NMR titration experiments showed that the sugar-binding site in subdomain a had a slow or intermediate exchange regime on the chemical-shift timescale Kd= 102 to 101mm, whereas that in s
Trang 1C-terminal domain of an R-type lectin from the earthworm Lumbricus terrestris
Hikaru Hemmi1, Atsushi Kuno2, Shigeyasu Ito2,3, Ryuichiro Suzuki2,3, Tsunemi Hasegawa3
and Jun Hirabayashi2
1 National Food Research Institute, National Agriculture and Food Research Organization (NARO), Tsukuba, Japan
2 Research Center for Medical Glycoscience, National Institute of Advanced Industrial Science and Technology (AIST), Tsukuba, Japan
3 Department of Material and Biological Chemistry, Yamagata University, Yamagata, Japan
Sugar-binding proteins, known as lectins, exist
ubiqui-tously in both animals and plants, but lectins from the
annelid phylum have rarely been reported A 29 kDa
lectin (EW29) was isolated from the earthworm
Lumbricus terrestris using affinity chromatography on
asialofetuin–agarose in the screening of galectin-like
proteins The protein consists of two homologous
domains (14 500 Da), i.e N- and C-terminal domains,
which show 27% identity with each other [1] Both
domains of EW29 form a tandem-repeat type structure
and contain triple-repeated QXW motifs [2,3] This short motif has been found in many carbohydrate-recognition proteins including the plant lectin ricin B-chain [4] The 3D structures of R-type lectins have been determined [2,5–19], and these proteins possess common b-trefoil fold structures, although their sugar-binding affinities differ depending on their ligand specificities
Recent biochemical data on EW29 and its truncated mutants showed it to be a single-domain type of
Keywords
earthworm Lumbricus terrestris;
hemagglutinating activity; NMR titration;
R-type lectin; sugar
Correspondence
H Hemmi, National Food Research
Institute, National Agriculture and Food
Research Organization (NARO), 2-1-12
Kannondai, Tsukuba, 305-8642 Ibaraki,
Japan
Fax: +81 298 387996
Tel: +81 298 388033
E-mail: hemmi@affrc.go.jp
(Received 1 December 2008, revised 30
January 2009, accepted 2 February 2009)
doi:10.1111/j.1742-4658.2009.06944.x
The R-type lectin EW29, isolated from the earthworm Lumbricus terrestris, consists of two homologous domains (14 500 Da) showing 27% identity with each other The C-terminal domain (Ch; C-half) of EW29 (EW29Ch) has two sugar-binding sites in subdomains a and c, and the protein uses these sugar-binding sites for its function as a single-domain-type hemagglu-tinin In order to determine the sugar-binding ability and specificity for each of the two sugar-binding sites in EW29Ch, ligand-induced chemical-shift changes in EW29Ch were monitored using 1H–15N HSQC spectra as
a function of increasing concentrations of lactose, melibiose, d-galactose, methyl a-d-galactopyranoside and methyl b-d-galactopyranoside Shift perturbation patterns for well-resolved resonances confirmed that all of these sugars associated independently with the two sugar-binding sites of EW29Ch NMR titration experiments showed that the sugar-binding site in subdomain a had a slow or intermediate exchange regime on the chemical-shift timescale (Kd= 10)2 to 10)1mm), whereas that in subdomain c had
a fast exchange regime for these sugars (Kd= 2–6 mm) Thus, our results suggest that the two sugar-binding sites of EW29Ch in the same molecule retain its hemagglutinating activity, but this activity is 10-fold lower than that of the whole protein because EW29Ch has two sugar-binding sites in the same molecule, one of which has a weak binding mode
Abbreviations
Ch (C-half), the C-terminal domain; EW29, earthworm 29 kDa lectin; a-Me-Gal, methyl a- D -galactopyranoside; b-Me-Gal, methyl
b- D -galactopyranoside; STD, saturation transfer difference.
