Fluorinated carbohydrates as lectin ligands: Dissecting glycan-cyanovirin interactions by using 19F NMR spectroscopy

Article abstract of DOI:10.1002/chem.201204070

NMR spectroscopy and isothermal titration calorimetry (ITC) are powerful methods to investigate ligand-protein interactions. Here, we present a versatile and sensitive fluorine NMR spectroscopic approach that exploits the ¹⁹F nucleus of ¹⁹F-labeled carbohydrates as a sensor to study glycan binding to lectins. Our approach is illustrated with the 11 kDa Cyanovirin-N, a mannose binding anti-HIV lectin. Two fluoro-deoxy sugar derivatives, methyl 2-deoxy-2-fluoro-α-D-mannopyranosyl-(1→2)- α-D-mannopyranoside and methyl 2-deoxy-2-fluoro-α-D-mannopyranosyl- (1→2)-α-D-mannopyranosyl-(1→2)-α-D-mannopyranoside were utilized. Binding was studied by ¹⁹F NMR spectroscopy of the ligand and ¹H-¹⁵N HSQC NMR spectroscopy of the protein. The NMR data agree well with those obtained from the equivalent reciprocal and direct ITC titrations. Our study shows that the strategic design of fluorinated ligands and fluorine NMR spectroscopy for ligand screening holds great promise for easy and fast identification of glycan binding, as well as for their use in reporting structural and/or electronic perturbations that ensue upon interaction with a cognate lectin. Copyright

Full text of DOI:10.1002/chem.201204070

DOI: 10.1002/chem.201204070
Fluorinated Carbohydrates as Lectin Ligands: Dissecting Glycan–Cyanovirin
19
Interactions by Using F NMR Spectroscopy
Elena Matei,[a] Sabine Andrꢀ,[b] Anja Glinschert,[c] Angela Simona Infantino,[c]
Stefan Oscarson,[c] Hans-Joachim Gabius,[b] and Angela M. Gronenborn*[a]
Abstract: NMR spectroscopy and iso-
thermal titration calorimetry (ITC) are
powerful methods to investigate
ligand–protein interactions. Here, we
present a versatile and sensitive fluo-
rine NMR spectroscopic approach that
exploits the 19F nucleus of 19F-labeled
carbohydrates as a sensor to study
glycan binding to lectins. Our approach
is illustrated with the 11 kDa Cyanovir-
mannopyranosyl-(1!2)-a-d-mannopy-
ranoside and methyl 2-deoxy-2-fluoro-
of the protein. The NMR data agree
well with those obtained from the
equivalent reciprocal and direct ITC ti-
trations. Our study shows that the stra-
tegic design of fluorinated ligands and
fluorine NMR spectroscopy for ligand
screening holds great promise for easy
and fast identification of glycan bind-
ing, as well as for their use in reporting
structural and/or electronic perturba-
tions that ensue upon interaction with
a cognate lectin.
AHCTUNGTRENNUNG
a-d-mannopyranosyl-(1!2)-a-d-man-
nopyranosyl-(1!2)-a-d-mannopyrano-
side were utilized. Binding was studied
by 19F NMR spectroscopy of the ligand
1
and H–15N HSQC NMR spectroscopy
Keywords: fluorine · fluoro-deoxy
sugars · lectins · 19F NMR spec
ACHTUNGTRENNUNG
in-N,
a mannose binding anti-HIV
AHCTUNGTRENNUNG
lectin. Two fluoro-deoxy sugar deriva-
tives, methyl 2-deoxy-2-fluoro-a-d-
Introduction
this need, having proved its value by providing high-resolu-
tion molecular structures in solution and valuable informa-
tion on ligand binding.[9–12]
The growing realization of the enormous potential of gly-
cans in information coding, embodied by the term “sugar
code”,[1] has led to define oligosaccharides as functionally
active units and provides the rational for identifying new
targets in drug design. Aided by the advances in chemical
synthesis of such determinants,[2] the detection of glycan re-
ceptors (lectins) and the analyses of the specificity/mecha-
nisms of protein–carbohydrate interactions are key steps in
this endeavor.[3] With glycan-receptor recognition critically
involved in viral and bacterial cell attachment, as well as in
Most biomolecular NMR applications employ proton/
carbon/nitrogen experiments and 2D heteronuclear NMR
spectroscopy is a powerful method to map ligand binding
sites on proteins and to quantify protein–ligand interac-
tions.[9–12] It has, however, some limitations. It is best suited
for medium size proteins (<50 kDa) since amide resonance
assignments are a prerequisite. In addition, for titration ex-
periments with low affinity ligands, large amounts of ligand
are needed to reach saturation, introducing uncertainty in
the estimation of affinity parameters, especially for synthetic
compounds of limited water-solubility. Thus, ligand-centered
methods hold some promise to overcome these problems.
Among ligand-detected NMR approaches, only few ex-
ploit the favorable properties of the fluorine nucleus.[13,14]
Mono- or trifluoro N-acetylglucosamines were used to meas-
ure affinity constants and map their binding environ-
ment.[15–18] By using fluorinated sugars,[19–21] saturation trans-
fer difference (STD) experiments in which magnetization
was relayed to the vicinal and geminal F-atoms in a mono-
saccharide were used in a competition screening assay in the
search for lead compounds in inhibitor design.[22,23] Analo-
gous to isothermal titration calorimetry (ITC), in which in
addition to titrating the ligand into the protein solution, a
second, reverse titration can be performed so that the pro-
tein is titrated into the ligand solution, ligand-based NMR
titrations provide the reciprocal data to information com-
host defense, immuneACHTNUGRTENUNG(dys)regulation and tumor progres-
sion,[4–8] versatile and sensitive analysis/screening tools in
this area are urgently needed. NMR spectroscopy can satisfy
[a] Dr. E. Matei, Prof. Dr. A. M. Gronenborn
Department of Structural Biology
University of Pittsburgh School of Medicine
Pittsburgh, PA 15260 (USA)
E-mail: amg100@pitt.edu
[b] Dr. S. Andrꢀ, Prof. Dr. H.-J. Gabius
Institute of Physiological Chemistry
Faculty of Veterinary Medicine
Ludwig-Maximilians-University Munich
Veterinꢁrstrasse 13, 80539 Munich (Germany)
[c] Dr. A. Glinschert, A. S. Infantino, Prof. Dr. S. Oscarson
Centre for Synthesis and Chemical Biology
UCD School of Chemistry and Chemical Biology
University College Dublin, Belfield, Dublin 4 (Ireland)
1
monly extracted from 2D H,15N heteronuclear correlation
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/chem.201204070.
spectra of the protein. If the correct model is used for data
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FULL PAPER
fitting, identical thermodynamic parameters should be ob-
tained, regardless of the direction of the titration.[24]
the interactions between di- and trimannoside with CV-N
and demonstrate that, based on the favorable properties of
the 19F nucleus, new features can be discerned.
Here we present a NMR study in which the 19F NMR
signal of a singly fluorinated small molecule ligand is moni-
tored upon protein addition. We compare these data to the
Bioactivity of the 19F-containing glycans: To ascertain that
substituting a fluorine at the 2’ or 2’’ position for the hydrox-
yl group does not alter the properties of the sugars signifi-
cantly and that the two 2’-19F-substituted dimannoside and
the 2’’-19F-substituted trimannoside are capable to act as spe-
cific and potent ligands, we tested their activity in blocking
the binding of lectin to cells. As shown in Figure 1, 2’-19F-
1
results obtained by recording changes in H,15N amide pro-
tein resonance frequencies upon ligand binding. For our
study we selected the 11 kDa antiviral lectin cyanovirin-N
(CV-N) as the sugar receptor, because its structure and
glycan-binding properties have been carefully analyzed by
NMR spectroscopy, crystallography, and titration calorime-
try.[25–28] CV-N harbors two carbohydrate-binding sites in its
pseudo-symmetric, bilobal structure, site 1 in domain B (res-
idues 39–89) and site 2 in domain A (residues 1–38/90–
101).[25–28] The minimal glycan recognition unit is a a-1,2-
linked dimannoside, either as the individual oligosaccharide
or as part of the terminal arms of the branched Man-8 and
Man-9 structures.[29,30] Domain A exhibits a slight preference
for trimannoside and domain B for dimannoside.[28,31,32] The
availability of detailed STD data for these ligands as well as
results with specific deoxy sugars allowed us to identify the
pivotal hydroxyl groups responsible for binding.[33,34] In par-
ticular, we determined that the 3’- and 4’-hydroxyl groups
are essential for the interaction, whereas the 2’- and 6’-hy-
droxyl groups of the ManaACTHNUTRGNENG(U 1-2)ManaACHUTNGTRENN(GUN Man2) terminal unit
are not directly involved in CV-N binding.[34] We therefore
prepared methyl 2-deoxy-2-fluoro-a-d-mannopyranosyl-(1!
