Catalytic Adaptation of Psychrophilic Elastase - ACS Publications

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Catalytic adaptation of psychrophilic elastase Jaka Socan, Masoud Kazemi, Geir Villy Isaksen, Bjorn-Olav Brandsdal, and Johan Åqvist Biochemistry, Just Accepted Manuscript • DOI: 10.1021/acs.biochem.8b00078 • Publication Date (Web): 04 May 2018 Downloaded from http://pubs.acs.org on May 5, 2018

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Biochemistry

Catalytic adaptation of psychrophilic elastase

Jaka Sočan,† Masoud Kazemi,† Geir Villy Isaksen,†,‡ Bjørn Olav Brandsdal‡ and Johan Åqvist†*

†

Department of Cell and Molecular Biology, Uppsala University, Biomedical Center, Box 596, SE-751 24 Uppsala, Sweden

‡

Hylleraas Centre for Quantum Molecular Sciences, Department of Chemistry, University of Tromsø, N9037 Tromsø, Norway

*Corresponding author: E-mail: [email protected], Phone: +46 18 471 4109

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Abstract The class I pancreatic elastase from Atlantic salmon is considered to be a cold-adapted enzyme in view of the cold habitat, the reduced thermostability of the enzyme and the fact that it is faster than its mesophilic porcine counterpart at room temperature. However, no experimental characterization of its catalytic properties at lower temperatures have actually been reported. Here we use extensive computer simulations of its catalytic reaction, at different temperatures and with different peptide substrates, to compare its characteristics with those of porcine pancreatic elastase, with which it shares 67% sequence identity. We find that both enzymes have a preference for smaller aliphatic residues at the P1 position, while the reaction rate with phenylalanine at P1 is predicted to be substantially lower. With the former class of substrates the calculated reaction rates for salmon enzyme are consistently higher than those of the porcine ortholog at all temperatures examined and the difference is most pronounced at the lowest temperature. As observed for other cold-adapted enzymes, this is due to redistribution of the activation free energy in terms of enthalpy and entropy and can be linked to differences in the mobility of surface exposed loops in the two enzymes. Such mobility changes are found to be reflected by characteristic sequence conservation patterns in psychrophilic and mesophilic species. Hence, calculations of mutations in a single surface loop show that the temperature dependence of the catalytic reaction is altered in a predictable way.

Keywords: elastase, cold adaptation, enzyme flexibility, molecular dynamics, free energy calculation


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Introduction The question of how enzymes of cold-adapted species are able to function near the freezing point of liquid water is a fundamental problem in biochemistry and structural biology. Psychrophilic species, which include both prokaryotes and eukaryotes, such as polar and deep sea fishes, have evolved enzymes that have considerable catalytic activity at low temperatures (near 0 °C).1-3 These psychrophilic enzymes are more heat-labile than their mesophilic orthologs and typically have their activity optima in the range of 25-40 °C, while mesophilic enzymes usually have their optima at higher temperatures.2-5 A major feature of cold-adapted enzymes is also that their catalyzed reactions consistently display a lower enthalpy of activation, which is more or less compensated by a more negative activation entropy.2-6 This universal phenomenon makes the reaction rate less temperature dependent, since it is the activation enthalpy term (and not the entropy) that causes the exponential damping of the rate as the temperature is lowered.7,8 Hence, by moving some of the activation free energy penalty from enthalpy to entropy the catalytic rate can be increased at low temperatures. What is particularly interesting here is that orthologous psychrophilic and mesophilic enzymes are often very similar both in terms of sequence and 3D structure. A sequence identity of ~70%, or more, is thus not uncommon and the active site is then usually fully conserved. This means that it is mutations outside and more or less distant from the active site that confer the temperature adaption effect to psychrophilic enzymes.9-12

Not all enzymes expressed by pyschrophilic organisms can, however, be classified as coldadapted enzymes. For example, in poikilothermic species such as Atlantic salmon, five different variants of trypsin (isoenzymes) have been found, where four are classified as anionic and one as cationic.13 The cationic salmon trypsin displays similar catalytic activity and stability as the bovine trypsin, which is also cationic, and does not have the characteristic


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fingerprint of cold-adapted enzymes. However, the anionic salmon trypsin characterized by Outzen et al.14 has higher catalytic activity at low to moderate temperatures and reduced thermal stability compared to the cationic salmon and bovine enzymes, and is thus classified as cold-adapted.

Figure 1. Multiple alignment of 5 mesophilic and 7 psychrophilic elastase sequences. PPE – Sus scrofa, M1 – Pan troglodytes, M2 – Erinaceus europaeus, M3 – Physeter catodon, M4 – Homo sapiens, SPE – Salmo salar, P1 – Oncorhynchus mykiss, P2 – Oncorhynchus kisutch, P3 – Notothenia coriiceps, P4 – Clupea harengus, P5 – Esox lucius, P6 – Labrus bergylta. Loop regions in the two protein domains are indicated.

