Proc. Nati. Acad. Sci. USAVol. 85, pp. 7443-7447, October 1988Biochemistry

Orthorhombic crystals and three-dimensional structure of thepotent toxin II from the scorpion Androctonus australis Hector

(structure-function relationships/sodium channels/lysine reactivity/x-ray crystallography)

JUAN C. FONTECILLA-CAMPS*t, CATHERINE HABERSETZER-ROCHAT*, AND HERVE ROCHATt

*Centre de Recherche sur les Mecanismes de la Croissance Cristalline-Centre National de la Recherche Scientifique, Campus Luminy, case 913, 13288

Marseille cedex 09, France; and tUnitd Associde 1179, Centre National de la Recherche Scientifique, Unite 172 Institut National de la Sant6 et de la

Recherche Mddicale, Laboratoire de Biochimie, Secteur Nord, Boulevard Pierre Dramard, 13326 Marseille cedex 15, France

Communicated by Jean-Marie Lehn, June 7, 1988

ABSTRACT Orthorhombic crystals (space group P212121,a = 45.94 A, b = 40.68 A, c = 29.93 A) of the potent scorpiona-toxin II from Androctonus australis Hector were grown usingsterile techniques. The structure was solved by a combinationof heavy-atom and model phasing. Subsequently, it was re-frned at 1.8 A resolution by a fast-Fourier restrained least-squares procedure. The crystallographic R factor is 0.152for data with 7.0 A > d > 1.8 A and F > 2.5o(F) and 0.177when all data are considered. Eighty-nine solvent moleculeshave been incorporated into the model. The dense core formedby the a-helical and antiparallel ,8-sheet moieties and three ofthe four disulfide bridges is similar in variant 3, a toxin purifiedfrom the North American scorpion Centruroides sculpturatus,and in toxin II. However, the two molecules differ markedly inthe orientation of loops protruding from the core. Toxin IIseems to contain several highly ordered solvent molecules.Eight of them occupy a cavity consisting of the C-terminalregion and a loop found only in scorpion a-toxins. The highlyreactive and pharmacologically important Lys-58 is found atone of the extremes of this cavity, where it establishes a seriesof hydrogen bonds with protein and solvent atoms. Thereactivities of the five lysine residues of toxin H are highlycorrelated with the formation of hydrogen bonds, hydrophobicinteractions, and salt links.

Scorpion toxins constitute a family of small basic proteinsthat are responsible for the neurotoxicity of the venoms (1-3). Although they show significant sequence homology (2-4),scorpion toxins display various degrees of toxicity towarddifferent animal classes. For example, venoms from Buthinaescorpions contain toxins that are preferentially directedagainst mammals, insects, or crustaceans (2, 5, 6). Mammal-directed toxins have been divided into two groups, a and f(7), depending on their mode of action, cooperative bindingwith other toxins, and dependence of membrane potential forbinding. The a-toxins are typically found in Buthinae venomsand they prolong the Na+ inactivation phase of the actionpotential (8). 8-Toxins, on the other hand, were first de-scribed in Centrurinae venoms and are known to affect theNa+ activation phase (7). As may have been expected,a-toxins and 8-toxins do not bind to the same site on the Na+channel (9-15).The three-dimensional structure of variant 3 from the

scorpion Centruroides sculpturatus Ewing has been deter-mined at 3 A resolution and refined at 1.8 A resolution (16,17). Although variant 3 is only weakly toxic, it is structurallyvery close to the potent p-toxins. Based on this structureFontecilla-Camps et al. (18) described a "conserved-hydrophobic" surface that they suggested should be impli-

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7443

cated in the binding of scorpion toxins to their receptor sites.This idea has received further support more recently (19-22).An earlier paper from this laboratory (23) reported the

crystallization of a monoclinic form of the most potentmammal-directed scorpion a-toxin known, toxin II fromAndroctonus australis Hector (AaHII). Subsequently, wehave succeeded in growing an orthorhombic form of thistoxin that diffracts to higher resolution than the monocliniccrystal form. We present here the crystallization protocol,the space group determination, and the three-dimensionalstructure of AaH II, and we consider structure-functionrelationships based on a series of studies performed on thiswidely studied molecule or on other a-toxins.