Trang 2hemagglutinin, differing from other tandem repeat-type
proteins in the R-type lectin family, such as ricin [2],
abrin [20] and Sambucus sieboldina agglutinin [21], the
exception being the ricin B1 domain which has two
sugar-binding sites in the same molecule [22,23] Based
on these structural features, R-type lectins generally
contain one sugar-binding site per domain, suggesting
that the truncated mutant, which comprises a single
domain, may have no hemagglutinating activity
[2,5,20] However, the C-terminal domain (Ch; C-half)
of EW29 (EW29Ch) bound to asialofetuin–agarose as
strongly as the whole protein and retained its
hemag-glutinating activity, although at a level 10-fold lower
than that of the whole protein [1] The crystal
struc-tures of the complex between EW29Ch and lactose or
N-acetylgalactosamine (PDB: 2ZQN or 2ZQO)
reported recently indicate that the overall structure of
EW29Ch resembles the characteristic pseudo-threefold
symmetry of the three subdomains designated a, b and
c, and that the protein has two sugar-binding sites in
subdomains a and c [24,25] Therefore, determination
of the sugar-binding ability and specificity for each of
the two sugar-binding sites in EW29Ch is expected to
elucidate the molecular basis of the carbohydrate
cross-linking properties of the lectin
In this study, the sugar-binding ability and
specific-ity of each of the two sugar-binding sites in EW29Ch
for certain sugars were determined using NMR
titra-tion experiments in combinatitra-tion with recent
crystallo-graphic studies [25] The NMR titration experiments
showed that the a sugar-binding site has a much
tigh-ter sugar-binding mode than the c sugar-binding site
Furthermore, saturation transfer difference (STD)– NMR experiments for a mixture of the protein with sugar revealed the epitope of the sugar for the sugar-binding protein Thus, our results suggest that the two sugar-binding sites of EW29Ch in the same molecule retained its hemagglutinating activity, but this activity was 10-fold lower than that of the whole protein because EW29Ch has two sugar-binding sites in the same molecule, one of which has a weak binding mode
Results
Resonance assignments Complete resonance assignments for EW29Ch have been reported elsewhere [26] In this study, we observed chemical shifts for some residues in sub-domain a as a pair of resonance signals in the unbound state and the bound state Furthermore, the resonance signal of residues Gly21 and Asn23 in sub-domain a, which was assigned because of lactose con-tamination in the previous study [26], disappeared in the completely sugar-free state (Fig S1) The reso-nance signals of EW29Ch in the completely sugar-free state were therefore reassigned using multidimensional and multinuclear NMR spectroscopy, as described elsewhere [26] Figure 1 shows the1H–15N HSQC spec-trum for EW29Ch in the completely sugar-free state The assignment data previously deposited in BMRB under accession number 6226 [26] were corrected and re-deposited under the same accession number
Fig 1 1 H– 15 N HSQC spectrum of the C-ter-minal domain of EW29 in the sugar-free state A 600 MHz 2D 1 H– 15 N HSQC spec-trum of the 0.9 m M C-terminal domain of EW29 at pH 6.1 and 298K in the sugar-free state Cross-peaks are labeled based on an analysis of through-bond connectivities The side chains of NH 2 resonances of aspara-gines and glutamines are connected by horizontal lines The side chains of NH resonances of tryptophan are marked by
‘sc’.