Another well characterized example of a cold-adapted enzyme is the class I pancreatic elastase from Atlantic salmon, where both the kinetics and a high resolution 3D structure have been determined.15,16 The salmon elastase also shows a typical reduced thermostability and loses its activity at 45-50 °C, compared to 55-60 °C for the mesophilic porcine ortholog.15 This enzyme has 67% sequence identity with the porcine pancreatic elastase I (Figure 1) and 4 ACS Paragon Plus Environment

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Biochemistry

the two 3D structures16,17 are very similar with a backbone root mean square deviation (RMSD) of only 1.8 Å (Figure 2). Particularly the core regions of the protein, excluding surface loops, are found to have a very small RMSD of 0.3 Å.16 With such a high similarity between the two enzymes, this psychrohile-mesophile pair provides an interesting case for exploring the structural mechanisms behind cold-adaptation of the salmon elastase.

Figure 2. Structural comparison of salmon16 (cyan) and porcine22 (yellow) elastase complexes with the Pro-Ile-Ala tripeptide (purple). The catalytic triad is shown in blue and surface loops discussed in the text are highlighted in red.

Computer simulations have frequently been employed to examine differences between coldand warm-active enzyme orthologs, but they have mostly focused solely on differences in flexibility and atomic positional fluctuations.18 Differences in protein flexibility are indeed believed to be involved in adaptation to low temperature and this seems to be supported by several molecular dynamics (MD) simulation studies.9-12,18,19 However, it should be obvious that in order to address the real problem of how the enzymatic rate is optimized to be higher at low temperatures, it is necessary to be able to characterize the energetics of the catalyzed 5 ACS Paragon Plus Environment

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chemical reaction by computer simulations. There are actually very few examples of this kind of calculations, namely for citrate synthase9, trypsin10,11, and triose phosphate isomerase (TIM)12, whereas most computational studies merely provide correlations between flexibility and cold-adapted structures, which can be viewed as circumstantial evidence regarding the role of increased mobility of certain protein regions. The reaction simulations for citrate synthase, trypsin and TIM, on the other hand, directly addressed the temperature dependence of the activation free energy barriers and reproduced the interesting activation enthalpyentropy phenomenon discussed above, which could also be interpreted in terms of differences in surface flexibility between the psychrophilic and mesophilic enzymes.8

Here we use molecular dynamics free energy perturbation simulations in combination with the empirical valence bond (EVB) method20,21 to describe the peptide hydrolysis reaction catalyzed by salmon (SPE) and porcine pancreatic elastase (PPE). Besides evaluating the activation free energy barrier at different temperatures, we also examine the substrate specificity of the two enzymes. The results show that the cold-adapted salmon pancreatic elastase is a faster catalyst at room temperature and that the proficiency over its mesophilic counterpart further increases as the temperature is lowered. As seen also in other cold-adapted enzymes, this is behavior found to originate from a redistribution of the activation free energy, where a significant part of the ∆‡ penalty has been moved to ∆ ‡. MD simulations of enzyme-substrate complex reveal a number of surface loops with increased mobility in the cold-adapted enzyme which can explain this behavior.

Methods MD simulation systems were constructed based on the atomic coordinates provided in the PDB entries 1ELT and 3HGP, for salmon16 and porcine22 pancreatic elastase, respectively.


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Biochemistry

The primary sequence of salmon pancreatic elastase in the PDB entry 1ELT was originally (1995) derived directly from the electron density8, relying in part on the porcine sequence to resolve ambiguities. We therefore needed to reconstruct the salmon pancreatic elastase model with the primary sequence obtained from genome sequencing of Salmo salar (UniProt entry A0A1S3LF92_SALSA). 18 amino acid residues were thus replaced and refitted to the electron density map of 1ELT using UCSF Chimera.23 Additionally, the PDB entry 1QNJ17 was consulted to model structural water molecules near the active site of porcine pancreatic elastase.

All MD/EVB simulations of were performed with the MD program Q.24 Spherical boundary conditions were applied with the MD simulation sphere centered at the centroid coordinate of the whole enzyme crystal structure. The systems were solvated using the TIP3P water model and the MD simulations utilized the OPLS-AA/M force field25 and were carried out with a 1 fs time step. Prior to production simulations, the systems were prepared by energy minimization in Maestro 10.1 (Schrödinger, LLC, New York, NY, 2011) and equilibrated for 140 ps with five temperature increments from 1 to 295 K. A two-state EVB model was used to describe the acylation reaction and trajectories were initiated from the tetrahedral intermediate (SI Methods).10 Reaction free energy profiles were calculated with the free energy perturbation (FEP) umbrella sampling approach, as described earlier.26 The FEP transformation between the tetrahedral intermediate and reactants encompassed 51 discrete windows. In order to obtain reliable free energy profile averages, the MD/EVB simulations were repeated multiple times with different initial conditions. We defined the reacting region in EVB model to include the side chains of His57 and Ser195, the Cα-atom of the substrate P1 residue and all atoms in the amide bond between P1 and P1’ (C, O, N and H).


