**4. Structure of elastase under inert gas pressure**

294 Current Trends in X-Ray Crystallography

regression model, this expansion would be reach for a pressure of 164 bar (Figure 4B). This pressure corresponds to about ten fold the pressure of 14 to 17 bar at which argon is known

Fig. 4. Linear regression model for cavity volume expansion as a function of occupancy (A)

0,00 5,00 10,00 15,00 20,00 25,00

**Expansion (%)**

0 20 40 60 80 100 120 140 160 180 **Pressure (bar)**

In addition, it should be mentioned that these estimated values for argon were also consistent with crystallographic data that have demonstrated that xenon at full occupancy (pressure of about 20 bar) produces a similar maximal expansion around 23-25 % of the gas binding site (Table 2). This is consistent with the fact that the ratio between the efficient estimated pressure of argon and the efficient experimental pressure of xenon at producing full occupancy and maximal expansion of the gas binding site (164 / 20 = 8.2) is similar to the ratio of their solubility in lipids (1.17 / 0.14 = 8.4) as predicted by the Meyer-Overton

Argon is narcotic only in hyperbaric condition. At ambient pressure, argon may thus have a very limited influence on its target function. Since one of the major effect of hydrostatic pressure is to contract the volume of internal cavities (Girard et al., 2010), it may explain

The three noble gases were bound to an identical location in urate oxidase, within an internal hydrophobic cavity. The gas occupancies increased in the sequence argon < krypton < xenon, as it was the case for T4 lysozyme (Quillin et al., 2000), who noticed that smaller gases do not bind as well as larger ones as a result of their attenuated polarizability. Xenon and krypton were bound also weakly in a secondary binding site, while argon was not

The main effect of the gas was to expand the cavity volume where it binds. The ratio of expansion was related to the narcotic potency of the gas, as evaluated by their MAC-awake or their solubility in lipids. The presence of xenon within the cavity induced an inhibition of the catalytic mechanism, with a relationship between gas-induced expansion and gasinduced inhibition, as shown by the comparison between xenon and nitrous oxide structural and functional effects. No activity assays were performed in presence of krypton or argon, but we can predict, based on the present structural results, that krypton should induce an inhibition of the catalytic mechanism of urate oxidase. However, krypton-induced inhibition should be lower than xenon-induced inhibition, according to their relative induced

to produces narcosis in rodents (Abraini et al., 1998; Koblin et al., 1998).

and as a function of pressure (B).

0,00 5,00 10,00 15,00 20,00 25,00

**Expansion (%)**

observed even at high pressure.

rule (Abraini et al., 2003; Campagna et al., 2003).

0 20 40 60 80 100 **Occupancy (%)**

why argon needs hyperbaric condition to exert its influence.

**3.5 Conclusion on urate oxidase structures under inert gas pressure** 

## **4.1 Introduction on elastase structures under inert gas pressure**

Pancreatic porcine elastase (EC 3.4.21.36) is a serine protease of 266 residues which hydrolyzes peptide bonds in proteins, its main substrate being elastine. The catalytic triad of elastase is composed, as for all serine proteases, of an activated serine (Ser 195) assisted by a proton relay (His 57), which acts as a general base, and stabilized through an hydrogen bond by an aspartic acid (Asp 102). Pancreatic porcine elastase crystallizes in the orthorhombic space group P212121 with one monomeric enzyme per asymmetric unit (cell : a = 51.4 Å, b = 58.0 Å, c = 75.3 Å, = = = 90°). In the crystallographic structure, a sulphate or an acetate ion is bound in the oxyanion hole, depending on the concentration of the precipitating agents. The primary specificity pocket S1 is a hydrophobic pocket located below the oxyanion hole and the Ser 195 which is specific for recognition of the peptidic substrate.

In the crystallographic structures of apo elastase, the S1 pocket was either empty, either filled by a water molecule hydrogen-bonded to a water molecule outside of the S1 pocket and to an oxygen atom of the sulphate ion (Figure 5A). When this water molecule (termed W-S1) was present, its B-factor was quite elevated. In the different crystallographic structures of elestase in complex with xenon deposited in the Protein Data Bank, 1C1M (Schiltz et al., 1995), 1L1G and 1L0Z (Panjikar et al., 2002), 1UO6 and 2A7C (Mueller-Dieckmann et al., 2004) and 2OQU (Kim et al., 2007), xenon was bound within the specificity pocket S1 in the active site of elastase (Figure 5B). This xenon binding site is moderately hydrophobic, lined with 60% carbons.

Fig. 5. Elastase shown with its solvent-accessible surface colored by atomic type. A. Native gas-less elastase with the water molecule W-S1 in the S1 pocket. B. Elastase in complex with xenon (shown as an orange sphere) or in complex with a peptidic inhibitor TFLA (shown in cyan in stick representation).

