculated from psychrophilic, mesophilic and thermo-philic protein structures for each family of the main dataset The number of total ion pairs at the interface was normalized by the number of residues composing the interface For each one of the five enzyme families considered, the number of ion pairs was higher in psy-chrophilic proteins than in mesophilic ones, whereas in three cases out of four (atomic coordinates of thermo-philic AP are not available), the number of ion pairs was higher in thermophilic proteins than in mesophilic ones, with the exception of the TIM enzyme family

No significant trend was found in the comparison of strong ion pairs between psychrophilic and meso⁄ ther-mophilic homologs (Table 3) The t-value for hydrogen bonds (Table 3) showed a significant decrease in these interactions at the oligomeric interface of psychrophilic enzymes when compared to their mesophilic counter-parts The trend held for the number of hydrogen bonds per unitary surface and per interface residue The inclusion of thermophilic enzymes in the compari-son increased both tendencies

The volume of the interface internal cavities, as well

as the amino acid packing at the interface, did not show any measurable difference between psychrophilic and mesophilic proteins, and therefore the results are not shown

Psychrophilic enzymes (Table 4) showed a significant decrease in the apolar fraction of interface in compari-son with their mesophilic counterparts (t-value of ) 2.12, P ¼ 3.44) The trend was strengthened by the inclusion of thermophilic enzymes in the comparison Figure 2 shows the percentage of apolar interface,

Table 3 Statistical analysis of the differences in the number of

interface ion pairs and hydrogen bonds The electrostatic interaction

quantities showed were normalized by mean interface surface.

psy, psychrophilic; mes, mesophilic; therm, thermophilic.

Interface structural property t-value P-value (%)

Strong ion pairs

Weak ion pairs

Total ion pairs

Hydrogen bonds

0 0.1 0.2 0.3 0.4 0.5 0.6 0.7

GAPDH AP

TIM

Fig 1 Normalized mean number of psychrophilic (white), mesophilic (gray) and thermophilic (black) interface total ionic interactions, calcu-lated from protein structures for each family of the main dataset The number of total ion pairs at the interface was normalized by the num-ber of residues composing the interface.

calculated from psychrophilic, mesophilic and

thermo-philic protein structures for each family of the main

dataset Each apolar interface area was normalized by

the total interface area With the exception of the CS

enzyme family, the percentage of apolar interface was

lower in cold-adapted enzymes than in mesophilic

ones, whereas in three cases out of four, the percentage

of apolar interface was higher in thermophilic proteins

than in mesophilic ones, with the exception of the

MDH enzyme family A similar significant trend was

found for the hydrophobic contact area at the interface (Table 4)

No significant trend was detected in the comparison

of the percentage of overall interface area between psychrophilic and mesophilic proteins However, this trend became significant upon inclusion of thermophilic enzymes (t-value of) 2.19) in the comparison (Table 4) Psychrophilic enzymes (Table 4) did not show signif-icant variation of the core interface area and of the core interface apolar atomic composition when com-pared to mesophilic counterparts and to meso⁄ thermo-philic counterparts

Table 5 reports the results of the validation of the ref-erence mesophilic dataset to exclude potential statistical bias on the t-tests applied to the psychrophilic proteins The number of randomized tests out of 1000 trials that resulted in a nonsignificant t-value were recorded for each structural properties On average, a structural property obtained a nonsignificant t-value in 820 out of

1000 randomized tests This suggests that the reference dataset appropriately represents the mesophilic proteins

in the main dataset

Discussion

This research was aimed at elucidating the adaptations that have occurred at the interface of oligomeric enzymes synthesized by psychrophilic microorganisms

We analyzed the structural differences between the oligomeric interfaces of psychrophilic and meso⁄ ther-mophilic homologs Psychrophilic oligomeric enzymes must maintain high structural flexibility and, at the same time, the correct quaternary structure Hence, the comparison was focused on those physicochemical characteristics of the interface that are related to struc-tural stability, namely: apolar contact surface; number

of ionic interactions and hydrogen bonds; atomic packing; presence of cavities; percentage of apolar

Table 4 Statistical analysis of the differences in the percentage of

apolar interface, the hydrophobic contact area of interface, and the

percentage of overall interface area psy, psychrophilic; mes,

meso-philic; therm, thermophilic.

