Domain-swapped structure of the potent antiviral protein griffithsin and its mode of carbohydrate binding.

Abstract

The crystal structure of griffithsin, an antiviral lectin from the red alga Griffithsia sp., was solved and refined at 1.3 A resolution for the free protein and 0.94 A for a complex with mannose. Griffithsin molecules form a domain-swapped dimer, in which two beta strands of one molecule complete a beta prism consisting of three four-stranded sheets, with an approximate 3-fold axis, of another molecule. The structure of each monomer bears close resemblance to jacalin-related lectins, but its dimeric structure is unique. The structures of complexes of griffithsin with mannose and N-acetylglucosamine defined the locations of three almost identical carbohydrate binding sites on each monomer. We have also shown that griffithsin is a potent inhibitor of the coronavirus responsible for severe acute respiratory syndrome (SARS). Antiviral potency of griffithsin is likely due to the presence of multiple, similar sugar binding sites that provide redundant attachment points for complex carbohydrate molecules present on viral envelopes.

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Structure 14, 1127–1135, July 2006 ª2006 Elsevier Ltd All rights reservedDOI 10.1016/j.str.2006.05.017
Domain-Swapped Structure of the Potent
Antiviral Protein Griffithsin and Its Mode
of Carbohydrate Binding
Natasza E. Zio ´łkowska,1Barry R. O’Keefe,2
Toshiyuki Mori,2,5Charles Zhu,2,3Barbara Giomarelli,2
Fakhrieh Vojdani,4Kenneth E. Palmer,4,6
James B. McMahon,2and Alexander Wlodawer1,*
1Protein Structure Section
Macromolecular Crystallography Laboratory
2Molecular Targets Development Program
Center for Cancer Research
3Werner H. Kirsten Student Internship Program
National Cancer Institute
NCI-Frederick
Frederick, Maryland 21702
4Large Scale Biology Corporation
3333 Vaca Valley Parkway
Vacaville, California 95688
Summary
The crystal structure of griffithsin, an antiviral lectin
from the red alga Griffithsia sp., was solved and re-
fined at 1.3 A˚resolution for the free protein and 0.94
A˚for a complex with mannose. Griffithsin molecules
form a domain-swapped dimer, in which two b strands
ofonemoleculecompleteabprismconsistingofthree
four-stranded sheets, with an approximate 3-fold axis,
of another molecule. The structure of each monomer
bears close resemblance to jacalin-related lectins,
but its dimeric structure is unique. The structures of
complexes of griffithsin with mannose and N-acetyl-
glucosamine defined the locations of three almost
identical carbohydrate binding sites on each mono-
mer. We have also shown that griffithsin is a potent
inhibitor of the coronavirus responsible for severe
acute respiratory syndrome (SARS). Antiviral potency
of griffithsin is likely due to the presence of multiple,
similar sugar binding sites that provide redundant at-
tachment points for complex carbohydrate molecules
present on viral envelopes.
Introduction
A number of lectins, particularly those that bind high-
mannose sugars, have been shown to exhibit significant
activity against human immunodeficiency virus (HIV), as
well as against a number of other viruses (Botos and
Wlodawer, 2005). For these reasons, several such pro-
teins, among them cyanovirin-N (Boyd et al., 1997) and
scytovirin (Bokesch et al., 2003), have been under active
development as topical antiviral therapeutics. Most re-
cently,anotherpotentantivirallectin,griffithsin,wasiso-
lated from the red alga Griffithsia sp., collected from the
watersoffNewZealand(Morietal.,2005).Griffithsinwas
shown to inhibit the cytopathic effects of different iso-
lates of HIV-1 at concentrations as low as 43 pM (Mori
et al., 2005), and its binding to viral envelope glyco-
proteins was shown to be directly dependent on their
glycosylation states. These properties make griffithsin
a promising potential candidate for development as a
future pharmaceutical agent.
Knowledgeofthethree-dimensionalstructureofputa-
tive therapeutic proteins is very helpful in their develop-
ment, yet the amino acid sequence of griffithsin was
initiallyreportedtobeunique,givingnocluestoitsstruc-
ture (Mori et al., 2005; Giomarelli et al., 2006). More care-
ful analysis of the sequence of griffithsin indicates that
this protein belongs to a diverse family of b-prism-I (or
jacalin-related) lectins (Raval et al., 2004) and shares
structural characteristics with proteins such as jacalin
(Aucouturier et al., 1987), artocarpin (Jeyaprakash
etal.,2004),andheltuba(Bourneetal.,1999)(Figure1A).
However, sequence identity between griffithsin and any
of these other proteins is less than 30% (Mori et al.,
2005) (see also Figure 1A). Thus, structural details, par-
ticularly of the carbohydrate binding site(s), cannot be
readilymodeled,necessitatingsolutionofanexperimen-
tal structure. We report here the structures of griffithsin
in three crystal forms, with and without bound carbohy-
drate molecules.
Since the antiviral activity of griffithsin against HIV-1
has already been proven (Mori et al., 2005), we have in-
vestigated other viral targets of this lectin. We have now
shown that griffithsin is a potent inhibitor of the life cycle
of the coronavirus that causes severe acute respiratory
syndrome (SARS), a life-threatening disease that first
manifested itself only a few years ago. Such broad anti-
viral activity makes this newly characterized lectin apar-
ticularly promising target for the development as a novel
antiviral agent.
Results
Description of the Fold of Griffithsin
The structure of griffithsin was solved by single-wave-
length anomalous diffraction (SAD) with selenomethio-
nine (SeMet)-labeled protein produced in E. coli and
was refined at high resolution with material grown in
plants. Three different crystal forms of griffithsin were
studied (Table 1). The fold of griffithsin corresponds to
the well-known b-prism motif (Chothia and Murzin,
1993), found mainly in a variety of lectins but also in
some other proteins (Shimizu and Morikawa, 1996).
