doi:10.1016/j.jmb.2004.12.039 j. mol. biol. - dtic · robert schwarzenbacher2, michael a. hanson1,...

doi:10.1016/j.jmb.2004.12.039 J. Mol. Biol. (2005) 346, 1083–1093

The Structure of the Neurotoxin-associated ProteinHA33/A from Clostridium botulinum Suggests aReoccurring b-Trefoil Fold in the Progenitor ToxinComplex

Joseph W. Arndt1, Jenny Gu1, Lukasz Jaroszewski2

Robert Schwarzenbacher2, Michael A. Hanson1, Frank J. Lebeda3 andRaymond C. Stevens1*

1Department of MolecularBiology, The Scripps ResearchInstitute, 10550 N. Torrey PinesRoad, La Jolla, CA 92037USA

2The Joint Center for StructuralGenomics, University ofCalifornia San Diego, 9500Gilman Drive, La Jolla, CA92093, USA

3Department of Cell Biologyand Biochemistry, ToxinologyDivision, U.S. Army MedicalResearch Institute of InfectiousDiseases Frederick, MD 21702USA

0022-2836/$ - see front matter q 2004 E

Present address: J. Gu, San DiegoCenter, University of California SanDrive, La Jolla, CA 92093, USA.Abbreviations used: HA, hemagg

non-toxic non-hemagglutinin; BoNTneurotoxin; NAP, neurotoxin-associroot-mean-square deviation; TeNT,E-mail address of the correspond

[email protected]

The hemagglutinating protein HA33 from Clostridium botulinum isassociated with the large botulinum neurotoxin secreted complexes andis critical in toxin protection, internalization, and possibly activation. Wereport the crystal structure of serotype A HA33 (HA33/A) at 1.5 Aresolution that contains a unique domain organization and a carbohydraterecognition site. In addition, sequence alignments of the other toxincomplex components, including the neurotoxin BoNT/A, hemagglutinat-ing protein HA17/A, and non-toxic non-hemagglutinating proteinNTNHA/A, suggests that most of the toxin complex consists of areoccurring b-trefoil fold.

q 2004 Elsevier Ltd. All rights reserved.

Keywords: neurotoxin; hemagglutinin; b-trefoil; progenitor toxin; sugar-
binding *Corresponding author
Introduction

The Clostridium botulinum neurotoxins (BoNTs)are among the most toxic bacterial toxins known.Exposure to toxin prevents the release of acetyl-choline at neuromuscular junctions and synapsesby cleaving one of the three neuronal proteins of thesoluble N-ethylmaleimide-sensitive-factor attach-ment protein receptor (SNARE) complex requiredfor synaptic vesicle membrane fusion resulting in

lsevier Ltd. All rights reserve

SupercomputerDiego, 9500 Gilman

lutinin; NTNHA,, botulinumated protein; RMSD,tetanus neurotoxin.ing author:

flaccid muscle paralysis.1–4 Ironically, these toxinsare used clinically to treat neuromuscular disorders.C. botulinum produces the 150 kDa BoNT con-

comitantly with a group of non-toxic neurotoxin-associated proteins (NAPs), forming complexesknown as progenitor toxins. Previous studieshave demonstrated that the NAPs protect BoNTfrom acid denaturation in the stomach and attackfrom a variety of proteolytic enzymes in thegastrointestinal tract.5–8 Seven distinct BoNTshave been identified and are referred to as serotypesA–G, with serotype A being the most virulent tohumans.9 BoNT/A is secreted as a progenitor toxincomplex in one of three sizes, 12 S (300 kDa), 16 S(500 kDa), or 19 S (900 kDa), depending on thetype and number of NAPs associated with thecomplex.3,10 The 12 S progenitor toxin complexconsists of a single BoNT molecule and one non-toxic non-hemagglutinin (NTNHA) protein, butlacks the associated proteins responsible for hemag-glutination activity. The 16 S progenitor toxin, in

