Eugénie Carletti

Nadine Aurbek

Emilie Gillon

Mélanie Loiodice

Yvain Nicolet

Juan-Carlos Fontecilla-Camps

Patrick Masson

Horst Thiermann

Dr Florian Nachon
email: fnachon@crssa.net

Franz Worek

Structure-activity analysis of aging and reactivation of human butyrylcholinesterase inhibited by analogues of tabun

Keywords: Butyrylcholinesterase, tabun analogues, X-ray crystallography, kinetics, aging, oxime reactivation AChE, Acetylcholinesterase, BChE, Butyrylcholinesterase, BSA, Bovine serum albumine, BTC, butyrylthiocholine, ChE, Cholinesterase, CHO, Chinese hamster ovary, DTNB,

Human butyrylcholinesterase (hBChE) naturally scavenges organophosphorus nerve agents (OPs). This bioscavenger is currently in Clinical Phase I for pretreatment of OP intoxication.

Phosphylated cholinesterases (ChEs) can undergo a spontaneous time-dependent process called « aging » during which the conjugate is dealkylated, leading to non-reactivable enzyme. hBChEs inhibited by phosphoramidates like tabun display a peculiar resistance to oximes mediated reactivation. We investigated the basis of oxime resistance of phosphoramidyl-BChE conjugates, by determining the kinetics of inhibition, reactivation (obidoxime, TMB-4, HLö 7, HI-6), aging, and the crystal structures of hBChE inhibited by different N-monoalkyl and N,N-dialkyl tabun analogues. The refined structures of aged hBChE conjugates show that aging proceeds through O-dealkylation of the P(R) enantiomer of N,N-diethyl and N-propyl analogues, with subsequent formation of a salt bridge preventing reactivation, similarly to previous observation made on tabun-ChEs conjugates. Interestingly, the N-methyl analogue projects its amino group towards the choline-binding pocket, so that aging proceeds through deamination. This orientation results from a preference of hBChE's acyl-binding pocket for larger than 2-atoms linear substituents. The correlation between the inhibitory potency and the N-monoalkyl chain length is related to increasingly optimized interactions with the acyl-binding pocket as shown by the X-ray structures. These kinetics and X-ray data lead to a structure-activity relationship that hightlights steric and electronic effects of the amino substituent of phosphoramidate. This study provides structural basis to design new oximes capable of reactivating phosphoramidyl-hBChE conjugates after intoxication, notably when hBChE is used as pretreatment or to design BChE-based catalytic bioscavengers.

Introduction

Human plasma butyrylcholinesterase (BChE; EC 3.1.1.8) is a toxicologically relevant enzyme because it hydrolyzes or scavenges a wide range of toxic esters, including heroin, cocaine, carbamate pesticides, organophosphorus pesticides and nerve agents [START_REF] Lockridge | Pesticides and susceptible populations: people with butyrylcholinesterase genetic variants may be at risk[END_REF][START_REF] Masson | Structure, activities and biomedical applications of human butyrylcholinesterase[END_REF].

Organophosphates (OPs) exert their acute toxicity through inhibition of acetylcholinesterase (AChE, E.C. 3.1.1.7) by phosphylation of the catalytic serine [START_REF] Masson | Structure, activities and biomedical applications of human butyrylcholinesterase[END_REF]. Subsequent accumulation of acetylcholine at neuronal synapses and neuromuscular junctions, results in paralysis, seizures and other symptoms of cholinergic syndrome [START_REF] Eddleston | The pathophysiology of organophosphorus pesticide selfpoisoning is not so simple[END_REF][START_REF] Konradsen | Pesticide selfpoisoning: thinking outside the box[END_REF]. Human BChE is a potential drug candidate that may be employed in several therapeutic fields. This is due to the unique structure of the enzyme that allows accommodation of a large variety of substrates and inhibitors, some of which are excluded from the catalytic gorge of acetylcholinesterase [START_REF] Saxena | Differences in active site gorge dimensions of cholinesterases revealed by binding of inhibitors to human butyrylcholinesterase[END_REF].

BChE-based biopharmaceuticals are under research and development as stoichiometric and catalytic bioscavengers for neutralization or detoxification of poisonous esters. Recombinant and plasma-purified hBChE, are in Clinical Phase I as the first stoichiometric bioscavengers for the pretreatment of OPs intoxication. In case of intoxication, reactivation of hBChE used in pretreatment will improve protection against high dose or repetitive OP exposure. For this reason it is essential to design efficient reactivators for hBChE. Phosphylated ChEs can be reactivated by specific nucleophiles such as oximes [START_REF] Worek | Kinetic analysis of interactions between human acetylcholinesterase, structurally different organophosphorus compounds and oximes[END_REF][START_REF] Worek | Kinetic analysis of reactivation and aging of human acetylcholinesterase inhibited by different phosphoramidates[END_REF]. The most effective oximes used for emergency treatment of nerve agent poisoning are the monopyridinium oximes (2-PAM) and bispyridinium oximes (TMB-4, MMB-4, obidoxime, HI-6 and HLö 7) [START_REF] Worek | Kinetic analysis of interactions between human acetylcholinesterase, structurally different organophosphorus compounds and oximes[END_REF]. Kinetic analysis of the interactions between different AChE-OP conjugates and different oximes shows that the most effective oximes are HLö 7 for phosphonylated AChE and obidoxime for phosphorylated AChE [START_REF] Worek | Kinetic analysis of interactions between human acetylcholinesterase, structurally different organophosphorus compounds and oximes[END_REF]. Unfortunately, a universal antidote, efficient against all nerve agents and capable of crossing the blood brain barrier, is not yet available.

Reactivation of inhibited ChEs is complicated by a side reaction. Indeed, phosphylated ChEs undergo a spontaneous, time-dependent process called « aging » during which the OP-ChE conjugate are O-dealkylated, leading to non-reactivatable enzyme [START_REF] Li | Aging pathways for organophosphate-inhibited human butyrylcholinesterase, including novel pathways for isomalathion, resolved by mass spectrometry[END_REF][START_REF] Shafferman | Aging of somanyl-acetylcholinesterase adducts: facts and models[END_REF][START_REF] Kovach | A self-consistent mechanism for dealkylation in soman-inhibited acetylcholinesterase[END_REF].

BChE inhibited by tabun and subsequent aging reaction of adduct are of interest, because tabun-ChE conjugates are particulary resistant to most oxime reactivators. Recent studies detail the mode of interaction of tabun with ChEs and show that tabun-ChE conjugates age through O-dealkylation [START_REF] Carletti | Aging of cholinesterases phosphylated by tabun proceeds through O-dealkylation[END_REF]. An extension of this study to various phosphoramidate anologues of tabun provide the basis of a structure-activity relationship analysis of phosphoramidate adducts. The analogues of tabun differs in their amino substituents (Fig. 1).

The reactivation of hBChE inhibited by tabun and its analogues was studied with obidoxime, TMB-4, HI-6 and HLö-7 (Fig. 2). This study provide a better understanding of the resistance of phosphoramidate-hBChE conjugates to oximes and give some insights to design new oximes capable of reactivating tabun-ChEs conjugates, and ChEs inhibited by related agents.

Material and Methods

Materials

5,5´-dithio-bis-2-nitrobenzoic acid (DTNB) and TMB-4 dibromide (TMB-4) were obtained from Sigma Chemical Co (St Louis, USA). Obidoxime dichloride (obidoxime) was purchased from Duphar. HI-6 dichloride (HI-6) was kindly provided by Dr. Clement (DRDC, Suffield, Canada) and HLö-7 dimethane-sulfonate (HLö-7) was a custom synthesis by Dr. Braxmeier (Chemisches Labor, Döpshofen, Germany). Tabun and its analogues (TA 1-6) (>98% by GC-MS, 1 H-NMR and 31 P-NMR) were made available by the German Ministry of Defence, all other chemicals were from Merck Eurolab GmbH. S-butyrylthiocholine iodide (BTCh) was obtained from Sigma.

Stock solutions of tabun and compounds 1 -6 (1% v/v; Fig. 1) were prepared in 2-propanol, stored at 4°C and were appropriately diluted in distilled water just before the experiment.

Oximes (200 mM) were prepared in distilled water, stored at -60°C and diluted as required in distilled water at the day of the experiment. Human plasma was obtained from heparinized whole blood by centrifugation (3000 rpm, 10 min, 4°C) and was stored in aliquots at -60°C until use.

Isolated plasma hBChE

Isolated human butyrylcholinesterase (hBChE; E.C. 3.1.1.8) was obtained from Sigma. This enzyme was used for kinetic assays.

Production of recombinant hBChE

Recombinant hBChE was produced in Chinese hamster ovary (CHO) cells and secreted into serum-free culture medium [START_REF] Nachon | Engineering of a monomeric and low-glycosylated form of human butyrylcholinesterase: expression, purification, characterization and crystallization[END_REF]. Briefly, recombinant hBChE was a truncated monomer containing residues 1-529, whereas the tetramerization domain at the C-terminus was deleted.

The glycan chain number was reduced by site-directed mutagenesis from 9 to 6. Enzyme purification was implemented by affinity and ion exchange chromatography as described. The active-site concentration of highly purified enzyme was determined using diisopropyl fluorophosphate as the titrant as described [START_REF] Amitai | Inhibition of acetylcholinesterase and butyrylcholinesterase by chlorpyrifos-oxon[END_REF]. This enzyme was used for crystallographic assays and for testing of enantioselectivity.

Enzyme assays

hBChE activities were measured spectrophotometrically (Cary 3Bio, Varian, Darmstadt) at 436 nm with a modified Ellman assay at 37°C [START_REF] Worek | Improved determination of acetylcholinesterase activity in human whole blood[END_REF]15]. Though, regular Ellman assay at 412 nm works as well. The assay mixture (3.16 ml) contained 1 mM BTCh as substrate and 0.3 mM DTNB as chromogen in 0.1 M phosphate buffer (pH 7.4).

Determination of inhibition rate constants (k i )

The inhibition rate constants were determined at pseudo-first-order reaction conditions as described before [START_REF] Worek | Kinetic analysis of interactions between human acetylcholinesterase, structurally different organophosphorus compounds and oximes[END_REF]. In brief, appropriately diluted OP was added to temperature-equilibrated (37°C) isolated hBChE at t = 0, an aliquot was removed after specified time intervals (1 -9 min) and transferred to a cuvette for the determination of residual hBChE activity. k i was calculated from equation 1:

ki = 1 [OP] t ln v0 vt eq.1
where [OP] is the initial concentration of the tested OP, v 0 and v t are the reaction rates at time zero and at time t, respectively. The concentration ranges of OP used in the experiment were:

Test of enantioselectivity for TA 4-6

Determination of inhibition enantioselectivity of recombinant hBChE was performed under second-order conditions as described [START_REF] Carletti | Aging of cholinesterases phosphylated by tabun proceeds through O-dealkylation[END_REF]. In brief, incubation was performed at 25 °C in 50 mM sodium phosphate buffer pH 7.0 containing 5% isopropanol, 1 mg/mL BSA, a

Oxime screening for the reactivation of OP-inhibited hBChE

Human plasma was incubated with a small volume (1% v/v) of appropriate concentrations of tabun and compounds TA 1-6, 30 min at 37°C to achieve an hBChE inhibition of >90%.