The motif consists of three repeats of an antiparallel
four-stranded b sheet that form a triangular prism. How-
ever, unlike other lectins whose sequences are com-
pared in Figure 1A, griffithsin forms an intimate dimer
(Figure 2) in which the first two b strands of one chain
(consisting of 18 residues located at its N terminus) are
associated with ten strands of the other chain (and
vice versa). That arrangement is unique among the fam-
ily members that have been described so far and is seen
in all crystal forms of griffithsin, despite the presence of
a significant nonnative N-terminal extension in one of
*Correspondence: wlodawer@ncifcrf.gov
5Present address: Takeda Pharmaceutical Company, 2-17-85 Juso-
homachi, Yodogawa-ku, Osaka 532-8686, Japan.
6Present address: Intrucept Biomedicine LLC, 707 West Monte
Vista Ave., Vacaville, CA 95688.

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them. Thus, griffithsin can be described as a domain-
swapped dimer, although it is not clear whether a corre-
sponding monomeric forms does (or even could) exist.
A considerable distance between the residues A18 and
B19 (10.5 A˚between their Ca atoms) is much larger
than a corresponding distance in the domain-swapped
proteins that have been shown to exist in both mono-
meric and dimeric forms, such as, for example, another
antiviral lectin, cyanovirn-N (Yang et al., 1999). In our
previous study that utilized size-exclusion chromatog-
raphy, griffithsin was shown to exist as a homodimer
in solution (Giomarelli et al., 2006).
Griffithsin contains three strictly conserved repeats
of a sequence GGSGG (Figure 1B). These repeats were
Table 1. Data Collection and Refinement Crystallographic Data
Crystal
Unliganded
Trigonal SeMet
Unliganded
Orthorhombic
Complex with
N-Glucosamine
Complex with Mannose
12
Data Collection
Cell parametersa = b = 66.87 A˚,
c = 68.20 A˚;
a = b = 90º,
g = 120º
a = 33.92 A˚,
b = 64.86 A˚,
c = 96.08 A˚;
a = b = g = 90º
a = 33.83 A˚,
b = 64.45 A˚,
c = 96.18 A˚;
a = b = g = 90º
a = 53.77 A˚,
b = 39.46 A˚,
c = 57.24 A˚;
a = g = 90º
b = 91.0º
P21
2
30–1.78
286,011
21,739
93.3 (73.7)
37.2 (6.2)
4.1 (17.6)
a = 53.74 A˚,
b = 39.44 A˚,
c = 57.16 A˚;
a = g = 90º
b = 91.0º
P21
2
30–0.94
1,416,707
148,669
95.5 (75.0)
14.16 (1.5)
6.4 (55.0)
Space group
Molecules/a.u.
Resolution (A˚)
Total reflections
Unique reflections
Completeness (%)b
Avg. I/s
Rmerge(%)c
P3221
1
20–2.0
260,626
22,075 (11,942)a
95.7 (66.6)
33.6 (1.7)
3.2 (34.9)
P212121
2
15–1.3
827,314
53,034
99.8 (99.1)
14.0 (2.5)
6.2 (38.2)
P212121
2
30–2.0
182,881
13,606
92.8 (56.0)
18.9 (2.2)
8.5 (36.1)
Refinement Statistics
R (%)d
Rfree(%)e
Rms deviations bond
lengths (A˚)
Rms deviations angles
(degrees)
PDB code
18.89
25.41
0.020
15.91
18.94
0.021
19.93
27.40
0.020
15.07
18.87
0.019
13.55
15.61
0.024
1.87 1.46 1.971.76 2.15
2gux2gty2gue 2guc2gud
aThe number of unique reflections is given first with Friedel pairs unmerged and after merging in parentheses.
bThe values in parentheses relate to the highest resolution shell.
cRmerge=PjI 2 <I>j/PI, where I is the observed intensity, and <I> is the average intensity obtained from multiple observations of symmetry-
related reflections after rejections.
dR =PkFoj 2 jFck/PjFoj, where Foand Fcare the observed and calculated structure factors, respectively.
eRfree= defined by Bru ¨nger (Bru ¨nger, 1992).
Figure 1. Comparison of the Sequences of
Griffithsin and Related Lectins
(A) Structure-based sequence comparison of
griffithsinandeightotherlectinswithasimilar
fold. Residues that are strictly conserved
among all nine proteins are shown on red
background, and those of a similar character
are highlighted in yellow. Residues that are
strictly conserved among the other lectins
but not in griffithsin are highlighted in green
in the former and magenta in the latter.
(B) Structure-based sequence alignment of
the three b sheets (blades) in griffithsin. Con-
served residues that interact directly with the
bound carbohydrates are highlighted in red,
whereas conserved residues that do not
make such contacts are shown on magenta
background.
Structure
1128 1128
Structure

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predicted to represent boundaries between subdo-
mains (Mori et al., 2005), since similar sequences are of-
ten reported for typical flexible linkers. However, in grif-
fithsin, these sequences are found in loops connecting
the first and fourth strand of each sheet rather than in
interdomain linkers and are very well ordered. As dis-
cussed below, the main chain amide of the last residue
of each of these sequences participates in creation of
a ligand binding site, and the strict conservation of this
sequencemayberelatedtothepresenceofthreemono-
saccharide binding sites on each molecule of griffithsin.
Comparisons with Related Lectins
The structure of griffithsin was compared to the struc-
tures of a number of proteins that display a similar over-
all fold, including mannose binding lectins. For the
purpose of such comparisons, we utilized a compact
domain of griffithsin derived from the coordinates of
the high-resolution, orthorhombic form of the carbohy-
drate-free protein. The domain used for the comparison
consisted of residues A1–A18 and B19–B121. For other
proteins used in these comparisons for which multiple
structures exist, we preferentially chose the structures
of complexes with carbohydrates and, in case of multi-
ple molecules in the asymmetric unit, arbitrarily chose
the first molecule present in a file if bound to a carbohy-
drate.