In the present study, structures of native elastase (gas-less) were determined in the same conditions than structures under inert gas pressure, i.e. at room temperature in a quartz

Protein-Noble Gas Interactions Investigated by Crystallography

**4.3 Structure of elastase under pressure of krypton** 

Krypton pressure (bar)

krypton-induced expansion of the S1 pocket in elastase.

expansion remained quite low, less than 10 %.

**4.4 Structure of elastase under pressure of argon** 

Argon pressure (bar)

of the water molecule W-S1 if it is present.

a same pressure, according to a lower polarizablity (Figure 6).

Resolution (Å)

Occupancy of krypton reached 70 % at the pressure of 30 bar (Table 6).

Resolution (Å)

enzymatic activity.

on Three Enzymes - Implication on Anesthesia and Neuroprotection Mechanisms 297

indeed structurally similar active site with the same catalytic triad which can be easily superimposed (Schiltz et al., 1995). The superimposition of the catalytic triad of elastase and of tPA suggested that xenon fitted perfectly in the S1 pocket of tPA, thus inhibiting tPA

Structures of elastase under pressure of krypton from 2 to 30 bar were determined in the present study. Krypton was bound within the S1 pocket, at the exact same location than xenon. The water molecule W-S1 if it was present was replaced by the krypton atom.

> 2 1.35 20 4.9 5 1.45 25 4.9 10 1.50 35 5.8 20 1.75 50 7.9 30 1.45 70 8.5

Table 6. Krypton pressure, resolution of the crystallographic structure, occupancy and

Krypton occupancy was always lower than xenon occupancy for a same pressure (Figure 6). Xenon which is more polarizable was bound with a higher occupancy than krypton. Since the gas binding site is only moderately hydrophobic, with a lot of polar atoms lining the gas, it is likely that dipole-induced interactions play an important role in gas binding. Like xenon, krypton induced an expansion of the S1 pocket where it binds. However, this

Structures of elastase under pressure of argon of 10 to 60 bar were determined in the present study. Argon was bound within the S1 pocket, at the exact same location than xenon and krypton. Argon occupancy reached 50 % for a pressure of 60 bar (Table 7). For technical conditions, it was not possible to reach higher pressure due to the risk of failure of the quartz capillary. Argon occupancy was always lower than krypton and xenon occupancy for

10 1.35 35 42.5 20 1.45 35 missing 30 1.50 40 40.1 40 1.35 40 missing 60 1.50 50 missing Table 7. Argon pressure, resolution of the crystallographic structure, occupancy and B-factor

Occ. (%) Kr-induced

Occ. (%) B factor

W-S1

expansion (%)

capillary. Three structures have been solved, at 1.38 Å, 1.7 Å and 1.45 Å resolution. In two native structures, the S1 pocket was empty while in one of them, there was the water molecule W-S1 with a B-factor of 36.3 Å2.

#### **4.2 Structure of elastase under pressure of xenon and comparison with** *in-vitro* **activity assays**

Structures of elastase under xenon pressure from 1 bar to 30 bar were determined in the present study. Whatever the applied pressure, xenon was bound to a unique site, within the specificity pocket S1, with an occupancy which increased with the applied pressure. Occupancy reached 100 % for a pressure of 30 bar (Table 5). The S1 pocket where xenon binds is moderately hydrophobic, with 60 % of atoms lining it being carbons. This gas binding site is less hydrophobic than the main gas binding site in urate oxidase, which was lined by 86 % carbons. The atom the closest to xenon is the side chain atom O of the catalytic Ser 195.


Table 5. Xenon pressure, resolution of the crystallographic structure, occupancy and xenoninduced expansion of the S1 pocket in elastase.

Whatever the pressure, there was no water molecules in the S1 pocket, so if the water molecule W-S1 was present in the native gas-less structure, it was not displaced but replaced by xenon. However, xenon did not take the exact location of the W-S1 and was closer to the O of the catalytic Ser 195 (3.4 Å instead of 3.8 Å).

The presence of xenon within the S1 pocket expanded its volume, its expansion rising with the applied pressure. However, since the gas was bound directly within the active site, the gas-induced inhibition is likely to be a direct inhibition and the expansion by itself has probably no functional relevance. Xenon took indeed the place of peptidic inhibitors, like the trifluroacetyl-leu-ala (TFLA) known to be an excellent inhibitor of elastase (Li de la Sierra et al., 1990) (Figure 5B).

To investigate the direct inhibition by xenon, we performed activity assays on elastase in presence either of air, either of 100 vol % xenon. Initial velocity in presence of xenon when compared to air (taken as 100 %) was 81.5 + 2.1 % revealing an inhibition of the catalytic activity of elastase of around 20 % by xenon. However, this inhibition was lower than xenon occupation in the range 5-10 bar (30 – 70 % occupation).