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4. TITLE AND SUBTITLE The structure of the neurotoxin- associated protein HA33/A fromClostridium botulinum suggests a reoccurring beta-trefoil fold in theprogenitor toxin complex, Journal of Molecular Biology 346:1083 - 1093

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6. AUTHOR(S) Arndt, JW Gu, J Jaroszewski, L Schwarzenbacher, R Hanson, MALebeda, FJ Stevens, RC

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7. PERFORMING ORGANIZATION NAME(S) AND ADDRESS(ES) United States Army Medical Research Institute of Infectious Diseases,Fort Detrick, Frederick, MD

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14. ABSTRACT The hemagglutinating protein HA33 from Clostridium botulinum is associated with the large botulinumneurotoxin secreted complexes and is critical in toxin protection, internalization, and possibly activation.We report the crystal structure of serotype A HA33 (HA33/A) at 1.5 A resolution that contains a uniquedomain organization and a carbohydrate recognition site. In addition, sequence alignments of the othertoxin complex components, including the neurotoxin BoNT/A, hemagglutinating protein HA17/A, andnon-toxic non-hemagglutinating protein NTNHA/A, suggests that most of the toxin complex consists of areoccurring beta-trefoil fold.

15. SUBJECT TERMS Clostridium botulinum, neurotoxin, BOT, hemagglutination, crystal structure, sequence

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Standard Form 298 (Rev. 8-98) Prescribed by ANSI Std Z39-18

1084 HA33/A Structure–High b-Trefoil Content in BoNT Complex

addition to the components found in the 12 Scomplex, contains three hemagglutinin (HA) pro-teins, HA70 (also referred to as HA3a and HA3bafter proteo-lysis), HA33, and HA17. The secretedand most toxic form 19 S complex is believed to be adimer of two 16 S toxins linked by an additionalHA33 protein.11

The HA positive progenitor toxins have beenshown to bind to glycolipids and glycoproteins ofthe intestinal microvilli through interactions witholigosaccharides, facilitating internalization andtransport in the bloodstream.12–14 Interestingly, theoral ingestion of the progenitor complexes displaysw100 times more toxicity than the naked BoNTprotein.15 Though the molecular details leadingto the higher efficacy of the progenitor toxincomplexes remain unknown, it has been proposedthat their improved effectiveness may be due tothe stabilization and protection of BoNT by theNAPs.15,16

The most prominent of the NAPs is HA33,comprising up to w30% of the 19S progenitortoxin complex mass, with binding specificity fordiverse carbohydrates, depending on the specificClostridium serotype and strain. For instance, theHA33 protein of serotype A (HA33/A) bindsglycolipids and glycoproteins containing galac-tose.13,17,18 In comparison, the HA componentsassociated with BoNT/C contain two distinctcarbohydrate-binding proteins, HA33/C (alsoreferred to as type C HA1) and the C terminus ofHA70/C (HA3b) that both recognize sialic acid-containing glycolipids and glycoproteins, albeitwith different specificities.19 Little is known aboutthe function of the other NAPs, HA17 and NTNHA.Here, we report the crystal structure of HA33/Afrom C. botulinum, the first structure of a serotype ANAP.

Results and Discussion

HA33/A structure

HA33/A was isolated from the progenitor toxincomplex of the C. botulinumHall strain and its X-raycrystal structure (Figure 1) was determined to1.50 A resolution by molecular replacement using

the HA33/C structure (PDB code 1QXM17) as thesearch model. Data collection, model building andrefinement statistics are summarized in Table 1. Thefinal model includes two protein molecules, chainsA and B (residues 10–293), and 564 water moleculesin the asymmetric unit. The final R-factor is 16.8%with an Rfree factor of 19.4%. The Ramachandranplot, produced by MolProbity,20 shows that allresidues lie within allowed regions. No electrondensity was observed for the first nine N-terminalresidues. This finding is consistent with theobserved post-translational modification, whichremoves the first five N-terminal amino acidresidues.21 It is not known whether this modifi-cation has any functional or serotype-specificrepercussions for HA33/A.