2)-a-d-mannopyranoside (19F-Mana
ACTHNUTRGNEUNG(1-2)Mana, hereafter
19F-Man2) and methyl 2-deoxy-2-fluoro-a-d-mannopyrano-
syl-(1!2)-a-d-mannopyranosyl-(1!2)-a-d-mannopyrano-
Figure 1. Cell-binding of monofluorinated di- and trimannosides. Human
colon adenocarcinoma cells (SW480) were stained with fluorescein-label-
led CV-N. Controls representing staining with CV-N (100%-value at
1 mgmLꢀ1; green line) and without (0%-value; shaded area) are included
in each panel (quantitative data for the percentage of positive cells/mean
fluorescence intensity are listed). Top panel: control experiment without
added glycan (green) and in the presence of 100 mm mannoside (left),
and in the presence of 4 mm or 8 mm 19F-Man2 (right). Bottom panel:
control experiment without added glycan (green) and in the presence of
100 mm mannoside (left), in the presence of 1 mm or 2 mm 19F-Man3
(right).
side (19F-Mana (1-2)Mana, hereafter 19F-Man3)
ACHTUNGTRENNUNG(1-2)ManaACHTUGNTRENNUGN
(see the Supporting Information, Scheme S1) for use in our
current work.
We show that fluorination at the 2’ or 2’’ position of the
sugar does not curtail its bioactivity in a cell-binding assay
in which 19F-Man2 and 19F-Man3 were used as inhibitors for
the interaction of CV-N with the cell surface. We deter-
mined affinity constants and exchange rates for 19F-Man2
and 19F-Man3 interaction with two of the CV-N variants in
which one of the binding sites had been impaired,[35–37] and
compared the results to the values derived from titration
calorimetry.
In addition, due to the inherent high sensitivity of the flu-
orine chemical shift to its local environment, it was possible
not only to distinguish between free and bound signals for
19F-Man2 and 19F-Man3, but also to identify two different
modes of binding for 19F-Man3.
substituted glycans strongly inhibited the binding of lectin to
human carcinoma cells, when compared with the natural
monosaccharide. Similar data were also obtained for a T
lymphoma (Jurkat) line (not shown).
Therefore, incorporation of the 19F sensor did not impair
bioactivity, confirming that the 2’ groups are not critical for
cell surface receptor binding on the two cell types tested,
which is in line with expectations based on results with the
2’-deoxy derivative of the dimannoside.[34] These results
prompted us to proceed in studying the interaction with the
lectin by using 19F NMR spectroscopy.
Results and Discussion
NMR spectroscopy is becoming a standard approach for
studying protein–ligand interactions because it provides
powerful means for mapping the structural details of binding
sites on the protein as well as the ligand. In addition, affinity
parameters can be determined. Here, we employed
19F NMR spectroscopy and fluorinated ligands to evaluate
Ligand-detected binding of fluorinated sugars to CV-N: For
ligand-detected NMR experiments we recorded the
1D-19F NMR spectra of 19F-Man2 (200 mm) and 19F-Man3
(50 mm) at 280 K. Spectra of 1:1 mixtures of sugar/protein
are provided in Figure 2A and 2B for [CVNDA ssm, CVNmDB
]
and CV-NP51G with 19F-Man2 and 19F-Man3, respectively.
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A. M. Gronenborn et al.
Figure 2. 1D-19F NMR spectra (at 280 K) of 19F-Man2/Man3 in the presence of [CVNDA
]
ssm, a variant that contains a single glycan binding site on domain
B, CVNmDB, a variant that contains a single glycan binding site on domain A, and CV-NP51G that is wild-type-like with both glycan binding sites present.
CV-N domains are represented by two elongated crescents, representing the two binding sites. Site 1 on domain B is colored pink and site 2 on domain
A in green. A) 19F-1D NMR spectra of 200 mm 19F-Man2 in the presence of [CVNDA
]
ssm, CVNmDB, and CV-NP51G; B) 19F-1D NMR spectra of 50 mm 19F-
] ]
ssm, CVNmDB and CV-NP51G. Bound-state and free-state signals are in slow exchange for [CVNDA (in C) and CVNmDB
ssm
Man3 in the presence of [CVNDA
(in D). In the titration curves (bottom panels) the bound fraction is derived from the signal intensity (1ꢀIfree/I0) during the titration, with Ifree the intensity
of the free ligand signal at each point in the titration, and I0 is the intensity of the free ligand signal at the beginning of the titration. In C) [CVNDA
]
ssm
was titrated into 50 mm 19F-Man3 (top panel), with molar ratios of protein/glycan: 0:1, 0.5:1, 1:1, 1.5:1. In D) CVNmDB was titrated into 50 mm 19F-Man3
(top panel), with protein/glycan molar ratios: 0:1, 0.5:1, 1:1, 2:1, and 3:1.
Two distinct resonances, separated by a Dd>1 ppm, cor-
respond to the protein-free and protein-bound conforma-
tions. The resonance corresponding to the protein-bound
conformation of 19F-Man2 when bound to domain A is shift-
ed downfield from the free resonance, whereas that corre-
sponding to the domain B protein-bound confirmation is
shifted upfield. The two resonances of 19F-Man3 when
bound to domain A and B both experience a downfield
shift, compared with the signal of the free sugar. Unambigu-
ous assignments to the domain A-bound or domain B-
Much of the power of fluorine NMR studies of biological
systems derives from the high sensitivity of the fluorine
shielding parameter to changes in local environment. Shield-
ing is observed when the fluorine is in close contact to H-
bond donors of the protein or solvent molecules.[38–40] On
the contrary, a fluorine nucleus in the vicinity of an electro-
negative atom shifts downfield. Also, deshielded fluorine
atoms are found in close contact with hydrophobic side-
chains.[38–40] Inspection of the crystal structure of a mutant
CV-N protein, in which the carbohydrate binding site in
domain A was abolished, complexed with a dimannoside
ligand, P51G-m4-CVN/Man2 (PDB accession code
2RDK),[41] revealed that in the binding site on domain B, a
2’-19F substitution could act as a H-bond acceptor with a vi-
cinal water molecule, causing an upfield shift. Unfortunately,
structural data for dimannoside binding in domain A are
not available, therefore we can only speculate that such H-
bond is not present for 2’-19F-Man2 interacting with domain
A. The small 19F downfield chemical shift observed in this
case is most likely caused by either anionic repulsion by an
electronegative protein atom or an interaction of the fluo-
bound state was achieved by using [CVNDA
]
and CVNmDB
,
ssm
variants of CV-N in which the binding site in domains A
and B were obliterated.[35–37] Gratifyingly, the two resonan-
ces corresponding to bound 19F-Man2 in the CV-NP51G wild-
type variant of CV-N that possesses both binding sites, ex-
hibit identical chemical shifts to those observed in the single
site mutants, confirming that the site that remains in the
mutant is identical to its wild-type counterpart. The same
holds for the 19F-Man3 resonances, although as pointed out
above, both bound resonances are downfield of the signal of
the free sugar.
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Fluorinated Carbohydrates as Lectin Ligands
FULL PAPER
rine atom with the hydrophobic protein-binding pocket. For
Man3, the crystal structures of wild-type CV-N bound to
Man9 (PDB accession code 3GXZ)[32] allows to delineate
residues in domain A that interact with the trimannoside on
the D1 arm of Man9. The negative Glu101 side-chain car-
boxylate causes electrostatic repulsion of the anionic 2’’-19F,
resulting in a downfield shift. A similar scenario or possibly
contacts with hydrophobic protein side-chains are likely for
19F-Man3 binding to domain B, although direct supporting
structural data are not available.