METHODSAaH II was purified as described (1). Solutions containing 5-10 mg of toxin per ml in 0.2 M NH4CH3COO (pH 6.8) weresterilized by Millipore filtration and collected in Nunc steriletubes. This operation was carried out in a laminar flow hood.Under these conditions crystals developed by slow evapo-ration after several months at 40C. Additional crystals wereobtained by seeding followed by slow concentration of thesesolutions. AaH II crystals, which grow as large prisms (0.6mmx 0.6 mm x 1.0 mm), belong to the orthorhombic space groupP212121 with a = 45.94 A, b = 40.68 A, and c = 29.93 A. Thereis one molecule in the asymmetric unit. Heavy-atom deriva-tives were prepared by soaking crystals in solutions containing20o polyethylene glycol (PEG) 6000 (Fisher) buffered with0.05 M sodium cacodylate (pH 7.0) and the heavy-atomcompound. The use of PEG was necessary to prevent crystaletching during the soaking procedure. Subsequently, thesecrystals were mounted in glass capillaries in the usual way andexamined by use of a sealed-tube x-ray generator or a rotatinganode generator and a precession camera. Of about 30 differ-ent heavy-atom salts tested, only 3 resulted in crystals thatshowed intensity changes on precession photographs, weremacroscopically intact, and gave useful derivatives. Thesecompounds were K2PtCl6, K2IrCl6, and AgNO3. However,the iridium salt and the platinum salt introduced significantchanges in the length ofthe three principal cell axes (a = 45.32A, b = 40.57 A, and c = 30.26A for the K2IrCl6 derivative anda = 45.73 A, b = 40.46 A, and c = 30.16 A for the K2PtCI6derivative). Furthermore, the platinum derivative cell dimen-sions had a tendency to change during data collection: shrink-ing of the b axis and expansion of the c axis gave final di-mensions of a = 45.73 A, b = 40.26 A, and c = 30.26 A. Ex-

Abbreviations: MIR, multiple isomorphous replacement; AaH I andAaH II, toxins I and II from the scorpion Androctonus australisHector.tTo whom reprint requests should be sent at present address:Laboratoire de Crystallographie et de Cristallisation de Macro-molecules Biologiques, Secteur Nord, Boulevard Pierre Dramard,13326 Marseille Cedex 15, France.

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Table 1. Heavy-atom centric refinement statisticsDerivative No. of sites Slope* Rt Et FH§ d, AK2PtCl6 3 0.37 0.57 177 248 3.0K2IrCl6 4 0.45 0.48 149 265 3.0AgNO3 3 0.44 0.55 41 66 5.0*Slope of FH' versus IFPH - FPI2 (28).tCentric R factor = (IFPH - FpI - FH)/IFpH - FpI.tLack-of-closure error for centric terms (25).§rms-calculated heavy-atom structure factor.

amination ofprecession photographs ofthe AgNO3 derivativeshowed the coexistence of a native crystal lattice and a highlynonisomorphous one. This suggested that the crystal was onlypartially substituted and that its core was not affected by thesoaking procedure. The nonisomorphism was evident beyond5.5 A resolution. Soaking times and concentrations for theheavy-atom derivatives were 5 days for 2.0 mM K2PtCl6, 10days for 1.0 mM K2IrCl6, and 18 hr for 0.5 mM AgNO3.Data for the native and derivative crystals were collected

with a CAD4 Enraf-Nonius diffractometer at room temper-ature, using an (-scan procedure and nickel-filtered Cu K-aradiation. Data were collected to 3.0 A resolution for theK2PtCI6 and the K2IrCI6 derivative crystals and to 5 Aresolution for the AgNO3 derivative crystal. Native data weremeasured to 1.8 A resolution from one crystal. In all cases,shells of -300 unique reflections were collected from high tolow resolution. The highest-resolution shell contained 91% ofobserved reflections (I > 2orI). Friedel mates were measuredat negative 20 values alternating with their unique counter-parts. The peak-fitting method of Oatley and French (24) wasthen used to obtain uncorrected intensities. Data werereduced in the usual way (25); absorption corrections wereapplied according to the method of North et al. (26). Thedecay in intensity in the x-ray beam for a set of standardreflections was 26% for the K2PtCI6 derivative, 22% for theK2IrC16 derivative, 3.4% for the AgNO3 derivative, and 8.6%for the native data. The overall quality of the data sets wasevaluated by comparing the intensities of Friedel mates. Wecomputed the reliability index:

