Structural and Functional Adaptations to Extreme Temperatures in Psychrophilic, Mesophilic, and Thermophilic DNA Ligases*

Psychrophiles, host of permanently cold habitats, display metabolic fluxes comparable to those exhibited by mesophilic organisms at moderate temperatures. These organisms have evolved by producing, among other peculiarities, cold-active enzymes that have the properties to cope with the reduction of chemical reaction rates induced by low temperatures. The emerging picture suggests that these enzymes display a high catalytic efficiency at low temperatures through an improved flexibility of the structural components involved in the catalytic cycle, whereas other protein regions, if not implicated in catalysis, may be even more rigid than their mesophilic counterparts. In return, the increased flexibility leads to a decreased stability of psychrophilic enzymes. In order to gain further advances in the analysis of the activity/flexibility/stability concept, psychrophilic, mesophilic, and thermophilic DNA ligases have been compared by three-dimensional-modeling studies, as well as regards their activity, surface hydrophobicity, structural permeability, conformational stabilities, and irreversible thermal unfolding. These data show that the cold-adapted DNA ligase is characterized by an increased activity at low and moderate temperatures, an overall destabilization of the molecular edifice, especially at the active site, and a high conformational flexibility. The opposite trend is observed in the mesophilic and thermophilic counterparts, the latter being characterized by a reduced low temperature activity, high stability and reduced flexibility. These results strongly suggest a complex relationship between activity, flexibility and stability. In addition, they also indicate that in cold-adapted enzymes, the driving force for denaturation is a large entropy change.


INTRODUCTION
The temperature range in which biological activity has been detected extends from -20°C, temperature recorded in the brine veins of Arctic or Antarctic sea ice (1), to 113°C, temperature at which the archae Pyrolobus fumarii is still able to grow (2). Although numerous investigations have been carried out on thermophilic microorganisms and on their molecular components, especially their enzymes, the efforts devoted to cold-adapted microorganisms have been comparatively limited despite their tremendous biotechnological (1,(3)(4)(5) and fundamental (1,(6)(7)(8) applications. Indeed, the biochemical and physiological bases of cold-adaptation which include, for example, regulation of gene expression by low temperatures, membrane adaptation in relation with the homeoviscosity concept and the activity-stability relationships sustaining the catalytic efficiency of cold-adapted enzymes, are still poorly understood.
In permanently cold-habitats, low temperatures have constrained psychrophiles to develop, among other peculiarities, enzymatic tools allowing metabolic rates compatible to life that are close to those of temperate organisms. Thermal compensation in these enzymes is reached, in most cases, through a high catalytic efficiency at low and moderate temperatures (for compilation, see (9,10)). The emerging picture is that this increased catalytic efficiency is attributed to an increase of the plasticity or flexibility of appropriate parts of the molecular structure in order to compensate for the lower thermal energy provided by the low temperature habitat. This plasticity would enable a good complementarity with the substrate at a low energy cost, thus explaining the high specific activity of psychrophilic enzymes. In return, this flexibility would be responsible for the weak thermal stability of cold-adapted enzymes. This relationship between activity, flexibility and stability constitutes a hot topic and represents a central issue in the adaptation of proteins to various environments. Moreover, it is believed that all proteins evolve through a balanced compromise between these features, i.e. structural rigidity allowing the retention of a specific 3D-conformation at the physiological temperature and in contrast flexibility, allowing the protein to perform its catalytic function. In the context of temperature adaptation of enzymes, it is assumed that high temperatures require stable protein structure and activity, whereas high enzyme activity is mandatory at low temperatures.

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Moreover, if the decreased stability of cold-adapted enzymes is well documented, there is however, no direct experimental evidence of an increased flexibility. Besides, controversial results were obtained when the flexibility of a few psychrophilic enzymes was investigated by measuring hydrogendeuterieum exchange rates. In the case of 3-isopropylmalate dehydrogenase (13), while the psychrophilic and mesophilic enzymes were found more flexible than the thermophilic counterpart, the psychrophile was however more rigid than the mesophile. Nevertheless, in this case, the technique suffered from the disadvantage of being a measure of the accessibility of deeply buried residues, and thus did not detect local flexibility, in particular that associated with the active site which is generally quite accessible. Using a similar technique, Fields (14) showed that while a psychrophilic and a mesophilic lactate dehydrogenases had similar flexibility at 2°C, the global flexibility of the psychrophile was significantly larger at 23°C. In addition to the lack of data about the flexibility of coldadapted enzymes, information on the thermodynamics of inactivation and unfolding is also missing and the few available reports are controversial. Indeed, Siddiqui et al. (15) proposed that the thermolability of psychrophilic enzymes is due to enthalpic effects, while some others (16)(17)(18) found this to be entropically driven. Further studies are thus required to resolve whether an unfavorable entropic or enthalpic contribution determines the irreversible unfolding and inactivation of these enzymes.