Thereafter, the treated samples were dialyzed (phosphate buffer, 0.1 M, pH 7.4) overnight at 4°C to remove residual inhibitor. The inhibitory activity of the samples was tested by 30 min of incubation at 37°C of treated plasma and control hBChE in order to verify the absence of residual inhibitor. OP-inhibited hBChE was incubated at 37°C with 1 mM obidoxime, TMB-4, HI-6 or HLö 7 and the hBChE activity was measured at specified time intervals (1 -120 min). Enzyme activities were referred to control activity and the % reactivation was calculated according to de Jong and Wolring [START_REF] De Jong | Effect of 1-(AR)alkyl-2-hydroxyiminomethylpyridinium salts on reactivation and aging of acetyl-cholinesterase inhibited by ethyl dimethylphosphoramidocyanidate (tabun)[END_REF]. Experiments were performed in duplicate.

Kinetics of obidoxime reactivation

Due to low reactivating potency, the reactivation kinetics were determined at 37°C by a discontinuous procedure as described before [START_REF] Worek | Kinetic analysis of interactions between human acetylcholinesterase, structurally different organophosphorus compounds and oximes[END_REF][START_REF] Wang | Semiquantitative determination of cholinesterase activity in human plasma: test-paper method[END_REF] which allowed the use of high obidoxime concentrations in incubation mixture (up to 5 mM). OP-inhibited hBChE was incubated with different obidoxime concentrations and 1 mM BTCh. Aliquots were transferred to cuvettes after specified time intervals (2 -45 min) for measurement of hBChE activity. The dissociation constant K D of enzyme-obidoxime complex, the reactivity rate constant k r and the second order reactivation rate constant k r2 were calculated by standard procedure [START_REF] Worek | Kinetic analysis of interactions between human acetylcholinesterase, structurally different organophosphorus compounds and oximes[END_REF].

E = E 0 E 0 OP 0 [ ] n E 0 OP 0 [ ] n e ( OP 0 [ ] n E 0 )k i t
Eight to ten obidoxime concentrations, 100 -5000 M with tabun-and TA3-inhibited BChE and 50 -3000 M with TA4 -TA6-inhibited enzyme, were used for the determination of reactivation rate constants.

Determination of rate constants for aging (k a ) and spontaneous reactivation (k s )

OP-inhibited hBChE was brought to 37°C (t = 0). Then, aliquots were taken after various time intervals for determination of BChE activity (spontaneous reactivation) and of the decrease of oxime-induced reactivation (aging). Hereby, small aliquots were incubated 1h with 1 mM obidoxime. Data from duplicate experiments were referred to control activities and the percentage reactivation (% react) was calculated. The pseudo first-order rate constants k s (spontaneous reactivation) and k a (aging) were calculated by a non-linear regression model [START_REF] Worek | Kinetic analysis of interactions between human acetylcholinesterase, structurally different organophosphorus compounds and oximes[END_REF].

Crystallization of nonaged TA1, TA4, TA5 and TA6-inhibited hBChE

The mother liquor was 0.1 M MES buffer, pH 6.5, with 2.1 M ammonium sulfate. Stock solutions of tabun analogues were 10 mM in isopropanol. Native enzyme was crystallized using the hanging drop method [START_REF] Nachon | Engineering of a monomeric and low-glycosylated form of human butyrylcholinesterase: expression, purification, characterization and crystallization[END_REF]. Crystals grew in one week at 20 °C. Then, the OP-hBChE conjugates were prepared by soaking crystals for 10 min in 0.1 M MES buffer, pH 6.5, with 2.1 M ammonium sulfate, containing 2 mM tabun analogue. The crystals were washed with a cryoprotectant solution [0.1 M MES buffer with 2.1 M ammonium sulfate, containing 20% glycerol], and then flash-cooled in liquid nitrogen.

Crystallization of aged TA1, TA4 and TA5-hBChE

Purified enzyme (9 mg/mL) was inhibited by 2 mM of tabun analogue compounds, in 10 mM Tris HCl buffer, pH 7.4. The inhibited enzyme was crystallized using the hanging drop method [START_REF] Nachon | Engineering of a monomeric and low-glycosylated form of human butyrylcholinesterase: expression, purification, characterization and crystallization[END_REF]. Hereby, crystals grew in one week at 20 °C. The time between phosphylation and data collection was sufficiently long (>2 week) to achieve completion of the aging reaction (aging half time < 70 h). detector. All data sets were processed with XDS and the structures were solved by use of the CCP4 suite [START_REF]The CCP4 suite: programs for protein crystallography[END_REF]. An initial solution model was determined by molecular replacement with

X-ray

MolRep [START_REF] Vagin | MOLREP: an automatedprogram for molecular replacement[END_REF], starting with the recombinant hBChE structure (PDB entry 1P0I) from which all ligands (butyrate, glycerol, ions) and glycan chains were removed. For all diffraction data sets, the model was refined as follows: an initial rigid-body refinement with REFMAC5 [START_REF] Murshudov | Refinement of macromolecular structures by the maximum-likelihood method[END_REF] was followed by iterative cycles of model building with Coot [START_REF] Emsley | Coot: model-building tools for molecular graphics[END_REF], and finally restrained refinement was carried out with REFMAC5 and Phenix [START_REF] Adams | PHENIX: building new software for automated crystallographic structure determination[END_REF]. The covalently bound inhibitors and their descriptions were built using the Dundee PRODRG2.5 server including energy minimization using GROMOS96.1 forcefield.

Determination of enantioselectivity

The time dependence of the inhibition of hBChE by TA4, TA5 and TA6, under second-order conditions, is shown in figure 3. Fitting the data to equation 2 yields ki'= 2.4±0.2 10 6 M - 1 .min -1 and n= 1.8±0.1 for TA4, ki'= 8.4±0.5 10 6 M -1 .min -1 and n= 2.0±0.1 for TA5, and ki'= 24±2 10 6 M -1 .min -1 and n= 2.0±0.1 for TA6. It follows that hBChE reacts preferentially with one enantiomer of tabun analogues. A similar enantiomeric preference was observed for inhibition of hBChE by tabun [START_REF] Carletti | Aging of cholinesterases phosphylated by tabun proceeds through O-dealkylation[END_REF]. In this regard, ki differs from ki' because it is determined under pseudo first order conditions and describe the reaction of both enantiomers. Moreover, experimental conditions concerning temperature, pH and isopropanol-concentration were different.

Reactivation of phosphoramidate-inhibited hBChE by oximes

The reactivation of OP-inhibited hBChE by oximes was tested with 1 mM obidoxime, TMB-4, HI-6 and HLö 7 (Fig. 4). Tabun-inhibited hBChE could be partially reactivated by oximes, the extent of reactivation decreased in the order obidoxime > TMB-4 > HLö 7 > HI-6. Oximeinduced reactivation was rather ineffective with hBChE inhibited by TA1 and TA2.

Interestingly, reactivation of hBChE inhibited by TA3 followed a different pattern. All oximes were capable of reactivating the inhibited enzyme, and after 120 min incubation the recovery in hBChE activity was in the range of 60 -85%.

In contrast all oximes reactivated hBChE inhibited by N-monoalkyl tabun analogues, i.e.

TA4, TA5 and TA6 completely, although there were some differences between oximes regarding velocity and extent of reactivation (Fig. 4).

The reactivation kinetics of hBChE inhibited by tabun and its analogues TA3 to TA6, was determined with obidoxime. The reactivation constants are in table 1. K D is the dissociation constant of oxime-OP-hBChE complex, kr the reactivation constant and kr2 describes the specific reactivity. No rate constants could be determined with hBChE inhibited by TA1 and TA2, due to low reactivation extent. TA3-inhibited hBChE was, compared to tabun, substantially more susceptible towards reactivation.

Reactivation of hBChE inhibited by N-monoalkyl tabun analogues, TA4, TA5 and TA6, resulted in comparable second-order rate constant k r2 (table 1). There was only little difference in K D and k r between TA5 and TA6 while both values were substantially lower with TA4.

Aging and spontaneous reactivation of phosphoramidate-inhibited hBChE

Aging and spontaneous reactivation constants are in table 1. Aging kinetics was not determined for TA1 and TA2 due to insufficient reactivation extent by oximes. Tabun and its N,N-di-i-propyl analogue (TA3) showed comparable aging kinetics with a k a of 0.1 h -1 and 0.094 h -1 , respectively. hBChE inhibited by N-monoalkyl tabun analogues TA4, 5 and 6, was susceptible towards aging and spontaneous reactivation. Hereby, the velocity of spontaneous reactivation increased with the chain length of the N-alkyl group, whereas the aging rates are comparable and noticeably slower than for tabun-inhibited hBChE (table 1).

X-ray structure of non-aged tabun analogue-hBChE conjugates

Data were collected from tetragonal crystals of space group I422 and refined to 2. ) within covalent bond distance of the catalytic serine was observed in the initial Fo -Fc map for each conjugates. This confirms that inhibitors were bound to the active site serine after 5-min soaking. The refinement yields an adduct that exhibits P(R) stereochemistry for the chiral phosphorus atom except for TA4 that exhibits P(S) stereochemistry (Fig. 5). Given the good resolution (2.15 Å) and the map quality there is no ambiguity in TA4 stereochemistry assignment. As a control, we also placed the presumably incorrect stereoisomer of TA4 and performed one round of structural refinement. Distinct positive and There is a strong peak of positive electron density in the initial Fo -Fc map close to Trp82. This density could not be reasonably modeled. It is most likely due to the presence of the small ligand that was previously observed in other structures of hBChE [START_REF] Nachon | Role of water in aging of human butyrylcholinesterase inhibited by echothiophate: the crystal structure suggests two alternative mechanisms of aging[END_REF][START_REF] Nicolet | Crystal structure of human butyrylcholinesterase and of its complexes with substrate and products[END_REF]. This unknown ligand appears stacked against Trp82 and interacts with water molecules. For TA1 there is no large blob of density, but electron density corresponding to two water molecules.

The phosphorus atoms were found at covalent bonding distance from the Ser198O atoms (about 1.6 Å) and the P-N distance were refined to values ranging from 1.6 Å to 1.7 Å. O2 of the phosphoramidate moiety is at hydrogen bonding distance of the main chain amide nitrogen of residues forming the oxyanion hole (Ala199/Gly116/Gly117). The N-alkyl subsituents were located in the acyl-binding pocket with the alkyl groups pointing toward the top of the gorge, except for TA4. For this later, the N-alkyl group projects towards the choline binding pocket and the ethoxy moiety, located in the acyl-binding pocket, is weakly interacting with Leu286 and Phe398. On the choline binding pocket side, ethoxy oxygen O2 of TA1, TA5, TA6 and N of TA4 are at hydrogen bonding distance of His438N 2.