Each HA33/A molecule (Figure 1) consists of asingle polypeptide chain of 284 (10–293) residueswith an overall shape reminiscent of a dumbbell.HA33/A contains two b-trefoil domains connectedby a short a-helix. The dimensions of the HA33/Amonomer are 70 A!40 A!37 A, with an overallsurface area of 10,800 A2. The total b-strand and a-helical content is 48% and 10%, respectively, whichis considerably lower as compared to the b-strandcontent predicted by FT-IR and far-UV circulardichroism (74–77%).22 Each b-trefoil domainconsists of three homologous b-trefoil repeats thatare arranged about a pseudo 3-fold axis to form a12-stranded anti-parallel b-barrel capped by threeb-hairpins. Each b-trefoil repeat is composed of fourb-strands with 1234 topology, with the second andthird strand separated by a b-hairpin and the othertwo strands connected by loops of variable length.These repeats, designated 1a, 1b, and 1g for theN-terminal domain and 2a, 2b, and 2g for theC-terminal domain, are composed of residues10–55, 56–102, 103–144, 151–197, 198–245, and 246–293, respectively. The two domains of HA33/A arehighly similar to each other and can be superposedwith a Ca RMSD of 1.07 A for 137 structurallyequivalent residues and 24% sequence identity,according to the program TOP.23

Inter-domain conformational plasticity

The structures of the two HA33/A moleculespresent in the crystallographic asymmetric unit,

Figure 1. Crystal structure ofHA33/A. Ribbon diagram ofClostridium botulinum HA33/AHall-A strain color-coded from Nterminus (blue) to C terminus (red)showing the two domains con-nected by a short helical linker.The b-strands (b1-b24) and a-helix(a1) are labeled. The putativecarbohydrate-binding site is indi-cated with an arrow.

Table 1. Summary of crystallographic parameters, data collection and refinement statistics for HA33/A (PDB: 1YBI)

Data collectionSpace group P21212Unit cell parameters (A) aZ104.08, bZ146.60, cZ35.71Wavelength (A) 0.9179Resolution range (A) 25.00–1.50Number of observations 158,552Number of reflections 88,654Completeness (%) 87.0 (78.6)a

Mean I/s(I) 13.6 (3.7)a

Rsym on I 0.066 (0.299)a

Sigma cutoff 0.0Highest resolution shell (A) 1.53–1.50Model and refinement statisticsResolution range (A) 21.22–1.50No. of reflections (total) 77,217No. of reflections (test) 3820Completeness (% total) 87.1Rcryst/Rfree 0.168/0.194Stereochemical parametersRestraints (RMS observed)Bond lengths (A) 0.009Bond angles (deg.) 1.24Average isotropic B-value (A2) 23.2ESU based on R value (A) 0.085Protein residues/atoms 568/4677Solvent molecules 564

ESU, estimated standard uncertainties;47,51 RsymZSjIiK hIiijj=SjIij where Ii is the scaled intensity of the ith measurement, and is the

mean intensity for that reflection. RcrystZSjjFobsjK jFcalcjj=SjFobsj where Fcalc and Fobs are the calculated and observed structure factor

amplitudes, respectively. Rfree as for Rcryst, but for 5.0% of the total reflections chosen at random and omitted from refinement.a Highest resolution shell.

HA33/A Structure–High b-Trefoil Content in BoNT Complex 1085

chains A and B, are slightly different, most likelydue to domain flexibility, as indicated by a Ca

RMSD of 1.91 A. However, the individual N and C-terminal domains found in these two molecules aremore similar and superpose with a Ca RMSD of0.52 A and 0.89 A, respectively, with the largestdifferences in the C-terminal domains occurring at

Figure 2. (a) Ribbon diagram of an N-terminal superpositio(b) Same superposition as (a), but superposition of HA33/A cused above, but containing a close-up view of the helical link

loops 223–228 and 243–248. The major disparity inthe overall structures of the two molecules is due tothe alternate packing of the two trefoil domainsrelative to the connecting helix linker that causes anapproximate 108 rotation of the C-terminal domainwith respect to the N-terminal domain (Figure 2(a)).The focal point of the domain rotation is located

n of HA33/A chain A (red) and HA33/A chain B (green).hain A (red) and HA33/C (white). (c) Same superpositioner region.