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As a reference reaction for the EVB calculations, we used a previously defined system describing amide bond cleavage in a water cage, in presence of the general base imidazole.10,27 Here, Pro-X-Ala tripeptide substrates were used with X=Ala, Val, Leu, Ile and Phe (X at P1 and Ala at P1’). These reference reactions were independently calibrated to fit ab initio data from Ref. 27, namely 26 kcal/mol for the activation barrier and 20 kcal/mol for the reaction free energy. This yielded the EVB parameters (∆, )20,21 for each P1 residue (Ala: 205.0, 115.1, Val: 192.6, 111.0, Leu: 207.0, 114.3, Ile: 195.0, 113.0, Phe: 197.0 113.0). The calibration procedure utilized 50 independent MD/EVB simulations (yielding 26 ns simulation time) of the reference reaction, solvated in a 25 Å radius sphere, in order to obtain a sample with a standard error of the mean < 0.1 kcal/mol for the activation free energy. Simulations of the enzyme reactions were carried out in 35 Å sphere, which encapsulates the entire enzyme. At least 50 repetitions were carried out at 295 K for each of five different substrates in both SPE and PPE. An additional 40 MD/EVB simulations were carried out for the selected four substrates at 285 and 305 K to examine the cold adaptation of catalytic rates. This yielded a total simulation time of 230 and 235 ns for SPE and PPE, respectively. The same simulation protocol was also used to evaluate the effects of the S218L/A221V and L218S/V221A double mutations on the reaction with Pro-Ile-Ala in SPE and PPE, respectively. In addition, 100 ns plain MD simulations of the reactant state of the two wildtype enzymes at 295 K were carried out in order to evaluate atomic fluctuations.

We further searched the UniProt database for taxonomically related pancreatic elastase sequences with either SPE or PPE as query. For sequence comparison, six psychrophilic pancreatic elastase sequences with at least 74 % identity to SPE were chosen, corresponding only to sequences extracted from fishes thriving in habitats requiring a considerable degree of cold adaptation (inferred temperature preference TP50 below 285 K).28 Similarly, we chose


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Biochemistry

four mammal pancreatic elastase sequences with at least 89 % identity to PPE, which are clearly mesophilic due to mammal homeothermy (Figure 1).

Results Free energy profiles for porcine and salmon elastases Like other chymotrypsin-like enzymes, a catalytic triad composed of His57, Asp102, Ser195 (trypsin numbering) constitutes the catalytic machinery in elastase (Figure 2).29 Here, the peptide bond is preferentially cleaved in substrates with aliphatic residues at the P1 position. Nucleophilic attack on the carbonyl group of the peptide bond between P1 and P1’ residues of the substrate is effected by the hydroxyl group of Ser195, assisted by the His57 general base. The prime role of Asp102 has been shown to be to stabilize the protonated form of His57,30,31 thereby facilitating the acylation reaction leading to formation of a transient anionic acylenzyme tetrahedral intermediate (TI). As in other members of the chymotrypsin family, the developing negative charge on carbonyl oxygen of this TI is also stabilized by the so-called oxyanion hole, which is comprised of the backbone amide groups of Gly193 and the catalytic Ser195.31

Formation of the TI from the initial enzyme-substrate complex, prior to expulsion of the Nterminal P1’ residue, is considered to be the rate limiting step of chymotrypsin-like proteolytic reactions.27,31 The acylation reaction has also been shown to be rate-limiting in the case of porcine pancreatic elastase (PPE) with the type of substrates considered here.32 We used here the MD/EVB approach20,21 to calculate free energy profiles for the transition from the reactant enzyme-substrate complex to the TI in both the porcine enzyme and salmon pancreatic elastase (SPE). Multiple independent free energy calculations were performed for each of five tripeptide substrates with sequence Pro-X-Ala, in the two elastases. In these


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substrates X denotes the P1 residue and corresponds to Ala, Val, Ile, Leu or Phe. We used the imidazole catalyzed TI formation in water as a reference reaction with an estimated activation free energy of 26 kcal/mol,27 which itself is approximately 6 kcal/mol lower than the activation free energy of the uncatalyzed reaction.33 This choice of reference reaction is particularly useful as it mimics the side chains of the catalytic histidine and serine and can also be modeled from the crystallographic structure of enzyme. The results of these MD/EVB calculations are shown as average free energy profiles for the different substrates in Figure

3A and summarized in terms of the predicted activation barriers in Figure 3B. The calculated activation free energies of 18-19 kcal/mol for the substrates with aliphatic sidechains coincide well with those obtained earlier for trypsin with native-like tripeptide substrates.10

In each of the two elastases, our calculations yield similar activation free energies for the ProX-Ala substrates with aliphatic sidechains at the P1-position (P1=Ala, Leu, Ile, Val), while the phenylalanine sidechain causes a 3-5 kcal/mol higher reaction barrier. Moreover, the P1Val and P1-Ile substrates are predicted to be slightly preferred over P1-Ala and P1-Leu in both SPE and PPE. This is in agreement with the experimentally observed sidechain preference at the P1-position for polypeptides cleaved by PPE, where isoleucine and valine were found to have the highest frequency, followed by alanine and leucine.34 This cleavage pattern also agrees with binding data for protease inhibitors with different P1-sidechains to human leukocyte elastase and PPE.35,36 Kinetic data for the different elastases is unfortunately only available for non-native chromogenic substrates, with p-nitroanilide or thiobenzyl leaving groups,15,37 and these groups appear to have relatively large effects on the measured rates. Nevertheless, it is interesting to compare our simulation results to the only available kinetic data for SPE, which corresponds to hydrolysis of a series of succinyl-Ala-Ala-Pro-Xp-nitroanilide substrates (Figure 3B).15 For these substrates alanine was found to be slightly