Interface structural property t-value P-value (%)

Percentage of apolar interface

Hydrophobic contact area

in the interface

Percentage of overall

interface area

Percentage of apolar

core interface

Percentage of overall

core interface area

0

10

20

30

40

50

60

70

80

GAPDH AP

TIM

Fig 2 Percentage of psychrophilic (white), mesophilic (gray) and

thermophilic (black) apolar interface, calculated from protein

struc-tures for each family of the main dataset Each apolar interface

area was normalized by the total interface area.

Table 5 Statistical validation of the reference mesophilic dataset.

Interface structural property

% not significanta

Hydrophobic contact area in the interface 83.7

Percentage of overall core interface area 84.5 a

Fraction of randomized trials that resulted in a nonsignificant t-value.

surface; and interface and core interface extension It

could be argued that cold interface adaptations may

be similar to those occurring at the level of the

mono-mer hydrophobic core and surface, which have already

been thoroughly studied However, it was previously

shown that hydrophobic interactions play a less

rele-vant role in protein binding than in protein folding

[31] Furthermore, in 1997, Xu and colleagues found

that hydrophilic bridges, established between charged

or polar atoms, can result in stronger stabilization in

monomer–monomer binding than in the interior of

monomers, due to the different environments to which

such interactions are exposed [32]

prompted one of our initial questions: are the

psychro-philic interface adaptations different from those

occur-ring at the level of the protein hydrophobic core and

surface? We believe that our comparative analysis can

correctly answer this question

However, one of the criticisms of such comparative

studies is the way in which the statistical significance

of the differences is assessed Therefore, in this

analy-sis, for each structural feature, a robust reference

dis-tribution of the differences observed in the comparison

of 148 mesophilic protein interfaces belonging to 43

oligomeric enzyme families was calculated Such a

dis-tribution answers the question of what difference

should be expected for the structural property if the

interfaces of two homologous mesophilic enzymes were

compared The t-test should then establish whether the

magnitude of the differences detected between the

psy-chrophilic enzyme and the meso⁄ thermophilic

counter-parts is significantly different from that expected from

a mesophilic–mesophilic comparison

The features that showed a significant difference

from the reference sample were: (a) increase in the

number of ionic interactions; (b) decrease in the

num-ber of hydrogen bonds; (c) decrease in the fraction of

apolar interface; and (d) decrease in the apolar contact

surface

However, the cold-adapted enzymes considered here

also showed a significant decrease in the overall

inter-face area, when compared with both mesophilic and

thermophilic homologs, but this trend disappeared

after comparison with the sole mesophilic oligomers

(Table 4) This suggests that the thermophilic enzymes

need a wider interface to maintain oligomer stability,

but psychrophilic counterparts obtain no advantage

from the shrinkage of the interface extension

The increase in the number of strong, weak and total

ion pairs at the interface of psychrophilic enzymes is

important in maintaining the quaternary structure,

whereas the strength of hydrophobic interactions is

diminished at low temperature It should be considered

that, at moderate temperatures, hydrophobic interac-tions are the most relevant forces for the preservation

of enzyme structure On the other hand, the trend for apolar surfaces to interact with other apolar surfaces, rather than with water, decreases at low temperatures, because solvation of nonpolar surface is thermodynam-ically favored at low temperatures This effect can lead

to cold-induced denaturation, particularly of the most hydrophobic proteins, as well as oligomeric enzymes [33] Moreover, it was previously observed [34] that psychrophilic enzymes are affected by the weakening of hydrophobic interactions at low temperatures Hence,

in these conditions, hydrophobic interactions are less relevant in maintaining the quaternary structure, and this phenomenon is reflected in the significant decrease

in apolar components at the interface that we have found for oligomeric psychrophilic enzymes Moreover, our analysis underlines a significant decrease in the interface apolar contact area when comparing psychro-philic and mesopsychro-philic enzymes or psychropsychro-philic and mesophilic plus thermophilic enzymes The same trend was previously found in the analysis of hydrophobicity

in core residues of psychrophilic proteins [35] Indeed, buried residues in psychrophilic enzymes show weaker hydrophobicity than those in their mesophilic homo-logs, making the protein interior less compact and more flexible Therefore, a lower degree of hydropho-bic interaction renders the role of salt bridges more relevant in stabilizing the protein quaternary structure