Exchange kinetics between free and protein-bound fluori-
nated glycans: The 19F NMR resonances for free and bound
states at 280 K are in slow exchange on the chemical shift
scale, as evidenced by exchange peaks in the 2D 19F-19F
NOESY spectra.
To extract the kinetic parameters for the exchange pro-
AHCTUNGTRENNUNG
cess, a series of 2D 19F-19F homonuclear NOESY spectra
with different mixing times were recorded. Representative
spectra of 19F-Man2 (200 mm) in the presence of [CVNDA
]
ssm
and CVNmDB (1:1 molar ratio) at 280 K are provided in Fig-
ures 3A and 3B. The exchange curves obtained from the in-
tensity ratios of the bound, diagonal peak, to the exchange
We also carried out 1D-19F NMR experiments at 298 K.
However, except for the resonance of the free fluorinated
glycan, no signal for the ligand-
bound to protein conformation
was detected in the case of 19F-
Man2, complexed with all CV-
N variants. This is probably due
to severe line broadening in the
intermediate exchange regime
at room temperature. For 19F-
Man3, on the other hand, both
ligand-free and ligand-bound
resonances were detected at
298 K
when
[CVNDA
] ,
ssm
CVNmDB or CV-NP51G was
added, although the ligand-
bound resonances were signifi-
cantly broader than the ones at
280 K (data not shown). The
1D 19F NMR titration data,
monitoring the 19F-Man3 reso-
nance intensities upon addition
of [CVNDA
]
ssm
and CVNmDB at
280 K, are provided in Fig-
ure 2C and 2D, respectively.
The spectra for 19F-Man3
(50 mm) are displayed in the top
panels and the resulting binding
isotherm in the bottom panel.
For both mannosides, the free
and bound resonances are in
F
slow exchange. The KD values
were extracted from the bind-
ing isotherms, monitoring the
intensity changes of the pro-
tein-bound ligand signal versus
the protein/ligand molar ratio.
The bound fraction (fb) was es-
timated from the intensity
changes of the free ligand reso-
nance during the titration using
Figure 3. 2D 19F-19F NOESY exchange spectra of 19F-Man2/Man3 in the presence of [CVNDA
]
and CVNmDB
ssm
protein; A) 19F-Man2 (200 mm) bound to [CVNDA
]ssm(left panel; tmix =0.1 s), and the exchange curve of bound/
bound over free/bound intensity ratio (IBB/IBF) versus mixing time (right panel) tmix: 0.05, 0.1, 0.3, 0.5 s; B) 19F-
Man2 bound to CVNmDB (left panel; tmix =0.05 s) and the exchange curve of bound/bound over free/bound in-
tensity ratio (IBB/IBF) versus mixing time (right panel) tmix: 0.025, 0.05, 0.1, 0.2 s; C) 19F-Man3 (50 mm) bound to
F
fb =1ꢀIfree/I0. The extracted KD
[CVNDA
]
(left panel; tmix =0.1 s) and the exchange curve of bound/bound over free/bound intensity ratio
ssm
values were 10.8ꢁ0.3 mm for
(IBB/IBF) versus mixing time (right panel) tmix: 0.1, 0.3, 0.5, 0.7, 0.9 s; D) 19F-Man3 (50 mm) bound to CVNmDB
(left panel; tmix =0.05 s) and the exchange curve of bound/bound over free/bound intensity ratio (IBB/IBF
[CVNDA
]
and 33.5ꢁ3.5 mm
ssm
)
for CVNmutDB binding to 19F-
versus mixing time (right panel) tmix: 0.05, 0.1, 0.15, 0.3 s. All spectra were recorded at 280 K for 1:1 molar
ratios. The auto-correlation (diagonal) peaks IFF and IBB arise from the free and bound states, whereas cross-
correlation (off-diagonal) peaks IFB =IBF report on the exchange between the free and bound states.
Man3, respectively.
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5367

A. M. Gronenborn et al.
F
cross-peak (IBB/IBF) as a function of mixing time are shown
next to the NOESY spectra. Analysis of the exchange curve
for 19F-Man2 when bound to the site on domain B yields a
free to bound exchange rate (kex) of 56.8ꢁ1.4 sꢀ1, whereas
that for binding to domain A yields a kex value of 99.2ꢁ
2.1 sꢀ1.
ed KD values of 960ꢁ91 mm and 1.37ꢁ0.075 mm, respec-
tively. The latter value is about a factor of two larger than
the one determined for Man2 binding to site A on CVNmutDB
(757ꢁ80 mm),[36] suggesting that the substitution of fluorine
at the 2’ position reduces the binding affinity only by a very
small amount. As noted previously, Man2 exhibits a slight
preference for Domain B over Domain A, respective-
ly.[28,31,32]
The equivalent data of 19F-Man3 (50 mm) in the presence
of [CVNDA
]
and CVNmDB (1:1 molar ratio) are shown in
ssm
Figures 3C and 3D and resulted in kex values of 2.29ꢁ
0.1 sꢀ1 and 10.5ꢁ2.9 sꢀ1 for the binding to domains B and
A, respectively. Thus, the exchange between the free and
bound states of the di- and trimannoside is somewhat faster
from domain A than domain B. Although easily observed at
280 K, no exchange peak was observed at 298 K for 19F-
Man2 due to intermediate exchange that caused line broad-
ening beyond detection. The peaks for 19F-Man3, on the
other hand, are still present at 298 K.
Equivalent NMR titration experiments, monitoring the
1
chemical shift perturbations in the H-15N HSQC spectra of
[CVNDA
]
and CVNmDB were carried out at 280 K with 19F-
ssm
Man3. A superposition of [CVNDA
]
(50 mm) spectra in the
ssm
absence (black contours) and presence (green contours) of 3
molar equivalents of 19F-Man3 is provided in Figure 4B. The
binding isotherm derived from the intensity changes of free
resonances is depicted in the right panel. The equivalent ti-
tration experiment was performed for CVNmDB (50 mm) and
the data are provided in the Supporting Information (Fig-
ure S1B). The dissociation constants were extracted as de-
Protein-detected binding between 19F-Man2 and 19F-Man3
with CV-N: Direct protein-observed NMR titration experi-
ments were carried out with 19F-Man2 at 298 K by following
scribed for 19F-Man2 (above) and binding to a single site on
F
domain B (in [CVNDA
]
ssm) yielded a KD value of 63.5ꢁ
1
chemical shift perturbations in the H-15N HSQC spectrum
5 mm, whereas the one for binding to a single site on domain
of 15N uniformly labeled [CVNDA
CVNmDB (400 mm, the Support-
ing Information, Figure S1) as a
function of added sugar. Spec-
tra in the absence (black con-
tours) and presence (green con-
tours) of 25 molar equivalents
of 19F-Man2 are provided in
Figure 4A and the Supporting
Information (Figure S1A), re-
spectively. In the 1H-15N
]
ssm
(75 mm, Figure 4) and
A (in CVNmDB) was 150.3ꢁ7 mm.
HSQC NMR
spectra
of
[CVNDA and CVNmDB titra-
]
ssm
tions at 298 K, resonances are
in fast exchange, which is in
contrast to the situation in the
19F NMR spectra in which reso-
nances at 298 K were in inter-
mediate exchange and broad-
ened beyond detection. Follow-
ing the chemical shift changes
throughout the titration yielded
the binding isotherm displayed
in the right panels of Figure 4A
and the Supporting Information
(Figure S1A), respectively.
Figure 4. NMR titration of [CVNDA
]
with 19F-Man2 recorded at 298 K and 19F-Man3 recorded at 280 K, re-
ssm
spectively. A) Perturbed amide resonances in the [CVNDA
]
spectrum clearly reside in domain B and are la-
ssm
The equilibrium dissociation
beled by amino acid name and number. The left panel display a superposition of 1H-15N HSQC NMR spectra
F
constant (KD
)
for 19F-Man2
of [CVNDA in the absence (black) and presence of 25 molar equivalents of 19F-Man2 (green). The right
]
ssm
binding was determined from
the titration shifts (chemical
shift Dd (ppm) vs. the ratio of
ligand to protein concentration)
by using several resonances.