We found artocarpin (PDB code 1vbo) (Jeyaprakash
et al., 2004) to be the closest homolog of griffithsin,
with an rms deviation of only 1.44 A˚between 110 pairs
of Ca atoms. The deviations for the closely related lec-
tins including banana lectin (2bmz) (Meagher et al.,
2005), heltuba (1c3m) (Bourne et al., 1999), Artocarpus
hirsuta lectin (1toq) (Rao et al., 2004), Maclura pomifera
agglutinin (1jot) (Lee et al., 1998), and jacalin (1ugw)
(Jeyaprakash et al., 2003) were only slightly larger, at
1.60 A˚(113 pairs), 1.64 A˚(112 pairs), 1.72 A˚(112 pairs),
1.78 A˚(111 pairs), and 1.80 A˚(113 pairs), respectively.
The deviation from molecule D of calsepa (PDB code
1ouw; that molecule chosen due to the presence of
bound mannose) is 2.05 A˚for 115 pairs, whereas the de-
viationforthefirstofthethreecovalently linkeddomains
of Parkia lectin was 1.97 A˚for 116 pairs. By comparison,
thedeviationfromthecoordinatesofvitellinemembrane
outer layer protein I (PDB code 1vmo) (Shimizu and Mor-
ikawa, 1996), a protein that may exhibit enzymatic rather
than strictly carbohydrate binding properties, was 3.0 A˚
for 116 pairs of Ca atoms.
The first of the repeated broad GGSGG loops in grif-
fithsin (residues 8–12) is located between strands 1
and 2 that are swapped between the two molecules
forming the dimer. This region is close to the principal
carbohydrate binding sites in this family of lectins, yet
it shows only partial structural conservation, limited to
glycines 8 and 12. Jacalin, Maclura pomifera agglutinin,
and Artocarpus hirsuta lectin contain two separate
chains, with a break between chains B and A falling in
exactly this area. The trace of that chain in other lectins,
on theotherhand, isverysimilar tothatingriffithsin(Fig-
ure 3), although the amino acid sequence is not very
highly conserved between them (Figure 1A). A loop con-
necting the two inner strands of the samesheet (Gly108-
Tyr110 in griffithsin), on the other hand, follows an al-
most identical path in all the compared structures.
This structural element interacts closely with the carbo-
hydrate molecules (or with a sulfate in the structure of
griffithsin solved in the absence of a sugar ligand), and
it is not surprising that its sequence is also very similar.
Griffthsin differs very considerably from all other
compared lectins in the conformation of the adjacent
Figure 3. Superposition of the Backbone
Tracing of Griffithsin and Other Related
Lectins
A compact domain of griffithsin was defined
as residues A1–A18 and B19–B121. The fol-
lowing colors were used: griffithsin, red; arto-
carpin (1vbo), blue; heltuba (1c3m), magenta;
parkia lectin (1zgs), yellow; calsepa (1ouw),
pink; banana lectin (2bmz), black; jacalin
(1ugw), violet; AHL (1toq), brown; MPA (1jot),
green.
Figure 2. A Ribbon Ca Trace of a Griffithsin Dimer, Based on the Co-
ordinates of the His-Tagged Version of the Protein
Molecule A is colored blue, and molecule B green, with the N-termi-
nal extension that resulted from the cloning procedure colored red.
Crystal Structure of Griffithsin
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b-hairpin, consisting of Gly66 and Asp67. This loop is
much shorter than in any other lectins, leaving this part
of the structure much more open. With the exception
of artocarpin, in which this loop is even longer, all other
lectins are similar in this area.
Another conserved broad loop with the GGSGG se-
quence, 86–90, is located near the secondary carbohy-
drate binding site in calsepa and follows a course quite
different than in banana and parkia lectins, yet similar to
the path in the other proteins. However, since that site is
createdin calsepa also by the previously described loop
that is significantly shorter in griffithsin, it is unlikely that
an analogous site could exist in the latter.
Another short inner loop formed by residues 26–28 in
griffithsin bears considerable similarity to the equivalent
loops in banana and parkia lectins as well as artocarpin
butisquitedifferentintheremainingproteins.Thatloop,
together with the last broad GGSGG loop (40–44) cre-
ates a second carbohydrate binding site in banana lec-
tin. The latter loop differs very significantly between
mostoftheseproteins,withonlyitspathinbananalectin
having any resemblance to its trace in griffithsin.
Only 12 residues are strictly conserved in all the com-
pared structures (Figure 1A). It appears that almost all, if
not all of them, are crucial in establishing the b-prism-I
fold. As mentioned above, Gly8 and Gly12 are part of
abroadloopinthefirstsheetandarefoundinthevicinity
of the principal carbohydrate binding site. Interestingly,
even though these residues are conserved also in the
two-chain lectins such as jacalin, they are located on
two separate chains that are not connected directly.
Glu56, Phe74, Thr76, and Asn77 are all part of a partially
buriedclusterthatfillsthespacebetweensheets2and3
and maintains their spacing. The side chains of Glu56
and Thr76 make a strong (2.6 A˚) hydrogen bond, and
this feature is also strictly conserved. Gly83, Pro84,
and Gly86 are part of an outer strand in sheet 3 and
are located in a region that shows surprising conserva-
tion, including the cis conformation of the Gly-Pro pep-
tide bond. Gly105 and Gly108 are found in a very highly
conserved loop connecting the inner strands of sheet 1,
whereas the nearby Asp112 may be the only side chain
of a residue directly involved in carbohydrate binding
to be found in all the structures.