Figure 3 (legend next page)Figure 3(a) (legend next page)


after the hydrophobic linker helix a1 (residues 145–150) that balances between the two conformationsobserved in the two HA33/A chains. In the A chain,the inter-domain interactions are less extensiveand mostly hydrophilic, such as the water-facilitated hydrogen-bonding of Glu143 andGlu196. While in the B chain hydrophobic inter-actions predominate, with Ile146 and Leu150 ofthe linker helix packing more deeply into ahydrophobic pocket of the C-terminal domain

formed by residues Ile192, Ile240, Pro242 andTyr250 than that observed in chain A. Additionalinteractions in the B chain include a bifurcatedhydrogen-bonding network between the side-chainof Asp144 of the N-terminal domain with the amidenitrogen atoms of Ile146 and Ile147 of helix a1 andan inter-domain salt-bridge between Arg47 andAsp247, which are not found in the chain Amolecule or the structure of HA33/C. Resultsobtained from a normal mode analysis using the

Figure 3. (a) Sequence alignment of HA33 homologs. The alignment shows strict sequence conservation in whiteletters and red background, and strong sequence conservation in red letters. The secondary structure elements of theHA33/A structure are labeled a (a-helix), h (310 helix), b (b-strand) and TT (turn). The solvent-accessibility of eachresidue in the HA33/A structure is indicated in the bar at the base of the sequences, with white representing buriedresidues, dark blue representing solvent-accessible residues and light blue representing an intermediate value. Theresidues at the putative carbohydrate-binding sites of HA33/A and HA33/C are indicated underneath with red starsand blue circles, respectively. This Figure was prepared using ESPript.49 (b) Surface representation of HA33/A showingconserved patches in green among HA33 homologs used in the sequence alignment in (a) indicating that the N-terminaldomain (top) is more highly conserved than the C-terminal domain (bottom); (c) same as (b), but rotated 1808; (d) same as(b), but looking down the N-terminal domain; and (e) same as (b), but looking down the C-terminal domain with theresidues Asp263, Tyr265, Gln276, Phe278, and Asn285 forming the putative carbohydrate recognition site in red. ThisFigure was prepared using the ConSurf server.50


ElNemo server24 confer HA33/A flexibilitybetween the trefoil domains at the helical linker.However, this inter-domain conformational flexi-bility is not seen in HA33/C, as the two moleculesin its asymmetric unit superpose with a Ca RMSD of0.53 A. In this light, further studies are necessary toconclude whether the two conformations observedin the X-ray structure of HA33/A are functionallyimportant or merely a crystal packing artifact.

Of particular note is that the inter-domainarrangement of the HA33/A molecules differssignificantly from that observed for the HA33/Cstructure.17 A comparison of the two serotypestructures reveals an RMSD of 2.6 A over 159aligned residues (out of the 284 possible residues)with 36% sequence identity.25 An approximately 608rotation of the C-terminal domain was observed inthe HA33/C structure as compared to the twoHA33/A molecules, despite the fact that the N-terminal domains of these two serotypes superposeclosely with an RMSD of 0.52 A (Figure 2(b) and(c)). The dissimilarity of the structures found forthese two serotypes is focused immediately beforethe linker helix a1, with the result that the twotrefoil domains of HA33/C congregate together.This domain orientation dissimilarity may beserotype-dependent, since HA33/C has a longerN terminus located at the interface of the b-trefoil

domains that does not undergo the post-translational cleavage (Figure 2(b)). The differencein the N termini may contribute to the serotype sizedifferences in the progenitor toxin complexes,particularly since the 19 S progenitor toxin complexis produced only by serotype A and not by the otherserotypes. N-terminal sequence analysis hasrevealed noteworthy serotype differences indicat-ing that HA33/C26 and HA33/D27 do not undergoprocessing like that observed for HA33/A andHA33/B, which are similarly proteolytically shor-tened at their N termini.21 Since BoNT serotypes Aand B are both involved in human botulism, thehigh level of sequence conservation of their HA33 smay reflect their similar specificities, activities, andimmuno-responses. Interestingly, the less toxicserotype A2,3 E28 and F29 strains lack the genesencoding the HA components, and thus produceonly 300 kDa 12 S toxin and have no ability toassemble with HA proteins while serotype G lacksonly the HA33 gene.30

