doi:10.1016/j.jmb.2009.07.037 J. Mol. Biol. (2009) 392, 614–629

Available online at www.sciencedirect.com

The Protective Component of Forms Functional Octameric Complexes

Alexander F. Kintzer1,2, Katie L. Thoren1, Harry J. Sterling1, Ken C. Dong1, Geoffrey K. Feld1, Iok I. Tang1, Teri T. Zhang3, Evan R. Williams1,2, James M. Berger2,3 and Bryan A. Krantz1,2,3⁎

1Department of Chemistry, The assembly of bacterial and factors is critical to their University of California, function, but the regulation of assembly during infection has not been Berkeley, CA 94720, USA studied. We begin to address this question using anthrax toxin as a model. The protective antigen (PA) component of the toxin assembles into ring- 2California Institute for shaped homooligomers that bind the two other components of the Quantitative Biomedical toxin, lethal factor (LF) and edema factor (EF), to form toxic complexes. To Research (QB3), disrupt the host, these toxic complexes are endocytosed, such that the PA University of California, oligomer forms a membrane-spanning channel that LF and EF translocate Berkeley, CA 94720, USA through to enter the . Using single-channel electrophysiology, we 3Department of Molecular & show that PA channels contain two populations of conductance states, Cell Biology, University of which correspond to two different PA pre-channel oligomers observed by California, Berkeley, electron microscopy—the well-described heptamer and a novel octamer. CA 94720, USA Mass spectrometry demonstrates that the PA octamer binds four LFs, and assembly routes leading to the octamer are populated with even-numbered, Received 16 June 2009; dimeric and tetrameric, PA intermediates. Both heptameric and octameric received in revised form PA complexes can translocate LF and EF with similar rates and efficiencies. 11 July 2009; Here, we report a 3.2-Å crystal structure of the PA octamer. The octamer accepted 13 July 2009 comprises ∼20–30% of the oligomers on cells, but outside of the cell, the Available online octamer is more stable than the heptamer under physiological pH. Thus, the 20 July 2009 PA octamer is a physiological, stable, and active assembly state capable of forming lethal toxins that may withstand the hostile conditions encountered in the bloodstream. This assembly mechanism may provide a novel means to control cytotoxicity. © 2009 Elsevier Ltd. All rights reserved. Keywords: anthrax toxin; cell-surface assembly; translocation; lethal toxin; Edited by J. Bowie oligomerization

Introduction cell surfaces to make noncovalent, toxic complexes (Supplementary Data Fig. S1). Protective antigen (PA) 1 is the 83 kDa, cell-binding B component that Anthrax toxin (Atx) is a binary toxin (A2B) comprised of three nontoxic that are secreted ultimately forms a translocase channel for the two ∼ — by and combine on eukaryotic host 90 kDa, enzymatically active, A components lethal factor (LF) and edema factor (EF). Following secretion, PA binds to the host cell via one of two *Corresponding author. Department of Chemistry, known Atx receptors, ATR12 and ATR23, and is then University of California, Berkeley, CA 94720, USA. cleaved by a -type to make the E-mail address: [email protected]. proteolytically activated form, called nPA. The Abbreviations used: Atx, anthrax toxin; PA, protective 63 kDa, receptor-bound portion of nPA then self- antigen; LF, lethal factor; EF, edema factor; WT, wild type; assembles into a ring-shaped homooligomer, or pre- EM, electron microscopy; nanoESI, nanoelectrospray channel, which has been shown to be heptameric.4-8 ionization; MS, mass spectrometry; dsATR, dimeric The pre-channel can bind LF or EF to make lethal or soluble Atx receptor; msATR, monomeric soluble ATR; edema toxins, respectively. These toxin complexes are D4, domain 4; MIL, membrane insertion loop; UBB, endocytosed and brought to an acidic compartment.9 universal bilayer buffer. Under acidic conditions, the PA pre-channel inserts

0022-2836/$ - see front matter © 2009 Elsevier Ltd. All rights reserved. Protective Antigen Forms Functional Octamers 615 into the membrane to form a translocase channel.10 LF assembly mechanism, since PA oligomers are and EF translocate through the channel to enter the believed to be odd-numbered and heptameric.23,24 cytosol, where they catalyze reactions that disrupt the ATR2 and Atx were recently implicated as factors that host cell (Supplementary Data Fig. S1).11 help the B. anthracis bacterium escape the acidic Analogous to the staphylococcal α-hemolysin phagolysosome following spore germination, sug- pore,12 PA assumes a similar mushroom-shaped gesting Atx components may assemble in hostile architecture8 and β-barrel transmembrane motif.13,14 environments as well.25 Another potentially interest- The β-barrel of the PA channel is similarly narrow ing extracellular assembly mechanism has become (∼15-Å in diameter8,15) but considerably longer apparent in the investigation of animals infected with (∼100 Å) than the α-hemolysin. The narrow channel B. anthracis, where it has been shown that anthrax requires LF and EF to unfold before translocation.16-18 toxin accumulates in the blood of animals.26 Specifi- Acidic endosomal conditions serve two purposes: cally, LF is found alongside proteolytically activated 27,28 first, they destabilize LF and EF by acid nPA, implying that the PA is potentiated for denaturation;18 and second, they drive translocation assembly. Relative to toxin produced in vitro, the toxin via a proton gradient (ΔpH).11 PA also contains a produced in vivo (i.e., isolated from the blood of required ring of phenylalanine side chains, or ϕ animals suffering from anthrax) forms unique assem- clamp, which catalyzes translocation,19 exemplifying blies, as evidenced by their unique resistance to how the structure of the channel is crucial to its antibody binding, and the in vivo-derived toxin is translocase function. more lethal.29 We probe assembly in various cellular Assembly is paramount to Atx function20,21 and its and extracellular contexts using electrophysiology, cellular internalization.22 ATR receptors are interna- electron microscopy, mass spectrometry, and crystal- lized slowly by the host cell, but PA-bound ATR can lography, and we conclude that the activity of the internalize rapidly once it dimerizes,22 making proper toxin may be regulated through assembly, potentially oligomerization a critical step in the internalization affecting the degree of cytotoxicity throughout the pathway. ATR is dimeric, further complicating the stages of anthrax.

Fig. 1. Heterogeneous PA chan- nel conductance distributions. (a) Two PA samples were analyzed: (i) PA is nicked by trypsin to make nPA; a20kDapiece(PA20) dissociates, allowing PA to oligomerize into the pre-channel on a Q-Sepharose col- umn, making QPA; and (ii) nPA is mixed with LFN to drive oligomer- ization, making nPA + LFN. Either pre-channel oligomer forms a channel upon inserting into the membrane. (b) Example of 200 Hz-filtered, sin- gle-channel data collected at a Δψ of 20 mV, 100 mM KCl, pH 6.60; γ values computed by γ=i/Δψ are listed next to each channel insertion. (c) Normalized histograms of the estimated single-channel γ values for the QPA and nPA + LFN samples. Data bins are 1 pS wide, and the number of channels, n,ineach sample are normalized for compar- ison. The samples, QPA (n =360; black bars) and nPA + LFN (n=107; red bars), are statistically distinct by a non-parametric, lower-tailed, Whitney-Mann test (p N0.95). (d) Histogram of all the pairwise differ- ences, δ, between measured γ values identified within the same mem- δ brane for the nPA + LFN sample. The histogram was fit to one (dotted line) and two Gaussian (continuous line) functions, δ σ √ π – δ2 σ2 σ √ π δ–μ σ 2 – δ μ σ 2 using A( )=A1/ 1 ( /2) exp( 2 / 1)+A2/ 2 ( /2) [exp(-2(( 2)/ 1) )+exp( 2(( + 2)/ 1) )], with R values of 0.89 and μ 0.96, respectively, yielding best-fit parameters: peak area A1 =470±80, A2 =140±40; mean, 2 =8 (±2) pS; and standard σ σ deviation, 1 =5.5 (±0.5) pS, 2 =9 (±2) pS. (See also Supplementary Data Table S2.) (e) Single-channel current records for smaller (black) and larger (red) PA channels in 100 mM KCl, pH 6.6. Arrows indicate the two respective channel sizes. Data were acquired at 400 Hz and filtered further with a 100 point/σ Gaussian filter to better reveal conductance sub-states. 616 Protective Antigen Forms Functional Octamers