19F-Man2 binding to the single
panel displays the titration curve (chemical shift difference vs. ligand/protein molar ratio), with the chemical
shift difference defined as: Dd=[(DdH)2 +(DdN ꢃ0.17)2]1/2, in which DdH and DdN are the observed chemical
1
shift changes for H and 15N, respectively. The system is in fast exchange, as evidenced by a single signal corre-
sponding to glycan-free and glycan-bound [CVNDA
]
;
B) Superposition of 1H-15N HSQC spectra of
ssm
[CVNDA
]
(50 mm) in the absence (black) and presence (green) of 3 molar equivalents of 19F-Man3 (left
ssm
panel). The right panel displays the titration curve at glycan/protein molar ratios: 0:1, 0.5:1, 1:1, 1.5:1, 2:1, and
3:1. The system is in slow exchange, as evidenced by separate signals for glycan free and glycan bound
sites on domains B and A yield- [CVNDA
ssm. The binding isotherm is derived from the relative signal intensities (1ꢀIfree/I0) (right panel).
]
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Fluorinated Carbohydrates as Lectin Ligands
FULL PAPER
ITC binding: A direct comparison of binding parameters
that were extracted from the different NMR titrations with
those from ITC measurements was performed.
fluorinated Man3,[28,31,32] possibly due to the 19F substitution.
The extracted dissociation constants for 19F-Man3 binding
are provided in Table 1.
In particular, reverse titrations in which 19F-Man3 (50 mm)
was placed into the cell and protein into the injector were
carried out. In this manner, an equivalent situation to the
one in 19F-glycan-observed NMR titrations at 280 K is creat-
ed. The measured titration heat as well as the derived bind-
ing isotherms are provided in Figure 5A and the Supporting
Table 1. Dissociation constants for the interaction of 19F-Man3 with
single binding site CV-N variants determined by ligand-observed
19F NMR spectroscopy, reverse ITC, protein-observed NMR, and direct
ITC experiments.
F
F
Protein
KD [mm]
KDF [mm]
reverse ITC
KDF [mm]
KD [mm]
19F NMR
1H-15N HSQC
direct ITC
Information (Figure S2A) for [CVNDA
]
ssm
and CVNmDB, re-
F
A
]
10.8ꢁ0.3
33.5ꢁ3.5
12.6ꢁ1.2
28.5ꢁ3.5
63.5ꢁ5
62.5ꢁ2
spectively. The extracted KD values were 12.6ꢁ1.2 mm for
ssm
CVNmDB
150.3ꢁ7
153.3ꢁ5
[CVNDA
]
and 28.5ꢁ3.5 mm for CVNmutDB, which are in ex-
ssm
cellent agreement with the values extracted from the 19F-
glycan-observed NMR titrations (see above).
We also carried out direct ITC titrations at 280 K with
Calorimetry is the only technique that directly permits to
evaluate the basic physical forces in sufficient detail be-
tween and within molecules by measuring heat quantities or
heat effects.[42] Therefore, a deeper understanding of the mo-
lecular basis of protein–ligand interactions can be gained by
thoroughly characterizing and quantifying the energetics
that govern complex formation.[24,42] Analysis of the reverse
ITC binding isotherm for the 19F-Man3 titrations with
19F-Man3 in the injector and [CVNDA
] CHTUNGTRENN(UNG 30 mm) and
ACHTUNGTRENNUNG(50 mm) in the cell. The observed binding isotherms
ssmA
CVNmDB
are provided in Figure 5B and the Supporting Information
F
(Figure S2B), yielding KD values of 62.5ꢁ2 mm and 153.3ꢁ
5 mm for [CVNDA
]
and CVNmDB, respectively. These values
ssm
are in good agreement with those obtained from the NMR
direct titration experiment for both [CVNDA ssm and CVNmDB
]
variants, which were 63.5ꢁ5 mm and 150.3ꢁ7 mm, respec-
tively. Interestingly, it appears that 19F-Man3 no longer pref-
erentially binds to domain A, as was observed for the non-
[CVNDA
]
yielded a DG value of ꢀ5.1 kcalmolꢀ1, DH=
ssm
ꢀ2.9 kcalmolꢀ1, and a binding entropy (TDS) of 2.2 kcal
molꢀ1, whereas for the direct ITC titration, a DG value of
ꢀ5.3 kcalmolꢀ1, DH=ꢀ5.1 kcal
molꢀ1, and TDS of 0.2 kcal
molꢀ1 was obtained. Similarly, a
DG value of ꢀ5.8 kcalmolꢀ1,
DH=ꢀ1.5 kcalmolꢀ1, and TDS
of 4.3 kcalmolꢀ1 was measured
in the reverse ITC titration of
19F-Man3 with CVNmDB, where-
as a DG value of ꢀ4.88 kcal
molꢀ1, DH=ꢀ4.08 kcalmolꢀ1,
and TDS of 0.8 kcalmolꢀ1 was
obtained for the direct titration.
A summary of all thermody-
namic data determined for 19F-
Man3 binding to [CVNDA
]
ssm
and CVNmDB in reverse and
direct ITC titrations is provided
in Table 2.
For both mutants, binding to
19F-Man3 was driven by en-
thalpic contributions (with neg-
ative DH values of ꢀ2.9 kcal
molꢀ1 and ꢀ5.1 kcalmolꢀ1 in
the case of [CVNDA
] , and
ssm
ꢀ1.5 kcalmolꢀ1 and ꢀ4.08 kcal
molꢀ1 for CVNmDB, for the re-
verse and direct ITC titrations,
respectively), with favorable en-
tropic contributions. Somewhat
larger positive TDS values
Figure 5. ITC titration experiments for the interaction between 19F-Man3 and [CVNDA
]
at 280 K. A) The
ssm
trace for titrating [CVNDA
]
into 19F-Man3 (50 mm; reverse titration) for 28 automated injections is shown
ssm
(top panel), and the derived binding isotherm is displayed in the bottom panel. The nonlinear least-squares
best fit to the experimental data using a one-site model is shown by the solid line; B) The ITC trace of titrat-
ing 19F-Man3 into [CVNDA
]
(30 mm; direct titrations) for 28 automated injections (top panel) with the de-
ssm
(2.2 kcalmolꢀ1 for [CVNDA
]
ssm
rived binding isotherm (bottom panel) is displayed. The nonlinear least-squares best fit to the experimental
data by using a one-site model is shown by the solid line.
and 4.3 kcalmolꢀ1 for CVNmDB
)
Chem. Eur. J. 2013, 19, 5364 – 5374
ꢂ 2013 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
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5369

A. M. Gronenborn et al.
Table 2. Thermodynamic parameters extracted from the ITC binding
data.
cause any errors associated with solubility that may result in
incorrect ligand concentrations are avoided.
Protein
Enthalpy
(DH)
Entropy
Free energy Stoichiometry
(TDS)
(DG)
(n)
Detection of a second binding mode in the interaction of
19F-Man3 with CV-N: The concentration of 19F-Man3 in the
ligand-detected 19F NMR experiments was 50 mm, whereas
for those with 19F-Man2 it was 200 mm. To directly compare
[kcalmolꢀ1
]
[kcalmolꢀ1
]
[kcalmolꢀ1
]
[CVNDA
reverse ITC
]
ꢀ2.9ꢁ0.05
ꢀ5.1ꢁ0.1
ꢀ1.5ꢁ0.03
2.2ꢁ0.04
ꢀ5.1ꢁ0.06
ꢀ5.3ꢁ0.1
ꢀ5.8ꢁ0.04
1.01ꢁ0.03
1.0ꢁ0.0
ssm
A
]
0.2ꢁ0.08
4.3ꢁ0.02
ssm
the results of [CVNDA ssm, CVNmDB and CV-NP51G binding to
]
direct ITC
CVNmDB
1.02ꢁ0.04
the two fluorinated glycans, we also recorded a ligand-de-
tected 19F-spectrum of 19F-Man3 (200 mm) with CV-N var-
iants (1:1 molar ratio). As can be observed from the com-
parison of the spectra illustrated in Figure 6, at higher 19F-
Man3 concentrations (>150 mm), additional resonances
emerge. These new resonances exhibit exactly the same
chemical shifts (Figure 6B) that were seen for the signals of
19F-Man2 when bound to domains A and B of CV-N, respec-
tively (Figure 6A).
reverse ITC
CVNmDB
direct ITC
ꢀ4.08ꢁ0.06 0.8ꢁ0.03
ꢀ4.88ꢁ0.05 1.0ꢁ0.0
The errors listed are standard deviations of the fits.
were obtained for the reverse ITC titrations, compared with
the direct ones (0.2 kcalmolꢀ1 for [CVNDA
]
and 0.8 kcal
ssm
molꢀ1 for CVNmDB), suggesting that water molecules are
more efficiently released from the complex surface in the
case of reverse titrations.[42] The overall DG values, however,
are not very different.