Sequence conservation of HA33 serotypes

An alignment of sequences containing HA33/Aand nine more from other HA33 serotypes andstrains is shown along with the secondary structureof HA33/A in Figure 3(a). There is substantially


greater sequence conservation observed at thesurface of the N-terminal domain as compared tothe C-terminal domain, though the majority of theconserved residues are solvent-inaccessible andare presumably responsible for maintainingthe hydrophobic core of the b-trefoil fold(Figure 3(a)–(e)), with the greatest conservationbeing localized between the 1a and 1b repeats (b-strands b4–b6) possessing 65% identity. Since it isnot clear which of the NAP components interactwith the BoNT, the greater sequence conservation intheN-terminal domainofHA33/Asuggests that thisregion is likely to be important for protein–proteininteractions in the progenitor toxin complexes.Previous studies have reported that HA33/Aaccounts for most of the immunogenic response ofthe progenitor toxin complexes,31 indicating that atleast part of the molecule is exposed in the complex.Furthermore, C-terminally truncated variants ofHA33/C lose their hemagglutination and erythro-cyte-binding activity, suggesting that the C-terminaldomain contains the sugar-binding site.18 The lowerlevel of sequence conservation in the C-terminaldomain and the fact that this domain likelypossesses the carbohydrate-binding site collectivelysuggest that the C-terminal HA33 domain issolvent-exposed and the likely major contributorto the distinct antigenicity of the serotypes.

Figure 4. (a) Close-up view of HA33/A super-posed onthe ricin lactose-binding site at the 2g repeat region.Putative carbohydrate-binding residues as observed inHA33/A (slate blue) and their counterparts as found inthe lactose-bound ricin structure (salmon). Residue labelsare indicated for HA33/A with those from the ricinstructure in parentheses. (b) Similar overlay as in (a), but aclose-up view at the 2g repeat of HA33/A (slate blue)superposed on HA33/C (white), with a modeled lactosemolecule (salmon) and with HA33/C labels inparentheses.

Putative HA33/A carbohydrate-binding site

Recently, HA33/A has been proposed to containa single sugar-binding site at the 2g trefoil repeatspecific for carbohydrates containing galactose, asdetermined by isothermal titration calorimetry andmutagenesis.17 In many instances, the b-trefoil foldhas been associated with oligosaccharide-bindingability, leading to a characteristic HA activity,including the plant toxin ricin, a prototypicalganglioside-binding protein, and other proteinslike the ribosome-inactivating and potential cancertherapeutic mistletoe lectin I. The ricin and mistle-toe lectin I structures revealed a domain architec-ture that are similar to HA33/A with two b-trefoildomains. The complex crystal structures of ricinbound to lactose and mistletoe lectin I bound togalactose revealed that only the 1a and 2g repeats ofthese two proteins are involved in carbohydraterecognition.32,33 Based on the structural comparisonof HA33/A to ricin (PDB code 2AAI, with anoverall sequence identity of 14%), the 2g trefoilrepeat (25% identity) of the HA33/A C-terminaldomain likely contains the sugar-recognition site.An overlay of the residues of the 2g repeat in theHA33/A structure with the residues in the hom-ologous ricin lectin chain shows a spatial coinci-dence in the lactose-binding site when residuescontained in a 4 A sphere around lactose areoverlaid (Figure 4(a)). The key residues of Asp234,Arg235, Glu239, Tyr248, and Asn255 of ricin havecounterparts in HA33/A residues Asp263, Tyr265,Gln268, Phe278, and Asn285, respectively. Thisputative carbohydrate-binding site in HA33/A

contains three structurally essential componentsfor galactoside-binding that have been proposed forthe ricin lectin chain.33,34 In support of the 2g sugar-binding site for HA33/A are mutants Asp263 andAsn285 to Ala that lose their ability to bindcarbohydrates.17