Trang 3Identification of sugar-binding sites
The interaction of 15N-labeled EW29Ch with lactose,
melibiose, galactose, methyl a-d-galactopyranoside
(a-Me-Gal) and methyl b-d-galactopyranoside
(b-Me-Gal) was monitored using 1H–15N HSQC
spectros-copy An overlay of 101H–15N HSQC spectra showed
progressive chemical-shift changes for some amide
resonances of EW29Ch upon the addition of lactose
Overlaid spectra showed two types of chemical
exchange (slow and fast) on the chemical-shift
time-scale (Fig 2A) Figure 2B shows the overall effect of
lactose binding by mapping the observed main- and
side-chain 15N chemical-shift changes on the crystal
structure of EW29Ch Residues showing a slow exchange regime in EW29Ch were located in subdo-main a, whereas those showing a fast exchange regime were located in subdomain c (Fig 2B) Larger chemi-cal-shift changes in the fast exchange regime were observed for backbone amides, as well as side-chain amide and indole groups, of residues within or adja-cent to the sugar-binding site of subdomain c, identi-fied from the crystal structure of lactose-liganded EW29Ch In the case of other sugars used in this study, chemical exchanges in subdomain a showed a slow exchange regime for b-Me-Gal and an intermediate exchange regime for melibiose, galactose and a-Me-Gal, whereas those in subdomain c showed a fast exchange
A
B
Unbound Bound Slow exchange Fast exchange
Fig 2 Chemical-shift changes in NMR titration of EW29Ch with lactose (A) Ten 1 H– 15 N HSQC spectra of 15 N-labeled EW29Ch in the pres-ence of protein ⁄ lactose molar ratios of 0 (red), 0.5 (green), 1.0 (blue), 2.0 (magenta), 4.0 (gold), 6.0 (orange), 10.0 (pink), 20.0 (purple), 40.0 (coral) and 80.0 (cyan) are overlaid Overlay spectra show that free and bound forms of some residues in the protein are in a slow exchange
on the NMR time scale and those of other residues are in a fast exchange Representative residues for slow and fast exchanges are labeled The arrow indicates the direction in which amide 1 H– 15 N peaks shift with the adding of sugar (B) Mapping of the 1 HNand 15 N chemical-shift changes upon the addition of excess lactose on a ribbon diagram of the crystal structure of EW29Ch (PDB: 2ZQN) generated by MOLMOL [53] Spheres represent15N atoms of the main chain and side chains of each residue in the protein Residues showing a slow exchange regime are in red and those showing a fast exchange regime and Dav⁄ D max > 0.2, where Davis the normalized weighted average of the 1 H and 15 N chemical-shift changes and Dmaxis the maximum observed weighted shift difference (0.549 p.p.m for side chain amide cross peak
of N124), are in green Residues showing a fast exchange regime and 0.1 £ D av ⁄ D max £ 0.2 are in light green, and others are in gray Key residues showing chemical-shift changes in slow and in fast exchange regimes are labeled.
Trang 4regime for all sugars used No chemical-shift changes
were observed for any sugars in subdomain b These
results showed that each of the two sugar-binding sites
(a and c) of EW29Ch had a distinct chemical exchange
on the chemical-shift timescale
Site-specific dissociation constants determined
by NMR
The site-specific binding constants and chemical
exchange regimes of EW29Ch with sugars used in this
study are given Table 1 Upon the addition of sugars,
the chemical-shift changes in subdomain a were in a
slow exchange regime or an intermediate exchange
regime, as described above For lactose and b-Me-Gal,
the second signal corresponding to the bound state
clearly appeared at [sugar]⁄ [EW29Ch] 0.3; the first
signal corresponding to the unbound state disappeared
completely at [sugar]⁄ [EW29Ch] 1.3 and only the
second signal was observed (Fig 3A) This
phenome-non indicated a stoichiometric interaction between
EW29Ch and lactose at a 1 : 1 ratio The
dissocia-tion constants (Kd) of the a sugar-binding site for
lactose and b-Me-Gal were calculated in a similar way
using NMR titration experiments From the theoretical
calculations of Kdfor the ratios of free to
sugar-bound peak intensities as a function of sugar
concentra-tion, the protein concentration was lowered to 0.05 mm
to obtain the Kd more precisely for the sugar-binding
site in the slow exchange regime However, the Kd of
the a sugar-binding site could not be calculated using
nonlinear regression fitting to the binding isotherm
because the protein concentration was too low to detect
the peak intensities accurately (Fig S2) Therefore, the
Kd values of the a sugar-binding site (residues Asp18, Ser28, Trp33 and Gln44) were approximated to 0.01– 0.07 mm for lactose and 0.02–0.08 mm for b-Me-Gal This was similar to the previously reported Kdvalue of 0.016 mm for lactose using total binding constants of the two sugar-binding sites in EW29Ch, measured by frontal affinity chromatography analysis [24]