Results significantly different (pN0.95). Finally, three separate QPA samples contained a consistently lower mean ∼ conductance of 95 pS relative to two other nPA + LFN PA can form two different channel sizes samples, which had a mean conductance of ∼98 pS. Thus, the method of assembly shifts the single- PA channels were inserted into planar lipid bilayers channel conductance distribution (Fig. 1c). following one of two different assembly methods Inherent variability in bilayer thickness, combined (outlined in Fig. 1a). A pre-oligomerized sample, with the presence of another larger-conductance called QPA (trypsin-nicked PA assembled on Q- conformation, likely contributes to the broad aggre- Sepharose anion-exchange resin10) was applied to gate distributions (Fig. 1c). To circumvent this the bilayer, and discrete single-channel steps were problem, we examined the discrete differences in observed (Fig. 1b). QPA channels had a mean channel conductance within each membrane by conductance of 95.5 pS (n =360). Single-channel tabulating the set of all the pairwise differences (δ) γ γ γ –γ ≠ conductance values ( ) for wild type (WT) PA in values/membrane, { i j}, where i j.Ahisto- – 30 δ channels were reported in the range 85 110 pS. We gram of values recorded for the nPA + LFN sample observed that the QPA sample contained two discrete shows that two sizes of channel conductance are sizes of channel: a prevalent, smaller one and a rarer, present, as the distribution fits best to a two-Gaussian larger one. The overall γ-value distribution recorded distribution (Fig. 1d). Based upon our fit, these two from many individual membranes was broad (Fig. 1c). populations of γ values differ from one another by 8 To determine if the method of PA assembly affected (±2) pS, or about ±10%, where the larger conductance the γ-value distribution, we studied trypsin-nicked state represents 25 (±6)% of the population. This result PA (nPA) samples assembled in the presence of the led us to conclude that PA forms two discrete amino-terminal domain of LF (LFN), called nPA + LFN. conductance states that may be populated differen- Again, larger-conductance channels appeared among tially, depending upon the method of assembly. smaller-conductance channels in two discrete sizes, We tested whether large-conductance channels were but the frequency of observing larger channels a substate of small-conductance channels due to a γ increased. The mean value for nPA + LFN (98 pS, conformational rearrangement. To test this possibility, ∼ n=107) increased relative to that observed for QPA. we measured 6 min recordings of single channels Moreover, a lower-tailed Whitney-Mann test (WMT) (Fig. 1e). While fluctuations to a more conducting showed that QPA and nPA + LFN distributions are substate are observed, these fluctuations are relatively

Fig. 2. EM studies of heptameric and octameric PA. EM images of negatively stained samples of WT PA oligomers assembled either in vitro (upper panels a–f) or in vivo on cell surfaces (lower panel). Representative class averages of octamers (left) and heptamers (right) are shown. The total number of particles assessed, n, and the relative percentages of heptamers and octamers are given. The proportions of heptamers and octamers are indicated by bars colored black and red, respectively. In vitro samples include: (a) nPA assembled on an anion-exchange column (QPA; n=12589; 98% heptamer; 2% octamer); (b) nPA assembled in the presence of soluble dimeric ATR2 at 4:1 stoichiometry (+dsATR; n=837; 74% heptamer; 26% octamer); (c) nPA assembled in the presence of soluble monomeric ATR2 (+msATR; n=9401; 99% heptamer; 1% octamer); (d) nPA assembled in the presence of LFN (+LFN; n=8409; 72% heptamer; 28% octamer); (e) nPA assembled in the presence of EFN;(+EFN; n=5363; 81% heptamer; 19% octamer); and (f) disulfide-bonded PA S170C assembled on an anion-exchange column (QPA S170C; n=2933; 78% heptamer; 22% octamer). Oligomers extracted from cells: (g) His6-PA assembled on cells expressing ATR2 (C-CHO; n=4729; 74% heptamer; 26% octamer), where three classes of octamers and heptamers are shown, resulting from reference-based analysis. The scale bar representing 5 nm (shown in a) is consistent for all images. The percentages of oligomers are means of reference-free and crystal structure-referenced alignments unless noted otherwise. (Specific percentages are listed in Supplementary Data Table S2.) Protective Antigen Forms Functional Octamers 617 small; and large-conductance channels (N102 pS) did species, 537,082 (±186) Da and 631,167 (±217) Da, b not interconvert to smaller ones ( 95 pS) during these corresponding to the oligomers, PA7(LFN)3 and PA8 and all other recordings. Therefore, either two unique (LFN)4, respectively, as well as two minor (and likely channel sizes exist in PA samples or the timescale of intermediate) complexes of 158,193 (±38) Da and the conformational rearrangements between the large- 315,395 (±27) Da, corresponding to PA2LFN and PA4 and small-conductance states is slow. (LFN)2, respectively (Supplementary Data Table S1). 6 The PA7(LFN)3 species has been reported. Moreover, Electron microscopy reveals two PA oligomers similar results were obtained when full-length LF and EF were used as ligands (data not shown). The PA8 ∼ Under the assumption that the 10% difference in (LFN)4 complex is novel and corroborates the presence conductance states may correspond to slow-timescale of the octameric PA species, and it demonstrates that structural changes in the channel diameter,19,31 we an octamer can carry a payload of four LFs or EFs— examined the QPA oligomers for structural hetero- one more than its heptameric counterpart. Finally, the geneity by electron microscopy (EM). Reference-free previously undetected forms, PA2LFN and PA4(LFN)2, analysis of ∼104 negatively stained particles identi- suggest a pathway of assembly for the octamer via fied two classes of ring-shaped oligomers—the even-numbered intermediates. heptamer and a novel octamer (Fig. 2a). We then probed the kinetics of PA assembly with nanoESI-MS (Fig. 3b). Here, oligomerization was Mass spectrometry reveals Atx heterogeneity initiated by mixing nPA and LFN in a 1:1 molar ratio, using the assembly method described in the To further establish the observed heterogeneity, we single-channel studies. The ion abundances in the ESI used nanoelectrospray ionization mass spectrometry mass spectra were recorded at several time points for ∼ (nanoESI-MS). We assembled WT nPA with WT LFN 1 h. The abundances of both PA7(LFN)3 and PA8 and then analyzed the mixtures by nanoESI-MS (LFN)4 increase concomitantly. Also, the appearance (Fig. 3a). We identified two large molecular mass of either oligomer is correlated to a decrease in the

Fig. 3. Mass spectrometry stu- dies of Atx assembly. (a) NanoESI mass spectrum of nPA co-assembled with LFN. Multiple charge-state dis- tributions are observed that corre- spond to five different PA-LFN complexes. Charge state and mole- cular mass were calculated as 69 described. The PA7(LFN)3 distri- bution clearly has the highest relative abundance and is shown above labeled with charge states. The insets show distributions of less intense oligomers PA7(LFN)2 and PA8(LFN)4, and low abundances of PA2LFN and PA4(LFN)2 that may be stable inter- mediates in the formation of the higher-order complexes. The mole- cular mass was assigned from the charge-state distribution that resulted in the smallest standard deviation in calculated molecular mass. (Supplementary Data Table S1 summarizes the observed masses and describes the solvent correction.) (b) Assembly kinetics for the 63 kDa PA monomer, PA7(LFN)3,PA8(LFN)4, and PA4(LFN)2 from a solution of nPA mixed with excess LFN.Datafor all but PA4(LFN)2 were fit with exponential functions to guide the eye. The appearance of the oligo- mers, PA7(LFN)3 and PA8(LFN)4, coincides with the disappearance of PA monomer. Data at early times indicate a rapid increase in the abundance of PA4(LFN)2 followed by slow decay for t≥5 min., suggesting it is an intermediate in the formation of the higher-order complexes; an interpolated ∼ line is given for PA4(LFN)2 also to guide the eye. All four analytes reach steady-state levels in 30 min. 618 Protective Antigen Forms Functional Octamers