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Biochemistry

favoured at the P1 position in both SPE and PPE (by a factor of 2-3) and P1-Phe showed no activity in SPE. In contrast, for a series of Ala-Ala-X-thiobenzyl substrates in PPE, P1-Leu yielded the highest values of kcat (by a factor of 11) while P1-Phe showed no reaction.37 Hence most experimental data for the pancreatic elastases, both regarding binding and catalysis show a strong discrimination against substrates with phenylalanine at the P1 position,16,35-37 an effect that is clearly captured by the present MD/EVB simulations. The basic reason for this discrimination is that the S1 cavity needs to expand with a phenylalanine sidechain bound to it, which forces a rotation of the Val216 sidechain and a slight backbone displacement around Cys191.


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Figure 3. (A) Calculated free energy profiles at 295 K for rate limiting step of peptide bond hydrolysis with Pro-X-Ala substrates in porcine pancreatic elastase (PPE) and salmon pancreatic elastase (SPE), based on 60 independent MD/EVB calculations per system. (B) Calculated activation free energy barriers in PPE and SPE compared to experimental free energy barriers, derived from kcat values for amide bond cleavage in Suc-Ala-Ala-Pro-X-pNA substrates15 (error bars – 1 s.e.m. for calculated values. The asterisk for the experimental values for P1-Phe in SPE denotes an assumed detection limit of kcat < 0.01s−1 as no activity was observed).

Salmon pancreatic elastase exhibits cold-adapted properties To investigate the temperature adaptation properties of the compared pancreatic elastases, we carried out 40 additional free-energy profile calculations for the substrates Pro-Ala-Ala, ProVal-Ala, Pro-Leu-Ala and Pro-Ile-Ala at temperatures 285 and 305 K. The MD/EVB simulations predict that SPE is the faster of the two enzymes, with lower activation barriers for all substrates at all temperatures examined (Table 1). The proficiency of the salmon compared to the porcine enzyme generally increases as the temperature is lowered, and it achieves a 4-24 times higher catalytic rate at 285 K, where the factor of 24 corresponds to the P1-Ile substrate. This is also in agreement with the experiments on the succinyl-Ala-Ala-ProX-p-nitroanilide substrates which show the largest rate ratio (SPE/PPE) for the isoleucine substrate.15 The effect of calculated activation barrier ( ∆ ‡ ) differences between the two enzymes can be appreciated from the relative rate constants (krel) compared to that of the highest temperature (305 K). Hence, the calculated values of krel for the cold-adapted elastase at 285 K predict that the enzyme retains 19-59% of its peptide bond cleavage activity at the lower temperature (Table 1). In contrast, the mesophilic elastase only retains 12-16% of the catalytic activity at the lower temperature. The lower percentage value here, in both cases, corresponds to the Pro-Ala-Ala substrate, while the relative rates for SPE at 285 K for the


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Biochemistry

other investigated substrates are around 50% of those at 305 K and considerably higher than those for PPE.

Table 1. Calculated activation barriers (kcal/mol) and relative turnover numbers (krel) for ProX-Ala substrates in salmon (SPE) and porcine (PPE) elastases at different temperatures.a SPE T (K)

X

∆G‡

285 290 295 300 305

Ala Ala Ala Ala Ala

19.12 19.23 19.31 19.54 19.49

285 290 295 300 305

Val Val Val Val Val

285 290 295 300 305 285 290 295 300 305

PPE ∆G‡

krel

0.19 0.28 0.44 0.53 1

19.94 20.19 20.17 20.44 20.08

0.12 0.14 0.27 0.31 1

17.79 17.92 18.40 18.48 18.63

0.47 0.67 0.51 0.75 1

18.82 18.76 19.13 19.25 19.00

0.14 0.29 0.27 0.38 1

Ile Ile Ile Ile Ile

17.42 17.76 18.01 18.14 18.36

0.59 0.56 0.63 0.86 1

19.22 19.30 19.25 19.08 19.51

0.16 0.26 0.50 0.38 1

Leu Leu Leu Leu Leu

18.91 18.85 19.17 19.29 19.71

0.39 0.79 0.81 1.16 1

19.70 19.82 19.90 20.13 19.91

0.13 0.20 0.32 0.39 1

krel

a

Values for kcat are obtained from transition state theory, with a transmission factor of unity, as = /ℎexp −∆ ‡ / , where and h are Boltzmann’s and Planck’s constants, respectively.