For TA1, N-ethyl group makes van der Waals contact with Trp231 and Phe329. N-diethyl substituent of TA1 imposes much more stress on the residue of the acyl-binding pocket such that we observed a concerted shift about 0.5 Å of Phe398, Phe329 and Leu286.

X-ray structure of aged tabun analogue-hBChE conjugates

The structures of aged forms were obtained by crystallization of inhibited enzyme in solution.

X-ray crystallographic data were collected from tetragonal crystals of space group I422, except for TA5 (P4212) and refined to 2.25 Å, 2.0 Å and 3.1 Å for TA1, TA4 and TA5, respectively. Corresponding data and refinement statistics are shown in table 2B. The orientation of aged conjugates is identical to the non-aged conjugates and confirms that the stereoselectivity of the inhibition is identical in cristallo and in solution (Fig. 5). This also confirms the initial stereochemistry of TA4. As expected in aged forms, the alkyl group projecting toward the choline-binding pocket is missing. This shows that TA1 and TA5 undergo classical O-dealkylation as previously observed for tabun [START_REF] Carletti | Aging of cholinesterases phosphylated by tabun proceeds through O-dealkylation[END_REF], phosphates and phosphonates [START_REF] Li | Aging pathways for organophosphate-inhibited human butyrylcholinesterase, including novel pathways for isomalathion, resolved by mass spectrometry[END_REF]. On the other hand, TA4 likely undergoes deamination. As a result of aging, O3 forms a salt bridge with protonated His438N 2 (2.7-2.8 Å). For each conjugates, the

Discussion

The higher inhibitory potency of phosphoramidocyanidates compared to homologous phosphoramidofluoridates (table 1) could be related to the electron-withdrawing effect of cyanide that is stronger than that of fluorine. Cyanide is also a mesomer acceptor in contrast to fluorine. It follows that the phosphorus atom of phosphoramidocyanidates is more electrophilic and more reactive. It is also possible that during the approach of the inhibitor, the cyanide substituent makes more favorable interactions with the enzyme than fluorine.

Non-aged and aged structures were obtained for tabun analogues TA1, TA4, TA5 and nonaged structures for TA6 (Fig. 5). We failed to obtain conjugates with TA3 and TA2, most likely because of their poor reactivity with hBChE in relation to their large size. The X-Ray structures and second order kinetic data show that there is enantioselectivity for reaction with fluorophosphoramidate analogues (Fig. 3, Fig. 5).

We observed that the N,N-dialkyl substituent of TA1 is located in the acyl-binding pocket, similarly to tabun [START_REF] Carletti | Aging of cholinesterases phosphylated by tabun proceeds through O-dealkylation[END_REF]. The bulky N,N-diethyl substituent of TA1 fits in the acyl-binding pocket, only inducing slight rotation of Leu286, and the phosphoramidate head is pushed toward the choline-binding pocket compared to TA5 and TA6 (Fig. 6). The steric hindrance resulting from N,N-diethyl group, forces the O-ethoxy substituent to orient toward the bottom of the choline-binding pocket. This notably prevents the binding of the unknown ligand, just leaving enough room for two water molecules (Fig. 5, Fig. 6). As for tabun, the orientation of TA1 is explained by delocalization of the N-lone pair and subsequent cation-interactions between the diethylamino and Trp231.

For N-monoalkyl analogues (TA4-6), the orientation of the OP follows different rules. The larger substituent, wherever the ethoxy or amino group, is located in the acyl-binding pocket, forming more van der Waals interactions than the smaller substituent and suggesting that cation-interactions of N-monoalkyl group and Trp231 is secondary in importance. Indeed, we expect weaker cation-interactions because delocalization of the nitrogen lone pair is less stabilized for primary amines compared to secondary amines, thus leading to a lesser positive partial charge on the amino group. For the N-monoalkyl analogues, the length of the substituent appears to prime on the interaction of N-methyl with the acyl-binding pocket.

Actually, this could result from preference of hBChE's acyl-binding pocket for larger than 2atoms linear substituents. Indeed, when the substituent is shorter, for example a single atom as for soman-aged hBChE conjugate (pdb code : 1p0q), it induces a rearrangement of the acylloop (Val279-Asn289). Thus, for TA4 the O-ethyl (3-atom chain) substituent is located in the acyl-binding pocket and the shorter N-methyl (2-atom chain) toward the choline-binding pocket (Fig. 5).

For TA5, we cannot conclude regarding the orientation, as both substituents are undistinguishable by X-ray crystallography. However, second order kinetics shows that hBChE reacts specifically with one enantiomer of TA5. Since van der Waals interactions should be equivalent for both substituents, we suppose that the ethylamino group interacts preferably with Trp231 through cation-interactions, whereas the ethoxy oxygen interacts with His438N 2.

Crystallographic data show that aging occurs through classical O-dealkylation for TA1, TA6 (Fig. 5), similarly to previous observation made on tabun-ChEs conjugates [START_REF] Carletti | Aging of cholinesterases phosphylated by tabun proceeds through O-dealkylation[END_REF].

Unexpectedly, there was also aging for TA4 although the ethoxy group is located in the acylbinding pocket (Fig. 5). These results show that aging of phosphoramidates systematically involves departure of the substituent in the choline-binding pocket. The mechanism differs, depending on the nature of the substituent O-alkyl or N-alkyl group (Fig. 7). When this substituent is the ethoxy, aging proceeds through dealkylation: His438 imidazolium stabilizes a developing negative charge on the C-O oxygen, and a water molecule activated by Glu197 attacks the carbocationic center that appears on the C + -O carbon. This leads to scission of the C-O bond, release of the ethoxy moiety, and subsequent formation of the salt bridge between the phosphorous O3-oxygen and the imidazolium. This is illustrated with TA1, TA6 and previous solved structures of aged hBChE inhibited by tabun, soman, DFP, echothiophate [START_REF] Carletti | Aging of cholinesterases phosphylated by tabun proceeds through O-dealkylation[END_REF][START_REF] Nachon | Role of water in aging of human butyrylcholinesterase inhibited by echothiophate: the crystal structure suggests two alternative mechanisms of aging[END_REF][START_REF] Nicolet | Crystal structure of human butyrylcholinesterase and of its complexes with substrate and products[END_REF].

For TA4, the N-methyl substituent projects towards the choline-binding site so that aging proceeds through deamination: His438 imidazolium protonates the methylamine group.

Extremely rapid deamination after nucleophilic attack by water can be assumed since a protonated methylamine is an excellent leaving group. This mechanism, like dealkylation, leads to the formation of a salt bridge with a short interatomic distance, as shown by the N,N-dialkyl compounds are generally less potent inhibitors of hBChE than N-monoalkyl analogues (table 1). N,N-dialkyl compounds are less reactive because steric hindrance from the two alkyl side-chains prevents a correct approach of the OP to the catalytic serine.

Moreover, the two alkyls chains have electron-releasing effect that favors delocalization of the nitrogen lone pair on the phosphorous atom, thus decreasing the electrophilicity of this later. Additionally, Trp231 could stabilize positive partial charges on the amino group when the dialkylamino substituent is in the acyl-binding pocket, thus further favoring delocalization of the N-lone pair.

The remarkable correlation between the inhibitory potency and the N-monoalkyl chain length is most likely related to increasingly optimized interactions with the acyl-binding pocket as shown by the X-ray structures (Fig. 5).

The electronic and steric effects have similar influence for the reactivation reaction by oximes of N,N-dialkyl compared to N-monoalkyl compounds (Fig. 4). In addition to the poorer reactivity of the phosphorus atom, the two alkyl side-chains prevents a correct approach of the oxime in apical position, as illustrated in figure 6.

Obidoxime, TMB-4, HLö 7, HI-6 have a similar efficiency on each phosphoramidyl-hBChE conjugates. The increase in chain length of N-monoalkyl compounds does not affect the affinity of oximes to hBChE conjugates by contrast to hAChE [START_REF] Worek | Kinetic analysis of reactivation and aging of human acetylcholinesterase inhibited by different phosphoramidates[END_REF], because the long chain can easily fit in the acyl-binding pocket.

These bispyridinium oximes are much less efficient than for phosphoramidyl-hAChE [START_REF] Worek | Kinetic analysis of reactivation and aging of human acetylcholinesterase inhibited by different phosphoramidates[END_REF],

despite Tyr124, that hinders the phosphorus atom of hAChE conjugates for an optimal apical approach, is not conserved in hBChE (Gln119). This is due to a lower affinity of oximes for phosphoramidyl-hBChE conjugates. Indeed, these bispyridinium oximes bind to the peripheral site of hAChE, through aromatic stacking and cation-interactions between one pyridinium moiety and peripheral site residues (Trp286, Tyr124 and Tyr72) [START_REF] Ekstrom | Novel nerve-agent antidote design based on crystallographic and mass spectrometric analyses of tabun-conjugated acetylcholinesterase in complex with antidotes[END_REF]. In hBChE, these aromatic residues are not conserved and bispyridinium oximes cannot interact in the same fashion. Considering that the OPs occupy the bottom of the active site gorge, and that the nucleophile function must be in apical position for optimal in-line attack, efficient reactivators must bind to the gorge entrance of hBChE. Efforts should be pursue to design such reactivators. Tables Table 1 : Rate constants for inhibition (k i ), obidoxime-induced reactivation (K D , k r , k r2 ), aging (k a ) and spontaneous reactivation (k s ) of hBChE phosphorylated with tabun and its analogues. hBChE inhibited by tabun and its analogues (Fig. 1) was incubated with 1mM oxime and the hBChE activity was determined after 120 min incubation. Data are given as % reactivation (means ± SD of 2 experiments). Electron density 2 Fo -Fc is represented by a blue mesh, contoured at 1.0 and Fo -Fc is represented by green/red mesh contoured at 3.5 . Licenced copy. Copying is not permitted, except with prior permission and as allowed by law.

Biochemical

  Journal Immediate Publication. Published on 15 Apr 2009 as manuscript BJ20090091 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20090091 A c c e p t e d M a n u s c r i p t Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. © 2009 The Authors Journal compilation © 2009 Portland Press Limited

Biochemical

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tabun 20 -

 20 80 nM; TA1 100-250 nM; TA1' 1200-1800 nM; TA2 200-500 nM; TA3 20-50 M; TA4 50-125 nM; TA5 70-100 nM; TA6 50-90 nM.