relative ion abundances for free PA monomer and PA4 were harvested, lysed in detergent and purified on (LFN)2 species. We observed an initial burst in the His6-affinity resin. SDS-PAGE of His6-pure extracts abundance of the PA4(LFN)2 species followed by a were Western blotted, confirming that the purified subsequent decrease with time; the decrease was fraction contained His6-PA in the 63 kDa form concomitant with the increase in the formation of PA7 (Supplementary Data Fig. S3). (LFN)3 and PA8(LFN)4.ThetrendinPA4(LFN)2 EM images of these extracts identified oligomeric abundance suggests that it is a stable intermediate rings consistent with the size and shape of PA in the formation of the higher-order complexes. PA8 oligomers; however, these complexes were less well (LFN)4, however, is not an intermediate species in the oriented than the other in vitro samples, perhaps due heptamerization pathway, since its relative abun- to the presence of cellular components, like full-length dance did not decrease during the experiment. ATR, which may form the observed extensions from the oligomeric structure. Several tilted class averages Stabilizing dimeric PA intermediates promotes were obtained to capture this heterogeneity. From octamer formation these, we determined that ∼20–30% of the oligomers were octameric (Fig. 2g). Control experiments show ∼ To further investigate how even-numbered inter- that His6-PA on its own forms 1% octamer mediates influence assembly, we conducted EM (Supplementary Data Fig. S2A), confirming that the studies using PA oligomers prepared in the presence His6 tag is not responsible for the high levels of of a dimeric soluble Atx receptor domain (dsATR), octamer observed on cells. We examined extracts from LFN,orEFN (EF's PA-binding, amino-terminal T-CHO cells (expressing ATR1) and from C-CHO cells domain). A dimeric ATR construct was also chosen that were treated with both PA and LFN;andwefound based upon evidence that the receptor may exist in a that octamer levels were 20∼30% under all cell-surface dimeric state on cell surfaces.24 PA pre-complexed to conditions tested (data not shown). Thus, PA forms a dsATR (Fig. 2b), but not to monomeric ATR (msATR) mixture of octamers and heptamers on cell surfaces. (Fig. 2c), showed increased proportions of octamers upon assembly. Further studies demonstrated that Crystal structure of the octamer when dsATR was loaded under less saturating conditions, the octamer levels decreased (Supplemen- Mutations were then introduced into PA to probe tary Data Fig. S2B). Thus the more saturating the molecular mechanism of assembly. The most conditions allow the dsATR sites to fully populate interesting mutations identified disrupted the inter- with PA before assembly, which increases the prob- face between two PA subunits at the interface of ability of forming the even-numbered, octameric form. domain 4 (D4) and the neighboring membrane LF or EF can form a ternary complex with PA insertion loop (MIL) in the adjacent PA subunit. One dimers,21 and we have observed this species in our mutant replaced the MIL (i.e., residues 305–324) with mass spectrometry experiments (Fig. 3a; Supplemen- a type II turn, deleting all possible hydrophobic tary Data Table S1). When LFN or EFN is used to interactions between L668 of D4 and F313 and F314 in ∼ ΔMIL assemble nPA into oligomers (Fig. 2d and e), 25% of the MIL. This type of PA oligomer (PA )was the population became octameric. This increase is 5- to reported to form heptameric rings;33 but our version 10-fold more than that observed for PA oligomerized made an enriched source of octameric rings, as in the absence of LF or EF . Also the S170C PA observed by EM. N N Δ mutant, which can form a disulfide-bonded homo- We used PA MIL oligomers as a concentrated source dimer (as modeled in Supplementary Data Fig. S2C), of octameric PA, and solved the crystal structure of the increases the proportion of octamers relative to octamer to 3.2-Å resolution (Fig. 4a; Supplementary unlinked WT PA (Fig. 2f). Finally, our reference-free Data Table S3). Molecular replacement identified eight EM analysis was supported, when possible, with PA monomers arranged as a ring in the asymmetric reference-based analysis, mass spectrometry, and unit. We find that the octamer is best described as electrophysiology (Supplementary Data Table S2). having fourfold noncrystallographic symmetry, Therefore, we conclude that the observation of an becausetherearetwotypesofPAmonomer increase in the proportion of octamers was the result conformations, called A and B (Fig. 4c), which occupy of increasing the population of even-numbered PA2- alternating positions around the ring (Fig. 4d). From precursor complexes. structural alignments, these two conformers mainly differ in the orientation of D4 (Fig. 4c). This confor- PA forms octamers on cells mational heterogeneity is notable, since D4 interacts with the MIL and may be a structural feature in the These in vitro results led us to probe the oligomer- assembly mechanism. The flexibility of D4 is also ization pathway on cell surfaces. We used a Chinese consistent with the higher than average B-factors hamster ovary (CHO) cell line, expressing ATR2, observed there and in the MIL of the heptamer 32 7 called C-CHO. PAwith a carboxy-terminal His6 tag, structure. Thus, plasticity in these two regions may called His6-PA, was added to the extracellular provide a mechanism for octamer formation, where medium of cultured C-CHO cells and incubated to modulation of the structure in these regions may assemble. Endosomal acidification was blocked with occur either (i) via ATR binding, which reorients D4 ammonium chloride to prevent the conversion of the relative to D27,34 or (ii) via exposure to a more acidic pre-channel oligomers to the channel state. The cells pH, which may alter the conformation of the MIL. Protective Antigen Forms Functional Octamers 619

Δ Fig. 4. X-ray crystal structure of PA in the octameric oligomerization state. Axial views of: (a) the PA MIL octamer (PDB 3HVD) side-by-side with (b) the WT PA heptamer (PDB 1TZO).7 Monomer subunit chains are colored uniquely. The MIL is depicted with spheres in the latter structure of WT heptamer. (c) A backbone alignment of two adjacent PA monomers, chains A and B, called conformation A (red) and B (blue), showing the displacement of D4. (d) Superimposed on a surface rendering of half an octamer is the square planar arrangement of symmetrically related A and B conformers calculated from the center of mass of each chain. Chains A, C, E, G are conformation A; and chains B, D, F and H are conformation B. Adjacent A-B pair center of masses are 64.3 (±0.1) Å apart at angles of 90 (±0.1)°. Domains are colored as: D1' (magenta), D2 (green), D3 (gold), and D4 (blue). (e) An A-B oligomerization interface split apart to compare relative differences in surface area burial at the oligomerization interface of the heptamer and octamer structure among the four domains. The domains (upper panel) are colored as in d. The relative degrees of surface area buried (lower panel) are colored as follows: green, surface buried equally (i.e., to within 10%) in either structure; red, surface buried 10% more buried in the heptamer; blue, surface buried 10% more in the octamer; and white, surface buried b75% in both structures. All molecular graphics were rendered using CHIMERA.64

Consistent with our single-channel data, the mean least 350 Å2 more surface area per monomer than the pore diameter of the octamer pre-channel is ∼10% heptamer when the MIL is present. Thus, WToctamer larger than that of the heptamer (46 (±4) Å and 40 (±4) will bury ∼6100 Å2 of additional surface relative to Å, respectively) (Fig. 4a and b). The residues lining the the heptamer, when including the eighth subunit and pre-channel of the octamer are similar to those in the additional increases in burial per monomer. heptamer; all types of chemistry are represented, The angles between n adjacent monomers arranged though the charge composition is more anionic symmetrically about a ring (θ) are ideally equal to overall. The octamer buries ∼3300 Å2 of solvent- 180–360/n, and these angles are widened by ∼6° for accessible surface area per monomer, which is the octamer (Fig. 4a) with respect to the heptamer ∼800 Å2 less than the heptamer; the MIL and its (Fig. 4b). We found this is accomplished by a subtle interactions with its neighboring docking groove in shift in the inter-monomer packing interfaces (Fig. 4e), D4 largely account for this difference. The octamer where the octamer buries more surface area in regions forms additional contacts (not found in the heptamer) proximal to the central channel. During assembly, the at sites more proximal to the central pore, accounting steric mass of the MIL may act as a nonspecific wedge for ∼350 Å2 of additional buried surface per dimer that effectively nudges the adjacent monomer toward interface (Fig. 4e). For WT octameric PA, the MIL amoreacuteθ in the heptamer. Thus, in the absence of Δ should form analogous interactions with this D4- this constraint, PA MIL is able to relax θ to achieve the docking groove; therefore, the octamer may bury at octameric configuration. The MIL has two functions: 620 Protective Antigen Forms Functional Octamers