As indicated from the calculated activation free energies at different temperature (Table 1), the salmon enzyme is generally faster and retains a higher activity at low temperature than its


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mesophilic counterpart. This is precisely the behavior that is usually found for cold-adapted enzymes and it has been seen to originate from a lower enthalpy of activation, which is partly compensated by a more negative activation entropy.8 Hence, although we have examined five temperatures here, but four substrates at each of these, these data can be used to qualitatively examine the activation enthalpy-entropy balance of the two enzyme reactions. Arrhenius plots of ∆ ‡ / versus 1/ (Figure S1) were therefore use to extract the thermodynamic activation parameters for each catalyzed reaction by linear regression (Table 2). Remarkably, we find that the activation enthalpies (∆‡ ) of SPE are consistently lower than those of PPE, with values between 5-13 kcal/mol compared to 14-17 kcal/mol, respectively. As observed for other previously analyzed orthologous pairs of psychrophilic and mesophilic enzymes,9-12 the lower activation enthalpies of the former are also here compensated by more negative ‡ activation entropy penalties, expressed as ∆ (Table 2). Interestingly, the differences in

thermodynamic activation parameters are found to be smallest for the Pro-Ala-Ala substrate, ‡ while the larger P1 sidechains show rather unform shifts of ∆‡ and ∆ of about 10 ‡ kcal/mol. Hence, it is clear that while the absolute values of ∆‡ and ∆ do depend

somewhat on the substrate, their differences between the psychrophilic and mesophilic enzymes consistenly obey the general activation enthalpy-entropy shift indicative of coldadaptation.


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Biochemistry

Table 2. Calculated thermodynamic activation parameters (kcal/mol) for peptide bond hydrolysis by salmon (SPE) and porcine (PPE) pancreatic elastase at 295 K. SPE

PPE

∆G‡

∆H‡

T∆S‡

Pro-Ala-Ala

19.4

13.2

-6.2

Pro-Val-Ala

18.2

5.0

Pro-Ile-Ala

18.0

Pro-Leu-Ala

19.2

∆G‡

∆H‡

T∆S‡

20.1

16.8

-3.3

-13.2

19.0

13.8

-5.2

4.6

-13.4

19.2

17.2

-2.0

7.3

-11.9

19.9

15.5

-4.4

S218L/A221V Pro-Ile-Ala

18.0

11.3

L218S/V221A -6.7

19.4

11.4

-8.0

Cold-adaptation is related increased flexibility of surface loops It has been shown that cold-adapted enzymes tend to be more flexible in comparison to their evolutionary related mesophilic counterparts. However, in contrast to earlier hypotheses, these flexibility differences appear to primarily be localized to regions outside of the highly conserved active site and, particularly, to surface exposed loops.9-12 To examine the mobility differences between SPE and PPE, we calculated the backbone positional root mean square fluctuations (RMSF), averaged per amino acid residue, for both enzymes in complex with the Pro-Ile-Ala substrate during 100 ns MD simulations. As noted above, this substrate shows the highest value of krel at low temperature in SPE also the largest rate ratio (a factor of 24) between the enzymes. Indeed, the MD simulations show a notably higher mobility for SPE relative to PPE in a number of loop regions (Figure 4A). However, the active site residues His57, Asp102 and Ser195 are localized in regions with a generally low and similar backbone mobility (RMSF < 0.5 Å), although the fluctuations are slightly higher also here for SPE. The overall atomic positional fluctuations, including side chains, were also examined for the three


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substrate residues (P2-P1-P1’) and the catalytic residues His57, Asp102 and Ser195 (Figure

4B). This again shows very similar mobilities of the active sites in SPE and PPE.

Among the loop regions with notably higher backbone mobility in the cold-adapted enzyme the first one is the Nβ3-Nβ4 loop, involving residues 56-64 (we use here the notation N or C, for the N- and C-terminal domains, respectively, together with the sequential numbering of βstrands in each domain). This loop shows an average increase in backbone mobility of 41% (Figure 4A) and contains two conserved mutations in several psychrophilic species compared to mesophilic mammals. These are the substitution of a Leu/Met/Lys residue at position 63 for an arginine, and the F65W mutation. Together with the I88V substitution in the adjacent

β5 strand, these changes affect the hydrophobic packing network at the end of the Nβ3-Nβ4 loop and alters the conformation of it, which also renders it more flexible (Figure 5A). It can be noted that the three aforementioned sequence positions appear strongly conserved in psychrophilic species which indicates that the loop behavior has indeed been optimized by evolution (Figure 1). This is in contrast to the subsequent Nβ4-Nβ5 loop which has a more or less identical mobility in the two enzymes and also retains the same conformation both in the X-ray structures and MD simulations (not shown).


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Biochemistry

Figure 4. Positional root mean square fluctuations averaged per residue for (A) the enzyme backbone and (B) entire residues of the Ala-Pro-Ile substrate and active side residues, from 100 ns MD simulations. Results for SPE and PPE are shown in blue and red, respectively, and the trypsin numbering is used.