In attempt to elucidate how stability, catalytic activity and conformational flexibility are connected in psychrophilic enzymes, we have investigated three structurally homologous NAD +dependent DNA ligases. The psychrophilic DNA ligase from the Antarctic bacterium Pseudoalteromonas haloplanktis (Phlig) has been overexpressed and characterized (19), highlighting an increased catalytic efficiency as well as an increased thermolability of the enzyme. Cold-adaptation of Phlig is believed to be due to a decreased level of arginine and proline residues, as well as an overall destabilization of its N-terminal domain (19). The mesophilic reference chosen is the DNA ligase form Escherichia coli (Eclig) (20) and the thermophilic homologue is the Thermus scotoductus DNA ligase (Tslig) (21). The three enzymes are of a similar size, share all the properties common to NAD + -dependent DNA ligases, but are adapted to different extremes of the temperature scale (19), therefore constituting an adequate series of homologous enzymes for temperature adaptation studies.
In the present work, the overall destabilization of Phlig is examined with the help of 3D-modelling and investigation of solvent-exposed hydrophobic clusters. In addition, the intricate relationship between Fluorescence measurements -Both intrinsic and ANS fluorescence measurements were recorded on an Aminco SLM 8100 spectrofluorimeter. Thermal denaturation (scan rate 1°C/min) of the DNA ligases was monitored by recording the fluorescence intensity change at 330 nm, using a protein concentration of 50 µg/ml (0.66 µM) in 20 mM phosphate sodium, 50 mM NaCl, pH 7.6, with excitation at 280 nm (2-nm band pass) and emission at 330 nm (4-nm band pass). Data were normalized using the pre-and post-transition baseline slopes as described (25) and were fit according to a three-state model (26).
where v represents the scan rate (K min -1 ), C p the excess heat capacity at a given temperature, ∆H cal the total heat of the process and Q the heat evolved at the given temperature.

Molecular Model of Adenylated Phlig, Eclig and Tslig -The amino acid sequences of Phlig,
Eclig and Tslig show 45%, 45% and 87% identity with T. filiformis (Tflig) DNA ligase, allowing us to build 3D-models from the known X-ray structure ( (Table II). As shown in Table II, the psychrophilic enzyme displays an increased exposure of hydrophobic residues to the solvent, whereas the thermophilic DNA ligase shows an increased hydrophilic accessible surface area, including an increase of the charged accessible surface. Such discrepancies probably lead to improved electrostatic interactions in the thermophilic ligase that are likely to stabilize the enzyme at high temperatures, whereas the excess of hydrophobic surfaces in Phlig represents an entropy-driven destabilizing factor.
ANS Fluorescence -It is well known that the interaction of hydrophobic fluorescent probes such as 8-anilino-1-naphtalenesulfonic acid (ANS) with the exposed hydrophobic sites on the surface of native protein results in a considerable increase of the dye fluorescence intensity and a blue shift of its fluorescence spectrum (34,35). As the 3D-modelling predicted an increased exposure of hydrophobic residues to the solvent in Phlig, the binding of ANS was investigated in the three adenylated DNA ligases.
As seen in Fig. 2A, no significant binding of ANS can be observed with the native adenylated Tslig. In the case of Eclig, a slight binding is detected, suggesting the exposure of some hydrophobic patches at the surface of the native enzyme. By contrast, strong enhancement of ANS fluorescence is recorded for Phlig, underlining a significant population of solvent-accessible non-polar clusters in the native enzyme. Changes in ANS fluorescence were also monitored during thermal denaturation. In the case of Phlig (Fig. 2B), a slight increase of ANS binding is observed when increasing the temperature, which reaches a maximum around 33°C (corresponding to a maximal surface exposition of hydrophobic clusters), and then decreases to an intensity lower than that of the native enzyme. Eclig displays a similar behavior (Fig. 2B), except that the increase in ANS fluorescence is more pronounced when increasing the temperature, reaches a maximum around 55°C and then decreases at higher temperatures, but still exhibits significant ANS fluorescence, at a level significantly higher than that of the native enzyme. Such result suggests that Eclig is more prone to form aggregates at high temperatures. Such aggregates are still able to bind ANS, leading to the observed ANS signal at high temperatures. The high stability of Tslig (see below) did not allow monitoring ANS changes occurring upon thermal denaturation.