For other sugars, the first signal corresponding to the unbound state began to broaden at [sugar]⁄ [EW29Ch] 0.5, the center of the broadened signals shifted to the position of the second signal correspond-ing to the bound state durcorrespond-ing titration ([sugar]⁄ [EW29Ch] = 0.5–3) and the resonance signal of the side-chain NH of Trp33 disappeared The broadened signals sharpened at the position of the second signal
at [sugar]⁄ [EW29Ch] = 3–4 and the resonance signal
of the side-chain NH of Trp33 appeared at the posi-tion of the second signal corresponding to the bound state (Fig 3B) Because the signals were in an interme-diate exchange, of which the Kdshould have a medium value between that in the slow exchange regime and that in the fast exchange regime, titration data indi-cated that the interaction with sugars had a Kd of
10)1mm Furthermore, at the a sugar-binding site, the binding specificity for an anomer was observed; the chemical exchange for lactose and b-Me-Gal was in the slow exchange regime, whereas that for melibiose and a-Me-Gal, as anomers of lactose and b-Me-Gal, was in the intermediate exchange regime Thus, the configuration at the hemiacetal carbon of galactose may affect the dissociation constants
By contrast, because chemical-shift changes upon the addition of sugars at subdomain c were in the fast exchange regime, Kd values describing the interaction
of lactose, melibiose, galactose, a-Me-Gal and b-Me-Gal with EW29Ch were calculated using nonlinear least-square fitting of the chemical shift titration data
to the binding isotherm [27] A plot of the weighted average chemical-shift changes of 1H and 15N reso-nances for the cross-peaks of Gly122, as a function of the molar ratio of each sugar to EW29Ch, is shown in Fig 4 The Kd values for each sugar were calculated from the titration curves measured for the main chain and amide proton groups of Ile102, Ile104, Cys115, Trp117, Lys118 and Gly122, and the side chain nitro-gen and amide proton group of Asn124 in the c sugar-binding site These residues, which exhibited the most significant perturbations in the 15N and amide proton chemical shifts upon sugar binding, all lie within or adjacent to the sugar-binding sites of EW29Ch identi-fied by the crystal structure of the complex Average c-site dissociation constants calculated for each sugar
Table 1 Average site-specific dissociation constants calculated for
EW29Ch with sugar ligands Data obtained at 25 C and pH 6.1 in
50 m M of potassium phosphate and a 10% D 2 O ⁄ 90% H 2 O
mix-ture The chemical exchange regime in parentheses is based on
the observed alterations in NMR signal positions and intensities.
Sugar
Kd(m M )
Melibiose 10)1(intermediate) 5.34 ± 0.81 (fast)
Galactose 10)1(intermediate) 3.89 ± 0.37 (fast)
a-Me-Gal 10)1(intermediate) 4.48 ± 0.38 (fast)
a Dissociation constants could not be calculated accurately due to
the slow or intermediate exchange regime, so the K d is shown as
an approximation b The reported Kdvalues are the average of the
those determined from the 15 N and H N chemical shift perturbations
of Ile102, Ile104, Cys115, Trp117, Lys118, Gly122 and Asn124 The
error range is the standard deviation.
Trang 5were analyzed in accordance with a simple model of
each of the two sugar-binding sites in EW29Ch
inter-acting with one sugar molecule in an independent or
non-cooperative manner, because this assumption was
supported by evidence (monophasic changes in
chemi-cal shifts upon the addition of each sugar and
crystal-lographic studies of the protein-sugar complex) similar
to that reported by Scha¨rpf et al [28] The Kd values
for the c sugar-binding site were 2–6 mm for all sugars
in this study (Table 1), so these results indicated that the a sugar-binding site of EW29Ch is a high-affinity site and the c sugar-binding site is a low-affinity site
Interactions of sugars with EW29Ch by STD–NMR experiments
STD–NMR experiments were conducted to determine the binding epitope of lactose and b-Me-Gal to
A
B
Fig 3 Close-up of the 1 H– 15 N HSQC regions showing the chemical exchange for Asp18 or Trp33sc with increasing amounts of some sug-ars Peak movements of main chain amide and amide proton of Asp18, and side chain amide and amide proton (marked by ‘sc’) of Trp33 in EW29Ch during the titration of lactose (A, top), b-Me-Gal (A, bottom), melibiose (B, top) and a-Me-Gal (B, bottom) Shown are regions of the
1 H– 15 N HSQC spectra corresponding to Asp18 and Trp33sc in EW29Ch at [sugar] ⁄ [EW29Ch] molar ratios indicated at the top The behavior
of Asp18 and Trp33sc during the titration of lactose and b-Me-Gal corresponds to a slow exchange regime (A) and those during titration of melibiose and a-Me-Gal correspond to an intermediate exchange regime (B).