Rsym =II(h) - I(h)I>I(h) + I(h)I

The RSym values for centric data were 0.016 for the nativedata, 0.021 for the Pt derivative, 0.024 for the Ir derivative,

and 0.019 for the Ag derivative. Before merging the Friedelsets HKL and FRE, they were scaled together using theprogram ANOSCL of Hendrickson and Teeter (27). Deriv-ative data were scaled to native data by equating FPH2 to Fp2in shells containing 200 reflections; FP corresponds to thenative structure factor and FPH corresponds to the heavy-atom derivative structure factor.The positions for the heavy atoms were determined from

isomorphous difference Patterson maps and cross-differenceFourier maps (25). PtCl42- was found to occupy two majorsites and a minor one. The K2IrCl derivative was modeledby four sites; the two major sites were unique to thisderivative, whereas the minor sites were the same as themajor ones ofthe platinum derivative. Soaking the crystals inthe AgNO3 solution resulted in a derivative with completelydifferent sites. All derivative sites were brought to a commonorigin by use of cross-difference Fourier maps. Since allphase calculations included anomalous scattering effects,these maps were also used to establish the correct enantio-morphic arrangement of heavy atoms (25).

Pt, Ir, and Ag positional and thermal parameters wererefined by the centric refinement method (28). Data concern-ing heavy-atom refinement statistics are presented in Table 1.The overall figure-of-merit (25) for data with d > 3 A was0.74.A series of electron density maps were calculated ranging

from d > 3.0 A to d > 5 A. The 5-A resolution map clearlyshowed the molecular boundaries except for one region ofclose contact between symmetry-related molecules. How-ever, inspection of maps calculated using higher-resolutiondata indicated that it would not be realistic to attempt thetracing of the complete polypeptide chain in the multipleisomorphous replacement (MIR) maps since they showedmany ill-defined features and poor connectivity in general. Asvariant 3 and AaH II were thought to share a basic corestructure, the following strategy was adopted: an a-carbonmodel of variant 3 was fitted interactively to the 5-A reso-lution map using the program FRODO (29). Subsequently,this model was fitted to an electron density map calculatedwith d > 3.5 A, where the amino acid sequence of variant 3was replaced by the sequence of AaH II; the differencesbetween the toxins due to amino acid insertions and deletions(Table 2) were accommodated in the best possible way. Theconventional crystallographic R factor for this preliminarymodel was 0.617 for the data with 5 A > d > 3.5 A. Atomic

Table 2. Amino acid sequences of AaH II and variant 3

Val Lys Asp Gly Tyr Ile Val Asp Asp ------ Lys Glu Gly Tyr Leu Val Lys Lys Ser

Gly Arg ---Leu Lys Leu

Lys --- --- LeuAla Lys Asn Gln

Pro Tyr Gly Asn--- --- --- Phe

Arg Thr Lys GlyPro Thr Tyr Pro

Gly2030LysGly3545AlaAla45

Leu60

20--- Asn Ala TyrGlu Asn Glu Gly

Gly Glu Ser GlyGly Ser Tyr Gly

Cys Tyr Cys TyrCys Trp Cys Glu

60

ProPro

--- Gly ArgAsn Lys Ser

10ValAspl0

CysCys2535TyrTyr40soLysGlyso

CysCys65

Asn Cys Thr TyrGly Cys Lys Tyr

25Asn Glu Glu CysAsp Thr Glu Cys

Cys Gln Trp AlaCys Tyr Ala ---

Leu Pro Asp HisLeu Pro Glu Ser

64His

Each sequence (4, 16) has been numbered independently. The gaps that have been introduced are based on the comparisonof the three-dimensional structures of both toxins.

AaH IIVariant 3

AaH IIVariant 3

AaH IIVariant 3

15Phe CysGly Cys15

ThrLys3040SerAaH II

Variant 3

AaH II

Variant 3

55ValThr55

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FIG. 1. a-Carbon backbone of scorpion toxin AaH II. Thecystine side chains are shown with their sulfur atoms labeled. Everyfifth a-carbon has been numbered. Figs. 2 and 3 have the sameorientation as this figure. All drawings were generated by using theprogram PLUTO [program available from Sam Motherwell (Cam-bridge Crystallographic Data Center, University Chemical Labora-tory, Lensfield Road, Cambridge, England)].