Thermodependence of activity -The thermodependence of activity of Phlig, Eclig and Tslig is shown in Fig. 3A. It can be seen that the maximal activity of Phlig (16°C) is shifted towards low temperatures (42°C for Eclig), reflecting its weak stability. Denaturation of the DNA substrate at high temperatures did not allow the determination of the optimal temperature for Tslig activity, but our results indicate that this value is higher than 55°C. This highlights a clear link between optimal activity acquisition and thermal adaptation. It should be noted in Fig. 3A that Phlig exhibits significantly higher reaction rates (k cat ) at low and moderate temperatures as compared to Eclig and Tslig. Comparison of the temperature dependence of activity (Fig. 3A) and thermal unfolding recorded by fluorescence emission (Fig. 3B) also reveals an interesting behavior of Phlig. Indeed, the maximal activity of Eclig closely corresponds to the beginning of the unfolding transition, therefore explaining the loss of activity at higher temperatures. By contrast, the maximal activity of Phlig is reached 10°C before unfolding and the enzyme is inactivated at the beginning of the transition. This suggests an extremely labile active site or catalytic intermediates in the cold-adapted enzyme.
Fluorescence quenching -The conformational flexibility of the three DNA ligases was probed by dynamic fluorescence quenching, using acrylamide as quencher. Fig. 4 shows the Stern-Volmer plots for Phlig (Fig. 4A), Eclig (Fig. 4B) and Tslig (Fig 4C) (Fig. 1C). Finally, Tslig contains 6 tryptophan residues; W135, W246, W274 and W 405 are rather buried, whereas W298 and W375 are relatively accessible to the solvent (Fig. 1D). Thermal stability of DNA ligases -Thermal unfolding of Phlig, Eclig and Tslig was monitored by differential scanning calorimetry (DSC). A non detergent sulphobetaine was added prior to DSC experiments (see Experimental Procedures), in order to limit aggregation (29) that distorts the calorimetric traces and impairs deconvolution processes. Fig. 5 illustrates the range of stabilities encountered within the DNA ligase family, and Table III provides the thermodynamic parameters associated with thermal unfolding recorded by microcalorimetry. As indicated by the large differences in transition temperature T max , the psychrophilic enzyme is by far the least stable when compared to its mesophilic and thermophillic homologues. In addition, the lowest calorimetric enthalpy, representing the total heat absorbed during unfolding, is recorded for Phlig, underlining its weak thermostability.
Deconvolution of the excess heat capacity (C p ) functions revealed one, two and three subsequent transitions for Phlig, Eclig and Tslig, respectively ( Fig. 5 and Table III), pointing out the presence in the enzymes of an increased number of domains of distinct stability in the order psychrophile→mesophile→thermophile, also corresponding to a decrease of unfolding cooperativity.
The absence of heat absorption effects on rescanning of denatured samples indicates that thermal unfolding of the three enzymes studied is irreversible. The deviation from a two-sate model was further confirmed by the fact that the ∆H cal /∆H eff ratio for the three enzymes exceeds unity (not shown). The irreversible conformational unfolding of DNA ligases is also highlighted by the scan-rate dependence of their apparent T max (Fig. 6A), revealing that the thermal denaturation of these proteins is kinetically controlled. Analysis of the thermodynamic parameters of activation for the denaturation process ( Fig 6B and Table IV) indicates that, as expected, the denaturation rate is highest for Phlig (higher k denat ) and correspondingly, the energy barrier, ∆G # , is lowest. However, Ea, ∆H # and T∆S # are all highest for the psychrophile, indicating that the lower thermostability of the cold-adapted DNA ligase is due to an unfavorable entropic contribution. The complex unfolding pattern of Tslig (Fig. 5) precludes such analysis of the kinetically-driven denaturation   (19) and provide further evidence for the structural determinants implicated in the adaptation to low temperatures of Phlig. Our results suggest that the active site of the cold-enzyme is destabilized by an excess of hydrophobic surfaces and contains a decreased number of charged residues compared to its thermophilic counterpart. These findings are in perfect agreement with the kinetic parameters determined for both enzymes (19). Indeed, k cat is believed to be the most important adaptive parameter for Phlig hence compensating for the effect of low temperatures on the catalytic rate, while for Tslig, the adaptive parameter is expected to be K m for nicked DNA which is prone to melting at high temperatures. In the latter, the increase of positively charged amino acids within the active site is likely to facilitate the interaction with the oppositely charged DNA substrate, leading to an improved K m . Determination of the composition of the accessible surface of the whole molecule also points out an increase of the charged surface in the order psychrophile→ mesophile→thermophile.