  Data Collection and Structure Solution of hBChE Diffraction data were collected at = 0.932 Å wavelength at the ID14-eh2 beamline of the European Synchrotron Radiation Facility (ESRF, Grenoble) with a MAR-Research CCD Biochemical Journal Immediate Publication. Published on 15 Apr 2009 as manuscript BJ20090091 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20090091 A c c e p t e d M a n u s c r i p t Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. © 2009 The Authors Journal compilation © 2009 Portland Press Limited

Biochemical

  Journal Immediate Publication. Published on 15 Apr 2009 as manuscript BJ20090091 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20090091 A c c e p t e d M a n u s c r i p t Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. © 2009 The Authors Journal compilation © 2009 Portland Press Limited negative difference density ( Fo -Fc map) peaks support the conclusion that the adduct intermediate observed in the structures is in the S conformation. There was no significant movement of residue side-chains compared to the native enzyme (pdb code: 1P0I).

Biochemical

  Journal Immediate Publication. Published on 15 Apr 2009 as manuscript BJ20090091 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20090091 A c c e p t e d M a n u s c r i p t Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. © 2009 The Authors Journal compilation © 2009 Portland Press Limited phosphoramidyl moiety has tilted toward His438 as a consequence of the salt bridge formation. For TA1, this also translates into a release of stress in the acyl-binding pocket so that only Phe329 remains shifted. For TA4 and TA5, van der Waals contacts with Trp231 and Phe329 are similar as in their nonaged form. The small ligand is still present stacked to Trp82 and interacts with O3 of the aged adducts of TA1, TA4 and is replace by two water molecules in structure of aged TA5-hBChE conjugate.

Biochemical

  Journal Immediate Publication. Published on 15 Apr 2009 as manuscript BJ20090091 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20090091 A c c e p t e d M a n u s c r i p t Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. © 2009 The Authors Journal compilation © 2009 Portland Press Limited

Biochemical

  Journal Immediate Publication. Published on 15 Apr 2009 as manuscript BJ20090091 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20090091 A c c e p t e d M a n u s c r i p t Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. © 2009 The Authors Journal compilation © 2009 Portland Press Limited crystal structure (His438N 2 and O3 = 2.7Å). Remarkably, aging rates of all mono-alkyl analogues are comparable despite the difference of mechanism.

Biochemical

  Journal Immediate Publication. Published on 15 Apr 2009 as manuscript BJ20090091 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20090091 A c c e p t e d M a n u s c r i p t In summary, this work provides a better understanding of inhibition of hBChE by phosphoramidates and aging of the conjugates, and provides a structural basis for rational design by molecular modeling of hBChE-based catalytic bioscavengers and oximes able to reactivate phosphoramidyl-hBChE. Great improvement in prophylaxis, decontamination and treatment of organophosphate poisoning are expected in the near future as hBChE is close to FDA approval.

Fig. 1

 1 Fig. 1 Chemical structures of tabun analogues (TA).

Fig. 2

 2 Fig. 2 Chemical structures of oximes used in this study.

Fig. 3

 3 Fig. 3 Irreversible inhibition hBChE by racemic tabun analogues performed under secondorder conditions. 29, 32.5 and 30 nM of hBChE was inhibited by various concentrations of TA4, TA5 and TA6, respectively, and the residual enzyme activity was monitored as a function of time.

Fig. 4

 4 Fig. 4 Reactivation of OP-inhibited hBChE by obidoxime (black column), TMB-4 (dark grey column), HLö 7 (horizontally striped column) or HI-6 (light grey column).

Fig 5

 5 Fig 5 Active site of non-aged and aged TA-hBChE conjugates. Key residues are represented as sticks with carbon in green for OP and blue for hBChE residues, nitrogen atoms in blue, oxygen atoms in red and phosphorus atom in orange. Water molecules are represented by red spheres and hydrogen bonds by red dashes.

Fig 6

 6 Fig 6 Superimposition of the active site of non-aged form of TA1-hBChE (red), TA5-hBChE (green) and TA6-hBChE (blue) conjugates.

Fig 7

 7 Fig 7 Aging mechanisms of phosphoramidyl-hBChE conjugates.

  Fig. 1

Biochemical

  Fig. 2

Fig. 3 Biochemical

 3 Fig. 3

Fig. 4 Biochemical

 4 Fig. 4

Fig. 5

 5 Fig. 5

1 Å

 1 Biochemical Journal Immediate Publication. Published on 15 Apr 2009 as manuscript BJ20090091 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20090091 A c c e p t e d M a n u s c r i p t Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. © 2009 The Authors Journal compilation © 2009 Portland Press Limited

  Fig. 6

  Fig. 7

  concentration of hBChE about 30 nM while that of tabun analogues ranged between 15 and 200 nM. Residual activity was monitored at 25°C and pH 7.0, according to Ellman's method using 1 mM BTCh and 0.5 mM DTNB. Under second order conditions, the concentration of

		Biochemical Journal Immediate Publication. Published on 15 Apr 2009 as manuscript BJ20090091
		residual active enzyme follows equation 2, as a function of time [11] :
	THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20090091	eq. 2 Where E is the concentration of residual active enzyme at time=t, E 0 the initial concentration of active enzyme, [OP 0 ] the initial concentration of racemic OP, n the number of enantiomers, and ki' the bimolecular rate constant of the most active enantiomer assuming the inhibition rate of the other enantiomer is negligible. Analysis of the kinetic data was performed using GOSA-fit, a fitting software based on a simulated annealing algorithm (BioLog, Toulouse, France; http://www.bio-log.biz), allowing simultaneous fitting of all the A c c e p t e d M a n u s c r i p t data at once.

Inhibition of hBChE by tabun analogues

	The bimolecular rate constants, k i , for inhibition of human BChE by tabun and its analogues
	(TA1 to TA6) in the absence of substrate are in table 1. Phosphoramidocyanidates react faster
	than homologous phosphoramidofluoridate: TA1 with cyanide as leaving group reacts 17
	times faster than TA1' with fluoride as leaving group. Hereby, the inhibitory potency was
	related to the chain length of the N-alkyl group. With tabun and its closest analogues, TA1
	and TA2, bearing a cyanide leaving group and dialkyl chains, k i values decreased with
	increasing chain length. In contrast, for TA 4, 5 and 6, bearing a mono-N-alkyl group, the
	inhibitory potency increases with chain length, each carbon addition doubling the bimolecular
	rate constant.

Biochemical Journal Immediate Publication. Published on 15 Apr 2009 as manuscript BJ20090091 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20090091 A c c e p t e d M a n u s c r i p t Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. © 2009 The Authors Journal compilation © 2009 Portland Press Limited

Results

  No spontaneous reactivation of inhibited hBChE activity during the observation period d Single experiment Biochemical Journal Immediate Publication. Published on 15 Apr 2009 as manuscript BJ20090091

	THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20090091	TA3 TA4 TA5 TA6 A c c e p t e d M a n u s c r i p t N 0.012 ± 0.001 920 d 0.09 d 0.098 d 0.09 ± 0.0001 HN 4 ± 0.5 370 ± 40 0.042 ± 0.001 0.117 ± 0.010 0.011 ± 0.0003 0.004 ± c 0.0001 HN 8 ± 0.6 630 ± 60 0.072 ± 0.003 0.114 ± 0.002 0.012 ± 0.0002 0.008 ± 0.0001 HN 17 ± 0.3 660 ± 50 0.069 ± 0.002 0.108 ± 0.002 0.015 ± 0.0005 0.019 ± 0.0004

a Failure of oximes to reactivate inhibited hBChE b Not feasible c Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. © 2009 The Authors Journal compilation © 2009 Portland Press Limited

Table 2A :

 2A Data Collection and Refinement Statistics

		TA1-hBChE	TA4-hBChE	TA5-hBChE	TA6-hBChE
			(non-aged)
	data
	space group	I422	I422	I422	I422
	unit cell axes, a=b, c (Å)	155.04, 126.21	155.01, 126.84	155.02, 127.22	155.24, 127.25
	no. of reflections	240 729	302 893	328 846	314 110
	unique reflections	34 103	41 918	45 161	45 258
	resolution (Å)	47.8-2.3 (2.4-2.3)	49.0-2.15 (2.2-2.15)	55.1-2.1 (2.2-2.1)	55.1-2.1 (2.2-2.1)
	completeness (%) Rmerge * (%)	99.3 8.5 (34.9)	99.7 8.1 (29.6)	99.9 8.6 (28.4)	99.6 9.7 (35.5)
	I/ó(I)	23.1 (5.3)	25.8 (4.3)	24.7 (4.4)	16.6 (3.4)
	Redundancy	7.1 (7.2)	7.2 (7.2)	7.3 (7.4)	6.9 (5.9)
	Refinement Statistics
	R-factor † (R-free ‡ )	18.9 (25.5)	18.9 (25.3)	20.0 (25.3)	21.3 (27.6)
	no. of atoms
	protein	4 240	4 227	4 213	4 208
	solvent	335	343	342	384
	others	167	163	162	164
	mean B-factor (Å 2 )	37.6	35.3	35.9	37.4
	RMS from ideality
	bond length (Å) angles (deg) chiral (Å 3 )	0.017 1.757 0.128	0.018 1.875 0.114	0.017 1.782 0.121	0.033 2.897 0.229

* Rmerge = ( | I -<I> |) / I, where I is the observed intensity and <I> is the average intensity obtained from multiple observations of symmetryrelated reflections after rejections. † R-factor= |Fo -|Fc|| / |Fo|, Fo and Fc are observed and and calculated structure factors ‡ R-free set uses 5% of randomly chosen reflections defined in Brunger et al. (38).

Table 2B :

 2B Data Collection and Refinement Statistics

		TA1-hBChE	TA4-hBChE	TA5-hBChE
			(aged)
	data
	space group	I422	I422	P4212
	unit cell axes, a=b, c (Å)	155.04, 126.21	154.9, 126.01	150.35, 139.43
	no. of reflections	248 419	391 729	167 928
	unique reflections	35 245	51 417	28 167
	resolution (Å)	47.7-2.25 (2.3-2.25)	48.9-2.0 (2.1-2.0)	48.4-3.1 (3.2-3.1)
	completeness (%) Rmerge * (%)	96.3 10.1 (48.1)	98.3 8.8 (37.5)	98.2 10.1 (30.5)
	I/ó(I)	15.3 (4.0)	24.8 (5.4)	29.2 (8.6)
	Redundancy	7.1 (7.3)	7.6 (7.4)	5.7 (5.9)
	Refinement Statistics
	R-factor † (R-free ‡ )	20.7 (27.5)	23.4 (22.0)	22.0 (25.3)
	no. of atoms
	protein	4 216	4 263	8 396
	solvent	369	396	136
	others	163	156	268
	mean B-factor (Å 2 )	38.01	27.9	73.7
	RMS from ideality
	bond length (Å) angles (deg) chiral (Å 3 )	0.025 2.238 0.218	0.011 1.351 0.101	0.012 1.415 0.087

Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. © 2009 The Authors Journal compilation © 2009 Portland Press Limited

© 2009 The Authors Journal compilation © 2009 Portland Press Limited

Acknowledgments

This work was supported by DGA grant 03co10-05/PEA 01 08 7 to PM, DGA/PEA 08co501 and ANR-06-BLAN-0163 to FN. The authors are grateful to T. Hannig and L. Windisch for their skilful and engaged technical assistance. Biochemical Journal Immediate Publication. Published on 15 Apr 2009 as manuscript BJ20090091 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20090091 A c c e p t e d M a n u s c r i p t Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. © 2009 The Authors Journal compilation © 2009 Portland Press Limited