Six residues that are conserved in all other structures
are not preserved in griffithsin (Figure 1). Four of them
(Arg5, Phe7, Ile63, and Tyr68) are part of a cluster be-
tween sheets 1 and 3. The strand containing the latter
two residues is shifted considerably in griffithsin com-
pared to all other structures and thus may need differ-
ently sized residues to maintain its location. Ile103
(Phe in all other structures) is deeply buried and its na-
ture depends on the other neighbors found in its vicinity.
The side chain of Tyr68 points at the unique carbohy-
drate binding site of griffithsin (see below), and this
residue may be partially responsible for the properties
of this lectin. In addition to these residues, a deletion
in the aligned sequences, located between residues 18
and 19 of griffithsin, corresponds to an isoleucine in all
other structures. Deletion of this residue may be related
to the domain-swapped nature of griffithsin that differ-
entiates this protein from all the other related lectins. It
should be noted that the site of domain swapping that
follows the second b strand is topologically different
than the location of the separation between chains A
and B of jacalin, the latter following the first b strand.
The structure presented here does not shed much
new light on the nature and/or importance of residue
31, which was reported not to be among the 20 standard
amino acids (Mori et al., 2005). An alanine inserted at
that position in the artificial genes used for the work
described here is buried, but the side chain of His38 at
which it is pointing (at a distance of 3.3 A˚to CE1) could
easily move if a larger residue were to substitute for
Ala31. Although this residue follows Asp30, the side
chain of which interacts directly with a bound sugar
(see below), it does not appear itself to be implicated
in carbohydrate binding since its side chain points into
a direction opposite to that of Asp30. The residues
that follow the other two aspartic acids involved in car-
bohydrate binding are asparagine and serine; thus the
identity of these residues does not appear to be an im-
portant contributor to the carbohydrate binding proper-
ties of griffithsin.
The Mode of Binding of Carbohydrates
Cocrystallization of griffithsin with mannose yielded a
new, monoclinic crystal form with excellent diffraction
properties,containingagriffithsindimerintheasymmet-
ric unit. A comparison of the coordinates resulting from
the two high-resolution structures of the unliganded
and mannose bound griffithsin yielded rms difference
of 0.46 A˚. The largest differences were observed for
SerA88,GlyB11,andGlyB53,aswellasintheorientation
of the side chains of Tyr28, Tyr68, Asp70, and Tyr110 of
both molecules. Native griffithsin incorporated SO422
ions in places corresponding to the localization of
Man1, Man4, Man5, and Man6 in the structure of the
complexwithmannoseandamoleculeofethyleneglycol
in place of Man2.
Seven mannose molecules were found to be bound to
eachdimerofgriffithsininthe1.8A˚structure butonlysix
in the atomic-resolution structure. The latter carbohy-
drates are very well ordered in both structures, whereas
the seventh molecule in the lower-resolution structure,
bound in a site created through intermolecular interac-
tions in the crystal, shows some indications of disorder.
It is likely that low-occupancy mannose might also be
present in this site in the atomic-resolution structure,
but such variability suggests that ligand binding in this
site is not very specific, and thus site 7 will not be con-
sidered further. However, the six principal sites are
arranged in two groups of three, one on molecule A
and the other on molecule B, and are very similar in
both structures. The only significant difference is that
whereas the electron density in the lower-resolution
structure (Figure 4) could be interpreted as resulting
from the presence of exclusively a mannose, clear indi-
cations of the presence of an admixture of b mannose
are seenintheatomic-resolution structure. Similarpres-
ence of both anomers of mannose was also indicated in
the Ralstonia solanacearum lectin, solved at atomic res-
olution (PDB code 1vyy; no other reference available).
This minor component will not be considered in the
further discussion.
The interactions between Man1, Man2, and Man3
with molecule A are virtually identical to the interactions
of Man4, Man5, and Man6 with molecule B. Man1
Structure
1130

Page 5

(and Man4) are found in a site that corresponds to the
major carbohydrate binding site described for a variety
of lectins from this family. Due to domain swapping
that exists in griffithsin, however, this site is formed by
the residues from both molecules A and B. The protein
side chain most responsible for the specific interactions
with the mannose is AspA112, which interacts through
its carboxylate with the hydroxyl oxygens O6 and O4
of the mannose. Oxygen O6 also accepts a hydrogen
bond from the main chain amide of TyrA110, whereas
O5 is a recipient of a bond from the N of AspA109.
Hydroxyl O3 accepts a proton from the main chain am-
ide of GlyB12, while O2 and O1 interact only with solvent
(water or glycol). The interactions for Man4 are the same
with the opposite chains. This pattern of interactions is
very similar for the other mannose residues. Man2 (and
Man5) bind in an area anchored by AspA30 and B30, re-
spectively, with the hydrogen bonds to the main chain
amides of residues 27, 28, and 44. Similarly, Man3 (and
Man6) interact primarily with Asp70, with the other hy-
drogen bonds supplied by the main chain amides of res-
idues 67, 68, and 90 (Figure 5).
The presence of three carbohydrate binding sites that
formanalmostperfectequilateraltriangleontheedgeof
the lectin (Figure 4) is unprecedented for this family, al-
though seen in lectins with b-prism-II fold (Hester
et al., 1995; Wright et al., 2000). However, the distances
between the sites in griffithsin are w14 A˚, whereas cor-
responding distances in Galanthus nivalis agglutinin are
w25 A˚. While the presence of site 1 was reported for all
b-prism-I lectins, site 2 has so far only been seen in ba-
nana lectin (Meagher et al., 2005). As seen in Figure 1,
Asp112 is strictly conserved in all lectins that were com-
pared, explaining the conservation of this site. Not sur-
prisingly, the only other lectin that has an equivalent of
Asp30 is banana lectin since that residue is crucial for
creation of site 2. Site 3, however, appears to be unique
to griffithsin, and Asp70 is not conserved at all in any
other b-prism-I lectin.