The presence of the second binding mode can also be ob-
served in the 2D 19F-19F exchange NOESY experiment re-
corded with a 50 ms mixing time on the sample of 19F-Man3
(200 mm) with [CVNDA
]
ssm
and CVNmDB (Figure 6C). Given
ITC also allows the stoichiometry of binding to be deter-
mined, independent of the binding affinity.[24] Since a 1:1
stoichiometry was obtained from the experimental (sigmoid)
curves in the cases of reciprocal ITC titrations, a one bind-
ing site model was used to fit the data, from which the bind-
ing-affinity parameters were extracted. In the case of direct
ITC titrations, since ꢂ1:1 stoichiometry was observed, a
that the additional peaks exactly match the chemical shifts
of the bound conformations of 19F-Man2 when sitting in do-
mains A and B of CV-N, and are only observed at relatively
high concentrations, it appears that each domain of CV-N
binds first to the Mana
methyl group in 19F-Man3 (high affinity binding mode: 1)
and subsequently to the non-reducing end 19F-Mana
(1-
2)Mana unit (low affinity binding mode: 1’).
ACHTUNGTRNE(NUNG 1-2)Mana unit that is closer to O-
ACHTUNGTRENNUNG
stoiACHTUNGTRENNUNGchiometry value of n=1 was fixed to fit the experimental
(parabolic) curves, consistent with the detection of a single
binding site for the equivalent, protein-observed 1H-15N
HSQC titrations (Figure 4B and the Supporting Informa-
tion, Figure S1B). Furthermore, the fully saturated samples
that resulted either at the end of protein-observed 1H-15N
HSQC or direct ITC titrations, were tested by using
1D-19F NMR, and indeed, only a single protein-bound peak,
corresponding to one binding site, was observed. Therefore,
the reverse titration allowed us not only to check the stoi-
chiometry, but also to ensure that a suitable binding model
was used to fit the ITC data. For a 1:1 binding stoichiometry,
the measured thermodynamic parameters are expected to
be invariant when changing the orientation of the experi-
ment.[24] However, this is rarely the case since one of the
two species may display greater aggregation or less solubili-
ty when concentrated.[24b] Indeed, a comparison of the data
To further substantiate our findings we also carried out a
competition binding experiment, by adding non-fluorinated
Man2 to a solution of 19F-Man3 (200 mm) and [CVNDA
]
ssm
(1:1 molar ratio) and monitored the change in 1D-19F NMR
ligand signal intensity as a function of Man2 addition. The
resulting data up to a 20 fold molar excess of Man2, are pro-
vided in Figure 6D.
As can be noted, the resonance that corresponds to the
bound conformation of the non-reducing end 19F-Mana
ACHTUNGTRENNUNG(1-
2)Mana unit is the one that is affected first by the addition
of non-fluorinated Man2, whereas the resonance corre-
sponding to the bound conformation of the second ManaACHTUNGTRENNUNG(1-
2)Mana unit (the one closer to O-methyl group) is affected
only at a higher Man2 addition. Such differentiation was not
observed when 19F-Man3 or non-fluorinated Man3 was used
in protein-detected titration experiments.
in Table 1 revealed that titrating 19F-Man3 into solutions of
1
[CVNDA
]
and CVNmDB by using protein-observed H-15N
19F NMR competition assay: To further highlight the advan-
tages of using 19F NMR spectroscopy, we also carried out a
quantitative competition binding experiment, titrating non-
fluorinated Man3 into a complex of 19F-Man3 (250 mm) with
relatively low concentration (25 mm) of CVNmDB (10:1 molar
ratio), which ensures that only the high affinity binding
mode is involved. Indeed, inspection of the 1D-19F NMR
spectra reveals only the presence of the peak corresponding
to the high affinity protein–ligand binding mode, whereas
no additional low affinity binding peak is observed.
ssm
HSQC spectra or direct ITC experiments yielded approxi-
F
mately five times higher KD values than those obtained
from ligand-observed 19F NMR and reciprocal ITC titra-
tions. Naturally, if aggregation occurs, this will happen at
high concentrations of the ligand. Therefore, it is advanta-
geous to measure the binding on the ligand, either by NMR
spectroscopy or reverse ITC, since lower ligand concentra-
tions are needed.
Thus, ligand-detected binding methods are preferable
compared with methods that measure protein signals, be-
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Fluorinated Carbohydrates as Lectin Ligands
FULL PAPER
Figure 6. 1D-19F NMR spectra of 19F-Man2/Man3 (200 mm) in the presence of different CV-N variants, which illustrates the power of 19F NMR spectrosco-
py for discriminating between different binding sites and modes. The presence of a second binding mode for the interaction of CV-N with 19F-Man3 is
clearly observed. All spectra were recorded at 280 K. A) 1D-19F spectra of 19F-Man2 (200 mm) in the presence of [CVNDA ssm, CVNmDB and CV-NP51G
] .
B) 1D-19F spectra of 19F-Man3 (200 mm) in the presence of [CVNDA
]
ssm, CVNmDB and CV-NP51G. Schematic depiction of 19F-Man2 and the two modes of
19F-Man3 binding to CV-N in the right-side panels of A) and B). The individual sugar units on the oligosaccharide are color coded according to their
linkage pattern; C) 2D 19F-19F NOESY spectra of 19F-Man3 in the presence of [CVNDA
]
and CVNmDB (200 mm; 1:1 molar ratio of glycan/protein). The
ssm
auto-correlation (diagonal) peak intensities IFF and IBB in the 2D 19F-19F NOESY spectra arise from the free and bound states, whereas the cross-correla-
tion (off-diagonal) resonances IFB =IBF and IFB’ =IBF’ arise from exchange between the two binding modes for CV-N and 19F-Man3; D) Competitive bind-
ing of Man2 to the complex of 19F-Man3/
[CVNDA
]
(200 mm;19F-Man3/
A
]ssm/Man2 molar ratios were: 1:1:0; 1:1:1; 1:1:5; 1:1:10; and 1:1:20). A
] is shown in the bottom panel. The Man2 competitor first displaces
ssm
ssm
schematic representation of Man2 competing with 19F-Man3 binding to [CVNDA
the 19F-Man3 bound to protein with the non-reducing 19F-Mana
bound with the unit closer to the O-methyl group (high affinity binding mode shown as a dashed arrow).
