Structural analysis of ricin-antibody interactions Structural Analysis of Single Domain Antibodies

28 Pages·2016·5.48 MB·English

JBC Papers in Press. Published on November 30, 2016 as Manuscript M116.758102
The latest version is at http://www.jbc.org/cgi/doi/10.1074/jbc.M116.758102
Structural analysis of ricin-antibody interactions

Structural Analysis of Single Domain Antibodies Bound to a Second Neutralizing Hot Spot on Ricin
Toxin’s Enzymatic Subunit

Michael J. Rudolph1,* , David J. Vance2, , Michael S. Cassidy1, Yinghui Rong2, and Nicholas J.
Mantis2,3,*

1New York Structural Biology Center, New York, New York 10027; 2Division of Infectious Diseases,
Wadsworth Center, New York State Department of Health, Albany, NY, 12208; 3Department of
Biomedical Sciences, University at Albany, Albany, NY 12201 United States

Running title: Structural analysis of ricin-antibody interactions
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*To whom correspondence should be addressed: Dr. Michael J. Rudolph, New York Structural Biology loa
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Center, 89 Convent Avenue, New York, NY 10027, Tel. [phone] and E-mail address: fro
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[email protected] and Dr. Nicholas J. Mantis, Division of Infectious Diseases, Wadsworth Center, h
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New York State Department of Health, 120 New Scotland Ave, Albany, NY 12208. Tel. [phone]. ://w
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E-mail address: [email protected] .jb
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Keywords: antibody, antigen, crystal structure, epitope mapping, toxin, neutralizing, ricin g
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ABSTRACT region 3 (CDR3), may also engage with RTB n
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Ricin toxin is a heterodimer consisting and potentially interfere with the high affinity ry
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of RTA, a ribosome-inactivating protein, and galactose-recognition element that plays a , 2
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RTB, a lectin that facilitates receptor-mediated critical role in toxin attachment to cell surfaces 19
uptake into mammalian cells. In previous and intracellular trafficking. The two other
studies, we demonstrated that toxin-neutralizing V Hs, E1 and V1C7, bind epitopes adjacent to
H
antibodies target four spatially distinct hotspots V5E1 but display only weak toxin-neutralizing
on RTA, which we refer to as epitope Clusters I- activity, thereby providing structural insights
IV. In this report we identified and into specific residues within Cluster II that may
characterized three single domain camelid be critical contact points for toxin inactivation.
antibodies (VHH) against Cluster II. One of these
V Hs, V5E1, ranks as one of the most potent
H
ricin-neutralizing antibodies described to date. Ricin toxin, a 65 kDa heterodimeric
We solved the X-ray crystal structures of each of glycoprotein from the castor bean plant (Ricinus
the three V Hs (E1, V1C7 and V5E1) in communis), is one of the most potent biological
H
complex with RTA. V5E1 buries a total of 1,133 toxins known. The toxin’s A subunit (RTA) is a
Å2 of surface area on RTA and makes primary ribosome-inactivating protein (RIP) that cleaves
contacts with α-helix A (residues 18-32), α- the N-glycosidic bond of a single conserved A
helix F (182-194), as well as the F-G loop. residue within sarcin-ricin loop (SRL) of 28S
V5E1, by virtue of complementarity determining rRNA, which is required for ribosomal
Copyright 2016 by The American Society for Biochemistry and Molecular Biology, Inc.
elongation (1,2). RTA (267 residues) is joined camelid library (referred to as “HobJo”) has an
via a single disulfide bond to RTB (262 estimated diversity of >107 independent clones
residues), a galactose-specific lectin that and was generated from two animals that had
attaches to glycoproteins and glycolipids on the been repeatedly immunized with a mixture of
surface of mammalian cells (3). RTB promotes recombinant non-toxic RTA, RTB and ricin
the uptake and retrograde trafficking of the toxin toxoid (16). Analysis to date has suggested that
to the endoplasmic reticulum (ER), where RTA there are (at least) four immunodominant
is liberated from RTB through the action of regions on RTA, which we refer to as epitope
protein-disulfide isomerase (PDI) (4). RTA is clusters I-IV. The four clusters are represented
then dislocated (retro-translocated) across the by the following toxin-neutralizing mAbs: PB10
ER membrane with the assistance of the ER- (I), SyH7 (II), IB2 (III) and GD12 (IV) (14).
associated protein degradation system (ERAD). Competition ELISAs with the four
Once in the cytoplasm, RTA triggers ribosome- representative mAbs enabled us to readily
inactivation and programmed cell death (5). categorize new mAbs and V Hs into their
H
Structurally, RTA is a globular protein that respective clusters (14-17).