A comparison of the 2g repeat of HA33/C withthe equivalent region in HA33/A (Figure 4(b))shows that they are highly similar, suggesting thatHA33/C contains the necessary components forcarbohydrate binding, but with noted differencesthat are likely to be important for ligand discrimi-nation of N-acetylneuraminic acid-containingmoieties by HA33/C. The residues of Asp263,Tyr265, Gln268, and Asn285 in HA33/A responsiblefor the putative carbohydrate recognition haveconserved counterparts in HA33/C residuesAsp256, Tyr258, Gln261, and Asn278, respectively.In addition, within strand b23 at location 278 inHA33/A (Hall) (Figure 3(a)) which has a Phe

Figure 5. (a) Scheme showing the b-trefoil content in the components of the 900 kDa progenitor toxin complex basedon the molecular composition of the type A toxin reported,11 although the exact stoichiometry is still under debate. (b)Sequence alignment of HA17/Awith HA33/A N and C-terminal trefoil domains. The alignment shows strict sequenceconservation in white letters and red background, and strong sequence conservation in red letters. The secondarystructure elements of the HA33/A N-terminal domain structure are labeled a (a-helix), h (310 helix), b (b-strand) and TT(turn). The solvent accessibility of each residue is indicated in the bar displayed at the base of the sequences, with whiterepresenting buried residues, dark blue representing solvent-accessible residues and light blue representing anintermediate value. The residues at the putative carbohydrate-binding sites of HA33/A and HA33/C are indicatedunderneath with red stars and blue circles, respectively. (c) Same as (b), but alignment of NTNHA with the b-trefoildomains of TeNTand BoNT/A (Hall-A strain) with the secondary structure elements of the BoNT/A (PDB code 3BTA).The residues at the receptor-binding sites of TeNT are indicated underneath with black circles.



residue, the sequences from the type C and DHA33s (except Yoichi) have an Asp residue. Thisvariation might account for the differences in thetypes of carbohydrates that bind to these twogroups of HA33s (i.e. A, B versus C, D). Furthersupport for the presence of a carbohydrate-bindingsite at the 2g repeat in HA33/C includes the factthat a C-terminally truncated variant of HA33/Clacking the 2g repeat loses sugar-binding activity.18

A superposition of the HA33 1a repeats for the Aand C serotypes (not shown) reveals that HA33/Ais not expected to bind carbohydrates at this site.Only the 2gHA33/A b-trefoil motif possesses all ofthe structural elements required for carbohydraterecognition. The absence of a second carbohydrate-binding site indicates that the HA activity of the16 S and 19 S progenitor toxin complexes requiresHA33/A oligomerization, either with itself or withother NAPs, in order to create multivalent sugar-binding sites as previously suggested.35

Role of HA33/A protein in the progenitor toxincomplexes

Previous studies suggest that the HA33/Aprotein is a dimer in solution as determined bygel filtration and mass spectrometry.22 Secondly,analysis of the protein stoichiometry in the 16 S and19 S progenitor toxins complexes led to the hypo-thesis that a HA33/A dimer links two molecules of16 S neurotoxin complex to form the 19 S progenitortoxin complex.11,36 However, an analysis of thecrystal packing of the HA33/A molecules revealsonly limited surface contacts between the twomolecules. This interface accounts for only 10.1%of the buried surface area, consisting of mostlyhydrophilic interactions such as residues Gln34,Asn76, Pro78, Thr79, Asn81, Gln85, His90, Lys128,and Thr131 of the N-terminal domain from oneHA33/A molecule interacting with residues Met64,Ile66, His67, Asp245, and Asp247 of both domainsfrom the other HA33/A molecule. Given such asmall and weak dimer interface, it is unlikely thatthe crystallographic interface is physiologicallysignificant and is unlikely to cross-link two mol-ecules of 16 S toxin to form the 19 S progenitor toxincomplex. Furthermore, to our knowledge thecrystallographic dimer interface is unlike that seenin other b-trefoil-containing structures. Nonethe-less, further functional studies are needed toconclusively determine the structural role ofHA33/A in forming the assembled progenitortoxin complexes.