Such increase could lead to an increase of the ion pairs at the surface of Tslig, that could take part to its increased stability. Indeed, ion pairs in solvent-exposed regions of thermophilic enzymes are believed to represent a major key in the adaptation of these enzymes at high temperatures (36,37).
Analysis of the N-terminal domain of Phlig also revealed a significant increase of the degree of exposure of hydrophobic residues to solvent, that is expected to destabilize the protein structure (19).
While the model of the entire psychrophilic enzyme confirmed this finding, it was experimentally demonstrated by ANS-binding experiments. Indeed, most native globular proteins do not bind ANS since their hydrophobic core is well protected from the solvent by the rigid packing of side chains (34).
However, when native proteins exhibit hydrophobic sites exposed to the solvent, a strong affinity for the dye is observed (34,35). In the case of Phlig, the strong enhancement of fluorescence intensity of ANS clearly demonstrates the exposure to solvent of hydrophobic clusters. The latter are likely to take part to an entropy-driven destabilization of the cold-adapted enzyme.
Activity at low temperatures -Our activity results point out that the cold-adapted Phlig is characterized by a shift of the optimal activity towards low temperatures and an increased specific activity at low and moderate temperatures compared to its mesophilic and thermophilic counterparts.
Such increase of activity seems to be due to a higher resilience of the structure, and of the active site. Phlig is more flexible than Eclig and Tslig, in a temperature range where the native state prevails.
Such flexibility can contribute to the high activity in the low temperature habitat, but also leads to a reduced stability of the molecular edifice. Investigation of the thermal stability of Phlig by DSC reveals that this enzyme possesses a fragile molecular edifice that is uniformly unstable (one deconvolution unit) and stabilized by fewer weak interactions (decreased ∆H cal ) than its mesophilic and thermophilic homologues. It is worth mentioning that Phlig displays the lowest calorimetric enthalpy ∆H cal as well as the lowest T m ever recorded for cold-adapted enzymes, underlining its strong psychrophilic character through a low conformational stability. Such decreased stability is also highlighted by GdmCl-induced unfolding in which Phlig is characterized by the lowest ∆G (H 2 O) and Cm values.
Further advances in the understanding of the activity-stability relationship can be drawn from the analysis of irreversible thermal unfolding of the cold-adapted DNA ligase and its mesophilic counterpart. Our results show an increase in free energies of activation ∆G # from Phlig to Eclig, reflecting that the same denaturation rate k denat is reached at increasing temperatures (see also Table   IV). The small differences in both ∆G # values arise however from large differences in the enthalpic and entropic contributions. Indeed, the low ∆G # value recorded for Phlig corresponds to the largest ∆H # and T∆S # contributions, and conversely for Eclig. Such trend has already been noticed for a few psychrophilic enzymes (17,18) and thus seems to be a common property of psychrophilic enzymes.
The low kinetic barrier (∆G # ) recorded for the psychrophilic enzyme allows in fact unfolding at a high rate, leading to a symmetrical and relatively narrow denaturation peak in microcalorimetry (Fig.   5), and consequently to high activation energy Ea and high ∆H # . This reflects a high cooperativity of unfolding for the psychrophilic enzyme, that probably originates from the lower number of interactions required to disrupt the active conformation. The large entropic contribution noticed in Phlig suggests that its transition state is more disordered than that of its mesophilic counterpart, probably due to the fact that at any specific temperature, more interactions are broken into the psychrophile. The entropy

CONCLUDING REMARKS
Until recently, the activity-flexibility-stability relationship within psychrophilic enzymes was still appreciated with caution. However, the results obtained for the three DNA ligases adapted to different thermal habitats clearly establish a link between activity, flexibility and stability. The cold-adapted DNA ligase is characterized by a high activity at low temperatures, a high flexibility and a low stability especially at the active site. These results are in perfect agreement with those recently obtained from studies performed on extremophilic α-amylases (18) and xylanases (17). Therefore, the emerging picture suggests that psychrophilic enzymes are characterized by increased catalytic efficiency attributed to an increase of the flexibility of appropriate parts of the molecular structure, in order to compensate for the lower thermal energy provided by the low temperature habitat. In return, this flexibility would be responsible for the weak thermal and chemical stabilities of cold-adapted enzymes.