Trang 6EW29Ch because NMR titration experiments of
EW29Ch with sugars showed that the chemical
exchange of the a sugar-binding site upon the
addi-tion of lactose or b-Me-Gal was in the slow exchange
regime The STD effect for sugar arose from the
con-tribution of both the a and c sugar-binding sites
because of the mixture of lactose and EW29Ch at a
ratio of 100 : 1 At first, 2D STD–TOCSY and 2D
STD–[1H,13C] HSQC spectra were obtained for
lactose with EW29Ch to assign the STD–NMR signals
completely, because the proton chemical shifts of the
galactose and glucose residues in lactose partly
over-lap In the STD–TOCSY and STD–[1H,13C] HSQC
spectra, the H1-Gal, H2-Gal, H3-Gal, H4-Gal,
H5-Gal and H6-Gal resonances were assigned
unambi-guously (Fig 5) In both 2D spectra, resonance signals
from the glucose residue in lactose were not observed
However, the crystal structure of EW29Ch with
lactose showed that the glucose residue of the lactose
molecule interacted with subdomain c of EW29Ch
[25] This interaction may be an artifact caused by the
crystallization of lactose-liganded EW29Ch because:
(a) in the other EW29Ch molecule of the crystal
structure (each crystal contained two molecules A and
B) the interaction between the glucose residue and
subdomain c of EW29Ch was not observed; (b) the
B-factor of the side chain of Lys105 was high,
indicat-ing that the side chain of Lys105 is flexible; (c) in this
NMR study, the Kd of the c sugar-binding site for
b-Me-Gal was the same as that for lactose; and (d)
the STD–NMR data in this study showed that the
epi-tope of lactose for EW29Ch is the galactose residue
Thus, these results showed that both sugar-binding
sites of EW29Ch only interact with the galactose residue
1D 1H STD–NMR was conducted for the EW29Ch–b-Me-Gal complex to quantitatively analyze the epitopes of sugars interacting with the protein, because the resonance signals of the glucose residue overlapping with those of the galactose residue within the lactose affected the subtraction of the free induction
0
0.1
0.2
0.3
0.4
0.5
[sugar] / [EW29Ch]
Fig 4 Dissociation constants (Kd) of EW29Ch for some sugars Kd
values of EW29Ch for some sugars were determined by nonlinear
regression fitting of the chemical-shift change versus the sugar
concentration to the binding isotherm describing the binding of one
ligand molecule to a single protein site using the Solver function of
EXCEL 2002 The weighted average of the 1 H and 15 N chemical-shift
changes of Gly122 given by D av = {(D NH2+ D N2⁄ 25) ⁄ 2} 1⁄ 2
[50] is plotted as a function of sugar ⁄ protein molar ratios of added lactose
()), melibiose (h), galactose (4), a-Me-Gal (·) and b-Me-Gal (s).
Fig 5 2D STD–TOCSY and STD–[ 1 H, 13 C] HSQC spectra for the mixture of lactose (5 m M ) and EW29Ch (50 l M ) (A) Reference TOCSY spectrum of the mixture of lactose and EW29Ch at a ratio
of 100 : 1 (B) The STD–TOCSY spectrum of the same sample was collected in an alternative fashion (C) STD–[ 1 H, 13 C] HSQC spec-trum at a 10-fold higher concentration of the same sample.