(i) to form the channel in the membrane; and (ii) to found that the sample could be purified by S400 gel- control the oligomerization number of pre-channel filtration, generating ∼90% pure octamer, as judged assemblies. by EM and nanoESI-MS (Fig. 5b; Supplementary Data Table S2). Therefore, we conclude that the heptameric The relative stability of the PA heptamer and form assembled and was subsequently inactivated by octamer aggregation under physiological conditions; how- ever, the octameric form persisted as a soluble To test whether the stability of octameric and complex. heptameric complexes was different, we incubated mixtures of heptameric and octameric PA at different Translocase activity of octameric channels temperatures and pH. Under mildly acidic conditions (pH 5.7), the heptameric form precipitated almost The pre-channel PA oligomer forms the translocase quantitatively as judged by both EM and nanoESI-MS channel state at acidic pH.10,33 The β-barrel, which (Fig. 5a; Supplementary Data Table S2); however, the penetrates the membrane, is comprised of the MIL octameric form maintained its solubility and per- and adjacent β-strands in D2. Our structure reveals sisted. This difference provided us with the means to that the contacts made in D1′ (solvent-accessible isolate the octameric form for crystallization. Since surface area of ∼1100 Å2) and those immediately Δ these experiments used the PA MIL construct, we adjacent to it in D2 (∼1400 Å2) are largely identical tested the relative stability of WT PA oligomers and are sufficient to form and maintain stable formed in the presence of LFN at physiological pH. oligomeric complexes even when the MIL is not After assembly under physiological conditions, we present. Therefore, the octamer may form channels,

Fig. 5. Octameric and heptameric PA stability and activity. Negatively-stained EM class-average images of PA heptamers and octamers before and after a reduction of pH and/or change in the temperature and solvent. The initial Δ conditions in a and b are pH 8 and 0 °C. (a) Heptameric and octameric PA MIL before (left) and after (right) exposure to pH 5.7, 7% ethanol, 4 °C. Before exposure, 25% octamer, 75% heptamer, n=10409. After exposure, 89% octamer, 11% heptamer, n=14516. (b) Heptameric and octameric WT PA oligomer complexes with LFN before (left) and after (right) exposure to pH 7, 37 °C. Before exposure, 28% octamer, 73% heptamer, n=8409. After exposure, 91% octamer, 9% heptamer, n=1084. Relative percentages are given by the bars at the right for heptamers (black) and octamers (red). (c) Ensemble translocation records measured using planar lipid bilayer electrophysiology. The panels compare the N relative translocase activities two types of samples: QPA + LFN, which is 95% heptameric (black); and nPA + LFN, which ∼ had been purified and shown to contain 90% octamer (red). Four substrates (LF, LFN, EF, or EFN) were used, and each was translocated at the indicated Δψ and ΔpH conditions. Records shown are the average of a set of three repetitions. The y-axes are normalized to the fraction of substrate-blocked channels that become unblocked due to translocation. Rates are Δψ Δ given as t½ (time for half of the substrate to translocate). LF translocated at =30 mV, pH=1 using heptameric Δψ (t½ =57 s) and octameric PA (t½ =96 s). LFN translocated at =50 mV using heptameric (t½ =16 s) and octameric PA Δψ Δ (t½ =12 s). EF translocated at =50 mV, pH=1 using heptameric (t½ =50 s) and octameric PA (t½ =66 s). EFN Δψ translocated at =60 mV using heptameric (t½ =137 s) and octameric PA (t½ =135 s). (D) Left: Single-channel translocations of LFN at 50 mV through either a large (red) or small (black) channel. Black arrowheads on either end of the translocation indicate the beginning and end of each translocation. Right: Histogram profiles of portions of the conductance levels of the large and small channels for comparison of the open-channel conductance levels. Protective Antigen Forms Functional Octamers 621 and the two Gaussian populations of conductance functional translocases, and both the octameric and levels observed in planar bilayers (Fig. 1b and c) may heptameric forms of the PA channel are functional. reflect octamers and heptamers, which have inserted stably into membranes. We then tested this further and asked whether Discussion octamers and heptamers possess similar translocase ∼ activity. Here, we compared QPA, which is 98% We suggest that the octameric form of PA has not ∼ 4-8 heptameric, to a sample, nPA + LFN, 90% enriched in been observed before, because the standard octamer. In an ensemble translocation experiment, method of PA assembly, which uses an anion- channels are first inserted into a membrane at 20 mV; exchange column,10 yields oligomers virtually devoid the channels are loaded with LFN, which blocks the of octamers (Fig. 2a). Assembly in the presence of conductance; excess LFN is removed by perfusion; ligand, LFN,EFN, LF, EF or dimeric ATR, produces a – and then the LFN is translocated at a higher voltage, or 25 30% population of octameric oligomers in vitro Δψ . We observe that LF, EF, LFN,andEFN translocate (Figs. 1, 2b, and d). This heterogeneous assembly through the two different PA oligomers with similar mechanism is of physiological significance, because a efficiencies and rates (Fig. 5c). (Efficiency is the similar proportion of octameric oligomers is observed measured amplitude translocated divided by the on the surface of cells (Fig. 2g). Our crystal structure of maximum theoretical amplitude; the rate is estimated the octamer is not a regular octagon, but rather is by the time it takes for half of the protein to composed of four PA dimer pairs arranged in a square translocate). planar symmetry. This symmetry suggests that To further verify that larger and smaller channels dimeric PA intermediates populate the assembly are capable of translocating protein, we performed pathway, and indeed mass spectrometry reveals single-channel translocation experiments (Fig. 5d). that dimeric PA species are general assembly inter- Here, we formed single PA channels at 20 mV and mediates (Fig. 3b). While heptameric and octameric then added LFN. Once the channel closed due to LFN channels have similar translocase activity, octameric binding, the voltage was raised to 50 mV. Transloca- oligomers are more stable under physiological pH tion events (n∼10) were recorded until the channel and temperature (Fig. 5). We propose that these two became inactive. This procedure was repeated on a different oligomerization states are functionally rele- second channel obtained in the same membrane, vant to anthrax pathogenesis; namely, in the two which had a∼10% larger conductance. Therefore, we different environments in which the toxin assembles conclude that large and small PA channels are (Fig. 6). (i) On cell surfaces, assembly bottlenecks may