The three carbohydrate binding sites of griffithsin are
formed from the parts of the structure with extensive se-
quence conservation. A tyrosine precedes the aspartic
acid by two positions, and a glycine by four. Either a leu-
cine or isoleucine is immediately preceding the aspar-
tate, and position 23 is occupied by an aspartic acid
or a serine. With the sequence and the structure of this
region highly conserved, it is not surprising that the
main chainamides ofthese residues canmakevery sim-
ilar interactions with the sugars in all three sites. The
sequence of site 1 is conserved among other lectins.
Gly108 is strictly conserved, and the positions corre-
sponding to Tyr110 and Leu111 in griffithsin are occu-
piedbysimilarresiduesinallb-prism-Ilectinscompared
in Figure 1A. Sequences corresponding to site 2 are not
wellconserved,exceptforbananalectinwhereaglycine
precedes by four positions the aspartic acid involved in
carbohydrate binding. Sequences corresponding to site
3 show also limited conservation. For example, Gly66 is
conserved in all other lectins except for banana lectin. A
positioncorresponding toTyr68ingriffithsinisoccupied
by valine, and the position of Ile69 is occupied by similar
residues in the other lectins.
The final interactions in the three sites are provided
by the main chain amides of Gly12, Gly44, and Gly90, re-
spectively.AlltheseglycinesaretheC-terminalresidues
of the strictly conserved sequence GGSGG, and this
observation may indeed explain the unprecedented
conservation of this feature of the primary structure of
griffithsin. Gly12, together with Gly8, is strictly con-
served in all compared lectins. The sequence GGSGG
is also present in banana lectin in site 2. Gly86 is strictly
conserved, and Gly90 is present in all lectins except for
heltuba.
While the carbohydrate molecules observed in the
cocrystal structure are remarkably clear, we have also
observed limited binding of monosaccharides in the
orthorhombic crystals soaked in either mannose or N-
acetylglucosamine, although in none of these structures
Figure 5. Details of the Interactions between
Man3 and Griffithsin
The locations and lengths of hydrogen bonds
are representative for all six principal carbo-
hydrate binding sites.
Figure 4. Mannose Binding Sites 1–3, Created Principally by Mole-
cule A of Griffithsin
The omitmap Fo2 Fcelectron density map was calculated at 1.8 A˚
resolution, based on the final coordinates of the structure of crystal
1 refined after the removal of the mannoses, and it was contoured at
the 2.7 s level.
Crystal Structure of Griffithsin
1131

Page 6

were all six principal sites occupied. Site 6 is clearly oc-
cupied by N-acetylglucosamine in a conformation simi-
lar to that of Man6 in the cocrystal structure, although
other sites are occupied by other ligands present in the
mother liquor. Ethylene glycol is found in site 5, sulfate
molecules are found in sites 1 and 4, site 2 is occupied
by only water molecules, whereas site 3 is involved in in-
termolecular contacts in this crystal form and is thus not
accessible to ligands. With the carbohydrate binding
sites at a variable distance from the symmetry-related
protein molecules, it is not certain whether the preferen-
tialbindingofacarbohydratemoleculeinsite6indicates
higher affinity or simply easier accessibility. The latter
explanation seems to be more probable in view of the
similarityofthesitesasshowninthecocrystalstructure.
To further test the antiviral activity of griffithsin, the
protein was submitted for testing against the coronavi-
rus that was responsible for SARS outbreak in 2004. It
had been reported previously that the coronovirus
bored a surface ‘‘spike’’ protein that was heavily glyco-
sylated and was able to bind to the monosaccharide-
specifichumanlectinDCSIGN (Yangetal.,2004).Based
on the demonstrated promiscuity of griffithsin binding
to oligosaccharides, it was hypothesized that griffithsin
should be capable of binding to the spike protein of the
SARS virus and inhibiting subsequent infection. The
results of three different antiviral assays with the SARS
virus showed that griffithsin could indeed inhibit both
viralreplicationandthecytopathicityinducedbythevirus
(Table 2). Though griffithsin was active against the
virus at nanomolar concentrations, it was not as active
against the SARS virus as it is against HIV (Table 3).
Discussion
Although both the His-tagged and the plant-expressed
griffithsin crystallized easily, crystals grown from the
plant-produced material (resembling more closely the
authenticprotein)diffractedsignificantlybetter.Crystals
of the His-tagged griffithsin contained only a single mol-
eculeintheasymmetric unit,whereasbothcrystal forms
grownfromtheplant-expressedmaterialcontainedtwo.
However, only the latter crystals diffracted to atomic (or
atleastnear-atomic)resolution,bothinthepresenceand
absence of bound carbohydrate. In all crystals that did
not contain bound carbohydrate, the binding sites ex-
hibited considerable disorder, with even the side chains
of the strictly conserved tyrosines 28, 68, and 110 show-
ingsignificantflexibility.Thepresenceofcarbohydrates,
whether soaked in or cocrystallized, stabilized the bind-
ing sites and significantly lowered the temperature fac-
tors of the residues involved in their binding.
Griffithsinisanunusualproteininmanyways,andcon-
sequently, the determination of its three-dimensional
structure is illuminating on many levels. These include
its unusual domain-swapped dimeric structure, its three
repeated domains, and its six independent binding sites
for monosaccharides. The antiviral lectin cyanovirin-N
has previously been reported to form domain-swapped
dimers but they differed from the griffithsin dimers in
that half of the each molecule was involved in swapping,
rather than two b strands out of 12. Both monomers and
dimers of cyanovirin-N could be isolated, whereas we
have seen no indications of the existence of griffithsin
ina monomeric form.In addition, only four carbohydrate
binding sites are present in each dimer of cyanovirin-N,
rather than six in griffithsin.