coordinates were then refined by using a fast-Fourier versionofPROLSQ (PROFFT) kindly provided to us by Barry Finzel(30, 31). The first six cycles of refinement brought the Rfactor to a value of 0.505. The model was kept relativelyunrestrained by setting target values for the standard devia-tions ofbond distances and bond angles that were about threetimes as high as the conventional ones (30). Subsequently,phases calculated from the refined coordinates were com-bined with the available MIR phases (32). An electron densitymap was calculated using MIR phases for the data betweeninfinity and 5 A resolution and combined phases for the datawith d < 5.0 A. The starting, rather than the refined, coordi-nate set was then fitted using FRODO and a new round ofrefinement was initiated. After cycling through this procedureseveral times, while gradually increasing the resolution, theR factor was 0.38 for the data with 5.0 A > d > 2.5 A anda normally restrained model (30). In the final stages, whendata with 3.0 A> d > 2.5 A were added to the map cal-culations, reflections in this resolution range were phased withphases calculated from the toxin model; they were given aconstant figure-of-merit value of 0.70. At this point a conven-tional refinement procedure using only calculated phases inmap calculations was started. A total of254 cycles ofPROFFTrefinement, during which 89 solvent molecules were added tothe model, reduced the R factor to a value of0.152 for the datawith 7 A > d> 1.8 A andF > 2.5o((F) and of0. 177 for all data.Only solvent molecules withB < 45 A2 were kept in the model;

their occupancies were not refined but were fixed at a value of1. The overall B value for the protein structure is 9.8 A2 andthat ofthe solvent atoms is 27.3 A2. We did not use the RTESToption of PROFFT when using the fast-Fourier algorithm.Although this resulted in a rather large number ofcycles, it wasmore efficient than estimating damping factors by conven-tional structure factor calculations with RTEST. The details ofthe crystallographic refinement will be published elsewhere.

RESULTS AND DISCUSSIONThe a-carbon structure of AaH II is depicted in Fig. 1 and astereo view of all non-hydrogen protein atoms is shown inFig. 2. The most prominent secondary structural features arean a-helical stretch comprising residues 19-28 and a three-stranded antiparallel p-sheet moiety containing residues 2-4,32-37, and 45-51. The p-sheet runs approximately parallel tothe a-helix. These two secondary structural elements areconnected by disulfide bridges linking Cys-22 to Cys-46 andCys-26 to Cys-48. A third disulfide bridge is found nearby; itconnects Cys-16 to Cys-36. There are three type I reverseturns (33) in the structure (residues 27-30, 40-43, and 52-55)and a 5-residue turn stabilized by Asp-8 (residues 8-12). Twoof the five lysine residues and the terminal a-amino groupseem to be implicated in salt links to acidic functions: N ofVal-1 to OD2 of Asp-53, NZ of Lys-28 to OE1 of Glu-25, andNZ of Lys-50 to OE2 of Asp-32. The structure is furtherstabilized by the presence of several solvent molecules thatseem to be integral parts of the toxin molecule (Fig. 3). Inparticular, the cavity formed by residue 13, part of the loopcontaining residues 39-43, and the C-terminal region is filledby a series of highly ordered water molecules (Fig. 4). Thesemolecules have temperature factors that range from 6.8 A2 to20.5 A2 (mean B = 15.8 A2). AaH II shows an interestingdistribution of aromatic residues at the molecular surface: thearomatic rings of tyrosine residues 42, 5, 47, and 49 interactin the "herringbone" fashion in a way that resembles thecrystal structure of L-tyrosine hydrochloride (34). This hasthe effect of minimizing the exposed surfaces of these large,hydrophobic residues.

Fig. 5 depicts the comparison of the a-carbon backbones ofvariant 3 and AaH II. Both molecules have a very similar,highly structured region that contains the a-helix, the three-stranded antiparallel p8-sheet moiety, and three of the fourdisulfide bridges. In contrast, loops protruding from thedense core of secondary structure differ appreciably in thetwo molecules. As expected, major differences are found forthe regions where amino acid insertions exist (Table 2).Surprisingly, however, the C-terminal stretch ofAaH II alsodiffers quite markedly from the corresponding region invariant 3. Its positioning contributes to the formation of thecavity filled by several solvent molecules (Fig. 3). Thisarrangement constitutes a particular feature of scorpion

FIG. 2. Stereo drawing showing all non-hydrogen atoms of AaH II. Sulfur atoms are shown as small black circles.