Fig. 6. Heterogeneous assembly mechanism may modulate toxin activity. (Top) On cells, PA may encounter dimeric ATR sites and assemble into PA2 and PA4 intermediates. Intermediates can combine to form either PA8 or PA7, which can load with EF and/or LF. (In principle, LF and EF may be involved in the mechanism as well to produce similar outcomes.) (Bottom) During extracellular or ATR-independent assembly, PA may encounter LF or EF, making the intermediates, PA2LFN and PA4(LFN)2, which then form either PA8(LFN)4 or PA7(LFN)3. Models of LF-PA complexes are derived from a theoretical model.70 Toxin activity is a combination of the oligomer's stability and translocase activity; instability may lead to the formation of inactive oligomeric complexes (*). 622 Protective Antigen Forms Functional Octamers be alleviated, allowing for proper of pathway was possible and the concentration of the PA functional complexes by having the two assembly monomer was low, then the dimeric ATR sites may routes. (ii) In blood plasma, PA may assemble before not be saturated, and assembly would be inhibited at reaching the cell surface and require a more stable the critical oligomerization step, allowing unas- oligomeric configuration, since the heptamer is sembled complexes to be endocytosed. On the other weakly stable under physiological conditions, espe- hand, supposing only the heptameric form was cially in the absence of its cellular receptor. possible and the concentration of the PA monomer was high, the dimeric ATR sites would be fully Cell-surface assembly and endocytosis saturated with PA, and assembly would be inhibited until one PA could dissociate from its ATR binding The current model for anthrax toxin assembly site, allowing for an odd number of subunits to proposes that PA assembles into a heptamer on cell assemble immediately before endocytosis. This latter surfaces expressing ATRs (Supplementary Data Fig. scenario is especially prohibited by knowing that the S1). Initially, PA binds a cell-surface receptor, ATR1 or dissociation lifetime for the PA–ATR2 interaction is of ATR2, is cleaved by a furin-type protease, and then the order of days.35 In summary, the mixed-oligomer- begins to assemble into the ring-shaped oligomer. ization mechanism alleviates these potential assembly While the current model predicts that these oligomers bottlenecks, allowing the toxin components to fully will be heptameric,1 the oligomeric states populated assemble and remain efficacious under the wide on cell surfaces have not been reported. We addressed dynamic range of extracellular concentrations of PA. this question specifically by extracting oligomers from cell surfaces and analyzing the distribution of Extracellular assembly in blood plasma oligomeric states by EM. We find that PA forms bothaheptamerandthenoveloctamerinratioof Anthrax toxin complexes were first identified in the ∼2:1, using cell lines expressing either ATR1 or ATR2. blood of B. anthracis-infected guinea pigs; and this What selection pressures on the toxin might maintain blood (after sterilization with antibiotic treatment) this dual-oligomerization-state mechanism? It is could be administered to a second uninfected animal thought that cell-surface assembly and endocytosis to impart its lethal affect.26 Throughout the later stages may be coupled processes. For example, the rate of of infection, both the proteolytically activated form of cellular internalization through endocytosis for either PA (nPA) and LF are found in the sera of infected free ATR or PA-bound ATR is slow; and this basal rate animal models.28 This form of PA, of course, may co- is accelerated only if the PA is pre-assembled into PA assemble with LF; however, the earlier study did not heptamers or PA-bound ATR subunits are aggregated test this possibility. We demonstrate here, using native by antibody cross-linking.22 Therefore, PA assembly gel electrophoresis, that PA can be activated proteo- and the corresponding aggregation of ATRs triggers lytically in bovine blood plasma, and stable toxin endocytosis. complexes can form (Supplementary Data Fig. S4). Our experiments extend the current understanding Under aqueous conditions, we have shown that of assembly and now we can improve the model. octamers form in the presence of LFN or EFN (Fig. 2d First, we assume that ATR1 and ATR2 are effectively and e). Thus, octamer formation is not limited to a dimeric on cell surfaces, because ATR-mediated receptor-dependent mechanism, and octamers may assembly in solution promotes only octameric assem- assemble extracellularly in blood plasma in an LF- or bly when the receptor is dimeric (Fig. 2bandc).A in an EF-dependent manner (Fig. 6). dimeric receptor model agrees with previous studies, which examined the aggregation state of ATR1's Toxin stability transmembrane helix.24 Therefore, we propose that the antibody cross-linking experiment reported by Recent work in vitro revealed that heptameric pre- Abrami et al.22 more likely involves a higher-order channel complexes are unstable under physiological aggregation of PA subunits beyond the formation of conditions, readily converting to the channel state.8 PA dimers. We anticipate this, because it is known that Here, we demonstrate that physiological tempera- LF and EF promote the dimerization of PA (Fig. 3);21 tures and pH lead to irreversible aggregation of the and an unintended consequence of this dimerization heptameric form, such that the octameric form is left may be that PA2LF or PA2EF ternary complexes are behind in stable, isolable complexes (Fig. 5a and b). endocytosed improperly before assembly into func- We think this effect may be linked to pH-dependent tional ring-shaped oligomeric complexes. Thus, an differences in the octameric pre-channel to channel efficient coupling of cell-surface assembly and endo- transition or inherent differences in the stability of cytosis would limit premature endocytosis of non- octameric toxin complexes. ATRs have been shown functional dimers and tetramers. previously to stabilize the pre-channel conformation A key function of the dual assembly mechanism under physiological conditions.7,33,36,37 Toxin com- may be to allow assembly under a range of plexes assembled in the bloodstream, however, do not concentrations of the PA monomer. If we consider benefit from ATR stabilization. Thus octameric com- the two extreme cases of either low or high plexes may represent a more stable toxin configura- concentrations of the PA monomer, assembly would tion that may persist in the blood of infected animals, be hindered if only one oligomeric state was possible. serving a primary role of maintaining cytotoxicity On one hand, supposing only the octameric assembly under physiological conditions. Protective Antigen Forms Functional Octamers 623

Octamer structure complexes may be inactivated under physiological conditions (Fig. 5a and b), whereas octameric toxin Our crystal structure shows that the octamer is a complexes may remain soluble and fully functional. ring of eight PA monomers, consisting of a square- We hypothesize that these physiological conditions planar arrangement of four PA dimers (Fig. 4c and d). are present when the toxin assembles in an ATR- This structure is in contrast to the near-perfect, regular independent manner (e.g., in the blood, lymph, or heptagon model of the heptamer.7 OurEMand phagolysosomal compartment25), and these condi- crystallographic studies of these toxin complexes tions may favor the octameric form. suggest that the octamer is composed of four pairs of PA subunits that are conformational heterodimers; Staphylococcal α-hemolysin and the heptamer may actually consist of three PA heterodimers and one asymmetric monomer (Fig. 2). The pathogenic factor produced by Our analysis of the precise symmetry of the heptamer aureus,calledα-hemolysin, is comprised of multiple by EM is limited by the resolution of the technique, copies of a 293 residue polypeptide.38 The assembled and further crystallographic studies are required. toxin ultimately forms a circular, β-barrel-type, ion- Nonetheless, the octamer and its tetrameric arrange- conducting pore with a mushroom-like architecture.12 ment of PA pairs suggests that dimeric PA inter- Interestingly, this pore-forming toxin is believed to mediates produced by interactions with LF, EF or a have multiple oligomeric states. EM,39-41 atomic force dimeric ATR subsequently assemble in a pairwise microscopy42 studies, and electrophysiology studies43 fashion to form the octamer (Figs. 2 and 6). determined that the α-hemolysin can be hexameric. On the basis of the crystal structure, we conclude However, chemical cross-linking studies,44 crystal- that the WT octamers should bury 6100 Å2 of lographic studies,12,44 single-molecule, photo-bleach- additional surface area per oligomer relative to the ing fluorescence methods,45 and electrophysiology heptamer, indicating that octamers may possess studies43 suggest the toxin forms a heptamer. How- greater inherent stability, preventing disassembly ever, the relative populations of the two oligomeric and/or premature conversion to the channel state. states and the conditions affecting these relative Each PA heterodimer, of course, is poised to bind an distributions have not been reported. LF/EF molecule (or a pair of ATRs), affording the octamer four binding sites and the heptamer three Pathogenesis (Figs. 3 and 6). The improved stability of octameric toxin complexes may result from full occupancy of Why may PA assemble into a mixture of oligomers? four heterodimeric, hydrophobic, LF-binding sites. B. anthracis optimizes its lifecycle and proliferate in its For the heptamer, however, only three such sites can host by the secretion of a toxin, which at the initial be occupied, leaving one PA subunit exposed. More- stages of pathogenesis, may first help germinated over, additional interfaces between adjacent LF escape from ,25 and second subunits in the octameric complex may create a allow a small population of bacteria to selectively novel ring of interfaces between adjacent LF subunits, suppress the . However, these toxin adding stability that cannot be attained in the conditions are nonlethal. During infection, B. anthracis heptameric complex (Fig. 6). Future structural studies secretes low, nearly undetectable concentrations of will elucidate how these two lethal toxin configura- the toxin components; at the latest stages of infection, tions may differ. We conclude that the added surface 100 μg/mL PA is detectable in the blood of infected burial and structural symmetry of the even-numbered animals.28 This large increase in the concentration of octameric configuration of toxin complexes may PA immediately precedes death in animal models.27 explain the improved stability over the heptameric Such a wide dynamic range in toxin levels suggests a configuration. relationship between toxin assembly and cytotoxicity. We propose that assembly of octameric oligomers on Toxin activity the cell surface alleviates a potential assembly bottle- neck imposed by ATR dimerization, while in the We propose that the general paradigm for the bloodstream, lymph, or phagolysosome25 the octa- aggregate physiological anthrax toxin activity is a meric toxin complexes may function as a more stable product of the catalytic rate of translocation and the species. Thus, heterogeneous assembly may function inherent stability of the two possible oligomeric in two key contexts: the known cell-surface assembly configurations: the heptamer and the octamer (Fig. pathway (Supplementary Data Fig. S1)andaputative 6). A secondary effect relates to the fact that the ATR-independent assembly pathway (Fig. 6; Supple- octameric configuration provides an additional LF- mentary Data Fig. S4). and EF-binding site per complex (Figs. 3a and 6), PA assembly pathways are further complicated by increasing the potential toxicity of saturated octa- dimerization of ATRs,23,24 and induced dimerization meric toxin complexes. We find in our planar lipid of PA subunits by LFN and EFN.Theheptameric bilayer translocation assays that the two oligomers assembly route may be efficient at low concentrations, translocate LF and EF at similar rates (Fig. 5c). where PA likely assembles predominantly from Therefore, we propose differences in toxin activity monomers. On the other hand, PA assembly would will likely result from intrinsic differences in oligomer be attenuated at high concentrations, where PA stability. Without ATR stabilization, heptameric toxin assembles via dimeric intermediates, in the absence 624 Protective Antigen Forms Functional Octamers of the octameric assembly route. Endocytosis coincides Soluble monomeric ATR2 22 with PA assembly, and a loss in toxin activity could A monomeric version of sATR2 (msATR) was made by occur if partially assembled complexes were endocy- subcloning residues 40–217 of ATR2 into pET15b (EMD tosed. Therefore, we reason that the observed hetero- Chemicals) via the NdeI and BamHI restriction sites, using a geneity (Fig. 2g) may serve to alleviate these potential pGEX expression clone.35 This construct has an amino- 34 assembly bottlenecks incurred during oligomerization. terminal His6 tag as described. The protein was similarly At later stages in anthrax infection, when the purified on a His6 affinity column and judged to be pure by concentrations of PA and LF are high, a significant SDS-PAGE, and it was confirmed to be monomeric by proportion of toxin complexes may assemble before nanoESI-MS. encountering cell surface ATRs. In fact, the blood of infected animals contains PA that is almost exclu- LFN and EFN sively proteolytically activated.28 Here, we demon- – Recombinant LFN (LF residues 1 263) and EFN (residues strate that PA, LF, and EF assemble into lethal and – 48 edema toxin complexes in bovine blood (Supplemen- 1 254 of EF) were over-expressed from pET15b constructs, tary Data Fig. S4). Assembly in the bloodstream and then purified from the cytosol using His6 affinity chromatography. LFN was further processed by incubating should produce a proportion of octameric toxin α with bovine -thrombin to remove the amino-terminal His6 complexes similar to that we observe in vitro (Fig. 2). tag and purified over Q-Sepharose anion-exchange resin as 19 However, we expect that octameric complexes could described. EFN was used with its His6 tag and not persist in these conditions, while heptameric com- processed any further. Each protein sample was verified plexes may form inactive aggregates. Therefore, by matrix-assisted laser desorption/ionization mass spec- octamer formation could provide a mechanism of trometry (MALDI-MS). overcoming these harsh, attenuating conditions encountered extracellularly (in the absence of an PA oligomerization on Q-Sepharose ATR). The proposed two-oligomer assembly model suggests that the anthrax toxin can modulate its PA monomers were first treated with trypsin at a 1:1000 aggregate activity through assembly due to these mass ratio for 15 min at room temperature, making nPA. The differences in toxin stability. Further structure/func- trypsin was blocked by the addition of a 1:100 mass ratio of soybean trypsin inhibitor and 1 mM phenylmethylsulpho- tion studies of heptameric and octameric oligomers nyl fluoride (PMSF). Then the small, 20 kDa fragment, will clarify this molecular mechanism. released by trypsinization was separated by anion-exchange chromatography, using Q-Sepharose High Performance resin (GE Healthcare), such that the remaining ∼60 kDa Materials and Methods portion oligomerized as described.10,35 This crude, oligo- meric PA mixture (QPA) was used throughout and contains mixtures of both heptameric and octameric complexes as Proteins judged by EM and nanoESI-MS.