The presence of three almost identical domains in
each molecule of griffithsin is a unique adaptation of
the b-prism-I family and an advance in our knowledge
of the structural complexity of this group of lectins. The
other structurally closely related lectins (Figures 1A and
3) contain either a single carbohydrate binding site, or
at most two such sites, in each molecule. Although grif-
fithsin shares structural homology with many mannose-
or N-acetylglucosamine-specific lectins, its reported bi-
ological activity against HIV was >1000-fold better than
those previously reported for several monosaccharide-
specific lectins (Charan et al., 2000). Of those lectins
most closely related to griffithsin, only jacalin has been
previously reported to exhibit anti-HIV activity (Corbeau
et al., 1995). Jacalin was, however, found to be relatively
weak in its anti-HIV activity, not reaching an EC50level at
a high dose of 10 mg/ml (227 nM) (O’Keefe et al., 1997) in
the same whole-cell assay system in which griffithsin
displayed an EC50= 43 pM (Mori etal., 2005). The source
of this enhanced potency and, presumably, higher affin-
ity for HIV envelope glycoproteins, is of considerable in-
terest. An explanation is suggested from the structure of
griffithsin, which offers no less than six separate binding
sitesformannoseinacompactdomain-swappeddimer.
The enhanced binding potential for the high-mannose
oligosaccharides present onthe HIVenvelope glycopro-
tein gp120 to be gained from this multivalency is signifi-
cant.Thesitesoneachmonomeraremuchcloserthanin
the lectins that belong to the b-prism-II family, presum-
ably allowing tighter binding of complex carbohydrates.
Similar enhancement was seen in previous studies on
the anti-HIV protein cyanovirin-N, which detailed the
increased relative binding affinity, due to multivalent
interactions, of this domain-swapped dimer for high
mannose oligosaccharides (Shenoy et al., 2002). Initial
attempts at the crystallization of griffithsin with larger
oligosaccharides have shown signs that similar multiva-
lent binding to these carbohydrate structures occurs
Table 2. The Anti-SARS Virus Activity of Grifithsin
Assay FormatEC50(mM) EC90(mM)
Visual cytopathic effect
Neutral red
Virus yield
0.28
0.96
n.r.
n.r.
n.r.
0.34
n.r., not recorded.
Table 3. Anti-HIV Activity of Several Lectins
LectinEC50(nM)
Jacalina(O’Keefe et al., 1997)
Concanavalin Aa(Charan et al., 2000)
Urtica diocia agglutinina(Charan et al., 2000)
P. tetragonolobus lectina(Charan et al., 2000)
N. pseudonarcissus lectina(Charan et al., 2000)
Myrianthus holstii lectina(Charan et al., 2000)
Scytovirin (Bokesch et al., 2003)
Cyanovirin-N (Boyd et al., 1997)
Griffithsina(Mori et al., 2005)
>227
98
105
52
96
150
0.3
0.1
0.04
aMonosaccharide-specific lectins.
Structure
1132

Page 7

with griffithsin, leading to rapid precipitation of the com-
plexes (data not shown).
The specificity of griffithsin for monosaccharides was
at first thought to be a disadvantage for this protein’s
potential as a prophylactic or therapeutic agent. How-
ever, in some ways, the specificity displayed by griffith-
sin may be an advantage. It was previously speculated
that griffithsin, due to its more promiscuous carbohy-
drate binding, might be active against viruses such as
the coronavirus responsible for the recent SARS out-
break (Mori et al., 2005). This prediction was based on
the ability of griffithsin to bind a broader spectrum of
oligosaccharide structures than other antiviral lectins
such as cyanovirin-N (Bolmstedt et al., 2001) and scyto-
virin (Adams et al., 2004). This prediction has proved to
betrueasrecentdatafromanti-SARSassayshasshown
that griffithsin is indeed active against this virus at nano-
molar concentrations, while both cyanovirin-N and
scytovirin were inactive (data not shown). Whether or
not this protein will prove to have any clinical utility as
a prophylactic or therapeutic agent for the treatment
or prevention of viral infections remains to be answered,
but its unique structure and interactions with carbo-
hydrates, in addition to its unusual potency against HIV,
provide ample justification for continued study.
Experimental Procedures
Cloning and Bacterial Expression of Griffithsin
His-tagged griffithsin was expressed and purified as previously re-
ported (Giomarelli et al., 2006). Protein labeled with SeMet (Calbio-
chem, San Diego, CA) was expressed in a similar manner. Briefly,
His-griffithsin expressing strain BL21(DE3) cells were grown over-
night at 37ºC on an LB agar plate containing kanamycin at a concen-
tration of 30 mg/ml. A single colony was picked from the plate and
inoculated in a 500 ml culture shake flask containing 125 ml Luria-
Bertani (LB) medium (Difco, Gaithersburg, MD). The overnight cul-
ture was grown at 37ºC with 250 rpm shaking for 9 hr. The total of
2 liters of Overnight Express Autoinduction Systems 2 medium
(Novagen, Madison, WI) was equally divided into five 2 liters baffled
culture shake flasks. Kanamycin was added to this medium to a final
concentration of 30 mg/ml. Each flask then received 8 ml of the orig-
inal seed culture and was grown at 37ºC with 300 rpm shaking. After
16hrofgrowth,cellswerecollectedforextractionandpurification of
SeMet-labeled His-griffithsin as previously reported (Giomarelli
et al., 2006). His-tagged protein produced in E. coli was used for
all of the in vitro studies on the antiviral activity of griffithsin against
the SARS virus.