Reoccurring b-trefoil fold in the progenitor toxincomplex

In addition to the b-trefoil domains found inHA33, BoNT/A and BoNT/B have previouslybeen shown to contain a single b-trefoil fold atthe C-terminal binding domain of the heavychain.37,38 Fold recognition methods alsodetect this b-trefoil domain in the NAPs HA17

and NTNHA (FFAS scores K23 and K208, respect-ively; scores below K9.5 typically indicate signifi-cant similarity with less than 3% of falsepositives).39 Based on this analysis, the b-trefoilmotif collectively forms nearly half of the massof the 900 kDa progenitor toxin serotype Acomplex (Figure 5(a)). The importance of sequenceconservation of BoNTwith this fold and gangliosiderecognition has been reported;40 however, thesignificance of the trefoil fold within the context ofthe NAPs and the progenitor complex has yet tobe addressed. Based on the HA33/A structure, asequence alignment of HA17/A with the N and C-terminal domains of HA33/A (each with an overallsequence similarity of 28%) allows the b-trefoilrepeats in HA17/A to be identified and assessed forsugar-binding conservation (Figure 5(b)). Most ofthe sequence conservation between HA17/A andHA33/A N and C-terminal domains is located inthe g repeats. Three residues that are importantfor carbohydrate recognition in the HA33/A C-terminal domain Asn263, Tyr265, and Asn285 areconserved with Asn113, Tyr115, and Asn138 inHA17/A. However, the three other residues ofGln268, Gln277, and Phe279 of HA33/A that formthe rest of the carbohydrate-binding site have noequivalent counterpart in Ile121, Leu129, andAsn131 of HA17/A suggesting that HA17/A lacksthe necessary molecular requirements for sugar-binding. Further support that HA17 does not bindcarbohydrates comes from experiments with GSTfusion proteins of HA17/A and HA17/C, whichdid not bind to erythrocytes and intestinal micro-villi.12,19 In addition, it has been reported thatNTNHA also does not possess HA activity and thatit does not bind to erythrocytes, but NTNHA is acritical component in formation of HA-positiveprogenitor toxin complexes.43 NTNHA showssignificant sequence similarity to the b-trefoil-containing binding domains of BoNT/A andtetanus neurotoxin (TeNT) from Clostridium tetaniwith sequence identities in the a and b repeats of16% and 18%, respectively, and an overall sequencesimilarity of 31%. The sequence comparison of theirb-trefoil subdomains (Figure 5(c)) offers clues intowhy NTNHA does not bind carbohydrates. Thecrystal structure of TeNT bound to a GT1bganglioside receptor analog (Gal4-GalNAc3) at theg repeat provides a structural prototype for thecharacterization of the ganglioside binding site,41

and has proven crucial in identifying the ganglio-side-binding site for BoNT/A and BoNT/B.37,42 TheTeNT key residues of Ser1287, Trp1289, and Tyr1290are conserved in BoNT/A with residues Ser1264,Trp1266, and Tyr1267, but are poorly conserved inNTNHA with corresponding residues of Asn1180,Asp1182, and Met1183.

The identification of the b-trefoil domain in amajority of the components of the 900 kDa serotypeA progenitor toxin suggests this fold is a result ofstructural domain duplication. It also allows one topostulate models in determining the molecularcomposition of the components of the progenitor


toxin complexes. With most of the molecular piecesof the proverbial progenitor toxin puzzle now athand, our future work will focus on determiningthe intimate protein–protein contacts that make upthe progenitor toxin complexes essential for BoNTprotection, uptake, and activation.