PA nPA oligomerization in the presence of LFN/EFN or Recombinant WT PA, carboxy-terminally His6-tagged ms/dsATR2 PA,46 and all other PA mutants described here were over- expressed in the periplasm of Escherichia coli BL21(DE3), and 35 nPA was prepared as described above and then diluted they were purified as 83 kDa monomers as described. A into an appropriate buffer containing stoichiometric modified QuikChange procedure47 using Pfu Turbo poly- amounts of LFN/EFN or ms/dsATR2. LFN/EFN assembly merase (Agilent Technologies, Santa Clara, CA) was ∼ ΔMIL experiments were done by diluting nPA to 1mg/mLin implemented to make a deletion construct (PA ) from 20 mM phosphate, 150 mM NaCl, pH 7.5, containing excess the PA expression vector, PA83 pET22b+ (EMD Chemicals, 13 ΔMIL – LFN/EFN. The assembly reaction was allowed to equilibrate Gibbstown, NJ). In PA , residues 305 324 were deleted for 2 h at room temperature to afford complete oligomeriza- and two point mutations (V303P and H304G) were tion, as assessed by native gel. The dsATR2- and msATR2- introduced simultaneously, leaving a type II turn in place 5 assembly experiments were carried out by diluting nPA to of the MIL (the residue numbering is that used in 1ACC.) ∼2 mg/mL in 20 mM cacodylate buffer, 150 mM NaCl, 1mMCaCl2, pH 7.5, containing the determined stoichio- Dimeric PA metric amount of msATR2 or dsATR2. The assembly reaction was allowed to equilibrate for 10 min at room The QuikChange procedure was used to engineer an temperature to afford complete oligomerization (as assessed S170C point mutation into WT PA. PA S170C, purified by native gel electrophoresis). Assembly reactions were under oxidizing conditions, and judged to be dimeric by purified on an S200 gel-filtration column equilibrated in SDS-PAGE. assembly buffer to remove unassembled components. msATR2 does not promote oligomerization of nPA, so it Soluble dimeric ATR2 was assembled as described above for QPA. Recombinant soluble anthrax toxin receptor domain (sATR2), residues 40–217,35 was expressed from a pGEX Isolation of WT octameric PA vector (GE Healthcare) as a glutathione-S-transferase (GST) fusion protein, affinity-purified on glutathione Sepharose as The nPA + LFN complexes were prepared as described described.35 The GST-sATR was shown to be fully dimeric above, dialyzed against 10 mM Tris, pH 8, and diluted to by nanoESI-MS and is called dsATR2. 2 mg/mL in 0.1 M cacodylate buffer, pH 7. The sample was Protective Antigen Forms Functional Octamers 625