ACHTUNGTRNE(NUNG 1-2)Mana unit (the low affinity binding mode shown as a solid arrow), and later the one
The intensity change in the 1D-19F NMR free ligand
signal was monitored as a function of Man3 addition. A
schematic representation of the competitive binding scenar-
io is illustrated in Figure 7A, and the resulting data are pro-
vided in Figure 7B. The intensity increase in the
1D-19F NMR spectrum of the free 19F-Man3 signal in com-
plex with CVNmDB, upon addition of the non-fluorinated
competitor (Man3), corresponds to the amount of non-la-
beled ligand bound to CVNmDB. The dissociation constant of
Man3 binding to CVNmDB was obtained from the relevant
competition titration data for the 19F-Man3/CVNmDB/Man3
complex (Figure 7B). To extract the binding constant kd of
Man3 interacting with CVNmDB, it is necessary to know the
F
KD value of 19F-Man3 binding to CVNmDB (Figure 7A). We
determined this value by 19F NMR spectroscopy and recip-
rocal ITC titrations (see above). The extracted kd value of
4.65ꢁ0.6 mm is consistent with our previous affinity data of
Chem. Eur. J. 2013, 19, 5364 – 5374
ꢂ 2013 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemeurj.org
5371

A. M. Gronenborn et al.
Figure 7. Competition of Man3 and 19F-Man3 for 19F-Man3 (250 mm) in complex with CVNmDB (25 mm). A) Schematic representation of the competitive
binding scenario. Cheng–Prusoff equation based on a competitive site binding model, in which [19F-Man3] and [Man3] are the concentrations of the fluo-
rinated and non-fluorinated ligand, and KDF the dissociation constants of 19F-Man3 interacting with the glycan-binding site on domain A of CVNmDB. The
data were fitted using the maximum increase in the free 19F-Man3 resonance intensity upon Man3 addition, with DImax, and kd the dissociation constant
for Man3 binding as adjustable parameters; B) Titration curve extracted from 19F NMR spectra that were recorded for 19F-Man3/CVNmDB/Man3 molar
ratios of 10:1:0, 10:1:0.25, 10:1:0.5, 10:1:0.75, and 10:1:1. In the titration curve, the bound fraction of Man3 is given by the difference in signal intensity
of the 19F-Man3 free signal DI=IfreeꢀI0 during the titration, in which Ifree is the intensity of the 19F-Man3 free signal at each point in the titration, and I0
the intensity of the 19F-Man3 free signal at the beginning of the titration.
the non-labeled Man3 with CVNmDB (3.4ꢁ0.05 mm), ob-
Experimental Section
tained from an ITC titration experiment.[36]
Lectins: The Cyanovirin-N wild-type stabilized variant CV-NP51G and the
two mutants [CVNDA
]
and CVNmDB that possess only one sugar-binding
ssm
site, either in domain B ([CVNDA ssm) or domain A (CVNmDB) were pre-
]
Conclusion
pared as described previously.[35–37] The two fluoro-deoxy sugar deriva-
tives, methyl 2-deoxy-2-fluoro-a-d-mannopyranosyl-(1!2)-a-d-manno-
pyranoside and methyl 2-deoxy-2-fluoro-a-d-mannopyranosyl-(1!2)-a-
d-mannopyranosyl-(1!2)-a-d-mannopyranoside were obtained using
synthetic procedures outlined in the Supporting Information
(Scheme S1).
We have presented an approach to quantitatively assess
glycan–lectin interactions by monitoring the changes in the
19F NMR resonances of fluorinated glycans upon protein ad-
dition. Our method exploits the relatively large 19F NMR
chemical shift range that results in well resolved resonances
in the 1D spectrum. Employing fluorinated ligands permits
the analysis protein–ligand interactions by 19F NMR spec-
Cell assays: CV-NP51G was fluorescently labeled by fluorescein isothiocya-
nate under activity-preserving conditions, separated from reagents by gel
filtration, and applied in cytofluorometry as described for other lectins
previously.[43]
NMR spectroscopy: 19F NMR spectra were recorded on
a Bruker
ACHTUNGTRENNUNGtrosACHTUNGTRENNUNGcopy, even for high molecular mass proteins (>100 kDa)
600 MHz AVANCE spectrometer, equipped with a Bruker CP TXO
and requires only minimal amounts of ligand. Therefore,
new avenues for screening in a facile fashion and relatively
short time can be envisaged.
triple-resonance, X-nuclei observe, z-axis gradient cryoprobe (Bruker
Bio
Bruker 600 MHz AVANCE spectrometer equipped with CP TCI triple-
spin, Billerica, MA). 1H-15N HSQC experiments were recorded on a
ACHTUNGTRENNUNG
resonance, z-axis gradient 1H detect cryoprobe (Bruker Bio
ACHTNUTRGNEsNUG pin, Billeri-
In particular we show that:
ca, MA). Spectra were processed with NMRPipe[44] and analyzed with
NMRview.[45]
1) Quantitative binding affinity data can be obtained using
19F NMR experiments with fluorinated glycans.
2) Information about protein–ligand binding exchange can
be extracted from 19F-19F NOESY experiments and we
determined the chemical exchange rate constant, kex, for
each of the two binding sites of CV-N.
NMR titrations: Monitoring the resonances of the fluorinated ligand:
Ligand-observed 19F NMR experiments were performed using fluorinated
deoxy-sugars, 19F-Man2 (200 mm) and 19F-Man3 (50 mm), and the single
binding site variants, [CVNDA
]
and CVNmDB, as well as the stabilized
ssm
CV-NP51G wild-type protein at 1:1 protein/ligand molar ratios, in sodium
phosphate buffer (20 mm), pH 6.0, 0.01% NaN3, 90% H2O/10% D2O.
For titration experiments with 19F-Man3 sugars (50 mm), 19F-spectra were
3) Different binding modes can be discerned for the two
monitored upon addition of increasing amounts of protein, [CVNDA
]
ssm
neighboring ManaACTHNUTRGNEUNG
(1-2)Mana units in 19F-Man3 when in-
and CVNmDB, (5 mm stock solution) in sodium phosphate buffer (20 mm),
pH 6.0, 0.01% NaN3, 90% H2O/10% D2O. A series of 1D-19F NMR
spectra were recorded at 280 K at protein/sugar molar ratios of 0:1, 0.5:1,
teracting with CV-N.
4) 19F NMR competition experiments can be employed for
quantitatively extracting affinity constants of non-fluori-
nated glycans with target lectins.
1:1, and 1.5:1 for [CVNDA
]
and 0:1, 0.5:1, 1:1, 2:1 and 3:1 for CVNmDB
.
ssm
Free and bound 19F ligand resonances throughout the titration are in
slow exchange on the chemical shift scale. The observed signal intensity
change during the titrations is directly proportional to the fraction bound
(fb) and is given by: fb =1ꢀff =1ꢀIfree/I0 =[PL]/[L], in which [L] is the
total concentration of ligand and [PL] the concentration of the resulting
ligand-protein complex; Ifree is the NMR peak intensity of the free ligand
signal at each point in the titration, and I0 is the peak intensity of the
free ligand signal at the beginning of the titration. Binding curves were
derived from the relative ratios of ligand resonance intensities (1ꢀIfree/I0)
In summary, utilizing 19F as a sensor for studying protein–
ligand interactions represents a promising perspective for
19F NMR spectroscopy, as a universal tool in drug discovery
for any target receptors for which fluorinated ligands are
available or can be synthesized.
F
versus the molar ratio (M) of protein/sugar, and apparent KD values
were obtained by non-linear best fitting of the titration curves using Ka-
leidaGraph (Synergy Software, Reading, PA), by using Equation (1).
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Fluorinated Carbohydrates as Lectin Ligands
FULL PAPER
sffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi
!
ꢀ
ꢁ
2ꢀ4M
mixing time (tmix), by using Equation (4), in which keq is the equilibrium
constant and kex is the free–bound exchange rate.[47]
Ifree
I0
KDF
½Lꢃ
KDF
½Lꢃ
*
ð1Þ
fb ¼ 1 ꢀ
¼ 0:5 M þ 1 þ
ꢀ
M þ 1 þ
keq½expðkex ꢄ tmixÞ þ 1ꢃ
IBB
IBF
Monitoring the protein resonances: The protein-observed NMR titration
experiments were performed on Bruker 600 MHz AVANCE spectrome-
ters, equipped with 5 mm, triple resonance, three-axis gradient probes or
ð4Þ
¼
½expðkex ꢄ tmixÞ ꢀ 1ꢃ
z-axis gradient cryoprobes, by using uniformly 15N-labeled [CVNDA
] -
ssm
Isothermal titration calorimetry: Calorimetric titrations were performed
using a VP-ITC isothermal titration calorimeter (MicroCal, LLC; North-
ampton, MA). Titrations were carried out at 280 K and all solutions con-
tained sodium phosphate buffer (50 mm), pH 7.4, 0.2m NaCl, 0.02%
ACHTUNGTRENNUNG ACHTUNGTRENNUNG
(75 mm) or CVNmDB(400 mm) with increasing amounts of 19F-Man2, in
sodium phosphate buffer (20 mm), pH 6.0, 0.01% NaN3, 90% H2O/10%
D2O. A series of 1H-15N HSQC spectra at 298 K were recorded after the
addition of sugar aliquots from a stock solution (10 mm) at sugar/protein
molar ratios of: 1:0, 2:1, 4:1, 6:1, 8:1, 10:1, 14:1, 18:1, 22:1, and 25:1 for
NaN3. ITC measurements were carried out with [CVNDA
]
and CVNmDB
ssm
solution in the cell (ca. 1.44 mL active volume) at 30 mm and 50 mm con-
centrations, respectively, and stirred at 310 rpm. 28 injections of 10 mL ali-
[CVNDA
]
and 0:1, 0.5:1, 1:1, 1.5:1, 2:1, 2.5:1, 3:1, 3.5:1, 5:1, 6:1, 7:1,
ssm
quots of 19F-Man3 (2.5 mm) in the case of [CVNDA
]ssm, and 35 injections
8:1, 9:1, 11:1, 12:1, 15:1, 16:1, 21:1, and 25:1 for CVNmDB
.