A second loop that was implicated in the cold-adaptation of trypsin10,11 is the Nβ5-Nβ6 loop, which also here shows a significantly enhanced mobility in salmon elastase, with a 35% increase in the average RMSF (Figure 4A). In trypsin, it was shown that the single N97Y mutation in bovine trypsin rendered the enzyme more psychrophilic in terms of ∆‡ and ∆ ‡ , and vice versa for the Y97N mutation of the salmon enzyme.10,11 In the elastases, however, the strongest mesophilic-specific sequence signal is Pro92 and Tyr93 at the beginning of the Nβ5-Nβ6 loop and Ala99 and its end, where the latter two residues are both


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replaced by glycines in the salmon enzyme (Figure 5B). Interestingly, Tyr93 in PPE shows an edge-to-face π-stacking with Tyr101 which, together with the more rigid backbone conformation of Ala99, renders the loop more stable than with two glycines at these positions. Also the P92N mutation contributes to reduced backbone mobility in tis region. Hence, the structural mechanism for attaining a higher flexibility of Nβ5-Nβ6 loop in SPE is rather different from that in cold-adapted trypsin, where mutations were seen to disrupt H-bond networks with surface bound water molecules.10

Figure 5. (A) Comparison of the Nβ3-Nβ4 loop crystal structure conformations in SPE (cyan) and PPE (yellow). Mutations in the salmon enzyme are indicated. (B) Conformations of the Nβ5-Nβ6 surface loop backbone in the crystal structures of SPE (cyan) and PPE (yellow). Key mutations in SPE at positions 92, 93 and 99B are indicated. (C) Comparison of the interdomain connector region (red) from crystal structures and three independent average MD structures of each enzyme. The SPE and PPE crystal structures are shown in cyan and yellow, respectively. The bound inhibitor in the PPE crystal structure on the opposite side of the connector region is shown in blue.

Interestingly, the long extended stretch of amino acids connecting the N- and C-terminal domains of elastase contains a region (118-124) of very low mobility, which is attained both by hydrophobic sidechain packing and a few backbone H-bonds to β-strands in each of the two domains in both enzymes. This interior region of the interdomain connector is flanked by


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more mobile regions, where the N-terminal part (110-116) is more flexible in SPE while the C-terminal part (125-130) is more flexible in PPE (Figure 4A). There are no strong sequence signals in the long connector region and, as the differential mobility seen in the MD simulations at the two ends tend to cancel if averaging over the whole region, it would appear that the connector is not subjected to any key adaptive mutations. This is also supported by the fact that it is located on the opposite side of the protein from the active site and that its conformation is virtually identical in the two enzymes, both in the average MD and X-ray structures (Figure 5C). Furthermore, the interdomain connector region did not either appear as a hotspot for differential mobility in earlier MD simulations cold- and warm-active trypsins.10 On the other hand, the trypsin simulations identified the Cβ1-Cβ2 (or autolysis) loop as a site of major differences between the salmon and bovine enzymes. Not only were the mobilities predicted to be significantly different for the Cβ1-Cβ2 loop, but its conformation in the corresponding trypsin crystal structures is also very different. MD/EVB calculations further showed that mutation of a single residue in this loop (S150D and D150S for bovine and salmon trypsin, respectively) could switch both the thermodynamic activation parameters and the structural conformation between warm- and cold-active characteristics. In contrast to those results, the present MD simulations show no major mobility difference in the Cβ1-Cβ2 loop (residues 143-149), although again the fluctuations are slightly higher for SPE than PPE (Figure 4A). The two crystal structures of SPE and PPE also have essentially identical conformations of the loop.16,22

The Cβ2-Cβ3 loop (residues 169-174) shows a considerably enhanced mobility in SPE where, particularly, residues Ser170A and Gly170B have a 58% higher RMSF than the corresponding two serines in PPE. Here, it can be seen both from the MD simulations and the crystal structure of PPE that the adjacent conserved Tyr171 is able to make water mediated H-


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bonds to the adjacent Cβ3-Cβ4 loop (Figure 6A), which is also much less flexible in the warm-active enzyme. The tyrosine residue is substituted for a conserved tryptophan in the set of cold-adapted fishes and this Trp171 sidechain is highly mobile with no preferred rotamer, due to lack of H-bonding capability of its six-membered ring. An additional strongly conserved mutation at the N-terminal (helical) part of the Cβ2-Cβ3 loop is Y165H, where the tyrosine in PPE similarly makes water mediated H-bonds to the backbone carbonyl groups of Gly173 and Val176. Another notable difference between cold- and warm-active elastase sequences is the consistent deletion of Asp186 in the former class. This residue is situated in the Cβ3-Cβ4 loop and the two conserved glycines flanking the deletion show a 35% increase in their backbone RMSF in the MD simulations of SPE compared to PPE (Figure 4A). The structural effect of Asp186 in the porcine enzyme is very clear, both from the crystal structures and MD simulations. It rigidifies the loop via H-bonding to the backbone nitrogens of Val187 and Arg188 and also has an ion-pair interaction with the sidechain of the latter residue (Figure 6B). Here, Arg188 is also a clear signature of the mesophilic mammal sequences, while the cold-adapted fishes consistently have Asn/Asp/Glu at this position, thereby removing the positive charge. These conserved mutations in the Cβ3-Cβ4 loop of differently adapted species appear to be a typical example of how a few distant mutations (~18-20 Å away from the active site) are used by evolution to alter the flexibility of a structurally seemingly unimportant surface loop.

