NMR Analysis of Type III Antifreeze Protein Intramolecular
Dimer
STRUCTURAL BASIS FOR ENHANCED ACTIVITY*
Kazunori
Miura
,
Satoru
Ohgiya
,
Tamotsu
Hoshino
,
Nobuaki
Nemoto§,
Tetsuya
Suetake
,
Ai
Miura
,
Leo
Spyracopoulos¶,
Hidemasa
Kondo
, and
Sakae
Tsuda
Bioscience and Chemistry Division, Hokkaido National
Industrial Research Institute, 2-17-2-1 Tsukisamu-Higashi, Toyohira,
Sapporo 062-8517, Japan, § Varian Japan, Varian Japan
Sumitomo Shibaura Building, 4-16-36 Shibaura, Minato-ku,
Tokyo 108-0023, Japan, and ¶ Department of Biochemistry,
University of Alberta, Edmonton, Alberta T6G 2H7, Canada
Received for publication, August 29, 2000, and in revised form, September 27, 2000
 |
ABSTRACT |
The structure of a new antifreeze protein (AFP)
variant, RD3, from antarctic eel pout (Rhigophila
dearborni) with enhanced activity has been determined for the
first time by nuclear magnetic resonance spectroscopy. RD3 comprises a
unique translational topology of two homologous type III AFP globular
domains, each containing one flat, ice binding plane. The ice binding
plane of the N domain is located ~3.5 Å "behind" that of the C
domain. The two ice binding planes are located laterally with an angle
of 32 ± 12° between the planes. These results suggest that the
C domain plane of RD3 binds first to the ice {10
0} prism
plane in the
0001
direction, which induces successive ice binding
of the N domain in the
0101
direction. This manner of ice binding
caused by the unique structural topology of RD3 is thought to be
crucial for the significant enhancement of antifreeze activity,
especially at low AFP concentrations.
 |
INTRODUCTION |
Inhibition of ice crystal growth by antifreeze proteins
(AFPs)1 is a unique
biological function identified in various organisms such as fishes (1,
2), insects (3-5), and plants (6). Antifreeze function is
macroscopically visible in aqueous solutions of AFP as the creation of
an unusual bipyramidal shape for ice crystals (7) and nonlinear
freezing temperature (Tf) depression (thermal
hysteresis; Ref. 8). However, it is not clear how AFPs cause these
phenomena. Indeed, quantitative analysis of the nonlinear profile of
Tf depression is not feasible, because it is
difficult to define the concentration of growing ice crystals and total
numbers of ice binding sites for AFP. Nevertheless, structural biology
has provided insight into the antifreeze mechanism. For example, types
I-IV AFP variants have the ability to bind specifically to the ice
crystal surface and inhibit its growth, albeit each variant is composed
of a different structural motif: type I AFP is composed of
alanine-rich, amphipathic
helices (Mr
~3,300-5,000); type II AFP (Mr ~14,000)
comprises cysteine-rich globular proteins related to C-type lectines;
type III AFP (Mr ~6,500) comprises compact
-sheet structures, and type IV AFP (Mr
~12,000) is assumed to form a four-helix bundle (9). Several new
variants identified from fish skins (10), worms (3-5), and plants (6)
are characterized by internal repetitions of a consensus peptide
sequence, which differ from the above four types of AFP. Concomitant
with the increasing library of structurally variant AFPs (1-10),
attention has been focused on the determination of a detailed
antifreeze mechanism and the molecular basis of antifreeze activity
enhancement with the ultimate goal of designing an industrial AFP with
improved activity.
For type III AFP monomer, the three-dimensional (3D) structure and the
ice-binding mechanism have been extensively examined in recent years.