of 8 mL aliquots of 19F-Man3 (4 mm) in the case of CVNmDB, were per-
formed at 2 min intervals from a 285 mL stirring syringe. Reverse ITC
measurements were performed as follows: the 19F-Man3 solution (50 mm)
was placed in the calorimeter cell (ca. 1.44 mL active volume), stirred at
Free and sugar-bound protein resonances throughout the titration are in
fast exchange on the chemical shift scale. The observed chemical shift
change during the titration is given by: Dd=[PL]/[P] (dbꢀdf), in which
[P] and [PL] are the concentrations of protein and ligand–protein com-
plex and db and df are the chemical shifts of protein resonances in the
free and fully bound state. The chemical shift difference was calculated
as: Dd=[(DdH)2 +(DdN ꢃ0.17)2]1/2, in which DdH and DdN represent the
310 rpm, while [CVNDA
]
or CVNmDB solution were placed in the injec-
ssm
tor. 10 mL aliquots of 0.6 mm [CVNDA
]
and 11 mL aliquots of 1 mm
ssm
CVNmDB were added at 2 min intervals from the 285 mL stirring syringe.
A total of 28 injections were performed for [CVNDA
and 25 injections
]
1
observed chemical shift changes for H and 15N, respectively. The dissoci-
ssm
for CVNmDB. Binding isotherms were fit using the Origin 7.0 software by
using a standard one-site model, to extract the apparent number of bind-
ing sites and affinity parameters. Values for the binding enthalpy, the ap-
parent number of binding sites and affinities were obtained from the fit
to the experimental data. Other thermodynamic parameters were calcu-
lated by using the standard expressions: DG=ꢀRT ln Ka; DG=
DHꢀTDS.
ation constant KDF, was obtained by best fitting the titration curve (chem-
ical shift change Dd vs. molar ratio M of sugar/protein) using Kaleida-
Graph software and Equation (2):
sffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi
!
ꢀ
ꢁ
KDF
½Pꢃ
KDF
½Pꢃ
*
ð2Þ
Dd ¼ 0:5 Ddmax M þ 1 þ
ꢀ
M þ 1 þ
2ꢀ4M
19F NMR spectroscopic competition binding assay: The binding affinities
of Man3 for the CVNmDB variant were quantified using a 19F NMR-based
competition assay. In this experiment, the non-labeled competitor ligand
Man3 was titrated into a solution of 19F-Man3 (250 mm) and CVNmDB
(25 mm) in sodium phosphate buffer (20 mm), pH 6.0, 0.01% NaN3, 90%
H2O/10% D2O. A series of 1D-19F NMR spectra were recorded at 280 K
with 19F-Man3/CVNmDB/Man3 molar ratios of: 10:1:0, 10:1:0.25, 10:1:0.5,
10:1:0.75, and 10:1:1. The intensity increase of the free 1D NMR signal
of 19F-Man3 complexed with CVNmDB, upon addition of the inhibitor
(Man3), is proportional to the amount of non-labeled ligand (Man3)
bound to CVNmDB. The apparent dissociation constants kd for Man3 bind-
ing to the Domain A of CVNmDB, was obtained from best fitting the re-
sponse curves with KaleidaGraph (Synergy Software, Reading, PA), by
using a competitive binding Cheng–Prusoff equation (see Equation in
Figure 7),[48] in which KDF is the dissociation constant of 19F-Man3 binding
to the Domain A of CVNmDB (see above).
Analogous NMR titration experiments were performed by using uni-
formly 15N-labeled [CVNDA (50 mm) and CVNmDB(50 mm) with 19F-
ssmA
]
G
ACHTUNGTRENNUNG
Man3, in sodium phosphate buffer (20 mm), pH 6.0, 0.01% NaN3, 90%
H2O/10% D2O. A series of 1H-15N HSQC spectra were recorded at
280 K after addition of sugar aliquots from a stock solution of 10 mm at
sugar/protein molar ratios of: 0:1, 0.5:1, 1:1, 1.5:1, 2:1, and 3:1 for
[CVNDA
]
and 0:1, 0.5:1, 1:1, 1.5:1, 2:1, 2.5:1, 3.5:1, and 4:1 for
ssm
CVNmDB. In this case, free and sugar-bound protein resonances through-
out the titration were in slow exchange on the chemical shift scale. The
observed signal intensity change during the titration is direct proportional
to the fraction bound (fb), given by: fb =1ꢀff =1ꢀIfree/I0 =[PL]/[P], in
which [P] is the total concentration of protein and [PL] the concentra-
tions of the protein–ligand complex. Ifree is the resonance intensity of the
ligand-free protein signal at each point in the titration, and I0 is the reso-
nance intensity of the ligand-free protein signal at the beginning of titra-
tion. Binding curves were derived from the relative ratios of ligand reso-
nance intensities (1ꢀIfree/I0) versus the molar ratio (M) of protein/sugar,
F
and apparent KD values were obtained by non-linear best fitting of the
titration curves using KaleidaGraph (Synergy Software, Reading, PA),
averaging over the eight titration curves by using Equation (3):
Acknowledgements
sffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi
!
We thank Mike Delk for NMR spectroscopy technical support. This
work was supported by a Science Foundation of Ireland Grant 08/IN.1/
B2067 (to S.O.), funding from the EC (contract no. 26060, GlycoHIT; to
H.J.G) and a National Institutes of Health Grant RO1GM080642 (to
A.M.G.).
ꢀ
ꢁ
2ꢀ4M
KDF
½Pꢃ
KDF
½Pꢃ
Ifree
I0
ð3Þ
fb ¼ 1 ꢀ
¼ 0:5 ꢄ M þ 1 þ
ꢀ
M þ 1 þ
2D 19F-19F NOESY exchange experiment: Two-dimensional exchange
spectroscopy (NOESY) represents powerful tool for probing the
a
ligand–receptor exchange.[46] NOESY experiments are particularly suita-
ble to evaluate chemical exchange processes that occur on a slow time
scale, in which the exchange rate has little effect on the line shape. A
series of 2D homonuclear exchange 19F-19F NOESY experiments were re-
corded at 280 K on a sample containing 200 mm 19F-Man2, complexed
[1] The Sugar Code. Fundamentals of glycosciences (Ed.: H.-J. Gabius,),
Wiley-VCH, Weinheim, Gemany, 2009.
[2] S. Oscarson in The Sugar Code. Fundamentals of glycosciences (Ed.:
H.-J. Gabius,), Wiley-VCH, Weinheim, Gemany, 2009, pp. 31–51.
[3] H.-J. Gabius, S. Andrꢀ, J. Jimꢀnez-Barbero, A. Romero, D. Solꢄs,
Trends Biochem. Sci. 2011, 36, 298–313.
[4] A. Imberty, Y. M. Chabre, R. Roy, Chem. Eur. J. 2008, 14, 7490–
7499.