Materials and Methods

Protein production and crystallization

HA33/A from the C. botulinumHall strain was purifiedas described,7 and kindly provided by the DasGupta/Johnson laboratories at the University of Wisconsin.Briefly, the ammonium sulfate-precipitated protein wasdissolved by dialysis against a buffer containing 10 mMsodium acetate (pH 5.5), 200 mM (NH4)2SO4 and con-centrated to 12 mg/ml by ultrafiltration. The protein wascrystallized by the hanging-drop, vapor-diffusion methodusing equal volumes of protein and reservoir solution.The crystallization reservoir solution contained 20% (w/v) PEG 4000, 15% (v/v) isopropanol, 200 mM Li2SO4, and0.1 M Hepes at pH 7.5. Crystals were stabilized in freshlyprepared reservoir solution containing 30% isopropanolfor approximately 15 seconds prior to cryo-cooling inliquid nitrogen. The crystals were indexed in theorthorhombic space group P21212 (Table 1).

Data collection

Diffraction data were collected at Stanford SynchrotronRadiation Laboratory (SSRL, Stanford, USA) on beamline9-1 (Table 1). Data were integrated, reduced, and scaledusing Denzo and Scalepack.44 Data statistics are sum-marized in Table 1.

Structure solution and refinement

Four homology models of the individual HA33/Ab-trefoil domains were constructed with programWhatif,45 based on the FFAS41 alignment with HA33/C(PDB code 1QXM, sequence identity 38%). Multiplemolecular replacement searches were carried out inprogramMOLREP46 on a 80 CPU Linux cluster expectingfour copies of a single HA33/A domain in the asymmetricunit. Out of 160MR trials only four obtained with a modelbased on the N-terminal domain of the HA33/C (chain B)structure had values of Rfree below 0.50 after rigid bodyand restrained refinement in Refmac5.47 Subsequentmanual rebuilding and refinement was carried out inprograms O48 and Refmac5. Refinement statistics aresummarized in Table 1. The final model includes twoprotein molecules and 564 water molecules. No electrondensity was observed for residues 1–9. Analysis of thestereochemical quality of the model was accomplishedusing the AutoDepInputTool†. Figures were preparedwith PYMOL (DeLano Scientific).

Ligand docking

The probable binding site for the carbohydrate sub-strate was obtained by superimposing the b-trefoildomain of the lactose-bound ricin structure34 with the

† http://deposit.pdb.org/adit/

C-terminal trefoil domain of HA33/A using the programTOP.23

Sequence alignments

NAP sequences were aligned using FFAS41 and Clustal-W.48 A gap opening penalty of ten, gap extension penaltyof 0.05, and gap separation distance of eight were usedwith the BLOSUM62 matrix. The alignment Figures wereprepared using ESPript using DSSP secondary structureassignments.49

Protein Data Bank accession code

Atomic coordinates and experimental structure factorsof HA33/A have been deposited with the PDB and areaccessible under the code 1YBI.

Acknowledgements

We thank Angela Walker and Dr Marianne Patchfor assistance in manuscript preparation andBibhuti DasGupta, Eric Johnson and Bill Tepp atthe University of Wisconsin for HA33 proteinsamples. This work was supported by contractDAMD17-00-C-0040 from the Department of theArmy and, in part, by National Institutes of HealthProtein Structure Initiative Grant GM62411 from theNational Institute of General Medical Sciences(http://www.nigms.nih.gov). Portions of thisresearch were carried out at the Stanford Synchro-tron Radiation Laboratory (SSRL), a national userfacility operated by Stanford University on behalf ofthe US Department of Energy, Office of Basic EnergySciences. The SSRL Structural Molecular BiologyProgram is supported by the Department of Energy,Office of Biological and Environmental Research,and by the National Institutes of Health (NationalCenter for Research Resources, Biomedical Tech-nology Program, and the National Institute ofGeneral Medical Sciences).

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Edited by M. Guss

(Received 28 October 2004; received in revised form 15 December 2004; accepted 16 December 2004)

doi:10.1016/j.jmb.2004.12.039 j. mol. biol. - dtic · robert schwarzenbacher2, michael a. hanson1,...

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