of oligomeric cold-adapted enzymes, particularly if we consider that, as the formation of ion pairs is an exo-thermic electrostatic interaction, they are particularly strong at low temperatures A similar hypothesis was put forward by Russell et al [17] with regard to cold-active CS in comparison with the hyperthermophilic homolog The authors observed an increase in psychro-philic intramolecular ion pairs, but paradoxically also a reduced number of interface ion pairs They concluded that a large number of intramolecular ion pairs may serve to counteract the reduced thermodynamic stabilization due to hydrophobic interaction at low temperatures, preventing the cold denaturation of psy-chrophilic CS However, the psypsy-chrophilic enzyme showed a reduction in the extent of intersubunit ion pairs in comparison with the hyperthermophilic homo-log Another comparative study of psychrophilic MDH and its thermophilic counterpart revealed the same trend: the cold-adapted enzyme had more intrasubunit and fewer intersubunit ion pairs [18] This is in appar-ent contrast with our results Indeed, in our analysis,

we observed a significant increase in interface ion pairs for psychrophilic enzymes when compared exclusively

to mesophilic homologs (Fig 1) The significance of

this trend decreased when psychrophilic enzymes were

compared to mesophilic plus thermophilic proteins

This suggests that, in general, psychrophilic enzymes

establish, on average, a number of ionic interactions at

the subunit interface that is slightly lower or

compara-ble with that observed in thermo⁄ hyperthermophilic

counterparts, but definitely higher than in mesophilic

proteins

It should be noted, therefore, that although

psychro-philic and thermopsychro-philic enzymes are adapted to

oppo-site temperature conditions, the structural adaptation

strategies, relying on ionic interactions, appear to be

similar A consistently higher number of interface salt

bridges, in comparison to that present in mesophilic

oligomers (Fig 1), could therefore be useful to

improve the cohesion between monomers and to avoid

both cold-induced and heat-induced unfolding in

psy-chrophilic and thermophilic enzymes, respectively

These results underline the fact that the comparative

analyses for determining the structural differences

related to thermal adaptation of psychrophiles should

always include both mesophilic and thermophilic

coun-terparts to ensure that the significant structural

differ-ences are appreciated

The role of hydrogen bonds in psychrophilic protein

adaptation is widely accepted Previous studies showed

that cold-adapted enzymes have fewer total hydrogen

bonds than their meso⁄ thermophilic homologs

Accordingly, we found a significant decrease in the

number of hydrogen bonds at the interface of

psychro-philic oligomers when compared with mesopsychro-philic

enzymes This trend increased when thermo⁄

hyper-thermophilic enzymes were included in the working

dataset Our findings underline the role of this kind of

electrostatic interaction in determining greater stability

of the quaternary structure in mesophilic and

thermo-philic proteins; in heat-labile cold-adapted enzymes,

the number of interface hydrogen bonds is lower At

the moment, no satisfactory mechanistic explanation

for the decrease in the number of interface hydrogen

bonds has been proposed

Other structural features analyzed did not show any

significant trend In particular, no significant trend was

detected in the comparison of the percentage of core

interface area and in the comparison of the core

inter-face apolar atomic composition between psychrophilic

and mesophilic proteins These results, showing that

the percentage of core interface area does not show a

significant difference, could be interpreted in the light

of the work of Bahadur et al [36] These authors

stud-ied the subunit interfaces of 122 homodimers, and

showed that the distribution of the area between the

rim and core interface varies widely from one oligomer

to another This could lead to a large value for the standard deviation of distributions of the rim and core interface extension, and, as a consequence, could lead

to the small t-value

An analysis of amino acid packing in mesophilic and thermophilic enzymes was performed by Karshikoff & Ladenstein [37] to determine the role of packing density

in thermostability They concluded that mesophilic and thermophilic proteins do not differ in the degree of packing Likewise, our analysis did not find any mea-surable difference in the amino acid packing at the interface of psychrophilic enzymes and in the total vol-ume of internal interface cavities, and for this reason the results are not shown