[5] J. Holgersson, A. Gustafsson, S. Gaunitz in The Sugar Code. Funda-
mentals of glycosciences (Ed.: H.-J. Gabius,), Wiley-VCH, Wein-
heim, Gemany, 2009, pp. 31–51.
with [CVNDA
]
and CVNmDB at 1:1 molar ratios and mixing times (tmix
)
ssm
of 0.05, 0.1, 0.3, 0.5 s, and 0.025 0.05, 0.1, 0.2 s, respectively. An equivalent
series was recorded for 50 mm 19F-Man3, complexed with [CVNDA
]
and
ssm
CVNmDB at mixing times (tmix) of 0.1, 0.3, 0.5, 0.7, 0.9 s and 0.05, 0.1, 0.15,
0.3 s, respectively. The exchange rate constant was extracted by fitting
the experimental intensity ratios (diagonal peak (IBB) of bound confor-
mation, to that of the bound to free exchange cross-peak (IBF)) versus
Chem. Eur. J. 2013, 19, 5364 – 5374
ꢂ 2013 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemeurj.org
5373

A. M. Gronenborn et al.
[6] M. Sperandio, C. A. Gleissner, K. Ley, Immunol. Rev. 2009, 230, 97–
113.
[29] A. J. Bolmstedt, B. R. O’Keefe, S. R. Shenoy, J. B. McMahon, M. R.
Boyd, Mol. Pharmacol. 2001, 59, 949–954.
[7] Y. M. Chabre, D. Giguere, B. Blanchard, J. Rodrigue, S. Rocheleau,
M. Neault, S. Rauthu, A. Papadopoulos, A. A. Arnold, A. Imberty,
R. Roy, Chem. Eur. J. 2011, 17, 6545–6562.
[8] H. Kaltner, H.-J. Gabius, Histol. Histopathol. 2012, 27, 397–416.
[9] G. M. Clore, A. M. Gronenborn, J. Magn. Reson. 1982, 48, 402–417.
[10] G. M. Clore, A. M. Gronenborn, Protein Sci. 1994, 3, 372–390.
[11] F. Ni, Y. Zhu, J. Magn. Reson. Ser. B 1994, 103, 180–184.
[12] V. Roldꢅs, F. J. Canada, J. Jimꢀnez-Barbero, ChemBioChem 2011,
12, 990–1005.
[30] L. G. Barrientos, A. M. Gronenborn, Mini-Rev. Med. Chem. 2005, 5,
21–31.
[31] C. A. Bewley, S. Kiyonaka, I. Hamachi, J. Mol. Biol. 2002, 322, 881–
889.
[32] I. Botos, B. R. OꢆKeefe, S. R. Shenoy, L. K. Cartner, D. M. Ratner,
P. H. Seeberger, M. R. Boyd, A. Wlodawer, J. Biol. Chem. 2002,
277, 34336–34342.
[33] C. Sandstrçm, O. Berteau, E. Gemma, S. Oscarson, L. Kenne, A. M.
Gronenborn, Biochemistry 2004, 43, 13926–13931.
[34] C. Sandstrçm, B. Hakkarainen, E. Matei, A. Glinchert, M. Lah-
mann, S. Oscarson, L. Kenne, A. M. Gronenborn, Biochemistry
2008, 47, 3625–3635.
[13] C. Dalvit, Prog. Nucl. Magn. Reson. Spectrosc. 2007, 51, 243–271.
[14] A. Vulpetti, U. Hommel, G. Landrum, R. Lewis, C. Dalvit, J. Am.
Chem. Soc. 2009, 131, 12949–12959.
[15] F. Millett, M. A. Raftery, Biochem. Biophys. Res. Commun. 1972,
47, 625–632.
[35] L. G. Barrientos, E. Matei, F. Lasala, R. Delgado, A. M. Gronen-
born, Protein Eng. Des. Sel. 2006, 19, 525–535.
[16] G. M. Alter, J. A. Magnuson, Biochemistry 1974, 13, 4038–4045.
[17] P. Midoux, J. P. Grivet, M. Monsigny, FEBS Lett. 1980, 120, 29–32.
[18] F. Jordan, H. Bahr, J. Patrick, P. W. Woo, Arch. Biochem. Biophys.
1981, 207, 81–86.
[36] E. Matei, W. Furey, A. M. Gronenborn, Structure 2008, 16, 1183–
1194.
[37] E. Matei, A. Zheng, W. Furey, J. Rose, C. Aiken, A. M. Gronen-
born, J. Biol. Chem. 2010, 285, 13057–13065.
[19] R. U. Lemieux, Chem. Soc. Rev. 1989, 18, 347–374.
[20] D. Solis, A. Romero, H. Kaltner, H.-J. Gabius, T. Diaz-Maurino, J.
Biol. Chem. 1996, 271, 12744–12748.
[21] D. Solꢄs, A. Romero, M. Menꢀdez, J. Jimꢀnez-Barbero in The Sugar
Code. Fundamentals of glycosciences (Ed.: H.-J. Gabius,), Wiley-
VCH, Weinheim, Gemany, 2009, pp. 233–245.
[22] T. Diercks, J. P. Ribeiro, F. J. Canada, S. Andrꢀ, J. Jimꢀnez-Barbero,
H.-J. Gabius, Chem. Eur. J. 2009, 15, 5666–5668.
[23] S. Andrꢀ, F. J. CaÇada, T. C. Shiao, L. Largartera, Eur. J. Org.
Chem. 2012, 4354–4364.
[24] a) R. OꢆBrien, J. E. Ladbury, B. Z. Chowdry in Protein-Ligand Inter-
actions: hydrodynamics and calorimetry (Eds.: S. E. Harding, B. Z.
Chowdry), Oxford University Press, Oxford, 2001, pp. 263–286;
b) A. Brown, Int. J. Mol. Sci. 2009, 10, 3457–3477.
[25] C. A. Bewley, K. R. Gustafson, M. R. Boyd, D. G. Covell, A. Bax,
G. M. Clore, A. M. Gronenborn, Nat. Struct. Biol. 1998, 5, 571–578.
[26] F. Yang, C. A. Bewley, J. M. Louis, K. R. Gustafson, M. R. Boyd,
A. M. Gronenborn, G. M. Clore, A. Wlodawer, J. Mol. Biol. 1999,
288, 403–412.
[38] C. Dalvit, A. Vulpetti, ChemMedChem 2011, 6, 104–114.
[39] C. Dalvit, A. Vulpetti, ChemMedChem 2012, 7, 262–272.
[40] A. Vulpetti, C. Dalvit, Drug Discovery Today 2012, 17, 890–897.
[41] R. Fromme, Z. Katiliene, B. Giomarelli, F. Bogani, J. Mc Mahon, T.
Mori, P. Fromme, G. Ghirlanda, Biochemistry 2007, 46, 9199–9207.
[42] R. Perozzo, G. Folkers, L. Scapozza, J. Recept. Signal Transduction
2004, 24, 1–52.
[43] S. Andrꢀ, M. Lahmann, H.-J. Gabius, S. Oscarson, Mol. Pharm.
2010, 7, 2270–2279.
[44] F. Delaglio, S. Grzesiek, G. W. Vuister, G. Zhu, J. Pfeifer, A. Bax, J.
Biomol. NMR 1995, 6, 277–293.
[45] B. A. Johnson, Methods Mol. Biol. 2004, 278, 313–352.
[46] G. Bodenhausen, G. Wagner, M. Rance, O. W. Soerensen, K. Wueth-
rich, R. R. Ernst, J. Magn. Reson. 1994, 59, 542–550.
[47] R. R. Ernst, G. Bodenhausen, A. Wokaun, Principles of nuclear
magnetic resonance in one and two dimensions, Claredon Press,
Oxford, 1987.
[48] Y. Cheng, W. H. Prusoff, Biochem. Pharmacol. 1973, 22, 3099–3108.
[27] S. R. Shenoy, L. G. Barrientos, D. M. Ratner, B. R. OꢆKeefe, P. H.
Seeberger, A. M. Gronenborn, M. R. Boyd, Chem. Biol. 2002, 9,
1109–1118.
[28] a) L. G. Barrientos, J. M. Louis, I. Botos, T. Mori, Z. Han, B. R.
OꢆKeefe, M. R. Boyd, A. Wlodawer, A. M. Gronenborn, Structure
2002, 10, 673–686.
Received: November 14, 2012
Revised: January 15, 2013
Published online: February 28, 2013
5374
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Chem. Eur. J. 2013, 19, 5364 – 5374