The last Cβ5-Cβ6 surface loop of elastase (residues 217-225), which is closer to the active site, also shows a significantly higher mobility in the salmon enzyme. It has a more than doubled average backbone RMSF value for the highly conserved Gly-Cys-Asn motif, which is thus the same in both psychrophilic and mesophilic species. Here, the cysteine residue also forms a disulphide bridge with another cysteine at the C-terminal end of the Cβ3-Cβ4 loop.


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The mobility difference in the Cβ5-Cβ6 loop, however, originates from hydrophobic stabilization of the loop in the porcine structure. That is, after the Gly-Cys-Asn motif is a conserved valine residue (Val221A) in the mesophilic species which packs against Leu218 in PPE, thereby rigidifying the loop structure (Figure 6C). In the salmon enzyme these two residues are replaced by an alanine and a serine, respectively, which affects the intra-loop hydrophobic interaction and renders it more mobile.

Figure 6. (A) View of Cβ2-Cβ3 loop in the porcine enzyme from the crystal structure (yellow) and average MD structure (orange). Tyr171, which is substituted to tryptophan in SPE, makes water mediated hydrogen bonds to the backbone of the adjacent Cβ3-Cβ4 loop both in the X-ray structure (waters shown as red spheres) and MD simulations. (B) Deletion of the PPE (yellow) residue Asp 186 in SPE (cyan), together with the R188N substitution, significantly increases the flexibility of the Cβ3-Cβ4 loop in the salmon enzyme. Both residues are highly conserved in mesophilic species. (C) View of the Cβ5-Cβ6 loop in the crystal structures of SPE (cyan) and PPE (yellow). The lower mobility of this loop in PPE is attributed to favourable hydrophobic packing between Val 221A and Leu 218.

In order to test the effect of mutations in a single surface loop on the thermodynamic activation parameters of the catalyzed reaction, we also carried out a series of MD/EVB simulations with the Pro-Ile-Ala substrate at the five different temperatures for the S218L/A221V double mutant of SPE and its counterpart L218S/V221A in PPE. These two double mutants thus replace the salmon with the porcine residues, and vice versa, in the 21 ACS Paragon Plus Environment

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above-mentioned Cβ5-Cβ6 loop thereby affecting its hydrophobic packing and mobility (Figure 6C). The results from these simulations show that the effects on the activation free energies at 295 K of both enyzmes are minute (Table 2), as would generally be expected for surface mutations ~16 Å away from the reaction center. Remarkably, however, pronounced ‡ effects are predicted for the activation enthalpies and entropies, where ∆ ‡ and ∆

change by about 7 kcal/mol in SPE, in the direction towards the porcine enzyme. Conversely, the PPE activation parameters change in the direction towards SPE although the magnitude of the effect is somewhat small in this case, about 6 kcal/mol. Hence the behavior of this surface loop can be directly linked to cold-adaptation, although it is almost certainly not the only part of the protein structure with such effects on the catalytic parameters.

Discussion The present MD/EVB simulations for salmon and porcine elastases yield activation free energy barriers close to those derived from experiments for the hydrolysis of five different tripeptides. Hence, among the examined peptides the calculations predict valine/isoleucine and phenylalanine to be the best and worst residues at the P1 position, respectively. Although no experiments have actually been reported for the temperature dependence of kcat of coldadapted salmon elastase, we carried out simulations here of both the SPE and PPE reactions with four of the tripeptide substrates at five different temperatures. For each of the examined substrates, with Ala, Val, Ile or Leu at the P1 position, the calculations predict that the salmon enzyme retains a higher catalytic rate as the temperature is lowered, as would be expected for a cold-adapted enzyme. The overall conclusion is also that SPE is a more highly optimized enzyme that is faster for all these substrates at all of the examined temperatures, although the ratios of kcat between SPE and PPE for any given substrate is not huge. These ratios are predicted to range between 4 and 24 at lowest temperature examined (285 K), where the


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highest value was calculated for the substrate with P1-Ile, which was also selected for indepth analysis of protein mobility differences. It is further noteworthy that extraction of thermodynamic activation parameters from Arrhenius plots in the temperature range 285-305 K in all cases yield a lower activation enthalpy and a more negative activation entropy for the salmon enzyme. This is perfectly in line with the usual behavior of cold-adapted enzymes and ‡ the differences in ∆‡ and ∆ are on the order of 10 kcal/mol for most of the substrates.

Such large differences between psychrophilic and mesophilic variants have also been observed experimentally for several other enzymes such as α-amylase, trypsin and endonuclease I.4,5,38

It should also be emphasized here that both enzymes are experimentally found to be folded and stable at at neutral pH at the lowest temperature examined in these experiments, which was 277 K.15 Likewise, in our simulations there are no signs of local unfolding at our lowest temperature (285 K). This is well in line with the fact that most globular proteins do not show signs of cold denaturation until well below the freezing point of water at normal pH values.39,40 Although there are interesting exceptions of small proteins with native low stability that show cold denaturation at around 280 K,41,42 this is generally not the case with typically sized globular enzymes. Calculations of solvent accessible surface areas (SASA) from our MD trajectories also show that this quantity is essentially constant over the examined temperature range, both for the overall protein surface and for the active site region (Figure S2).