Sabrina Gazzarrini

Ming Kang

Alessandra Abenavoli

Giulia Romani

Claudio Olivari

Daniele Gaslini

Giuseppina Ferrara

James L Van Etten

Michael Kreim

Stefan M Kast

Gerhard Thiel

Anna Moroni
email: anna.moroni@unimi.it

Chlorella virus ATCV1 encodes a functional potassium channel of eighty-two amino acids

Keywords: viral K + channel, Kcv, yeast complementation, Chlorella virus ATCV-1, K + /Rb + selectivity

de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Introduction

Phycodnaviruses [START_REF] Iyer | Common origin of four diverse families of large eukaryotic DNA viruses[END_REF][START_REF] Iyer | Evolutionary genomics of nucleocytoplasmic large DNA viruses[END_REF][START_REF] Raoult | The 1.2-megabase genome sequence of Mimivirus[END_REF][START_REF] Villarreal | Viruses and the Evolution of Life[END_REF] consist of a genetically diverse, but morphologically similar, group of large dsDNA-containing viruses (170 to 560 kb) that infect algae from both fresh-and marine-waters [START_REF] Dunigan | Phycodnaviruses: a peek at genetic diversity[END_REF][START_REF] Wilson | Lesser Known Large dsDNA Viruses[END_REF]. The most studied phycodnaviruses are the plaque-forming chlorella viruses that infect certain freshwater, unicellular, eukaryotic, chlorella-like green algae [7,[START_REF] Yamada | Chlorella viruses. Advances in Virus Research[END_REF]. Chlorella viruses encode up to 400 proteins of which 35 to 40% resemble entries in the public databases. Many chlorella virus encoded proteins are either the smallest or among the smallest representatives of their functional class. Consequently, some of the chlorella virus encoded proteins are the subject of intense physical and biochemical investigations. One example is the chlorella virus PBCV-1 encoded 94 amino acid K + channel protein, named Kcv, which forms functional channels in Xenopus oocytes [START_REF] Plugge | A potassium channel protein encoded by chlorella virus PBCV-1[END_REF], chinese hamster ovary cells [START_REF] Gazzarrini | The viral potassium channel Kcv: structural and functional features[END_REF], and human embryonic kidney cells [START_REF] Moroni | The short N-terminus is required for functional expression of the virus-encoded miniature K + channel Kcv[END_REF]. The PBCV-1 Kcv structure is truly minimal; the channel monomer consists of two transmembrane domains, a pore loop and a 12 amino acid cytoplasmic N terminus. Like other K + channels, PBCV-1 Kcv forms a functional tetramer [START_REF] Pagliuca | Molecular properties of Kcv, a virus encoded K + channel[END_REF] and electrophysiology experiments established that PBCV-1 Kcv has many properties similar to more complex K + channels, including gating, selectivity, sensitivity to voltage and susceptibility to channel blockers [START_REF] Plugge | A potassium channel protein encoded by chlorella virus PBCV-1[END_REF]. These results imply that these functional properties are inherent in the structure of the protein. Kcv-like genes were cloned and expressed from 40 additional viruses that infect the same host, Chlorella NC64A; 16 amino acid substitutions occurred among the 94 amino acids, producing six new Kcv-like proteins that formed functional K + selective channels in Xenopus oocytes. However, the biophysical properties of some of these Kcv channels differed from PBCV-1 Kcv, including altered current kinetics with K + and Rb + and altered sensitivity to channel blockers [START_REF] Kang | Small potassium ion channel proteins encoded by chlorella viruses[END_REF][START_REF] Gazzarrini | Long distance interactions within the potassium channel pore are revealed by molecular diversity of viral proteins[END_REF]. Even more diversity is found between K + channel proteins from chlorella viruses with different hosts. Virus MT325, which infects Chlorella Pbi, encodes a Kcv channel that is similar in size (95 amino acids) to PBCV-1 Kcv. However, the predicted architecture of MT325-Kcv differs significantly from PBCV-1 Kcv. Whereas PBCV-1 Kcv has a short amino acid cytoplasmic N-terminus and no cytoplasmic C-terminus, MT325-Kcv is organized differently; it lacks a cytoplasmic N-terminus but has 10 amino acids at the cytoplasmic C-terminus [START_REF] Gazzarrini | Chlorella virus MT325 encodes water and potassium channels that interact synergistically[END_REF]. Therefore, the minimal structure and genetic variability of the K + channels encoded by the chlorella viruses provide excellent model systems to study K + channel structure-function relationships.

the other viral K + channels [START_REF] Plugge | A potassium channel protein encoded by chlorella virus PBCV-1[END_REF][START_REF] Kang | Small potassium ion channel proteins encoded by chlorella viruses[END_REF][START_REF] Gazzarrini | Chlorella virus MT325 encodes water and potassium channels that interact synergistically[END_REF]. In the current manuscript we describe the characteristics of this putative K + channel. The results indicate that an 82 amino acid protein forms a functional K + channel.

EXPERIMENTAL Expression in Saccharomyces cerevisiae-Cloning

ATCV-1 kcv gene was cloned into a yeast expression vector pYES2/CT at BamHI and XhoI sites using primer sequences: 5' primer CGGGGATCCATGTTGCTGCTTATCATA, 3' primer ATTCTCGAGCTACCACGGAAACGTGAA. pYES2/CT (Invitrogen) carries a URA3 gene as a selectable marker for positive transformants in ura negative hosts. The exogenous gene is controlled by the GAL1 promoter and is expressed in the presence of galactose.

Both the empty vector and the vector which carries ATCV-1 kcv gene were transformed into a potassium uptake deficient yeast strain SGY1528 with the lithium acetate procedure. SGY1528 has the following genotype: MATα ade2-1 can1-100 his3-11,15 leu2-3,112 trp1-1 ura3-1 trk1::HIS3 trk2::TRP1 (a gift from Dr. Daniel Minor, University of California at San Francisco). Transformants were selected on low potassium medium using the methods described by Minor et al. [START_REF] Minor | Transmembrane structure of an inwardly rectifying potassium channel[END_REF] and Chatelain et al. [START_REF] Chatelain | The pore helix dipole has a minor role in inward rectifier channel function[END_REF]. For the drug inhibition experiments, CsCl was added to the medium to produce the expected concentration before the medium was solidified with agar; BaCl 2 was added to the 1 cm sterile paper discs at the indicated concentrations, the discs were placed on top of yeast cells spread on the agar plates. Plates were kept at 30 0 C for 72 h or until the yeast had reached adequate growth.

Pichia pastoris transformation

The ORF encoding the ATCV-1 kcv gene was amplified from ATCV-1 kcv -pSGEM by the Polymerase Chain Reaction (PCR) (forward primer: 5' CAAGGACCGAGCAGCCCCTCCTTGCTGCTTATCA TACATATC 3'; reverse primer 5' ACCACGGGGAACCAACCCTTATTACCACGGAAACG TGAAGCC 3'). The PCR fragment was subcloned into a modified P. pastoris expression vector pPICZ A (Invitrogen) containing a Kozak consensus sequence, a His 7 tag, a proteolytic site for the H3C protese and a Ligation Independent Cloning site (LIC) on the N-terminus of the protein sequence. The construct was confirmed by DNA sequencing (Cogentech, IFOM-IEO, Milano). P. pastoris cells (SMD 1163 strain) were transformed with 3 µg of the PmeI linearized construct by using the Pichia Easy compt Invitrogen™ kit as described by the manufacturer (Invitrogen). Positive colonies were selected from YPDS (10 g/l Bacto Yeast, 20 g/l Bacto Peptone, 20 g/l Dextrose) agar plates containing 50 µg/ml zeocin.

Protein induction

Single colonies were grown in MGYH medium [1.34% (w/v) yeast nitrogen base, 1% (v/v) glycerol, 4 x 10 -5 % (w/v) biotin and 0.004% histidine] to an absorbance of 4 O.D. at 600 nm (A 600 ). After centrifugation at 3000 x g for 10 min at 4°C, the pellet was re-suspended to an A 600 of 1 O.D. in MMH medium [1.34% (w/v) yeast nitrogen base, 4 x 10 -5 % (w/v) biotin, 0.004 % histidine and 0.5% (v/v) methanol] and grown for 24 h. P. pastoris membrane preparation-One gram of cells were suspended in 22.5 ml of breaking buffer [50 mM NaH 2 PO 4 (pH 7.4), 1 mM EDTA, 5% glycerol and 5 mM DTT] containing 1 mM phenylmethanesulfonyl fluoride (PMSF), 0.1 mg/ml trypsin inhibitor type II-O, 1 mM benzamidine and 0.1 mM Pefablock. An equal amount (1 g) of ice-cold, acid washed glass beads (0.25-0.5 mm diameter) was added and mixed vigorously on a vortex for 20 min by alternating 30 sec of vortexing and 30 sec on ice. The cell homogenate was centrifuged at 3000 x g for 10 min at 4°C to remove glass beads, unbroken cells and cell debris. The supernatant was removed and centrifuged at 30,000 x g for 45 min at 4 °C. The resulting crude membrane pellet (microsomes) was suspended in 0.3 ml of ice-cold breaking buffer.

Immunochemistry

For immunoblotting, SDS-PAGE-separated proteins were electrotransferred to a nitrocellulose (0.2 µm-Hybond) membrane, probed with anti-poly-His monoclonal antibody and visualized with alkaline phosphatase-coupled secondary antibody.

Expression of ATCV-1 kcv and PBCV-1 kcv genes in oocytes-Kcv cDNAs were cloned into a pSGEM vector (a modified version of pGEM-HE, courtesy of M. Hollmann, Max Planck Institute for Experimental Medicine, Göttingen, Germany). In vitro transcription was performed on linearized plasmids using T7 RNA polymerase (Promega) and cRNAs were injected (50 ng/oocyte) into Xenopus laevis oocytes, prepared according to standard methods [START_REF] Plugge | A potassium channel protein encoded by chlorella virus PBCV-1[END_REF]. Electrophysiological measurements were made 2-4 days after injection.

Site-directed in vitro mutagenesis

Point mutations were created using the Quikchange Site-directed Mutagenesis Kit (Stratagene, La Jolla, CA, USA), and confirmed by DNA sequencing (Cogentech, IFOM-IEO, Milano).