Trang 7decay values with on- and off-resonance protein
satu-ration Figure 6 shows the 1D 1H NMR spectrum of
b-Me-Gal incubated with EW29Ch at a ratio of
100 : 1, and the corresponding 1D STD spectrum
The integral value of the H4 proton, the largest signal
of b-Me-Gal, was set to 100% Figure 6C shows the
relative degree of saturation of individual protons
normalized to that of H4 The H3, H5, H6a and H6b
protons had similar STD intensities of between 41%
and 54% The H2 proton had a smaller STD intensity
of 30% The lowest intensities corresponded to the
H1 proton and protons of the O-methyl group, which
reached only 18% and 11%, respectively These
results indicated that EW29Ch recognizes the region
from H2 to H6a⁄ H6b, particularly H4, and barely
interacts with the region of H1 and the O-methyl
group The crystal structure of EW29Ch with lactose
showed that the O2, O3 and O4 atoms of the
galac-tose residue of lacgalac-tose formed hydrogen bonds with
EW29Ch, and the C3, C4, C5, C6, O3 and O6 atoms
of the galactose residue formed stacking interactions
with both the a and c sugar-binding sites of EW29Ch
[26] Therefore, our results confirmed that GalO2–
GalO6 of the galactose residue are epitopes for
bind-ing to EW29Ch
Discussion
Binding of an individual lectin site (monovalent bind-ing) to a monosaccharide is extremely weak, with Kd values typically in the range of 0.1–10 mm [29–31] In the R-type lectin family, the dissociation constants of ricin and RCA120 have been determined mainly by equilibrium dialysis studies and fluorescence polariza-tion studies [32–42] Ricin has at least two binding sites in its molecule The Kdvalues of ricin for lactose
at 4 C are 0.03 mm for the high-affinity site and
0.3 mm for the low-affinity site Recently, a third binding site has been found in ricin; thus, the ricin B1 domain of the ricin B chain has two sugar-binding sites in the same molecule [22,23] Because sugars bound at the third sugar-binding site of the ricin B chain were not observed, it is speculated that Kd for the third sugar-binding site of the ricin B1 domain is more than one order of magnitude larger than that for the low-affinity site of ricin, like the Kd value for the
c sugar-binding site of EW29Ch In this study, Kd of the a sugar-binding site of EW29Ch for lactose at
25C was 0.01–0.07 mm and that of the c sugar-bind-ing site was 2.66 mm (Table 1) Kd for the a sugar-binding site of EW29Ch was almost the same as that for the high-affinity sites of ricin and RCA120, whereas Kdfor the c sugar-binding site of EW29Ch is very similar to that for the third binding site of the ricin B chain and has the lowest binding ability Although previous structural studies using X-ray crystallography have clearly shown the mechanism of galactoside-binding to the two binding sites, it is still unclear why one site binds lactose more strongly [2]
In this study, we observed slight differences between the binding modes of the a and c sugar-binding sites
in EW29Ch from the sugar complex structure of EW29Ch [25] The residue at the a sugar-binding site, Gln22, interacts with GalO2 of lactose, but the corre-sponding residue in the c sugar-binding site was not observed Lys36 in subdomain a is replaced by His120
in subdomain c, and one hydrogen bond toward the O2 atom was deduced These results suggested that the
a sugar-binding site has a tighter interaction with lac-tose than the c sugar-binding site because of the num-ber of intermolecular hydrogen bonds and of residues interacting with lactose Our results agreed well with those from the crystal structure of the complex between EW29Ch and lactose [25] However, it remains unclear why the a sugar-binding site binds to lactose much more strongly Future studies will aim to determine both the refined sugar-free structure and the refined complex structure of EW29Ch with lactose
in a solution state by using residual dipolar coupling
Fig 6 1D STD–NMR spectrum for the mixture of b-Me-Gal (5 m M )
and EW29Ch (50 l M ) (A) Reference NMR spectrum of the mixture
of b-Me-Gal and EW29Ch at a ratio of 100 : 1 (B) STD–NMR
spec-trum of the same sample Prior to acquisition, a 30 ms spin–lock
pulse was applied to remove residual protein resonance (C)
Struc-ture of b-Me-Gal and the relative degree of saturation of individual
protons normalized to that of the H4 proton as determined from
the 1D STD–NMR spectrum (B).