Comparison of the active site mobilities, in terms of RMSF for the three catalytic enzyme residues (His57, Asp102 and Ser195) and the three residues of the Pro-Ile-Ala substrate, shows no significant differences between the cold- and warm-active enzyme (Figure 4B).


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This again supports the conclusion from other studies that it is not the active site mobility that primarily differs between psychrophilic and mesophilic enzymes.9-12,18,19,43,44 The protein backbone RMSF, averaged per residue, overall shows a very similar pattern of mobility and rigidity which reflects the identical topology and secondary structure of the two enzymes. However, there are distinct differences which appear as flexibility hot spots in the coldadapted enzyme, and these are exclusively located at surface exposed loops of the protein structure. Interestingly, all of these loops that differ in flexibility show distinct sequence signatures in psychrophilic and mesophilic elastases. Hence, sequence differences in these regions are largely conserved in the two classes of enzymes. This clearly indicates that evolution has found a way to operate on these regions, which are generally distant from the catalytic site, in such a way that the temperature dependence of the chemical reaction is altered. This is confirmed by our simulations of the two double mutants of SPE and PPE which bring about a change in activation enthalpy and entropy from one enzyme towards the orther. Moreover, the recipe is clearly to increase loop mobility in cold-adaptation, which is directly reflected in the thermodynamic activation parameters for the catalytic rate constant.8,11 That is, the computational experiment of artificially restraining the protein surface in Ref. 11 provided unambiguous proof of this connection between surface flexibility and reation thermodynamic activation parameters. It should further be mentioned that earlier MD simulations of cold- and warm-active elastases also identified one or two of the surface loops discussed herein as regions of increased flexibility in the former.18 However, those calculations were done on the apo-enzymes, for which mobility differences have less to do with effects on kcat, and they also had the incorrect amino acid sequence. As discussed in the Methods section, the sequence of the PDB entry 1ELT was only derived from the electron density, relying on the porcine sequence and is now known to be wrong.


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The repertoire of structural mechanisms for increasing surface loop flexibility is seen to be rather wide. Here, in the case of elastases, we can identify disruption of hydrophobic sidechain packing, insertion of glycine residues, disruption of H-bonded networks including surface bound water molecules and deletion of intraloop H-bonds and ion-pairs as examples of such mechanisms. As the elastases and trypsin share the same overall fold, it is not surprising that several of the surface exposed loops can be identified as cold-adaptation hotspots in both cases.10 What is perhaps more surprising is that different structural mechanisms for increasing the flexibility of the same loop (e.g., Nβ5-Nβ6) can occur in the two types of enzymes. This presumably reflects the divergent molecular evolution of the two types of enzymes (elastase and trypsin) which, once sequence motifs have been fixated, will require different types of amino acid substitutions in order to achieve a change in temperature dependence. Several of the surface loop in elastase and trypsin, such as Nβ5-Nβ6 and Cβ1Cβ2, also have distinctly different conformation in the two enzyme subfamilies.

The emerging picture of the principles behind enzyme cold-adaptation is thus that evolution cannot generally operate on the immediate active site environment for this purpose, since active site mutations with very high probability will be detrimental to catalytic throughput for an enzyme that has already been optimized by evolution. Instead, more remote mutations are selected for, provided that they confer slightly higher activity at lower temperatures. Such mutations apparently tend to be localized in surface loops of the protein rather than in its interior. This is probably because the rigid interior usually has well defined secondary structure with optimized hydrogen bonding and hydrophobic packing and mutations there are likely to have larger (detrimental) effects on the overall structure. In surface loop regions, on the other hand, structural changes induced by mutations can be significant, but tend to be more localized and can clearly have pronounced effects on loop mobility. Increased loop


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mobility also directly translates into a loss of surface rigidity in cold-adapted enzymes. It is now becoming clear that this enhanced surface flexibility affects catalysis by shifting the activation enthalpy-entropy balance, since the protein response to reactive events in the active site is softer and associated with a lower enthalpic penalty, that is partly compensated by entropy. Importantly, since it is the enthalpic contribution to the activation free energy that gives rise to exponential damping of rates as the temperature is lowered, this will lead to more efficient catalysis. Moreover, as protein melting is not really a problem in the low temperature regime, the evolutionary pressure on protein stability decreases and, hence, the enzyme melting temperature will naturally tend to drift towards lower values.

Associated Content Supporting Information Extended methods description and figures of Arrhenius plots and SASA calculations.

Author information Corresponding author *E-mail: [email protected]

Funding Support from the Swedish Research Council (VR), the Knut and Alice Wallenberg Foundation and the Research Council of Norway is gratefully acknowledged. Computational resources were provided by the Swedish National Infrastructure for Computing (SNIC).

Notes The authors declare no competing financial interest.


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Biochemistry

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