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FIG. 3. Stereo pair depicting the tightly bound solvent molecules ofAaH II. The solvent molecules, shown as dark circles, have been sortedaccording to ascending values of the temperature factor as determined at the end of the refinement procedure. Their numbering correspondsto their position among the 89 solvent atoms found and it starts at solvent atom W65.

a-toxins that may be at least partially responsible for theirspecific mode of action.The hydrophobic character of the conserved-hydrophobic

surface was determined mostly by the presence of a clusterofaromatic residues at the molecular surface ofvariant 3 (18).AaH II also shows a greater concentration of aromaticresidues on the side ofthe molecule thought to be responsiblefor the biological effects (Fig. 2). Clusters of hydrophobicresidues have also been described in the case of the cardio-toxin from Naja mossambica mossambica (35); they may begenerally implicated in the interaction of toxins with mem-brane components.Recent results (H. Darbon, H.R., and J.C.F.-C., unpub-

lished) indicate that the lysine residues of AaH II can beclassified according to their relative reactivities in the fol-lowing way: Lys-58 > Lys-2 > Lys-30 ¢ Lys-28 > Lys-50.An analysis of the environment of the lysine residues showsthat the most reactive ones, Lys-58 (36, 37) and Lys-2, formseveral hydrogen bonds, are well-ordered, and are not readilyaccessible to the solvent environment: Lys-2 is at hydrogenbond distance of the carbonyl oxygen of Val-55, of the OG ofThr-57, and of two solvent molecules (not shown). Lys-58seems to form hydrogen bonds with the carbonyl oxygens ofAsn-11 and Gly-61 and with two solvent molecules. Three ofthese interactions are depicted in Fig. 4. Booshard (38) hasoffered a possible explanation for their enhanced reactivity.This author argues that partially buried chains are found inhydrophobic environments, where the uncharged side chainis more stable and the PKa values of the E-amino group arelower. The less reactive Lys-30 is fully exposed to the solvent

and consequently may display the normal pKa value of thee-amino group under the conditions of the reaction. Lys-28and Lys-50, on the other hand, form salt links and their lowreactivities are most likely due to the abnormally high pKavalues of their E-amino groups expected from the ionicinteraction of the salt link (39). Thus, the relative reactivitiesof the lysine residues ofAaH II can be readily accounted forby the environment of each of these side chains.There is a drastic loss of pharmacological activity and

binding to synaptosomes (<0.1% residual binding) whenLys-58 is biotinylated (or when the equivalent Lys-56 ofAaHI is acetylated) (21, 37). In addition to the chemical reactionsaffecting Lys-58, only modification of the basic residue inposition 2, or removal of the N-terminal dipeptide, can resultin comparably low residual activities (-1%). These obser-vations, and the fact that Lys-58 and Lys-2 are partiallyburied, seem to indicate that these residues play a preferen-tially structural role and that, in both cases, modification ofthe E-amino group results in major effects on the orientationof several neighboring residues. This conclusion is supportedby recent results on the chemical modification of tyrosine andarginine residues of AaH II that showed that no specificmodification provoked >70-80% loss of binding to synapto-somes (40).Comparison of the x-ray models of a neurotoxin and a

cardiotoxin from snake venom (35) shows a common featurewith scorpion a- and f3-toxins: in both cases modulation ofthespecific toxin activity seems to be partly determined bychanges in the positioning of loops coming from a highlyconserved core structure.

FIG. 4. Stereo drawing of the solvent-filled cavity formed by residues 13, 42, and 43, and 62-64. Possible hydrogen bonds (d < 3.2 A) areshown as dashed lines. Side chains of residues 14 and 62 have been omitted for clarity. The solvent atoms are depicted as starred circles. Thepossible hydrogen bond between Lys-58 and the carbonyl oxygen of Asn-11 is not shown.

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FIG. 5. Comparison of the a-carbon backbones of AaH II (thick lines) and variant 3.

It has become clear that, even in the case of a family ofsmall proteins, a great deal of structural and functionaldiversity can be observed. We now must concentrate our

effort on the crystallographic study of insect-specific toxinsthat belong to a third structural group of molecules present inscorpion venom. They are characterized by different posi-tioning of one of the four disulfide bridges typical of scorpiontoxins (41). In this manner, hopefully a clearer picture of themechanisms involved in the evolution of scorpion toxins inparticular and of proteins in general may be obtained.

Part of this work was carried out during a short sabbatical leave ofone of us (J.C.F.-C.) in Dr. C. E. Bugg's laboratory. We thank himand his group for valuable advice and encouragement. We also thankDr. Hj. Darbon for fruitful discussions and Dr. R. Griffin-Shea forcritical reading of the manuscript. This research was partiallysupported by Grant 84C-1052 from the Centre National de laRecherche Scientifique.

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