Tissue-type plasminogen activator (tPA), the only approved treatment for thrombolysis after an ischemic stroke, is also a serine protease. As in the case of elastase, xenon inhibited tPA enzymatic activity (David et al., 2010). This inhibition is likely to be a direct inhibition with xenon binding directly in the S1 pocket in the active site of tPA. Serine proteases have indeed structurally similar active site with the same catalytic triad which can be easily superimposed (Schiltz et al., 1995). The superimposition of the catalytic triad of elastase and of tPA suggested that xenon fitted perfectly in the S1 pocket of tPA, thus inhibiting tPA enzymatic activity.

### **4.3 Structure of elastase under pressure of krypton**

296 Current Trends in X-Ray Crystallography

capillary. Three structures have been solved, at 1.38 Å, 1.7 Å and 1.45 Å resolution. In two native structures, the S1 pocket was empty while in one of them, there was the water

Structures of elastase under xenon pressure from 1 bar to 30 bar were determined in the present study. Whatever the applied pressure, xenon was bound to a unique site, within the specificity pocket S1, with an occupancy which increased with the applied pressure. Occupancy reached 100 % for a pressure of 30 bar (Table 5). The S1 pocket where xenon binds is moderately hydrophobic, with 60 % of atoms lining it being carbons. This gas binding site is less hydrophobic than the main gas binding site in urate oxidase, which was lined by 86 % carbons. The atom the closest to xenon is the side chain atom O of the

1 1.40 15 3.0 2 1.45 25 3.3 5 1.50 30 7.8 10 1.50 70 9.4 20 1.60 90 12.7 30 1.65 100 13.6 Table 5. Xenon pressure, resolution of the crystallographic structure, occupancy and xenon-

Whatever the pressure, there was no water molecules in the S1 pocket, so if the water molecule W-S1 was present in the native gas-less structure, it was not displaced but replaced by xenon. However, xenon did not take the exact location of the W-S1 and was

The presence of xenon within the S1 pocket expanded its volume, its expansion rising with the applied pressure. However, since the gas was bound directly within the active site, the gas-induced inhibition is likely to be a direct inhibition and the expansion by itself has probably no functional relevance. Xenon took indeed the place of peptidic inhibitors, like the trifluroacetyl-leu-ala (TFLA) known to be an excellent inhibitor of elastase (Li de la

To investigate the direct inhibition by xenon, we performed activity assays on elastase in presence either of air, either of 100 vol % xenon. Initial velocity in presence of xenon when compared to air (taken as 100 %) was 81.5 + 2.1 % revealing an inhibition of the catalytic activity of elastase of around 20 % by xenon. However, this inhibition was lower than xenon

Tissue-type plasminogen activator (tPA), the only approved treatment for thrombolysis after an ischemic stroke, is also a serine protease. As in the case of elastase, xenon inhibited tPA enzymatic activity (David et al., 2010). This inhibition is likely to be a direct inhibition with xenon binding directly in the S1 pocket in the active site of tPA. Serine proteases have

Occ. (%) Xe-induced

expansion (%)

**4.2 Structure of elastase under pressure of xenon and comparison with** *in-vitro*

Resolution (Å)

molecule W-S1 with a B-factor of 36.3 Å2.

Xenon pressure (bar)

induced expansion of the S1 pocket in elastase.

Sierra et al., 1990) (Figure 5B).

closer to the O of the catalytic Ser 195 (3.4 Å instead of 3.8 Å).

occupation in the range 5-10 bar (30 – 70 % occupation).

**activity assays** 

catalytic Ser 195.

Structures of elastase under pressure of krypton from 2 to 30 bar were determined in the present study. Krypton was bound within the S1 pocket, at the exact same location than xenon. The water molecule W-S1 if it was present was replaced by the krypton atom. Occupancy of krypton reached 70 % at the pressure of 30 bar (Table 6).


Table 6. Krypton pressure, resolution of the crystallographic structure, occupancy and krypton-induced expansion of the S1 pocket in elastase.

Krypton occupancy was always lower than xenon occupancy for a same pressure (Figure 6). Xenon which is more polarizable was bound with a higher occupancy than krypton. Since the gas binding site is only moderately hydrophobic, with a lot of polar atoms lining the gas, it is likely that dipole-induced interactions play an important role in gas binding. Like xenon, krypton induced an expansion of the S1 pocket where it binds. However, this expansion remained quite low, less than 10 %.

#### **4.4 Structure of elastase under pressure of argon**

Structures of elastase under pressure of argon of 10 to 60 bar were determined in the present study. Argon was bound within the S1 pocket, at the exact same location than xenon and krypton. Argon occupancy reached 50 % for a pressure of 60 bar (Table 7). For technical conditions, it was not possible to reach higher pressure due to the risk of failure of the quartz capillary. Argon occupancy was always lower than krypton and xenon occupancy for a same pressure, according to a lower polarizablity (Figure 6).