Purification of Griffithsin from Nicotiana Tissue
Nicothiana benthamiana plants (24 days postsowing) were grown in
a controlled environment (Shivprasad et al., 1999) and inoculated
with infectious recombinant tobamovirus vectors 1 that carried a
synthetic cDNA of griffithsin. Twelve days post inoculation (dpi), in-
fected leaf and stem material was harvested at one leaf above the
inoculation leaves, and tissue was homogenized in 20 mM acetate
buffer (pH 4.0) containing 250 mM NaCl, 15 mM ascorbic acid, and
5.3 mM sodium meta-bisulfite at tissue-to-buffer ratio of 1:1.25 w/v.
Green juice was recovered by passing the homogenate through
four layers of cheesecloth. Recovered green juice was adjusted to
pH 4.0 and then clarified by centrifugation at 7500 3 g for 30 min
(S1fraction).S1fractionwasdialyzedovernightat4ºC againstphos-
phate-buffered saline (PBS) (pH 7.4) with 7000 MWCO tubing and
centrifuged at 75,000 3 g for 30 min to remove insoluble proteins
and particles. Prior to column chromatography, the pH of the S1
fraction was adjusted to 4.0 by using HCl and filtered through
a 0.45 mm filter. Clarified S1 was loaded on a SP Sepharose Fast
Flow (GE Healthcare, Piscataway, NJ) column equilibrated with 20
mM acetate buffer (pH 4.0) (solution A). After washing the unbound
proteins from the column, griffithsin was eluted with a linear NaCl
gradient from 0 to 250 mM in solution A, with subsequent pH adjust-
ment of eluted protein fractions to pH 7.0 by using 1 M phosphate
buffer (pH 8.0) to a final concentration of 20 mM. Fractions were an-
alyzed by SDS-PAGE with 16% Tris glycine SDS-Page gels and by
matrix-assisted laser desorption-ionization time of flight (MALDI-
TOF) mass spectrometry with a PerSeptive Biosystem Voyager-DE
STR (Applied Biosystems, Foster City, CA) to verify fractions for
pooling. Protein was then loaded on a Source 15RPC column (GE
Healthcare, Piscataway, NJ) equilibrated with 20 mM phosphate
(pH 7.4) (solution B) and eluted with a 2.5% n-propanol gradient, fol-
lowed by a 6% linear n-propanol gradient in solution B. Fractions
containing >99.5% pure GRFT were pooled and dialyzed against
PBS (pH 7.4) by using 7000 MWCO dialysis tubing.
The plant-expressed griffithsin did not bear a His-tag, but was
acetylated on its N terminus. This material, like the E. coli produced
griffithsin, contained an alanine residue at position 31 to replace the
unidentified amino acid reported in the original publication (Mori
et al., 2005).
Severe Acute Respiratory Syndrome Virus Assays
Three types of assays were used to assess the activity of griffithsin
against the severe acute respiratory syndrome (SARS) coronavirus.
Inhibition of Viral Cytopathic Effect
Griffithsin was tested for its ability to inhibit the cytopathic effect
(CPE) of the SARS virus (strain 200300592). For the CPE assay,
four log10 dilutions of griffithsin (e.g., 1000, 100, 10, 1 mg/ml) were
added to triplicate wells of a 96-well flat-bottomed microplate con-
taining the Vero 76 cell monolayer; within 5 min, the SARS virus was
added, the plate was sealed and incubated at 37ºC, and CPE was
read microscopically when untreated infected controls developed
a 3 to 4+ CPE (approximately 72 to 120 hr). A known positive control
drug (interferon a-n3) was evaluated in parallel with griffithsin. The
data were expressed as 50% effective concentrations (EC50).
Increase in Neutral Red Dye Uptake
This test was run to validate the CPE inhibition seen in the initial test
and utilizes the same 96-well microplates after the CPE has been
read. Neutral red (NR) was added to the medium, and the resulting
dye uptake by the Vero 76 cells was read on a computerized micro-
plate reader as previously published (McManus, 1976). An EC50was
also determined from this dye uptake.
Decrease in Virus Yield Assay
Following positive CPE inhibition andNRdye uptake results, griffith-
sin was retested both by CPE inhibition and, using the same plate,
for effect on reduction of virus yield assay. Frozen and thawed elu-
ates from each cup were assayed for virus titer by serial dilution
onto freshmonolayers of Vero76cells. Developmentof CPEinthese
cells is an indication of presence of infectious virus. As in the initial
tests, interferon a-n3 was run in parallel as a positive control. The
90% effective concentration (EC90), which is the test drug concen-
trationthatinhibitsvirusyieldby1log10,wasdeterminedfromthese
data.
Crystallization of Unliganded Griffithsin
Plant-expressed griffithsin and an E. coli-expressed SeMet deriva-
tive were purified as reported earlier (Mori et al., 2005; Giomarelli
et al., 2006). The protein samples were concentrated to about
20 mg/ml. Crystallization was carried out by the hanging drop vapor
diffusion method (Wlodawer and Hodgson, 1975). The plant-ex-
pressed protein and SeMet derivative crystallized under identical
conditions (0.2 M ammonium sulfate and 30% PEG 4000 at low
pH), with the largest crystals reaching the size of 0.2 3 0.2 3
0.05 mm. Before flash freezing, the crystals were transferred into a
cryoprotectant solution containing 5% ethylene glycol. X-ray data
were collected at the SER-CAT beamline 22-ID, the Advanced Pho-
ton Source (APS), Argonne, Illinois, on a MAR300 CCD detector. All
data were processed with the HKL2000 package (Otwinowski and
Minor, 1997) (Table 1).
We have obtained two different crystal forms of unliganded grif-
fithsin. The protein expressed in E. coli crystallized in the trigonal
space group P3221, with a single molecule in the asymmetric unit.