In conclusion, the answers to our initial questions reveal that the interfaces of oligomeric psychrophilic enzymes are significantly different from those of their mesophilic and thermophilic homologs The most vari-able features are the increase in the number of ionic interactions, the decrease in the number of hydrogen bonds, the decrease in the fraction of apolar interface, and the decrease in the apolar contact surface There-fore, the structural adaptations observed are similar to those occurring at the monomer core and surface, with the notable exception of the increase in the number of ionic interactions Indeed, it has been reported that, in general, the flexibility of the monomeric structure is often achieved via a reduction of electrostatic inter-actions in psychrophiles Our results suggest that the interfaces of oligomeric psychrophilic enzymes need to

be stabilized by the introduction of additional ion pairs

It should be considered that relatively few structures

of oligomeric psychrophilic enzymes are presently available Therefore, although the conclusions reported here are correct from the statistical point of view, par-ticularly considering the robust testing procedure adopted, the results may change with the availability

of significantly more data To confirm the results described here, the analysis should be repeated when more structures of psychrophilic oligomeric enzymes are available

Several other analyses of the structural basis of enzyme cold adaptation have recently appeared in the literature For example, Jahandideh et al [38] reported

a statistical analysis of the sequence and structural parameters enhancing adaptation of proteins to low temperatures Their work was aimed at the detection

of variations in structural properties for the entire enzyme molecule, without any focus on the subunit interface Indeed, they considered both monomeric and oligomeric enzymes in their dataset, which included

13 pairs of homologous psychrophilic and mesophilic

proteins The structural properties tested were residue

frequencies, helical and tight turn content, backbone

hydrogen bonds, and disulfide bonds They assessed

the significance of the differences between the average

values of each structural property taken into account,

calculated over 13 psychrophilic and mesophilic

homo-logs Moreover, they utilized a t-test with a significance

threshold lower than that which we used in our

analy-sis, corresponding to a P-value equal to 0.1 with 24

degrees of freedom These differences make it difficult

to relate the results presented here to those reported

by Jahandideh et al [38], as well as to those of

previ-ous analyses Indeed, to our knowledge, the analysis

described here is the first systematic study of cold

adaptation at the level of the subunit interface

None-theless, Jahandideh et al [38] came to the conclusion

that the number of hydrogen bonds and, generally, the

number of electrostatic interactions are decreased in

psychrophilic proteins

It should be noted that, although each enzyme

fam-ily has its own strategy to increase flexibility by using

one or a combination of the above alterations in

struc-tural features [1], even with a relatively limited number

of psychrophilic enzyme structures available, some

general trends involved in the maintenance of both

structural flexibility and quaternary stucture in

oligo-meric psychrophilic enzymes can be appreciated by

comparative analysis

In conclusion, this kind of comparative analysis can

contribute to the elucidation of structural determinants

of adaptation of proteins to extreme conditions, and

can give useful hints on how to modulate, through

protein engineering, the stability and catalytic features

of enzymes of biotechnological interest

Experimental procedures

Collection of main dataset

The crystallographic structures of the available cold-active

oligomeric enzymes were found in the Brookhaven Protein

Data Bank [39] The search was carried out with the

key-words ‘psychro’, ‘cold’, ‘arctic’, ‘antarctic’ and the like

Only psychrophilic enzyme structures, for which exceptional

high cold activity and low thermostability have previously

been shown, were considered The protein structures

corre-sponding to the biological units were collected from the

Protein Quaternary Structure databank [40] Homologous

structures from mesophilic and thermophilic organisms

were subsequently retrieved from the Protein Data Bank

and Protein Quaternary Structure databank by means of

the program blast [41] To ensure structural homology,

psychrophilic sequence were considered Only unique tures were retrieved, and when there were alternative struc-tures for the same protein, only those displaying the best resolution and without point mutations were collected Pro-teins from plants were not taken into consideration, owing

to the ambiguous definition of ‘optimum temperature’ for such organisms

In order to assess the structural similarity within each collected family, we performed a structural alignment using the ce-mc program [42] Sequences of the selected proteins were aligned to each psychrophilic homolog The align-ments were then manually corrected by inspection of the superimposed structures

All the programs were written in PERL language and

LINUX 4.0 operating systems

Crystallographic structure quality assessments

All structures showing a resolution worse than 2.85 A˚ were excluded from the main dataset All the incomplete interface side chains were rebuilt using the program biopolymer of the insightii package (version 2005; Accelrys, San Diego,