Table 7. Argon pressure, resolution of the crystallographic structure, occupancy and B-factor of the water molecule W-S1 if it is present.

Protein-Noble Gas Interactions Investigated by Crystallography

**5. Structure of lysozyme under inert gas pressure** 

**5.1 Introduction on lysozyme structures under inert gas pressure** 

argon of 35 % at 10 bar.

beyond 25 % in GBS II.

symmetrical lysozymes.

other deposited structures.

but not in the crystallographic pocket (GBS I).

**GBS II GBS II GBS I** 

**A B**

on Three Enzymes - Implication on Anesthesia and Neuroprotection Mechanisms 299

small inhibition of the enzymatic reaction. Argon-induced inhibition of the catalytic reaction is likely to be quite low and similar to krypton-induced inhibition, with an occupation of

Egg white lysozyme C (EC 3.2.1.17) is an enzyme of 129 residues which hydrolyse peptidoglycans. It crystallizes in tetragonal space group P43212 with one monomeric enzyme per asymmetric unit (cell: a = 79.2 Å, b = 79.2 Å, c = 37.9 Å, = = = 90°). In the different crystallographic structures of lysozyme in presence of xenon deposited in the Protein Data Bank : 1C10 (Prangé et al., 1998), 2A7D (Mueller-Dieckmann et al., 2004) and 1VAU (Takeda et al., 2004), xenon was bound mainly in a pocket at a crystallographic interface (gas binding site I or GBS I in Figure 8) and weakly in a small internal cavity (GBS II in Figure 8). In the native gas-less structure, a water molecule was present in GBS I, and GBS II was empty. GBS I is moderately hydrophobic, lined with 55 % carbons, and GBS II is very hydrophobic, lined with 83 % carbons. The occupancy of xenon ranged between 30 and 60% in GBS I and stayed

Fig. 8. Xenon binding sites in lysozyme, shown with two symmetrical subunits. A. Xenon was bound at the crystallographic interface between two symmetrical monomers (GBS I) and a second xenon was bound within a small internal cavity buried within each monomer (GBS II) (xenon shown as orange spheres, and lysozyme with its C chain as ribbon, one symmetric is colored in green, the second in blue). B. Solvent-accessible surface of two

**GBS II GBS II GBS I** 

In the structure 1C10, a third xenon was located in the active site, where either two water molecules or one water molecule and one chloride ion were found in the other deposited structures. In the structure 1VAU, a third xenon was located in another crystallographic pocket at the interface between two monomers, where a water molecule was found in the

In the crystallographic structure of lysozyme under a 55 bar pressure of krypton 1QTK, (Schiltz et al., 1997; Prangé et al., 1998), krypton was bound in the internal cavity (GBS II)

In the present study, the structure of a native gas-less lysozyme was determined in the same condition than structures under inert gas pressure, i.e. at room temperature in quartz capillary. Two native structures have been solved, at 1.6 Å and 1.55 Å resolution. In both structures, the internal cavity GBS II was empty and there was a water molecule in GBS I which had a B-

Fig. 6. Xenon, krypton and argon occupancies as a function of pressure.

Argon occupancy was higher in elastase than in urate oxidase (Table 4), probably because of the smaller size of the gas binding site in elastase which would allow a better binding of argon.

Argon-induced expansion of the S1 pocket was very low and not very significant (less than 5 %), probably due to the small size of the argon atom.

Contrary to xenon and krypton cases, when the water molecule W-S1 was present in the native gas-less structure, this water molecule remained visible close to the argon electronic density (Figure 7), with a lower occupation factor. The distance between the argon atom and the W-S1 molecule was about 2.8 Å.

Fig. 7. *2Fo-Fc* electron density map of elastase under an argon pressure of 10 bar, contoured at 1 .

#### **4.5 Conclusion on elastase structures under inert gas pressure**

In elastase, there is one unique gas binding site, located within the S1 pocket in the active site. Contrary to urate oxidase where the gas binding site was an empty cavity, there might be a water molecule in the moderately hydrophobic S1 pocket. When present, this water molecule was replaced by xenon and krypton, but remained visible close to argon.

Xenon inhibited directly elastase catalytic activity by taking the place of the substrate, even if its inhibition stayed lower than its occupancy. It is also likely that krypton inhibited elastase catalytic activity. Since krypton occupancy was lower than xenon occupancy, krypton-induced inhibition is expected to be lower than xenon-induced inhibition. In the range 5-10 bar, krypton occupancy ranged between 25 and 35%, inducing probably a rather small inhibition of the enzymatic reaction. Argon-induced inhibition of the catalytic reaction is likely to be quite low and similar to krypton-induced inhibition, with an occupation of argon of 35 % at 10 bar.