can be divided into three distinct folding To date we have reported the X-ray crystal
domains (6). Folding domain I (residues 1-117) structures of seven different Cluster I V Hs in
H
is dominated by a six-stranded β-sheet that complex with RTA (18) and complemented D
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terminates in a prominent solvent exposed α- these studies with epitope mapping of three n
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helix, known as α-helix B (residues 97-108). different Cluster I mAbs by hydrogen deuterium ad
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dFoomldiinnagt eddo mbya ifniv IeI α(r-ehseidliuceess 1(C18-G-2)1, 0w) iitsh α-helix eVxoclhkainn,g aen (dH NX.- MMSan) t(iRs,. mTaonthu,s Dcr.i pWt einis , D. d from
E being at the core of the molecule. Folding preparation). Another group has reported the X- http
domain III (residues 211-267) forms a ray crystal structure of a Fab fragments in ://w
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protruding element that associates with RTB complex with cluster I (19). At the structural w
through a disulfide bond as well as extensive level, cluster I epitopes encompass α-helix B .jbc
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nonpolar interactions (6). Five residues, Tyr80, (residues 98-106), β-strand h (residues 113-117), rg
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Tyr123, Glu177, Arg180, and Trp211, define and the C-terminus of α-helix D (residues 154- y
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RTA’s active site, which consists of a shallow 156). In general, we found that within cluster I ue
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pocket in the central portion of the protein (7). ricin toxin-neutralizing activity correlated with t o
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With the exception of the region spanning α- the degree of interaction between the VHH and Jan
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helices F (residues 182-194) and G (residues RTA, particularly α-helix B; that is, larger ary
202-219), which is rich in arginine residues and interfaces (i.e., greater buried surface area 7, 2
thought to promote ribosome binding through values) corresponded to greater toxin- 01
9
electrostatic interactions, no other functionality neutralizing activity (17,18). Moreover, the
has been ascribed to RTA’s secondary elements interactions between the different V Hs and
H
or solvent exposed surface. RTA were largely dictated by the CDR3
Establishing a comprehensive molecular B elements. Finally, our original assumption that
cell epitope map of RTA is of importance in neutralizing and non-neutralizing antibodies
terms of facilitating design of more effective would occupy spatially distinct structural
RTA-based immunotoxins for cancer therapy (8- epitopes on the surface of RTA was proven
10) and ricin toxin subunit vaccine antigens for incorrect. Instead, we found several examples in
use in the military and in certain civilian which non-neutralizing antibody epitopes were
populations in the event that ricin were to be nested within the footprints of neutralizing
used as a biological weapon (11-13). Towards antibodies.
this end, we have generated a large collection of In the present study, we have embarked on
murine monoclonal antibodies (mAbs), as well an effort to define structural B cell epitopes
as single domain heavy-chain only camelid within Cluster II. As noted above, Cluster II is
antibodies (V Hs) against linear and defined by SyH7, a toxin-neutralizing mAb that
H
discontinuous epitopes on RTA (14-16). The was first identified based on its reactivity with a
2
peptide spanning RTA residues 187-198 (15). potent inhibitor of ricin (IC < 1 nM), while E1
50
In addition to its in vitro activities, SyH7 is able and V1C7 were classified as weak inhibitors
to passively protect mice against systemic 10x (IC s >100 nM) (Figure 1B)
50
lethal dose fifty (LD ) ricin challenge. We The three V Hs, E1, V1C7 and V5E1, each
50 H
subsequently identified three additional toxin- recognized receptor bound ricin, as well as ricin
neutralizing mAbs, TB12, PA1 and PH12 that that had been captured with representative
recognize discontinuous epitopes within cluster cluster I (PB10, WECB2), III (IB2) and IV
II (14). The epitopes recognized by SyH7, (GD12) mAbs (Figure 2). However, the V Hs
H
TB12, PA1 and PH12 are distinct and have been were inhibited from binding to ricin that had
tentatively localized by HX-MS (R. Toth, D. been captured by cluster II-specific antibodies
Weis, D. Volkin, and N. Mantis, manuscript in SyH7, PA1, PH12, and TB12 (Figure 2A;
preparation). Because all four cluster II mAbs Figure 3). V5E1 was interesting in this regard
neutralize ricin in vitro and in vivo at low because binding to ricin holotoxin was inhibited
toxin:mAb stoichiometric ratios, we have by SyH7, PA1 and TB12, but not by PH12
proposed that their epitopes represent a (Figure 2B), revealing a previously
particular site of vulnerability on the toxin. We unrecognized spatial complexity within Cluster
report here the X-ray crystal structures of three II. In pairwise competition ELISAs, E1 and
different Cluster II V Hs in complex with RTA, V1C7 inhibited each other from binding to ricin, D
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including one VHH whose epitope possibly but had little or no effect on V5E1 (Figure 4). nlo
spans the interface between RTA and RTB. The Conversely, V5E1 had little or no effect on E1 a
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cinutror etnhte sntuatduyr ea foffo redpsi ttohpee f cirlsuts stetrru IcIt uanradl rienvseigahlst ofrro Vm1 bCi7n,d ainltgh oriucginh. i tI nd isdu (momf caoryu,r steh)e itnhhriebei t itself d from