Electrophysiological measurements

Two-electrode voltage clamp experiments were performed using a GeneClamp 500 amplifier (Axon Instruments) and a digitized at 50 kHz with a Digidata 1200 (Axon Instruments). Data acquisition and analysis were done using the pCLAMP8 software package (Axon Instrument). Electrodes were filled with 3M KCl and had resistance of 0.2-0.8 MΩ in 50 mM KCl. The oocytes were perfused at room temperature (25-27°C) with a bath solution containing: 50 mM KCl (or RbCl, CsCl, NaCl, or LiCl as indicated in the figure legend and text), 1.8 mM CaCl 2 , 1 mM MgCl 2 , 5 mM HEPES, adjusted to pH 7.4 with KOH (or RbOH, CsOH, NaOH, or LiOH) at a rate of 2 ml/min. Mannitol was used to adjust the osmolarity of the solution to 215 mosmol. BaCl 2 and CsCl diluted from 1 M stocks were added to the various solutions as indicated. The standard clamp protocol consisted of steps from the holding voltage of -20 mV to voltages in the range +80 mV to -160 mV; tail currents were measured at -80 mV. Instantaneous and steady state currents were sampled after 10 ms and at the end of the voltage step (usually 800 ms), respectively.

Single channel measurements by patch clamp

Patch pipettes were pulled from thin-walled borosilicate glass capillaries, coated with Sylgard (Dow Corning Corporation) and fire-polished to a final resistance of 8 -15 MΩ for single-channel experiments. Single-channel recordings were made in a cell-attached configuration. Pipette and bath solutions contained 100 mM KCl, 1.8 mM CaCl 2 , 1 mM MgCl 2 and 10 mM HEPES (pH adjusted to 7.4 with KOH). When indicated, 100 mM RbCl was used instead of KCl. Experiments were performed at room temperature after removal of the vitelline membrane in a hyper-osmotic solution (200 mM NaCl). Currents were recorded with a Dagan 3900 amplifier; data were low-pass filtered at 2 kHz and digitized at a sampling rate of 10 kHz. Single-channel analysis was done using pCLAMP 9 (Axon Instruments) with the threshold based algorithm.

Ion Permeability-Permeability ratios (P X /P K ) were calculated according to the equation

∆E rev = E revX - E rev,K = RT/zF ln P X [X] o / P K [K] o . E rev is

RESULTS and DISCUSSION Virus ATCV-1 codes for a channel protein with 82 amino acids.

The chlorella virus ATCV-1 genome was recently sequenced and annotated [START_REF] Fitzgerald | Sequence and annotation of the 288-kb ATCV-1 virus that infects an endosymbiotic chlorella strain of the heliozoon Acanthocystis turfacea[END_REF]. Among the 860 ORFs, one (ORF Z585R) had all the hallmarks of a K + channel. The derived amino acid sequence of Z585R, termed ATCV-1 Kcv, is compared with the sequence of the prototype viral K + channel, PBCV-1 Kcv in Fig. 1A. The ATCV-1 Kcv sequence is only 82 amino acids, 12 residues smaller than PBCV-1 Kcv. In spite of ATCV-1 Kcv's small size, the protein has the essential features of a K + channel. Structural prediction programs TMHMM (http://www.cbs.dtu.dk/services/TMHMM) and Phobius (http://phobius.cbr.su.se) suggest two transmembrane domains, TM1 and TM2, and a pore helix (Phobius) preceding the filter region; it has a canonical selectivity filter sequence (TTVGYGD) and the aromatic couple, YF, in the pore helix. The two proteins have considerable sequence identity and similarity (Fig. 1A) but presumably differ in structure as shown by the superimposed structural models in Fig. 1B. ATCV-1 Kcv lacks the "slide helix" at the N terminus (amino acids 1-14 in PBCV-1 Kcv) and most of the external loop connecting TM1 to the pore helix, the "turret" (amino acids 36-48 in PBCV-1 Kcv ). The C-terminal transmembrane segments, TM2, of ATCV-1 Kcv are longer and the intracellular mouth of this channel is probably narrower in comparison with PBCV-1 Kcv. The finding that ATCV1 Kcv lacks the slide helix is of particular interest since this domain is essential for PBCV1 Kcv activity [START_REF] Moroni | The short N-terminus is required for functional expression of the virus-encoded miniature K + channel Kcv[END_REF].

ATCV-1 Kcv forms a functional K + channel.

The first indication that ATCV-1 Kcv forms a functional K + channel was obtained by complementation of a Saccharomyces cerevisae ∆trk1, ∆trk2 mutant that lacks endogenous K + uptake systems [START_REF] Minor | Transmembrane structure of an inwardly rectifying potassium channel[END_REF][START_REF] Chatelain | The pore helix dipole has a minor role in inward rectifier channel function[END_REF]. Expression of ATCV-1 Kcv protein in the mutant rescued yeast growth in low K + (0.5 mM), while yeast transformed with the empty vector only grew in high K + (100 mM) (Fig. 1C). These results indicate that ATCV-1 Kcv forms a functional K + channel presumably at the yeast plasma membrane. Growth of the ATCV-1 Kcv expressing yeast was inhibited by Ba 2+ and Cs + , two known K + channel blockers (Fig. 4A and Fig. Supplementary 1A).

ATCV-1 Kcv forms a stable tetramer.

Since functional K + channels are tetrameric [START_REF] Doyle | The structure of the potassium channel: molecular basis of K + conduction and selectivity[END_REF], the oligomeric state of ATCV-1 Kcv protein was examined. The channel was expressed in the yeast Pichia pastoris with an N-terminal His tag [START_REF] Pagliuca | Molecular properties of Kcv, a virus encoded K + channel[END_REF]. The microsomal proteins were analyzed by SDS-PAGE; one band, with an apparent molecular mass of 32.5 kDa, was detected by anti-His antibody (Fig. 1D, lane 1). The observed mass is roughly 4 times that of the monomer (6.5 kDa) obtained after heating the sample at 100 °C for 3 minutes (Fig. 1D, lane 2); this result indicates that ATCV-1 Kcv can form a stable tetramer.

ATCV-1 Kcv has characteristic electrical properties.

ATCV-1 Kcv was expressed in Xenopus oocytes and subjected to electrophysiological investigation by the two-electrode voltage clamp procedure. Macroscopic currents occurred (Fig. 2A) that clearly differ in amplitude and kinetics from the endogenous currents (water-injected oocyte). The ATCV-1 Kcv-induced currents have features typical of other chlorella virus Kcv channels [START_REF] Plugge | A potassium channel protein encoded by chlorella virus PBCV-1[END_REF][START_REF] Kang | Small potassium ion channel proteins encoded by chlorella viruses[END_REF][START_REF] Gazzarrini | Long distance interactions within the potassium channel pore are revealed by molecular diversity of viral proteins[END_REF][START_REF] Gazzarrini | Chlorella virus MT325 encodes water and potassium channels that interact synergistically[END_REF]. The standard voltage clamp protocol, (holding potential -20 mV, steps from 140 to -200 mV) evoked large instantaneous currents, which resulted in the current-voltage (I/V) relationship shown in Fig. 2B. The channel conductance is approximately linear between +/-80 mV but shows a pronounced negative slope at extreme negative voltages. Panel 2A shows, as an example, that the instantaneous current level recorded at -200 mV is smaller than that at -120 mV. Macro currents of Fig. 2A also show a slow kinetic component at negative potentials that is interpreted as an inactivation of ATCV-1 Kcv conductance.

ATCV-1 Kcv is K + selective. ATCV-1 Kcv channel currents were recorded in bath solutions with different ionic compositions to examine its selective properties. An increase of KCl concentration from 6 to 100 mM resulted in a progressive negative shift of the current reversal voltage. The insert in Fig. 2B A c c e p t e d M a n u s c r i p t logarithmic relationship between the measured current reversal potential and the external K + concentration. Linear regression analysis resulted in a straight line with a slope of 53.9 +1.4 mV (n≥3). This value is close to the theoretical value of 59.2 mV calculated with the Nernst equation [START_REF] Hille | Ionic Channels of Excitable Membranes[END_REF] and indicates that ATCV-1 Kcv is a K + channel.

Single channel properties.

To examine the single channel activity which underlies the ATCV-1 Kcv conductance, we measured the current in the plasma membrane of oocytes by the patch clamp method in cell-attach configuration. A typical example of single channel fluctuations monitored in oocytes expressing the viral protein is shown in Fig. 2C. The current-voltage relationship of the unitary conductance (Fig. 2D) has a pronounced outward rectifying shape and closely resembles that of the macro currents reported in 2B. The nonlinearity of the single channel I/V curve is a typical feature of this as well as of other Kcv channels and is due to a voltage-dependent reduction in the apparent channel conductance. At voltages more negative than -50 mV, the I/V curve progressively bends because the unitary currents become smaller even though the driving force is increasing. The reason for this voltage-dependent decrease in unitary conductance was not analyzed further; however, since the life-time of the channel openings becomes shorter with negative voltages it is likely due to a fast gating mechanism. Note that the full openings are not recorded at very negative voltages. In the linear range ± 50 mV, the estimated single channel conductance was 80 pS. This value is similar to that of PBCV-1 Kcv which, under the same ionic conditions (100 mM symmetric K + ), has a unitary conductance of about 100 pS [12 and manuscript submitted].

In summary, the membrane conductance generated by ATCV-1 Kcv is due to the activity of a canonical ion channel that displays a characteristic voltage-dependent decrease in the unitary channel conductance. ATCV-1 Kcv thus resembles PBCV-1 Kcv that also exhibits a voltage-dependent single channel I/V. This result indicates that the structural elements that underlay the gating mechanism are shared by both channel proteins.

Permeability to monovalent cations.

To study the permeability of ATCV-1 Kcv, we performed two electrode voltage clamp experiments with different monovalent cations in the external solution. Substituting 50 mM K + with Rb + , Cs + , Na + , or Li + resulted in the macroscopic currents shown in Fig. 3A from which the corresponding I/V relations were produced (Fig. 3B). Only Rb + permeates the channel among the tested cations, as shown by the presence of an inward current. Rubidium induced a negative shift in the current reversal potential, E rev , of 16 mV (more clearly visible in Fig. 5A : E rev K + = -20 + 1.08 mV; E rev Rb + = -36.2 + 0.86 mV, n = 7). A qualitatively similar result was obtained when the permeability to rubidium was tested at the single channel level. Fig. 3E shows examples of single channel currents of ATCV-1 Kcv recorded in a cellattach configuration with 100 mM KCl or RbCl in the pipette. Replacing external K + with Rb + resulted in a reduction in inward current amplitude and a negative shift of the reversal potential (Fig. 3F). These results indicate that Rb + has a lower permeability than K + and confirms the results obtained with macro currents. Taking the constant field model as a basis for explaining selectivity [START_REF] Hille | Ionic Channels of Excitable Membranes[END_REF], we have used macroscopic current E rev to estimate the permeability ratios reported in Fig. 3E (P X /P K where X is the tested cation) by using a simplified version of the Goldman equation (see Experimental Procedures). ATCV-1 Kcv is compared with PBCV-1 Kcv to highlight its peculiar behavior; ATCV-1 Kcv is indeed the only Kcv channel found so far with a P X /P K <1 [START_REF] Kang | Small potassium ion channel proteins encoded by chlorella viruses[END_REF]. The permeability sequence of ATCV-1 Kcv is K + > Rb + > Cs + >> Na + ~ Li + while that of PBCV-1 Kcv is Rb + >K+>Cs + >> Li + >Na + [13 and Table I). A closer view of the currents recorded with different cations indicates that the cation-composition of the medium affects other aspects of the current/voltage relations of the channel in addition to the reversal potential. Replacement of K + with Na + or Li + not only reduces the inward current of the ATCV-1 Kcv generated conductance but also abolishes its outward current. This result is consistent with a blocking effect of external Na + and Li + on the outward K + current. A reasonable explanation for this effect is that Na + or Li + enters the filter in the absence of K + and that this entry results in a destabilization of the filter 6 geometry [START_REF] Doyle | The structure of the potassium channel: molecular basis of K + conduction and selectivity[END_REF]. A similar effect by Na + , but not Li + , was previously reported for the prototype PBCV-1 Kcv channel [START_REF] Plugge | A potassium channel protein encoded by chlorella virus PBCV-1[END_REF]. The present data indicate that Li + behaves like Na + in the ATCV-1 Kcv channel meaning that structural differences between the two channels could allow the small Li + ion to enter the pore. In contrast, Cs + does not have the same effect on the outward K + currents, which resemble the control condition (with external K + ).