incubated for 5 min at 37 °C, concentrated, and purified on Single-channel recordings of the nPA+LFN sample an S400 gel-filtration column. The purity and oligomeric homogeneity of the resulting complexes were assessed by We also analyzed the nPA + LFN sample, which was EM and MS. formed by taking 0.1 mg/ml of nPA and adding a stoichiometric equivalent of LFN. The mixture was incu- Electrophysiology bated at room temperature for 1 h. The assembled complex (as judged by native gel electrophoresis, gel-filtration chromatography, and EM) was applied to planar lipid An Axopatch 200B amplifier (Molecular Devices Corp., bilayers membranes at ∼10-14 M. We initially observed Sunnyvale, CA) was used in the voltage clamp, capacitor Δψ – channel insertion at a of 20 mV to preclude the LFN feedback mode. The amplifier was interfaced to a Cyber- moiety from binding within the channel and blocking the Amp 320 signal conditioner (Molecular Devices), which conductance. Note that because the PA sample was diluted typically filtered the data at 200 Hz via a low-pass, 4-pole, 109-fold, newly inserted channels could be shifted back to a Bessel section. The filtered, analog signal was typically Δψ of +20 mV to record their currents once the LFN recorded by computer at 400 Hz using a Digidata 1440A dissociated from the channel. analog-to-digital converter (Molecular Devices) and AXO- CLAMP software (Molecular Devices). Most data analysis, post-acquisition filtering, and curve fitting used a combina- Electrophysiology-based translocation assay tion of CLAMPFIT (Molecular Devices), ORIGIN6.1 (Origi- nLab Corp., Northampton, MA), and custom Perl scripts. For translocation experiments, a universal bilayer buffer Relative macroscopic membrane insertion activities for (UBB) was used (10 mM oxalic acid, 10 mM Mes, 10 mM WT PA and mutant forms were assayed as follows. Planar phosphoric acid, 1 mM EDTA, 100 mM KCl). The pH of the 49 lipid bilayers were painted using a 3% (w/v) solution of the UBB is either 5.6 or 6.6, depending on whether a ΔpH will be lipid, 1,2-diphytanoyl-sn-glycerol-3-phosphocholine formed during the translocation assay. Once a membrane (DPhPC; Avanti Polar Lipids, Alabaster, AL), in n-decane was formed, PA + LF or PA + LF was added to the cis μ Q N n N as solvent. The bilayer was painted inside either a 100 mor compartment (at pH 5.6); the cis compartment was held at a μ a200 m aperture of a 1 mL, white delrin cup while bathed in Δψ of±20 mV with respect to the trans compartment. (i.e., in Δψ – aqueous buffer S (100 mM KCl, 1 mM EDTA, 10 mM succinic the case of nPA + LFN,the was held at 20 mV to allow acid, pH 6.6). The cis (the side to which the PA oligomer is for channel insertion to be observed, because at +20 mV,LFN added) and the trans compartments were bathed in would block the channel). When nPA + LFN was used, the symmetric buffer S. For macroscopic current measurements, excess LFN was perfused away, and residual bound LFN PA oligomer (25 pM) was added to the cis compartment, was then translocated at 80 mV to clear the channels before Δψ Δψ which was held at a of +20 mV. ( ,themembrane initiating further translocation experiments. After the Δψ Δψ –ψ ψ ≡ potential, is defined as = cis trans,where trans 0mV.) ensemble channel population was established, LF, LFN,EF, Channel insertion increased over a period of minutes and or EF were added to the cis compartment. The progress of – N stabilized after 20 30 min. Macroscopic currents were then substrate binding to PA63 channels was monitored by the measured at this point or the channel-inserted membrane continuous fall in conductance. After the conductance block wasusedintranslocationexperimentsasdescribedbelow. of PA channels was complete, excess substrate was removed Single-channel measurements were obtained using from the cis compartment with perfusion with UBB, using a μ DPhPC/decane films formed on a 100 m aperture in a push–pull, hand cranked 10 mL syringe pump. The cis white delrin cup. Single-channel conductance measure- compartment was perfused with 10 ml of UBB at a flow rate Δψ ments were carried out at a of +20 mV in symmetric of 2 ml/min and Δψ held at +20 mV. Translocation of LF, buffer S. Single-channel current recordings were determined LF ,EF,andEF were initiated by jumping the Δψ to a ∼ -14 N N by adding a dilute solution ( 10 M) of PA oligomer to the higher positive voltage and/or jumping the ΔpH to a higher – cis chamber, stirring briefly, and then waiting 5 30 min until positive value where: discrete steps in current were observed. The clamping voltage and current responses were acquired at 400 Hz DpHupHtrans À pHcis under low-pass filtering at 200 Hz. To observe subtle, slow- Δ timescale fluctuations in the single-channel current records, The pH is induced by adding predetermined amounts of we implemented a Gaussian-filter algorithm; this filter does 0.4 M phosphoric acid to the cis chamber. The final pH is not cause ”overshoot” during channel opening and closing determined using a pH meter following the completion of transitions. The filter's Gaussian kernel was defined with σ the translocation recording. having a width of 100 data points. A Perl script was written to apply the Gaussian filter to our datasets. Electron microscopy To calculate the mean unitary conductance levels, time courses not treated with the Gaussian filter were analyzed All the samples were prepared for EM in a similar manner. by fitting Gaussians to 0.003–0.03 pA binned histograms of The PA oligomer at 20–30 nM monomer was incubated in the current responses, using either ORIGIN6.1 or CLAMP- buffer E (20 mM Tris, 100–250 mM NaCl, pH 8) for 5 min. FIT. Some records contained multiple, but readily separable, The 400-mesh copper grids were covered successively by a current steps up to as many as 10 single channels. The holey carbon film and a continuous carbon film. A 4 μlof means, μ, from Gaussian curve fits were then subtracted sample was applied to a freshly glow-discharged support from similar fits to the noise observed at “zero” current to grid for 30 s and then stained with five successive drops obtain each single-channel current i. The single-channel (75 μl each) of either 1% (w/v) uranyl formate (Structure conductance γ is calculated from the single-channel current i Probe, Inc., West Chester, PA) or 2% (w/v) uranyl acetate and voltage V by: (Sigma-Aldrich, St. Louis, MO). g = i=V Negatively stained EM images were recorded with a Tecnai 12 (FEI Company, Hillsboro, OR) operated at 100 kV Errors from μ were propagated to establish errors in γ for or at 120 kV at a magnification of either 49,000× or 50,000×. each measurement, which were ∼0.5 pS. In some cases, data were collected on Kodak SO163 films 626 Protective Antigen Forms Functional Octamers

(Eastman Kodak, Rochester, NY) at 600–800 nm underfocus. capillaries (1.0 mm O.D./0.78 mm I.D., Sutter Instruments, Film images were digitized with a Nikon Super Coolscan Novato CA) to a tip I.D. of∼1 μm with a Flaming/Brown 8000 (Nikon USA, Melville, NY) at a 12.7 μm pixel size, micropipette puller (model P-87, Sutter). The instrument resulting in 2.54 Å/pixel at the specimen scale. In other was calibrated with CsI clusters formed by nanoESI using a cases, data were collected directly on a CCD camera, 24 mg/mL solution of CsI in 70:30 (v/v) Milli-Q water:2- resulting in 2.13 Å/pixel at the specimen scale. Particle propanol before mass measurement. The protein solution images were selected for each data set by automatic or for the stoichiometry determinations was prepared as manual particle picking with boxer in EMAN.50 described above and then concentrated to 10 μM followed Reference-free processing was done with the software by dialysis into 10 mM ammonium bicarbonate, pH 7.8. package IMAGIC (Image Science Software, Berlin, Ger- Immediately before mass analysis, the solution was diluted many) or .51 Images were subjected to three 1:1 (v/v) with 200 mM ammonium acetate, pH 7.8. A successive cycles of multi-reference alignment, multivariate platinum wire (0.127 mm diam., Sigma, St. Louis, MO) was statistical analysis, and classification.52,53 The last classifica- inserted through the capillary into the solution and tion was done using only the lowest order eigenvectors as electrospray was initiated and maintained by applying 1– described,54 to separate the data by size and the heptameric 1.3 kV to the wire (relative to instrument ground). The raw and octameric oligomerization states. data were smoothed three times using the Waters MassLynx A second method of image processing was used whereby software mean smoothing algorithm with a window of reference images were made from 2D projections of low- 50 m/z (mass/charge ratio). resolution density maps generated from the crystal struc- The reactions for assembly kinetics were initiated by tures of the PA heptameric,7 and octameric pre-channels, mixing 10 mM ammonium bicarbonate solutions of 51 using SPIDER. Boxed images were then subjected to purified nPA and LFN monomer in a 1:1 (v/v) ratio to reference-based alignment and classification using the initiate oligomerization, and mass spectra were acquired lowest order eigenvectors as stated above. Final class- continuously for 50 min. Variation in the voltage applied average images were inspected manually for their oligomer to the nanospray capillary and new capillaries at ∼19 min number, designated as either heptamer or octamer, and and ∼30 min were required to maintain ion current. Mass tabulated to produce the final percentages of heptamers and spectra were averaged for 5 min intervals and smoothed octamers. Each method of classification, reference-free or three times using the Waters MassLynx software mean crystal structure-referenced, produced similar results (Sup- smoothing algorithm with a window of 50 m/z. Each plementary Data Table S2). peak in a given charge-state distribution was integrated and the peak areas summed to give an absolute Extraction of PA oligomers from CHO cells abundance for the corresponding analyte. Relative abun- dances were calculated as a fraction of the total abundances of the four analytes of interest monitored C-CHO and T-CHO cells were a kind gift from Arthur during the experiment. Frankel. The cell line was created from a spontaneous ATR- deficient CHO cell mutant line (PR230-CHO). The C-CHO and T-CHO lines were derived from stable transfections Protein crystallization with the human ATR2 and ATR1 expression clones, 55 respectively. The cell lines were grown to confluence in Δ Purified PA MIL oligomer (judged to be rich in octa- Ham's F12 medium (Invitrogen), 10% (v/v) fetal bovine Q meric complexes by EM) was prepared in buffer X, which serum (Invitrogen), 100 units/mL of penicillin, 100 μg/mL contained 74 mM sodium acetate, 7 mM Tris, 0.62 M NaCl, of streptomycin (Sigma-Aldrich) in a humid 5% CO 2 37 mM tetrabutylammonium bromide, 7% (v/v) ethanol, atmosphere at 37 °C, as described.32 Confluent cells were 0.07% (w/v) n-dodecyl-β-D-maltopyranoside, pH 5.7, treated for 1 h with 50 mM ammonium chloride, 50 mM 4(2- centrifuged to remove precipitated protein, and concen- hydroxyethyl)-1-piperazineethanesulfonic acid (Hepes), pH trated to 13 mg/mL. Initial crystallization conditions were 7.3 in Ham's medium to inhibit endosomal acidification, established by sparse-matrix crystallization screens,57 preventing conversion of PA to the channel state.56 His -PA 6 except our screens contained ∼1000 unique conditions. A monomers (WT PA with a carboxy-terminal His tag) were 6 Mosquito nanoliter, liquid-handling robot (TTP Labtech, applied to cells in the same medium at 100 μg/mL. Cells Cambridge, MA) was used to form 200 nL drops in 96-well were incubated with His -PA for 1 h at 4 °C, washed with 6 format at 18 °C, using the hanging-drop, vapor-diffusion five volumes of ice-cold PBS to remove unbound PA, and method.58 Diffraction-quality crystals were formed with lysed in buffer L (20 mM Tris, 0.35 M NaCl, 10 mM 1 μL hanging drops using the Mosquito (at 18 °C), imidazole, 1% (v/v) Nonidet P-40 (or IPEGAL), 0.25% (w/v) Δ containing a 1:1 (v/v) mixture of 13 mg/ml PA MIL deoxycholic acid, 1 mM PMSF, pH 8) as described.55 Cell Q oligomer in buffer X with the reservoir solutions (18–30% debris was removed by centrifugation and the supernatant (v/v) t-butanol, 0.1 M Tris, pH 7.5–8.5). Often irregular, was incubated with 0.5 mL of His -affinity resin (Ni-NTA 6 rectangular-prism-shaped crystals formed overnight and Superflow, Qiagen, Valencia, CA) overnight at 4 °C with grew as large as ∼200 μm×200 μm×75 μm. Crystals were stirring. The resin was washed with five volumes of buffer L harvested in a 30% (v/v) polyethylene glycol (PEG) 400 and eluted in buffer L supplemented with 300 mM cryoprotectant, where the PEG only replaced the water in imidazole. The elution was applied to electron microscopy the mother liquor, and immediately flash-frozen in liquid grids for analysis. nitrogen.