possible mechanisms by which antibodies cluster II-specific VHHs, E1, V1C7 and V5E1 http
directed against this region of RTA neutralize likely represent two distinct structural epitopes ://w
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the toxin. within cluster II: E1 and V1C7 bind epitope(s) w
associated with weaker toxin-neutralizing .jb
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RESULTS activity and V5E1 binds an epitope associated rg
Isolation of VHHs against epitope cluster II with stronger neutralizing activity. by g/
on RTA--As part of this study, the HobJo phage- The observation that two V Hs (E1 and u
H e
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displayed single domain VHH library was V1C7) with weak toxin-neutralizing activities t on
panned against several different ricin-derived recognized epitopes within cluster II prompted Ja
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targets, including RTA directly immobilized on us to investigate whether they might ua
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polystyrene plates and ricin holotoxin captured competitively interfere with the ability of 7
, 2
via the surrogate receptor (ASF). However, to Cluster II mAbs to neutralize ricin. To test this 0
1
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specifically enrich for cluster II-specific hypothesis, SyH7, PA1, PH12, or TB12 were
antibodies, the library was panned against ricin mixed with molar excess of E1 or V1C7 and
captured via JIV-F5 to broadly mask epitope then tested in a cell-based toxin-neutralizing
cluster I and in the presence of saturating assay. We observed that E1 and V1C7
amounts of JIV-F6 to occlude epitope cluster III interfered (to varying degrees) with the
(16) (D. Vance, C. Shoemaker, and N. Mantis, neutralizing activities of PA1, PH12, and TB12,
manuscript in preparation). The screening but had only a marginal effect on SyH7 (Figure
approaches yielded three different RTA-specific 5). For example, Vero cell viability was 100%
V Hs: E1, V1C7 and V5E1. The antibodies in the presence of ricin and 0.8 nM PA1. In
H
were unique from all other previously identified contrast, upon addition of molar excess amounts
V Hs based on the DNA sequence of their of E1 and V1C7, Vero cell viability in the
H
respective CDR 1-3 elements (Figure 1A). The presence of 0.8 nM PA1 declined to < 20%
V Hs also differed in binding affinities (K ) for (Figure 5B). For reasons that are not
H D
ricin toxin, as determined by SPR; V5E1 had the immediately apparent, SyH7 was largely
highest affinity (K 20 pM), followed by E1 refractory to the effects of E1 and V1C7, as
D
(690 pM) and V1C7 (3.17 nM) (Table 1). In a evidenced by only a slight change in SyH7’s
cell-based toxin-neutralizing assay, V5E1 was a IC upon addition of E1 and V1C7.
50
3
X-ray crystallography of V H-RTA contains SyH7’s linear epitope, residues 187-
H
complexes-To elucidate the exact epitopes 198, is in close proximity to secondary structural
recognized by the three Cluster II V Hs, we elements that are likely to constitute the epitopes
H
solved the X-ray crystal structures of E1, V1C7 of other cluster II mAbs like PA1, PH12, and
and V5E1 in complex with RTA at 3.1Å, 1.8 Å TB12, as well as E1, V1C7 and V5E1 (Figure
and 1.7 Å, respectively (Tables 1-2; Figure 6). 3). These structural elements include six α-
In all three complexes, the VHHs assumed a helices (A, C-G), three loop regions between α-
classical immunoglobulin fold consisting of nine helices C-D, D-E, and F-G, a single 3 -helix
10
β-strands arranged in two β-sheets with all three between α-helices C and D (referred to as 3 -
10
CDRs on one face of the molecule (Figure 7). helix C-D), the loops between α-helix A and β-
E1 displayed the canonical disulfide bond
strand a and β-strands e and d (referred to as
between Cys22 and Cys96 that links FR1 to FR3
loops A-a and e-d) (6,21,22).
(20), whereas V1C7 did not (Figures 1, 7). We
Analysis of the V H-RTA crystal structures
H
postulate that the absence of this disulfide bond
revealed that E1 and V1C7 recognize
in V1C7 is due to the low pH (pH 4.6) of the
overlapping but distinct epitopes on RTA. E1
crystallization conditions of the V1C7-RTA
makes contact with α-helix C (residue 130), α-
complex. Low pH is known to favor the
helix D (residues 142, 145-147) and α-helix E
reduction of disulfide bonds and this was the D
(residues 166, 167, 170). E1 also interacts with o
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only relative condition that differed among the n
loops e-d (residues 65-67), C-D (residues 131- lo
three VHH-RTA complexes. 132), D-E (residues 156,158), F-G (residues ade
V5E1, on the other hand, has two 197,199), as well as the 310-helix C-D (residues d fro
intramolecular disulfide bridges: the canonical m
135-136) (Figure 8A). V1C7’s interface with h
tdhiesu llefsids ef rbeoqnude nbtelytw oebesne rCveyds2 d2i saunldfi dCey bs9o6n,d a nd RTA is limited to α-helix D (residues 141-142, ttp://w
145-146, 149, 153) and α-helix E (residue 163), w
between Cys50 and Cys109 that links CDR2 and w
CDR3 (Figure 1). Two other potent ricin toxin- along with several additional interactions with .jbc
loops e-d (residues 65-68), C-D (residue 138), .o
nseecuotrnadliazriyn gd iVsuHlHfids,e Eb5o nadn d( 1F75,1, 8ex),h wibhiti cthh ims saakmese D-E (residues 157-160), and F-G (residues 195, byrg/
197) (Figure 8B). As compared to V1C7, E1 g
it tempting to speculate that stabilizing CDR2- u
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forms as many as 10 additional hydrogen bonds s
CDR3 through a covalent linkage positively t o