The aforementioned results imply that the cations interact with the channel protein. Hence permeation through Kcv channels is more complex than predicted by constant field theory. The non-linearity of the I i /V relations, i.e. in Rb + and K + , furthermore implies that the permeability ratios could be voltagedependent, as shown by Eisenmann and Horn [22]. For this reason the E rev value alone is not sufficient to provide an absolute measure of Kcv channel permeability. We also estimated the conductance of the channel for different ions away from equilibrium [22]. This relative conductance, G X /G K , provides additional information on the selectivity of the channels beyond the constant field theory.

The overall inward current in the presence of Rb + is smaller than in K + , as expected from the lower permeability of the channel in Rb + . This results in a low conductance ratio (Table I: G Rb /G K = 0.3, conductance estimated in the linear portion of the I/V relation) and again confirms a difference in behavior between ATCV-1 Kcv and PBCV-1 Kcv, which has a conductance ratio value largely higher than 1 (Table I: G Rb /G K = 1.8). Finally it has to be mentioned that the kinetics of the channel change as a consequence of the transported substrate. While the inward current in K + has a slow inactivating component, the current activates in a slow manner when Rb + is present in the medium. This kinetic effect was not analyzed further in this study.

ATCV-1 Kcv is blocked by conventional K + channel blockers.

As mentioned previously, the yeast complementation experiments indicate that ATCV-1 Kcv is sensitive to the two K + channel blockers Ba 2+ (Fig. 4A) and Cs + (Fig. S1A). Figure 4A shows that, in selective conditions (0.5 mM KCl), growth of yeast expressing ATCV-1 Kcv is strongly reduced by 1 mM Ba 2+ . To confirm and further study the Ba 2+ block, ATCV-1 Kcv macro currents were recorded in oocytes with and without Ba 2+ in the external solution. Fig. 4B shows the current response of an oocyte expressing ATCV-1 Kcv before and after addition of 1 mM Ba 2+ to an external solution containing 50 mM K + . Barium produces a voltage-dependent block of the ATCV-1 Kcv conductance. It abolishes the inward current and also slightly affects the outward current. The overall effect of Ba 2+ is summarized in the mean current/voltage curves obtained with four different Ba 2+ concentrations, 0.01, 0.1, 1 and 10 mM (4C); ATCV-1 Kcv is partially blocked by sub-millimolar concentrations of Ba 2+ . The dose-response relationship of the Ba 2+ block was obtained at different test potentials and plotted in Fig. 4D. The plotted values of fractional current, I f , could be fitted by the Hill equation as follow:

I f = Kd n /(Kd n + [Ba 2+ ] n ) (1)
where Kd is the concentration of Ba 2+ for half inhibition and n is the Hill coefficient. Since the best fit of n was between 1.03 and 1.07, we fixed n at 1. The resulting Kd for Ba 2+ changed with voltage and was 31, 40, 55 and 85 µM at -100, -80, -60 and -40 mV, respectively. Due to the voltage-dependent nature of the block the apparent Kd obtained from the dose-response curves varies in a linear fashion with voltage when plotted in a semilogarithmic scale (Fig. 4E). The extrapolated value of Kd at 0 mV was 0.16 mM and decreased e-fold by 120 mV hyperpolarization, confirming that the Ba 2+ block is facilitated by hyperpolarization. Collectively these data show that the smaller version of a Kcv-type channel behaves similarly to its larger orthologs. Other Kcv channels are also blocked by Ba 2+ in a voltage dependent manner with Kd values in the range of tens of µM [9; Moroni, unpublished data].

The same procedure was used to examine Cs + -sensitivity of the ATCV-1 Kcv channel. Addition of mM Cs + inhibits yeast growth when expressing the ATCV-1 Kcv channel (Fig. S1A). The electrophysiology data in Fig. S1B-E Structural basis for the difference in Rb + selectivity. The macroscopic and microscopic current/voltage data in Figure 3 reveal that the ATCV-1 Kcv generated channel is better at conducting K + than Rb + . This behavior is unusual for Kcv-type channels since all Kcvlike channels characterized to date [START_REF] Kang | Small potassium ion channel proteins encoded by chlorella viruses[END_REF] conduct Rb + better than K + . In search of a structural explanation for this difference we noted that ATCV-1 Kcv has a Tyr in the conserved selectivity sequence of the filter (GYG) while PBCV-1 Kcv has a Phe (GFG) (Fig. 1A). To determine if this substitution in the filter region is responsible for the difference in Rb + conductance and permeability, we prepared mutant channels in which we exchanged the two residues, i.e., ATCV-1 Kcv Y49F and PBCV-1 Kcv F66Y.

Both mutants were functional in oocytes and produced macrocurrents that were tested for K + selectivity; both exhibited Nernstian behavior when tested in buffers with different KCl concentrations (Supplementary figure 2). The I/V relationships obtained from wild type and mutant channels in the presence of K + and Rb + are presented in Fig. 5 (mean data for wild type ATCV-1 Kcv of Fig. 5A include the data set of Fig. 3B). For the aforementioned reasons we again used both the shift of E rev (∆E rev = E rev Rb -E rev K) and the ratio in conductance (G Rb /G K ) as parameters to estimate the selectivity of the channel for K + over Rb + . In the case of ATCV-1 Kcv, the Y49F mutation results in a dramatic increase in Rb + conductance and a small negative ∆E rev of -7mV (insert of 2B shows an enlargement of the currents at E rev : K + = -21.52 + 1.13 mV n=5; Rb + = -28.13 + 1.11 mV n=4). The conductance ratio, G Rb /G K , increases from 0.33 in the wild type to 2.8 in the mutant (Table I), while the permeability ratio, P Rb+ /P K+, estimated from the shift in E rev , is increased from 0.54 ± 0.01 in the wild type to 0.76 ± 0.02 in the mutant (Table I). These results imply that Phe and Tyr serve an important role in determining the selectivity between the two similar cations Rb + and K + . However this amino acid alone cannot explain the entire selectivity. The reverse mutation F66Y in PBCV1-Kcv does not cause a complete reversion of Rb + /K + selectivity. But both parameters G Rb /G K and ∆E rev change in the mutant in a direction which is consistent with a reduced selectivity of the channel for Rb + over K + : while the replacement of K + by Rb + causes a mean shift of the reversal voltage in the wild type of +10 mV (E rev K + = -21.39 + 0.70 mV; E rev Rb + = -11.23 + 1.06 mV, n = 5), the same operation results only in a +6 mV shift in the mutant highlighted by the enlargement of panel 5D ( E rev K + = -19.97 + 0.31 mV; E rev Rb + = -14.07 + 0.39 mV, n=5). Also, while the G Rb /G K ratio is 1.8 in the wild type channel, it is reduced to 1.5 in the mutant (Table I).

Altogether these results are consistent with the idea that Phe in the selectivity filter favors Rb + over K + conductance. However, the data also indicate that the overall selectivity of a filter can only be understood in the context of other structural parts of the channel protein.

CONCLUSION

The present data show that a Kcv protein from chlorella virus ATCV-1, which has the hallmarks of a K + channel, forms a functional K + channel. Even though the monomer is only 82 amino acids long it still forms a tetramer. This tetrameric channel has many features typical of more complex K + channels. ATCV-1 Kcv can discriminate between cations and it has some voltage dependent features; furthermore, ATCV-1 Kcv has a distinct voltage-dependent sensitivity to typical K + channel blockers such as Ba 2+ and Cs + . All of these functional features must be inherent in the basic structure of this miniature channel protein. A comparison of the ATCV-1 Kcv functions with another viral channel, PBCV-1 Kcv, indicates that it differs significantly because it is more permeable to K + than Rb + . This result can be partially explained by a Tyr residue in the ATCV-1 Kcv selectivity filter; in contrast PBCV-1 Kcv has a Phe.

FIGURE LEGENDS

Fig. 1. Sequence alignment, homology modeling, and protein expression of ATCV-1 Kcv in Saccharomyces cerevisiae. A. ATCV-1 Kcv sequence alignment with the sequence of the prototype chlorella virus K + channel PBCV-1 Kcv by ClustalW (http://www.ebi.ac.uk/tools/clustalw2). First and second transmembrane domains (TM1, TM2), pore helix (Pore) and selectivity filter (Filter) sequences of ATCV-1 Kcv are highlighted by horizontal lines. Assignment of these regions follows from a consensus among prediction programs TMHMM and Phobius as well as structural data of the molecular dynamicsrefined model of PBCV-1 Kcv [START_REF] Tayefeh | Molecular dynamics simulation of the cytosolic mouth in Kcv-type potassium channels[END_REF]. Asterisk and colon indicate identical and conserved residue respectively. B. Superimposition of ribbon representations of the PBCV-1 Kcv (light grey) and ATCV-1 Kcv (dark grey) structures, transverse section with two opposing monomers (left) and side view (right), visualized by MOLCAD [START_REF] Brickmann | Interactive visualization of molecular scenarios with MOLCAD/SYBYL[END_REF] (http://www.molcad.de). The C-terminal residue of PBCV-1 Kcv is shown explicitly. Starting with the alignment of ATCV-1 Kcv and PBCV-1 Kcv, 3D modeling has been performed by Modeller, [START_REF] Marti-Renom | Comparative protein structure modeling of genes and genomes[END_REF] using the structure of PBCV-1 Kcv [START_REF] Tayefeh | Molecular dynamics simulation of the cytosolic mouth in Kcv-type potassium channels[END_REF] Note the negative shift of the reversal potential in the presence of Rb + . E. Permeability ratios, P X /P K , calculated from mean reversal potentials of macroscopic currents obtained from n≥5 oocytes (Table I). F. Conductance ratios, G X /G K , calculated from slope conductance of macroscopic current-voltage relationships in the linear range from -50 to -100 mV. Mean values are from n=4 oocytes (Table I). Table I -Permeability and conductance of wt and mutant channels to different monovalent cations. All cations were 50 mM as chloride salts. P X /P K + , permeability of ion X + relative to K + , calculated from current reversal potentials (see Material and Methods). G X /G K + , ratio of chord conductances calculated at -100 mV. Mean ± s.e., n= number of oocytes.