Trang 8constants by NMR to analyze the interaction between
the protein and lactose in a solution state
As mentioned above, one of two sugar-binding sites
of EW29Ch, the a sugar-binding site, has a tight
bind-ing mode, but the c sugar-bindbind-ing site has a weak
binding mode This manner of binding reflected the
dissociation constants of EW29Ch in previous frontal
affinity chromatography analysis and corresponds to
that of the higher binding site by NMR, even if the
sugar-binding ability of one of the two binding sites is
much higher than that of the other However, the
hemagglutinating activity of lectin is related to its
mul-tivalency for the cross-linking of cells This means that
the hemagglutinating activity depends on the weaker
of the two sugar-binding sites because both must bind
to sugars on the surface of cells to cross-link and
agglutinate cells Consequently, EW29Ch retains its
hemagglutinating activity but at level 10-fold lower than
that of the whole protein, whereas EW29Ch binds to
asialofetuin–agarose as strongly as the whole protein
A common feature of lectins is their multivalent
binding properties As a consequence, lectin binding to
cells leads to cross-linking and aggregates of
glycopro-tein and glycolipid receptors Thus, the carbohydrate
cross-linking properties of lectins are a key feature of
their biological activities [30,43,44] R-type lectins are
reported to have physiological functions such as
enzyme targeting and glycoprotein hormone turnover
[45] The physiological function of EW29, however,
remains unknown Clarification of the sugar-binding
ability and specificity of the two sugar-binding sites,
which was determined by NMR titration studies and
STD–NMR experiments, is expected to provide clues to
understand the precise physiological function of EW29
Experimental procedures
Sample preparation
15
N-labeled or13C,15N-labeled EW29Ch was expressed and
purified using 15N-labeled or doubly labeled CHL medium
(Chlorella Industry Co., Tokyo, Japan) as described
else-where [26] In this study, purified EW29Ch was dialyzed in
distilled water many more times than had been done
previously, because a pair of resonance signals in the
sugar-free state and the sugar-bound state were observed for
some residues in the a subdomain owing to lactose
contamination from affinity chromatography using lactose–
agarose The final product contains the full-length 131
amino acid EW29Ch sequence of Lumbricus terrestris
(resi-dues Lys130)Glu260 in EW29) [1], with an N-terminal
methionine residue (total length of 132 amino acids)
Residues are numbered from the N-terminal methionine
residue (Met1–Lys2–Pro3 .) Residue Lys2 of EW29Ch in this study corresponds to residue Lys130 of EW29 or residue Lys130 of the crystal structure of EW29Ch (PDB: 2ZQN or 2ZQO) [25]
NMR spectroscopy Purified EW29Ch was dissolved in 50 mm of potassium phosphate buffer (pH 6.1) and a protease inhibitor cocktail (Sigma Chemical Co, St Louis, MO, USA) in either a 90%
H2O⁄ 10% 2
H2O mixture or 99.96% 2H2O The final con-centration of the protein was 0.9 mm NMR spectra were acquired at 25C on Bruker DRX600 and Avance 800 NMR spectrometers All1H dimensions were referenced to internal 4,4-dimethyl-4-silapentane-1-sulfate, and 13C and
15
N were indirectly referenced to 4,4-dimethyl-4-silapen-tane-1-sulfate [46] All multidimensional NMR spectra were acquired in the phase-sensitive mode using the States–time-proportional phase increment method [47] or the echo-anti-echo mode [48] Shifted sine-bell window functions were applied to NMR data prior to zero-filling and Fourier transformation NMR data were processed using felix2000 software (Accelrys, San Diego, CA, USA) or the nmrpipe package [49], and analyzed using sparky software (God-dard and Kneller, sparky 3, University of California, San Francisco, CA, USA) 1H, 13C and 15N assignments were obtained from standard multidimensional NMR methods
as described elsewhere [26]
Titration of EW29Ch with sugars monitored
by NMR The binding of each of sugar; lactose, melibiose, galactose (all from Wako Chemicals, Tokyo, Japan), a-Me-Gal and b-Me-Gal (both from Seikagaku Co., Tokyo, Japan), to EW29Ch at 25C (pH 6.1) was measured quantitatively using1H–15N HSQC NMR spectroscopy Each sugar stock solution used in this study was prepared by weight in a sample buffer of 50 mm of potassium phosphate (pH 6.1) Aliquots of these solutions (starting protein concentration