with RTA (12 vs. 22), depending on which of n
impacts toxin-neutralizing activity. Finally, we Ja
the six RTA-E1 complexes within the nu
noted that RTA assumed a very similar a
asymmetric unit (asu) is used to analyze the ry
conformation in all three RTA-V H complexes, 7
H RTA-E1 interface (Table 1; Figure S1). E1 , 2
as evidenced by RMSD values of 0.5 to 0.6 Å 0
establishes a buried surface area (bsa) with RTA 19
when Cα atoms of RTA (PDB: 1RTC) were of ~2,000 Å2, comprised of 63% non-polar and
superimposed onto each of the RTA-V H crystal
H 37% polar atoms. This is in contrast to V1C7,
structures. Thus, RTA does not undergo any
which has a bsa of 1,440 Å2 with RTA,
significant conformational changes upon
consisting of 59% non-polar and 41% polar
complex formation with E1, V1C7 or V5E1. As
atoms (Figure 8D,E). Altogether, the larger E1-
an aside, shape complementarity is not a strong
RTA interface with more H-bonds likely
predictor of either relative binding affinity or
contributes significantly to the five-fold stronger
toxin-neutralizing activity, as evidenced by the
binding affinity of E1 for ricin toxin, as
fact that V5E1 had a shape complementarity
compared to V1C7 (Table 1). E1 and V1C7
score of 0.66, as compared to E1 and V1C7,
each form one salt-bridge with RTA: E1 residue
which had higher shape complementarity scores
Lys114 interacts with Arg166 of RTA, while
of 0.76 and 0.75, respectively (data not shown).
V1C7 residue Asp59 binds Arg197 in RTA. It is
Thus, other factors must account for differences
unclear at this time whether the differences in
in toxin-neutralizing activity observed among
toxin-neutralizing activity between E1 and
the three different V Hs.
H V1C7 are associated with E1’s greater degree of
Molecular interactions between RTA and
interaction with RTA, different contact residues
V Hs E1 and V1C7- The region of RTA that
H
4
on RTA, and/or its enhanced affinity for ricin from the surface of RTA, making it accessible
holotoxin. for potential interactions with other molecules,
Molecular interactions between RTA and notably RTB. Indeed, superpositioning the RTA
V5E1-There is little overlap between the subunit within the RTA-V5E1 complex onto
structural epitope recognized by V5E1 and the RTA within ricin holotoxin (PDB: 2AAI)
E1 and V1C7 epitopes, even through all three reveals that V5E1 could contact RTB residues
antibodies are, by definition, situated within 147-149, 237-239, and 254, resulting in a
cluster II. The distinctiveness of V5E1’s potential gain of 237Å2 of bsa, as compared to
epitope, as compared to V1C7 and E1, is most the V5E1-RTA complex (Figure 10). The
apparent when the RTA subunit in the V5E1- association with RTB would also create an
RTA crystal structure is independently additional H-bond between V5E1 CDR3 residue
superpositioned onto the RTA subunits within Arg110 and RTB residue Ala237, which may be
the corresponding structures of RTA-V1C7 and consequential in terms of V5E1’s toxin-
RTA-E1 (Figure 4 D, E). V5E1 makes contact neutralizing activity. Arg110 is ~4.0 Å from
with loops A-a (residues 14-20) and e-d (residue RTB residue Ser238 of RTB, which, in turn,
65), α-helix D (residue 141,145) and loop F-G forms an H-bond with Asn255 (inset, Figure
(residues 192-196) (Figure 8C). The interface 10). Asn255 is involved in direct interaction
between V5E1 and RTA contains 55% non- with Gal/GalNAc ligands (23). Thus, D
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polar and 45% polar atoms and is relatively perturbation of the position of Asn255 by n
small (bsa =1,133 Å2), considering its tight Arg110 in V5E1 would be expected to interfere load
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pbointednint gto axfifnin-niteyu ftorarl irzicining (cKapd a=c 0it.y0 2(I CnM ) =a n0d.5 i ts wsuibths tRraTteBs.’ s ability to adhere to Gal/GalNAc d from
nM) (Table 2). V5E1 forms a total of 5100 To address this experimentally, we http
hydrogen bonds and two salt bridges with RTA. performed a solid phase binding assay in which ://w
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The salt bridges occur between CDR1 residue microtiter plates coated with ASF were probed w
Arg28 and RTA residue Glu145 and CDR3 with biotin-ricin in the absence or presence of .jbc
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residue Glu101 with RTA residue Arg193. V5E1. V5E1 reduced ricin-ASF interactions in a rg
A closer examination of V5E1’s interaction dose-dependent manner with maximal inhibition by/
g
with RTA residues 187-198 is warranted being ~60% when antibody concentrations were u
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s
considering that these residues are proposed to equal to or in excess of 30 nM (Figure 11A). In t o
n
constitute the core of SyH7’s epitope (15). V5E1 contrast, V1C7 (squares) had no effect on the Ja
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buries 481Å2 of this region of RTA and ricin-ASF interaction, even at 330 nM. We also ua
ry
establishes 3 H-bonds between framework performed toxin-binding assays in which 7
, 2
residue Gln3 and RTA residues Tyr194 and fluorescently-labeled ricin was mixed with 0
1
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Asn195, as well as the salt bridge between V5E1, applied to THP-1 monocyte cells on ice
CDR3 residue Glu101 and RTA residue Arg193, (to enable binding but not internalization) and
as noted above (Figure 9C). By comparison, then subjected to flow cytometry. Similar to the
V1C7 forms only a single H-bond with this ASF assay, V5E1 demonstrated a dose-
region (i.e., CDR2 residue Ser57 interacts with dependent reduction (~60%) in ricin binding to
RTA residue Arg197) and buries just 241 Å2 cell surfaces (Figure 11B).