Thisprotein construct was prepared with an N-terminal 6-His affinity
tag followed by a putative thrombin cleavage site, extending the se-
quence by 17 amino acids (Mori et al., 2005; Giomarelli et al., 2006).
However, enzymatic removal of the tag proved to be impractical and
Crystal Structure of Griffithsin
1133

Page 8

thus the crystallized protein consisted of 138 amino acids, rather
than the 121 that are present in plant-expressed griffithsin. Isomor-
phous crystals were grown for both the plant-expressed protein as
defined above and for a similarly produced SeMet derivative.
Determination of the Structure of Unliganded Griffithsin
The structure of griffithsin in the trigonal crystal form was solved
by single-wavelength anomalous diffraction (SAD), initially with
HKL3000 (Minor et al., 2006), followed by density modification with
RESOLVE (Terwilliger, 2003) and by automated tracing with ARP/
wARP (Perrakis et al., 1999). The latter program built 123 out of 138
residues expected in this construct, with only a single one-residue
break at Asn67. That residue as well as one residue each on the N
andCterminiwereaddedwiththeprogramO(JonesandKjeldgaard,
1997) during subsequent refinement and rebuilding. The structure
was refined with REFMAC5 (Murshudov et al., 1997) utilizing data
extending to 2.0 A˚resolution (Table 1). The resulting model contains
all 121 residues expected in the structure of authentic griffithsin, as
well as additional five residues derived from the N-terminal exten-
sion. The rest of the N-terminal extension, including the His-tag,
was found to be disordered.
The second crystal form, grown from material expressed in plants
and containing only 121 residues in a protein chain, was orthorhom-
bic (P212121) and contained two griffithsin molecules in the asym-
metric unit. These crystals diffracted better than the trigonal ones,
and data were collected to near-atomic resolution. The structure
was solved by molecular replacement with PHASER (Storoni et al.,
2004) and was refined with REFMAC5 (Murshudov et al., 1997) at
1.3 A˚resolution. Both chains could be traced end-to-end, and
even their terminal residues, including acetylated N-terminal ser-
ines, were clearly observed in the electron density.
Crystallization and Structure Determination of Carbohydrate
Complexes
A complex of griffithsin with N-acetylglucosamine was prepared by
soaking the orthorhombic crystals described above in a reservoir
solution supplemented with 50 mM N-acetylglucosamine. X-ray
data were collected on a MAR345 image plate detector mounted
on a Rigaku rotating anode X-ray source. CuKa radiation was fo-
cused by an MSC/Osmic mirror system. Data were processed with
the HKL2000 package (Otwinowski and Minor, 1997) (Table 1). The
structure of the complex was refined with REFMAC5 (Murshudov
et al., 1997) at 2.0 A˚resolution, with the structure of unliganded grif-
fithsin in the orthorhombic crystal form as the initial model. A com-
plex of griffithsin with mannose was prepared by soaking in a similar
manner and data were collected, but that structure was not refined
further when cocrystals became available.
A distinct crystal form of a complex of griffithsin with mannose
was obtained by cocrystallization by the hanging drop vapor diffu-
sion method. The crystals were grown in 1.8 M MgSO4, 0.1 M MES
(pH 6.5) with 1:10 molar ratio of griffithsin monomers to mannose.
Crystals belonged to space group P21(Table 1) and diffracted to
1.8 A˚resolution on the home source described above and to 0.9 A˚
on a synchrotron. Full data sets were collected on two crystals of
the complex. A complete data set extending to 1.8 A˚resolution
was collected on the home source from crystal 1. High-resolution
data were collected at the synchrotron to a nominal resolution of
0.9A˚fromcrystal2,butsincethatcrystalsufferedfromthepresence
of considerable ice rings, these data were merged with another low-
resolution data set collected on crystal 1, this time on the synchro-
tron. Data from these two crystals were scaled together with
HKL2000 package (Otwinowski and Minor, 1997), retaining reflec-
tions extending only to 0.94 A˚. The structure that utilized home
data was solved with PHASER (Storoni et al., 2004) by using the
orthorhombic structure of unliganded griffithsin and was refined
with REFMAC5 (Murshudov et al., 1997). The synchrotron structure
was refined starting directly from that model (Table 1).
Acknowledgments
We would like to thank Dr. W. Minor for permission to use the ex-
perimental version of HKL3000 for this work, Dr. Z. Dauter and
M. Li for help in structure determination, K.M. Schumaker for help
in protein expression, and R. Sowder II for MALDI-TOF analysis.
Drs. D. Barnard and R. Sidwell at Utah State University and the
National Institute of Allergy and Infectious Disease Antiviral Testing
Program provided the SARS virus assay data. We acknowledge the
use of beamline 22-ID of the Southeast Regional Collaborative
Access Team (SER-CAT), located at the Advanced Photon Source,
Argonne National Laboratory. Use of the APS was supported by
the U.S. Department of Energy, Office of Science, Office of Basic
Energy Sciences, under contract number W-31-109-Eng-38. The
project was supported by the Intramural Research Program of the
National Institutes of Health, National Cancer Institute, Center for
Cancer Research. The authors declare no competing direct financial
interests, with the exception of K.E. Palmer and F. Vojdani of Intru-
cept Biomedicine LLC, a company involved in licensing griffithsin
from the U.S. government.
Received: April 5, 2006
Revised: May 22, 2006
Accepted: May 24, 2006
Published: July 18, 2006
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Accession Numbers
Coordinates and structure factors have been deposited in the PDB
withaccessioncodes2gux(SeMetstructureoftheE.coli-expressed
griffithsin), 2gty (plant-expressed, unliganded griffithsin), 2guc
(a complex with mannose at 1.8 A˚resolution), 2gud (mannose com-
plex at 0.94 A˚resolution), and 2gue (N-acetylglucosamine complex).
Crystal Structure of Griffithsin
1135