Mass spectrometry X-ray diffraction data collection, solution and refinement Mass spectra of the protein complexes were acquired using a quadrupole time-of-flight (Q-TOF) mass spectro- X-ray diffraction data were collected at the Advanced meter equipped with a Z-spray ion source (Q-Tof Premier, Light Source in Lawrence Berkeley National Lab, Beamline Waters, Milford, MA). Ions were formed using a nanoelec- 8.3.1,59 using a Quantum 315r CCD area detector (ADSC, trospray (nanoESI) emitters prepared by pulling borosilicate Poway, CA). The crystals diffracted to 3.2 Å in the triclinic Protective Antigen Forms Functional Octamers 627 space group P1 with unit cell dimensions of a=125.60 Å, Supplementary Data b=125.67 Å, c=125.82 Å, α=106.64°, β=110.82°, and γ=110.98° (Supplementary Data Table S3). The diffraction data were indexed and scaled in HKL2000.60 The scaled Supplementary data associated with this article dataset was 98.7% complete to 3.2 Å. The self-rotation can be found, in the online version, at doi:10.1016/ function in the CCP4 suite61 revealed strong peaks at a χ j.jmb.2009.07.037 angles of 45°, 90° and 180°. Molecular replacement was done with PHASER62 in CCP4, where the search model was a References loop-stripped chain A from 1TZO.7 This molecular replace- ment solution placed eight PA monomer chains in the 1. Young, J. A. & Collier, R. J. (2007). Anthrax toxin: asymmetric unit. The molecular replacement solution was receptor binding, internalization, pore formation, and refined with rigid-body constraints defined by the known 76 – 63 translocation. Annu. Rev. Biochem. , 243 265. domain boundaries in PA, using PHENIX. Noncrystallo- 2. Bradley, K. A., Mogridge, J., Mourez, M., Collier, R. J. graphic symmetry was established by first making struc- 64 & Young, J. A. (2001). Identification of the cellular tural alignments of individual monomers in CHIMERA, receptor for anthrax toxin. Nature, 414, 225–229. and then calculating the center of mass of each chain to 3. Scobie, H. M., Rainey, G. J. A., Bradley, K. A. & Young, compute the geometric arrangement of the chains about the J. A. (2003). Human capillary morphogenesis protein 2 oligomeric ring. We found the oligomer was an irregular functions as an anthrax toxin receptor. Proc. Natl Acad. octagon, and pairs of monomers of two different conforma- Sci. USA, 100, 5170–5174. tions, A and B formed the sides of a regular, square, planar 65 4. Milne, J. C., Furlong, D., Hanna, P. C., Wall, J. S. & tetramer. Subsequent rounds of model building in COOT Collier, R. J. (1994). Anthrax protective antigen were followed by coordinate and B-factor refinement, with forms oligomers during intoxication of mammalian 4-fold noncrystallographic symmetry restraints, using J. Biol. Chem. 269 – – – cells. , 20607 20612. PHENIX. 2Fo Fc and Fo Fc omit electron density maps 5. Petosa, C., Collier, R. J., Klimpel, K. R., Leppla, S. H. & were recalculated after iterations of model building and Liddington, R. C. (1997). Crystal structure of the refinement, where Fo and Fc are the observed and calculated anthrax toxin protective antigen. Nature, 385, 833–838. structure factors, respectively. MOLPROBITY66 and 67 6. Mogridge, J., Cunningham, K. & Collier, R. J. (2002). PROCHECK were used to validate the structure's Stoichiometry of anthrax toxin complexes. , geometry and stereochemistry during model building. 41, 1079–1082. Surface burial calculations were made using 7.Lacy,D.B.,Wigelsworth,D.J.,Melnyk,R.A., GETAREA1.1.68 Molecular graphics renderings were com- 64 Harrison, S. C. & Collier, R. J. (2004). Structure of puted using CHIMERA. heptameric protective antigen bound to an anthrax toxin receptor: a role for receptor in pH-dependent 101 accession code pore formation. Proc. Natl Acad. Sci. USA, , 13147–13151. 8. Katayama, H., Janowiak, B. 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Protein translocation through the anthrax toxin transmembrane pore is driven by a proton gradient. A.F.K. and K.C.D. crystallized the octamer; A.F.K. 355 – and B.A.K. refined and analyzed the octamer with J. Mol. Biol. , 968 979. guidance from J.M.B. A.F.K. did the ensemble 12. Song, L., Hobaugh, M. R., Shustak, C., Cheley, S., Bayley,H.&Gouaux,J.E.(1996).Structureof translocations. B.A.K, A.F.K., J.M.B, E.R.W., and staphylococcal α-hemolysin, a heptameric transmem- H.J.S. wrote the manuscript. We thank J. Mogridge, brane pore. Science , 274, 1859–1866. K. Bradley, and R.J. Collier for discussions; E. Nogales 13. Benson, E. L., Huynh, P. D., Finkelstein, A. & Collier, and P. Grob for EM studies and advice; J. Liphardt for R. J. (1998). Identification of residues lining the advice on single-molecule data; A. Frankel for the gift anthrax protective antigen channel. Biochemistry, 37, of the CHO cell lines; J. Fraser and S. 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