(Figure 9A). E1 forms 2 hydrogen bonds with Finally, we reasoned that if V5E1 has an
this area of RTA (i.e., CDR1 residue Arg31 with increased binding affinity for ricin holotoxin (as
RTA residue Arg197, and CDR3 residue Lys100 compared to RTA) then soluble ricin would be
with RTA residue Arg197) and buries 260 Å2 more effective than soluble RTA at
(Figure 9B). We speculate that contact with competitively inhibiting V5E1 attachment to
RTA residues 193-195 may be critical for toxin- surface-immobilized ricin. V5E1 was mixed
neutralizing activity for reasons that will be with equimolar amounts of RTA or ricin in
described in the Discussion. solution then subjected to SPR using a ricin-
Another striking feature of the RTA-V5E1 coated sensor. In the absence of soluble ricin or
complex is that the entire central region of the RTA, V5E1 elicited an RU value of 140 (Figure
CDR3 (residues 105-111) is positioned away S2). When V5E1 was mixed with 60 nM RTA
5
or 60 nM ricin the RU values dropped to 100 V5E1’s structural epitope, on the other hand, is
and 40, respectively, indicating that on a molar focused on the N-terminus of α-helix A
basis ricin holotoxin is more effective than RTA (residues 18-32) and C-terminus α-helix F (182-
at competitively inhibiting V5E1 binding. The 194), as well as residues in the F-G loop. Thus,
RU values were reduced to 40 and <5 when we tentatively conclude that differences in the
V5E1 was mixed with 120 nM RTA or ricin, structural epitopes recognized by E1, V1C7 and
further indicating that V5E1 preferentially V5E1 are primarily responsible for the observed
associates with soluble ricin over RTA. In differences in toxin-neutralizing activities
contrast, soluble ricin and RTA were equally among the three antibodies.
effective at inhibiting VHH E1 from binding to V5E1 ranks as one of the most potent toxin-
plate bound ricin (Figure S3). We conclude neutralizing single chain antibodies in our
from these studies that V5E1 is able to collection (16,24,25). V5E1 was isolated from
functionally interfere with ricin attachment to the so-called HobJo phage-displayed single
cellular receptors, possibly through an domain camelid antibody library, which was
interaction with residues within domain 2γ of generated from two alpacas (Vicugna paco) that
RTB. had been repeatedly immunized with RiVax,
RTB and ricin toxoid in the presence of
DISCUSSION aluminum salts and CpG as adjuvants (16). D
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We have previously proposed that ricin Although it is estimated that the HobJo library n
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toxin-neutralizing antibodies are directed against consists of >107 unique VHH clones (C. ade
four distinct “hotspots” on the surface of RTA, Shoemaker, unpublished results), exploiting the d fro
which we refer to as epitope clusters I-IV. diversity of the antibody repertoire has not been m
h
Cluster II is defined by SyH7, a mAb that trivial. The first ricin-specific VHHs isolated ttp
recognizes a linear epitope within RTA’s α- from the HobJo libraries were based on affinity ://w
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helix F (residues 187 to 198). Three additional enrichments using RTA or RTB bound directly w
mAbs, PA1, PH12 and TB12 were grouped to polystryrene surfaces (16). These initial .jbc
.o
within Cluster II based on their abilities to screens were biased towards epitopes in Cluster rg
interfere with SyH7 binding to ricin toxin, as I, as evidenced by the fact that 12 out of first 18 by/
g
determined by ELISA and SPR (Figure 3) (14). anti-RTA VHHs that we characterized were ues
TB12, PA1 and PH12 are some of the most competitively inhibited from binding to RTA by t o
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potent toxin-neutralizing mAbs in our collection PB10 (16-18). To circumvent this issue, we Ja
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(Figure 3C) and each has been shown to devised five different screening/masking/capture a
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passively protect mice against ricin challenge. strategies, which will be detailed in a separate 7
, 2
Thus, so-called epitope Cluster II appears to manuscript (D. Vance, C. Shoemaker, and N. 0
1
9
represent a particularly vulnerable region on Mantis, manuscript in preparation). V5E1 was
ricin toxin. isolated from a screen in which ricin holotoxin
In the current study, we provide the first was captured with a Cluster I-specific antibody
structural insights into epitope Cluster II, as well (V H F5) and masked with a Cluster III
H
as clues to contact points on RTA that are antibody (V H F6) (16,17). It is unlikely that
H
associated with toxin-neutralizing activity. We V5E1 would have been isolated without
solved the X-ray crystal structures of three masking Cluster I and, possibly, Cluster III
different Cluster II-specific V Hs, each in epitopes.