of 300–350 lm) were added directly to uniformly 15 N-labeled EW29Ch contained in an NMR tube For each titration, 20 1H–15N HSQC spectra were recorded consecu-tively with increasing concentrations of each sugar For the progressive chemical-shift changes of EW29Ch under condi-tions of fast exchange on the chemical-shift timescale, 15N and1HNchemical-shift changes in EW29Ch were calculated using the equation Dav= {(DNH2+ DN2⁄ 25) ⁄ 2}1 ⁄ 2, where
DNH is the chemical-shift change of the amide proton and
DN that of the nitrogen [50] Sugar-binding constants (Kd) were calculating using the Solver function of excel 2002 (Microsoft, Redmond, WA, USA) for the c sugar-binding site by nonlinear regression fitting of the chemical-shift change versus sugar concentration to the binding isotherm
Trang 9describing the binding of one ligand molecule to a single
protein site [27] Similarly, assuming that sugar binding
to EW29Ch is a reversible single-step transition under
conditions of slow exchange on the chemical-shift timescale,
the dissociation constant, Kd, is given by
Kd¼ ½P½L=½PL
Here, [P], [L] and [PL] are the respective concentrations
of free EW29Ch, free sugar and the EW29Ch–sugar
com-plex [P]⁄ [PL] ratios were determined as a function of [L]
from free and bound peak intensities [51], because the two
signals in sugar-free and sugar-bound forms were observed
separately under the slow exchange regime Kd values were
also calculated using the Solver function of excel 2002 for
the a sugar-binding site by nonlinear regression fitting of
the ratio of free and bound peak intensities versus sugar
concentration to the binding isotherm Throughout the
titration for calculating Kd values under the slow exchange
regime, the concentration of EW29Ch was maintained at
0.05 mm and lactose or b-Me-Gal was added incrementally
from 0 to 0.15 mm
STD–NMR experiments
Non-labeled EW29Ch was expressed using Luria–Bertani
medium and purified using the same method as the
prepa-ration of labeled EW29Ch [26] To a sample of EW29Ch in
phosphate-buffered solution (50 mm of potassium
phos-phate buffer pH 6.1 and a protease inhibitor cocktail in
99.96% 2H2O) was added lactose or b-Me-Gal The final
sugar concentration was 5 mm at a sugar-to-protein ratio
of 100 : 1 1D and 2D STD–NMR spectra were obtained
as described previously [52] The time dependence of the
saturation transfer was determined by recording 1D STD
spectra with 1000 scans and saturation times from 0.25 to
6.0 s The irradiation power in all STD–NMR experiments
was set to 0.15 W Relative STD values were calculated
by dividing STD signal intensities by the intensities of the
corresponding signals in a reference spectrum of the same
sample recorded with 64 scans All STD–NMR spectra for
epitope mapping were acquired using a series of equally
spaced 50 ms Gaussian-shaped pulses for saturation with
1 ms intervals and a total saturation time of 2 s
On-res-onance irradiation of the protein was conducted at a
chemi-cal shift of –0.4 p.p.m and off-resonance at a chemichemi-cal
shift of 30 p.p.m where no protein signal was present Free
induction decay values with on- and off-resonance protein
saturation were recorded in an alternative fashion
Subtrac-tion of the 1D STD spectra was achieved via phase cycling
Protein resonance was suppressed by the application of a
30 ms spin–lock pulse before acquisition 2D STD–TOCSY
and STD–[13C,1H] HSQC spectra at natural abundance
with on- and off-resonance protein saturation were
recorded with 128 scans or 512 scans per t1increment in an
alternative fashion The 2D spectra were acquired with spectra widths of 10 p.p.m in1H and 80 p.p.m in13C, and
128 (t1) and 2048 (t2) complex points or 64 (t1) and 1024 (t2) complex points for STD-TOCSY or STD-[13C, 1H] HSQC spectra An MLEV mixing time of 100 ms was applied in STD–TOCSY spectra
Acknowledgements
We thank Drs Rintaro Suzuki and Toshimasa Yama-zaki (National Institute of Agrobiological Sciences) and Dr Chojiro Kojima (Nara Institute of Science and Technology) for help in calculating sugar-binding constants from NMR titration data We also thank
Ms Sachiko Unno (AIST) for the preparation of the proteins This work was supported in part by Grant-in-Aid for Scientific Research (C) (18580342 and 20580373) from the Japan Society for the Promotion
of Science (to HH and AK)
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