CA, USA) The side chain rotamer displaying the lowest nonbond energy was kept and treated as experimental Ligands (cofactors, inhibitors, substrate analogs, etc.) and solvent molecules were always removed from the structures The quality assessment of the crystallographic structures was carried out using procheck software [43] Only two structures, 4gpd and 1ypi, did not pass the procheck stereochemical quality check, showing an overall average

acceptable quality For these, an energetic minimization was performed using the program modeller [44] of the

After the energy minimization, the quality of the two struc-tures was evaluated using the program prosaII [45] The refined structures showed overall average G-factors of ) 0.47 and ) 0.02, respectively

Identification of interface residues

In each oligomeric enzyme, the interface region was defined

as being composed of those residues that change their solvent accessibility area in the monomeric and in the oligo-meric state (Fig 3) Solvent accessibility computation [47] was performed with naccess [48] The change in solvent accessibility area for each residue in the monomeric state and in the oligomeric state was calculated using a PERL script The interface residues were defined as those residues that show a change in solvent accessibility area upon monomer association Those residues for which the change was more than 90% were defined as composing the core interface [36]

The structural similarity of the subunit interfaces within

each protein family was evaluated on the basis of the

multi-ple structure alignment To ensure that the interface was

structurally conserved within each family and the selected

of each mesophilic and thermophilic member were

superim-posed on the equivalent atoms from the psychrophilic

con-sidered to be similar (Fig 4) This threshold is within the

expected structural variation corresponding to the range of

sequence similarities of the multiple structure alignments

[49] Indeed, the expected value of rmsd for a pair of

homologous proteins whose sequence identity is 30% is

equal to 1.42 A˚ rmsds were calculated using the deepview–

Surface characteristics

overall surface composing the interface and the core inter-face, the percentages of polar and nonpolar atomic contri-butions to the interface, and the percentages of polar and nonpolar atomic contributions to the core interface The overall hydrophobic contact area between residues of different monomers was calculated using the program

areas between apolar atoms using a set of 512 points located

on a sphere around each atom The sphere interaction radius

of each atom is equal to the sum of the van der Waals radius

of the atom type plus the radius of a water molecule Then, for each atom, the closest interacting atom is found for every point that is not buried by other atoms of the same residue

Hydrogen bonds and ion pairs

Hydrogen bonds were calculated using hbplus [52] with the default parameters, except for the maximum distance between donor and acceptor, which was set to 3.5 A˚ instead of 3.9 A˚, to be closer to that proposed in 1984 by Baker & Hubbard (3.1–3.2 A˚) [53]

Ion pairs at the interface were identified using a PERL script, on the basis of calculation of atomic distance Two residues with opposite charges are considered a strong ion pair if the distance between charged atoms is less than 4 A˚ This distance threshold is generally accepted after system-atic analysis on a sample of protein structures [54] Weak ion pairs, which are established up to a distance of 8 A˚ [16], were also considered Positively charged atoms were arginine and lysine side chain nitrogens Negatively charged atoms were side chain carboxylate oxygens of glutamate and aspartate Complex salt bridges, defined as ion pair interactions joining more than two side chains, and simple salt links involving two side chains but more than two charged atoms were not considered in the calculations as a single interaction; rather, each individual atomic interaction (single ion pair) was counted [14] Ion pairs involving histi-dine were not considered because of the ambiguous assign-ment of its protonation state in the proteins Once all interactions were found, only those between residues of dif-ferent monomers were considered

Packing density, cavity volume and cavity internal surface

The atomic packing of interface residues was computed using the os program [55] The os package calculates the

Fig 3 Interface and core interface of the dimer of thymidylate

syn-thase from Lactobacillus casei (2tdm) The rim interface residues,

in green, show a change in solvent accessibility area upon

mono-mer association that is smaller than 90% Core interface residues,

in red, show a change greater than 90% The remaining

solvent-accessible residues are shown in gray Drawn with PYMOL [46].

Fig 4 Structural superimposition of 14 TIMs Interface regions are

in red Drawn with INSIGHTII (version 2005, Accelrys, Cambridge, UK).