H
complex with RTA. The two V Hs with weak The X-ray crystal structure of the V5E1-
H
toxin-neutralizing activities, E1 and V1C7, have RTA complex offers several clues as to the
overlapping structural epitopes on RTA that possible mechanism(s) underlying V5E1’s
primarily encompass residues within α-helix D toxin-neutralizing activity. First, V5E1 contacts
(residues 141-152) and α-helix E (residues 161- loop F-G (residues 192-196), a region located
180). E1’s slightly lower dissociation constant within an electrostatically positive patch of RTA
may explain its marginally better toxin- involved in mediating contact with ribosomes
neutralizing activity as compared to V1C7. (21,26). In a yeast model, site-directed
mutagenesis has implicated Arg193 and Arg196
6
as being important in ribosome binding (27). It hypermutation in response to ricin toxoid
is therefore notable that V5E1 forms a salt vaccination, not in response to either of the
bridge with Arg193, as well as H-bonds with individual subunits. The implications for
Tyr194 and Asn195. α-helix F and loop F-G vaccine design are significant considering that
also constitutes the core of SyH7’s epitope, as the two candidate vaccines being tested are
initially suggested by pepscan analysis (15) and derivatives of RTA, without RTB. RiVax is a
confirmed by hydrogen deuterium recombinant full-length version of RTA with
exchange/mass spectroscopy (R.Toth, D.Weis, point mutations at residues Tyr80 and Val76 to
and D.Volkin, manuscript in preparation). In an abolish RTA’s RNA N-glycosidase activity and
in vitro translation assay, SyH7 reduces RTA’s eliminate the protein’s propensity to induce
ability to inactivate ribosomes by >1,000-fold, vascular leak syndrome (VLS), respectively
presumably because the mAb buries RTA’s (10,32). RVEc is a truncated derivative of RTA
electrostatic positive patch and/or physically that lacks a small hydrophobic loop in the N-
occludes RTA-ribosome contact (15). terminus (residues 34-43), as well as C-terminal
Preliminary evidence indicates that V5E1 also residues 199-267 (33-35). Thus, neither RiVax
partially interferes with the ribosome- nor RVEc would be expected to elicit V5E1-like
inactivating properties of RTA in vitro (T. antibodies. In an effort to overcome this issue,
Czjaka, D. Vance, M. Rudolph and N. Mantis, we reconstituted RiVax with RTB in vitro and D
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unpublished results). used this novel toxoid immunogen to vaccinate n
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Second, superpositioning of the X-ray mice, as well as two additional alpacas (D. ad
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able to contact RTB’s subdomain 2γ, near one of still ongoing, preliminary analysis of the http
two galactose binding pockets involved in ricin antibody responses in mice suggest that RiVax ://w
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uptake into host cells (23). RTB’s two galactose- toxoid does indeed boost toxin-neutralizing titers w
binding pockets are located on opposite ends of as compared to RiVax alone. .jbc
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the molecule (28). While the two elements are While the crystal structure of V5E1-RTA rg
homologous to each other, they have slightly complex offers clues as to the mechanisms by by/
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different substrate specificities and affinities; which V5E1 neutralizes ricin, it is not obvious ue
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subdomain 1α is specific for galactose (Gal) and from the E1-RTA and V1C7-RTA structures t on
is of low affinity, while subdomain 2γ why E1 and V1C7 have only weak (not strong) Jan
recognizes Gal and N-acetylgalactosamine neutralizing activity. They each recognize uary
(GalNAc) and is of higher affinity (29,30). Our structural epitopes nested within Cluster II, as 7, 2
experimental results are consistent with V5E1 defined by competition ELISAs with SyH7, 01
9
PA1, TB12 and PH12 (Figure 2). In the case of
affecting the lectin activity of subdomain 2γ, as
V1C7, one possible explanation relates to its
the antibody reduced the attachment of ricin to
relatively low binding affinity for ricin, as
plate bound ASF and cell surface receptors by
compared to V5E1 (3.2 nM versus 0.02 nM). It
~60%. It is also possible that V5E1, by virtue of
is tempting to speculate, for example, that a 10-
its potential interaction with subdomain 2γ,
fold improvement in V1C7’s binding affinity
alters the dynamics of ricin uptake and/or
would result in a corresponding increase in
intracellular transport (29,31).