4 Biochemical

 4 the value in mV of the current reversal potential measured in the presence of the different monovalent cations in the external solution (either X + or K + ); [X] o and [K] o are the cations concentrations in the external solution; and R, T, z and F have their usual thermodynamic meanings. Journal Immediate Publication. Published on 06 Mar 2009 as manuscript BJ20090095 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20090095 A c c e p t e d M a n u s c r i p t

  shows the semi-5 Biochemical Journal Immediate Publication. Published on 06 Mar 2009 as manuscript BJ20090095 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20090095

Biochemical

  Journal Immediate Publication. Published on 06 Mar 2009 as manuscript BJ20090095 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20090095 A c c e p t e d M a n u s c r i p t

  , can be summarized as follows: ATCV-1 Kcv is inhibited by submillimolar 7 Biochemical Journal Immediate Publication. Published on 06 Mar 2009 as manuscript BJ20090095 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20090095 A c c e p t e d M a n u s c r i p t concentrations of Cs + in a strong voltage-dependent manner. The K d value is strongly voltage-dependent and decreases e-fold for 31 mV hyperpolarization.

8

  Biochemical Journal Immediate Publication. Published on 06 Mar 2009 as manuscript BJ20090095 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20090095 A c c e p t e d M a n u s c r i p t A c c e p t e d M a n u s c r i p t

  Fig.1. Sequence alignment, homology modeling, and protein expression of ATCV-1 Kcv in Saccharomyces cerevisiae. A. ATCV-1 Kcv sequence alignment with the sequence of the prototype chlorella virus K + channel PBCV-1 Kcv by ClustalW (http://www.ebi.ac.uk/tools/clustalw2). First and second transmembrane domains (TM1, TM2), pore helix (Pore) and selectivity filter (Filter) sequences of ATCV-1 Kcv are highlighted by horizontal lines. Assignment of these regions follows from a consensus among prediction programs TMHMM and Phobius as well as structural data of the molecular dynamicsrefined model of PBCV-1 Kcv[START_REF] Tayefeh | Molecular dynamics simulation of the cytosolic mouth in Kcv-type potassium channels[END_REF]. Asterisk and colon indicate identical and conserved residue respectively. B. Superimposition of ribbon representations of the PBCV-1 Kcv (light grey) and ATCV-1 Kcv (dark grey) structures, transverse section with two opposing monomers (left) and side view (right), visualized by MOLCAD[START_REF] Brickmann | Interactive visualization of molecular scenarios with MOLCAD/SYBYL[END_REF] (http://www.molcad.de). The C-terminal residue of PBCV-1 Kcv is shown explicitly. Starting with the alignment of ATCV-1 Kcv and PBCV-1 Kcv, 3D modeling has been performed by Modeller,[START_REF] Marti-Renom | Comparative protein structure modeling of genes and genomes[END_REF] using the structure of PBCV-1 Kcv[START_REF] Tayefeh | Molecular dynamics simulation of the cytosolic mouth in Kcv-type potassium channels[END_REF] as template. PBCV-1 Kcv's Ctermini have been extended by matching the corresponding KirBac1.1 helices (PDB code 1P7B), yielding a helical template structure that is long enough to superimpose ATCV-1 Kcv's C-terminus. Due to the shorter turret and N-terminal regions of ATCV-1 Kcv vs. PBCV-1 Kcv, ATCV-1 Kcv is overall smaller than PBCV-1 Kcv, although the C-terminal transmembrane segments of ATCV-1 Kcv are longer. The N-

Fig. 2 .

 2 Fig. 2. Properties of ATCV-1 Kcv currents at the macroscopic and single channel level. A. Exemplary macroscopic current traces recorded by two electrode voltage clamp from an oocyte injected with water for control and from an oocyte injected with cRNA of ATCV-1 Kcv. Clamp protocol: holding potential -20 mV, steps from 140 to -200 mV. External solution contained 50 mM KCl. Arrows indicate instantaneous current level at -120 and -200 mV to show reduction at extreme negative voltages. B. Current-voltage (I/V) relationship of instantaneous (measured 3 ms after the voltage step) currents shown in A. Current values are expressed in arbitrary units (a.u.) and are the mean of n≥5 different oocytes ± s.e. (error bars smaller than the symbol are not visible) normalized to the current value recorded at -100 mV. Insert in B: mean values (n=3, ± s.e.) of reversal potential [K + ] out plotted as a function of log [K + ] out . [K + ] out was 6, 10, 50 and 100 mM. [K + ] in was assumed to be 108.6 mM [26]. The slope of the linear relationship is 53.9 mV. C. Exemplary single channel fluctuations recorded at different voltages in cellattach configuration from an oocyte expressing ATCV-1 Kcv. The pipette solution contained 100 mM KCl. Dotted line marks the closed channel level of current. D. Open state I/V relationship of the single channel shown in C.

Fig. 3 .

 3 Fig. 3. Permeability and conductance properties of ATCV-1 Kcv to different monovalent cations. A. Currents recorded by a two electrode voltage clamp experiment with 50 mM KCl, RbCl, CsCl, NaCl, and LiCl in the external solution. Dotted line indicates zero current. B. I/V relations of the currents reported in A. The plotted values are of instantaneous current (I i ), measured 3 ms after the voltage step. C. Comparison of single channel recordings of ATCV-1 Kcv measured in cell-attach experiment at +40 and -40 mV with either 100 mM RbCl or KCl in the pipette solution. Dotted line marks the closed channel level of current. D. Corresponding I/V relation of the experiment from which traces at +/-40 mV are displayed in C.Note the negative shift of the reversal potential in the presence of Rb + . E. Permeability ratios, P X /P K , calculated from mean reversal potentials of macroscopic currents obtained from n≥5 oocytes (TableI). F. Conductance ratios, G X /G K , calculated from slope conductance of macroscopic current-voltage relationships in the linear range from -50 to -100 mV. Mean values are from n=4 oocytes (TableI).

Fig. 4 .

 4 Fig. 4. ATCV-1 Kcv is blocked by external Ba 2+ . A. Complementation assay performed on a ∆trk1, ∆trk2 yeast mutant transformed with empty vector (control) and ATCV-1 Kcv. Yeast growth was tested by adding various Ba 2+ concentrations (water for control) to the paper discs in the plates. B. Effect of 1 mM Ba 2+ on ATCV-1 Kcv currents recorded in 50 mM K + . C. I/V relationship of ATCV-1 Kcv currents recorded at different external Ba 2+ concentrations. Steady state current values (Iss, measured at the end of the voltage pulse) are expressed in arbitrary units (a.u.) because normalization was performed at the current value at -100 mV to allow comparison of different oocytes (n =9). When not visible, error bars (± s.e.) are smaller than symbols. D. Dose-response curve, obtained by plotting fractional current, I f , as a function of [Ba 2+ ] out . Curves were fitted with the Hill equation (see text) at fixed n=1. Kd = 24, 32, 45 and 75 µM at -100, -80, -60 and -40 mV respectively. E. Semi logarithmic plot of the Kd against the membrane potential. Kd was obtained from the dose-response curve obtained from data plotted in C,

Fig. 5 .

 5 Fig. 5. Effect of a mutation in the selectivity filter on conductance and permeability to Rubidium. A. ATCV-1 Kcv sequence GYG was changed to GFG in the Y49F mutant displayed in B. C. PBCV-1 Kcv sequence GFG was changed to GYG in the F66F mutant displayed in D. Measurements performed in oocytes by two electrode voltage clamp, applying the standard voltage protocol (see Experimental procedures) and in the presence of 50 mM external KCl (square symbols) or RbCl (circle symbols). The plotted values are of instantaneous current (I i ), measured 3 ms after the voltage step. Each set of data is normalized to the value of current at -100 mV to include measurements from different oocytes and is expressed in arbitrary units (a.u.). Mean values ± s.e. (n = 7 for ATCV-1 wt, n=8 for ATCV-1 Y49F, n= 5 for PBCV-1 wt and n=10 for F66Y). A. includes a set of data already displayed in Figure 3B. Insert of B and D show enlargement of the I/V relationship to allow better appreciation of shifts in current reversal potentials induced by Rb + : -7 mV for ATCV-1 Kcv Y49F (E rev K + = -21.52 + 1.13 mV; E rev Rb + = -28.13 + 1.11 mV) and + 6 mV for PBCV-1 Kcv F66Y (E rev K + = -19.97 + 0.31 mV; E rev Rb + = -14.07 + 0.39 mV).

  Biochemical Journal Immediate Publication. Published on 06 Mar 2009 as manuscript BJ20090095 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20090095

	Li + Na + Cs + Rb + K +	9 10 5 7 -n ATCV-1 0.03 ± 0.01 0.04 ± 0.01 0.20 ± 0.01 0.54 ± 0.01 1 P X /P K +	4 4 4 4 -n a n u s c r i p t < 0.01 10 0.05 ± 0.01 4 < 0.001 5 0.03 ± 0.01 4 0.06 ± 0.01 8 0.24 ± 0.01 4 1.82 ± 0.17 5 1.72 ± 0.08 4 1 -1 -G X /G K + n P X /P K + n PBCV-1 M < 0.001 < 0.001 0.02 ± 0.01 0.33 ± 0.01 1 G X /G K +
	< 0.01 < 0.01 < 0.01 2.80 ± 0.45 1 G X /G K + A c c e p t e d 5 0.02 ± 0.01 Li + 5 0.04 ± 0.01 Na + 7 0.03 ± 0.01 Cs + 8 0.76 ± 0.02 Rb + -1 K + n P X /P K + ATCV-1 Y49F	4 4 4 4 -n	7 6 8 10 -n PBCV-1 F66Y 0.05 ± 0.01 0.04 ± 0.01 0.13 ± 0.01 1.26 ± 0.02 1 P X /P K +	< 0.01 < 0.01 0.03 ± 0.01 1.53 ± 0.13 1 G X /G K +	4 4 4 4 -n
				13

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ACKNOWLEDGMENT

We thank Dan Minor, University of California San Francisco, for the mutant yeast strain SGY1528 and for help with the complementation protocol. This investigation was supported in part by Public Health Service grant GM32441 and NIH grant P20RR15635 from the COBRE program of the National Center of Research Resources to J.V.E.; by grant 2005052099 PRIN 2005 of the Ministero dell' Università e Ricerca and by project EDICT (European Drug Initiative on Channels and Transporters) EU PF7 (Grant number 201924) to A.M. and by a grant of the Deutsche Forschungsgemeinschaft to G.T.