toxin-neutralizing activity. In a separate study,
The V5E1-RTA crystal structure reveals a
we have identified three V1C7-like family
potential shortcoming of two candidate ricin
members from the HobJo V H library that differ
toxin subunit vaccines, RiVax and RVEc (13). H
in their relative affinities and toxin-neutralizing
As noted above, V5E1 appears to recognize a
activities. The three new family members were
quaternary epitope in which CDR1 makes
subjected to homology modeling by Rosetta
contact with RTA, while CDR3 makes contact
using the V1C7-RTA complex as a template, to
with RTA and RTB. From an immunological
identify residues in CDRs 1, 2 and 3 that
perspective, V5E1 most certainly arose as a
account for differences in affinity (A. Bazzoli,
consequence of affinity maturation and somatic
D. Vance, M. Rudolph, J. Karanicolas, and N.
7
Mantis, manuscript in preparation). Our long some modifications (16). JIY-E1 (referred to as
term goal is to engineer higher affinity variants E1) was identified in a pan against RTA-coated
of V1C7 as a means to parse out the relative immunotubes, essentially as described (16).
contributions of affinity and epitope specificity V1C7 was identified using a screen in which
in ricin toxin-neutralization. ricin was captured by surrogate receptor
asialofetuin (ASF) in order to retain proper
EXPERIMENTAL APPROACHES conformation of ricin. V5E1 was identified in a
Materials- Ricin toxin (RCA-II), biotinylated pan against ricin captured by the Cluster I
ricin (b-ricin) and RTA were obtained from specific V H JIV-F5 (16), in order to mask that
H
Vector Laboratories (Burlingame, CA). HRP- immunodominant region. Cluster III was also
anti-E-tag antibodies (Bethyl, Montgomery, TX) masked with an excess of V H JIV-F6. The
H
and HRP-anti-M13 Ab were purchased from GE details of the screens are described elsewhere
Healthcare (Piscataway Township, NJ). All (D. Vance, J. Tremblay, C. Shoemaker and N.
other chemicals and reagents were purchased Mantis, manuscript in preparation).
from Sigma-Aldrich (St. Louis, MO) unless Vero cell cytotoxicity assays- The Vero
noted otherwise. Epitope tagged VHHs were cell cytotoxicity assay has been described in
produced and purified as described.(24). detail elsewhere (37). Vero cells grown in
DMEM containing 10% FBS were seeded D
o
ELISA, surface plasmon resonance and flow (5x104 per mL) in 96 well cell culture plates and wn
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cytometry- The basic ELISA protocol has been incubated at 37°C overnight. The cells were a
d
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(dTehscerrimbeodS c(1ie6n)t. iNficu,n Scw-Imedmesubnoor op,l aNteJs) were tthheen a bosveenrlcaei do rw pirthes reinccine (o1f0 5 n-fgo/lmd Ls,e r1i5a0l dpiMlut)i oinn s d from
coated overnight at 4°C with target antigen (1 of VHHs, and incubated at 37°C for 2 h. The http
µg /mL), blocked for 2 h with 2% goat serum in cells were then washed and fresh medium ://w
w
0.1% PBST and then incubated for 1 h with five- applied. Cell viability was assessed 45-48 h w
fold serial dilutions of VHHs and then anti-E- later using CellTiter-Glo (Promega, Madison, .jbc
.o
HRP antibodies diluted 1:10000. For WI). In cytotoxicity assays in which VHH was rg
competition assays, murine IgGs (1 ug/mL) were competed against mAb, VHH concentration was by/
g
coated onto wells overnight, then blocked for 2 held steady while the mAb concentration was u
e
s
h. In a separate dilution plate, VHHs were diluted 2-fold. t o
n
diluted into biotinylated-ricin at the fixed EC90 Cloning, expression, and purification of Jan
concentration for each individual coated mAb. untagged VHHs for crystallization- PCR ua
ry
These mixtures were then transferred into the amplicons corresponding to E1 (residues 1-134), 7
, 2
mAb coated plates and allowed to bind for 1 V1C7 (residues 1-136), V5E1 (residues 1-134), 0
1
9
hour. After washing, bound biotinylated-ricin and RTA (residues 1-267) were subcloned into
was detected with Streptavidin-HRP (1:1000) the N-terminally deca-histidine maltose binding
(ThermoFisher), and developed with SureBlue protein tagged MCSG9 expression vector using
Peroxidase Substrate (KPL). The reaction was a standard ligase independent cloning protocol.
quenched with 1M phosphoric acid and All 4 proteins were expressed in E. coli strain
absorbance was read at 450 nm using a BL21(DE3)-pRARE. The transformed bacteria
VersaMax Microplate Reader (Molecular were grown at 37°C in TB medium and induced
Devices, Sunnyvale, CA). VHH affinity for at 20°C with 0.1 mM IPTG at an OD600 of 0.6
ricin was determined by surface plasmon for ~16 hours. After induction, cells were
resonance (SPR) using the ProteOn XPR36 harvested and resuspended in 20 mM Tris-Cl pH
(Bio-Rad Inc. CA, USA), as described 7.5 and 150 mM NaCl. The cell suspension was
previously(18). Ricin binding assays to THP-1 sonicated and centrifuged at 30,000 x g for 30
cells was described previously (36) minutes. After centrifugation, the protein-
containing supernatant was purified by nickel-
Identification of Cluster II V Hs- The affinity and size-exclusion chromatography on
H
RTA-specific V Hs were identified using the an AKTAxpress system (GE Healthcare), which
H
basic panning strategy described previously with consisted of a 1 mL nickel affinity column
8
followed by a Superdex 200 16/60 gel filtration collected at the 24-ID-C and 24-ID-E beamlines
column. The elution buffer consisted of 0.5M at the Advanced Photon Source, Argonne
imidazole in binding buffer, and the gel filtration National Labs.
buffer consisted of 20 mM HEPES pH 7.6, Structure determination and refinement-
150mM NaCl, and 20mM imidazole. Fractions The structures of each RTA-VHH complex was
containing protein were pooled and subject to solved by molecular replacement using the
TEV protease cleavage (1:20 weight ratio) for 3 program Phaser (38). Molecular replacement
hours at room temperature in order to remove calculations were performed using the
the decahistidine-maltose binding protein tag. coordinates of the ricin A chain as a search
The cleaved protein was passed over a 1mL Ni- model for RTA (PDB: 1RTC) in all three RTA-
NTA agarose (Qiagen) and 1 mL Amylose- VHH complexes. The VHH coordinates used as
agarose gravity column to remove the added a search model for all three RTA-V H
H
TEV protease, cleaved residues, and uncleaved complexes was D10 (PDB: 4LGR) with all three
fusion protein. In order to generate RTA-VHH of the CDRs removed from the search model.
protein complexes, after purification RTA was For the RTA-V1C7 and RTA-V5E1 structures,
mixed in a 1:1 stoichiometry with the purified the resulting phase information was used to
VHH and incubated on ice for 1 hour. Purified autobuild most of the model for the both RTA-
RTA-V H complex was concentrated to a final VHH structures using the program ARP (39). D
H o
w
total concentration of 10 mg/ml for Some additional manual building of each model n
lo
crystallization experiments. was performed with COOT (40). For the RTA- a
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3 RTA-VCrHys tcaollmizpalteioxn c raynsdta dlsa twa ecroel lgercotwionn -b Ay ll Eus1e dst riuncseturtr eth, eth Ee 1r esseuqlutienngc pe haansde minafonrumalalyti obnu iwlda s d from
H
sitting drop vapor diffusion at 20°C using a the mode in COOT. All structural refinement http
protein to reservoir volume ratio of 1:1 with was done employing the PHENIX package (41). ://w
w
total drop volumes of 0.4 µL. Crystals of the Twinned refinement was performed for the w
RTA-E1 complex were grown against RTA-E1 complex using the twin operator -k,-h,- .jb
c
.o
crystallization buffer containing 1.5 M l with a twinning fraction of 0.015. During rg
Ammonium Sulfate and 25% glycerol. Crystals refinement a cross-validation test set was created by/
g
of the RTA-E1 complex nucleated within 24 from a random 5% of the reflections. Data u
e
s
hours and grew slowly to full size of ~60 µm collection and refinement statistics are listed in t o
n
over a period of 10-14 days. Crystals of the Table S1. Molecular graphics were prepared Ja
n
RTA-V1C7 complex were grown with a using PyMOL (Schrodinger) (DeLano Scientific ua
ry
crystallization buffer containing 100 mM LLC, Palo Alto, CA). 7
, 2
sodium acetate pH 4.6 and 8% PEG4000. Antibody-antigen shape correlation- We 0
1
9
Crystals of the RTA- V1C7 complex nucleated examined the shape correlation parameter Sc
within 24 hours and grew to full size of ~100 µm [16] to see whether there was a correlation
within 5 days. Crystals of the RTA- V5E1 between toxin-neutralizing capacity, binding
complex were grown using a crystallization affinity (Kd), and shape complementarity within
buffer containing 100 mM Tris pH 7.0 and 20% the RTA-VHH interfaces described here.
PEG 1000. Crystals of the RTA-V5E1 complex Accession Numbers- The structures
took 8 days to nucleate and continued growing generated in this study were deposited in the
very slowly for another 7 days until they reached Protein Data Bank (PDB;
a full size of ~60 µm. All crystals were flash http://www.rcsb.org/pdb/) under accession
frozen in liquid nitrogen after a short soak in the numbers 5BOZ (E1), 5J56 (V1C7), and 5J57
appropriate crystallization buffers supplemented (V5E1) as described in Table 1.
with 20-25% ethylene glycol. Data were

Acknowledgements:We gratefully acknowledge the 24-ID-C and 24-ID-E beamline staff at the
Advanced Photon Source for their assistance in data collection and Dr. Jon Robertus (University of
Texas, Austin) for providing us with a plasmid encoding RTA. We thank the Wadsworth Center’s
9
Immunology Core for assistance with flow cytometry and SPR. We thank Amanda Poon for assistance
with the ricin binding assays to THP-1 cells.

Conflicts of Interest: The authors declare that they have no conflicts of interest with the contents of this
article.

Author Contributions: MJR, DJV, MC and YR conducted experiments described in paper; MJR, DJV
and NJM analyzed the results and wrote the paper. All authors reviewed the results and approved the final
version of the manuscript.

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