The high-resolution NMR (modified HPLC-12 or QAE isoform;
Refs. 11, 12) and x-ray (QAE isoform and HPLC-3; Refs. 13, 14)
structures revealed that type III AFP exhibits a compact fold with
several short and irregular
-sheets and one
-helical turn. A
remarkably flat and amphipathic surface plane that encompasses residues
9-21 and 41-44 is thought to be an essential ice binding site. The
polar atoms of the putative ice binding residues Gln9,
Asn14, Thr15, Ala16,
Thr18, and Gln44 are located to form hydrogen
bonds with oxygen atoms of the ice lattice {10
0} prism plane
(11-14). RD3 is an activity-enhanced variant of type III AFP that is
an intramolecular dimer (134 residues, Mr
15,800; Ref. 15). In RD3 a 9-residue linker sequence
(D65GTTSPGLK73) connects the two type III AFP
domains in tandem and locates them in specific orientations, which
determines in part the antifreeze activity of the intact molecule.
Together with activity measurements, the present structural
determination of RD3 will improve understanding of the ice binding
mechanism and will provide insight into the potential for production of
AFP with enhanced activity.
 |
EXPERIMENTAL PROCEDURES |
Sample Preparation and Activity Measurement--
Preparations of
unlabeled, 15N-labeled-, and
13C/15N-labeled-RD3 were briefly described
previously (16). Escherichia coli JM105 cells were
transformed with a pKK223-3UC-based expression plasmid containing
synthesized DNA encoding RD3 and were cultured in M10 minimal media.
The media contained 15N-labeled NH4Cl for the
expression of 15N-labeled RD3 and both
15N-labeled NH4Cl and 13C-labeled
glucose for expression of 13C/15N-labeled-RD3.
The transformed cells were lysed by sonication, and the lysate was
centrifuged at 21,000 × g for 30 min at 4 °C. The
precipitate was suspended in TEN buffer (50 mM Tris-HCl, pH 7.5, 1 mM EDTA, 300 mM NaCl, and 0.1 mM phenylmethylsulfonyl fluoride). Inclusion bodies in the
precipitate were purified by centrifugation in 40% (w/v) sucrose and
washed with TEN buffer containing 1.0% Triton X-100. RD3 was extracted
from the inclusion bodies with extraction buffer
(acetonitrile:isopropanol:water, 46.7:23.3:30) containing 0.1%
trifluoroacetic acid at 22 °C. After centrifugation at 16,000 × g for 10 min at 4 °C, the supernatant was diluted with
acetic acid buffer (pH 3.8) and loaded onto a fast protein liquid
chromatography High-S column (Bio-Rad). Bound RD3 was eluted from the
column with a linear gradient of aqueous ammonium sulfate. Fractions
containing RD3 were further purified by reverse-phase liquid
chromatography using a TSKgel ODS-80Ts column (Tosoh, Tokyo, Japan).
The RD3 sample was lyophilized after check of the purity by
Tricine-SDS-polyacrylamide gel electrophoresis with Coomassie brilliant
blue staining. Approximately 50 mg of unlabeled RD3 and 15 mg of
15N- and 13C/15N-labeled RD3 were
prepared from 2.4- and 6.0-liter cultures, respectively.
The thermal hysteresis measurements were performed using an osmometer
(model OM 802, VOGEL GmbH) for lyophilized RD3 and type III AFP
monomer (RD3-Nl) samples. RD3 and RD3-N1 samples were dissolved in 0.1 M ammonium bicarbonate (pH 7.9) to give final concentrations of 0.05, 0.1, 0.2, 0.3, 0.4, 0.5, 1.5, and 2.0 mM. Thermal hysteresis measurements were repeated three
times using fresh solution for each point. The antifreeze activity of RD3 was further examined by observation of the ice crystal morphology using a Leica DMLB 100 photomicroscope equipped with a Linkam LK600
temperature controller. RD3 (2 µl) was dissolved in 0.1 M
ammonium bicarbonate (pH 7.9, 0.1-1.0 mg/ml), momentarily frozen (approximately
22 °C), and warmed to 0 °C on the sample stage of the photomicroscope in order to create several ice crystal seeds in
the solution. This solution was then cooled to approximately
1 to
5 °C, and growth of ice crystal seeds was monitored.
Structure Determination--
A total of 1575 NMR-derived
experimental restraints were obtained using a 500-MHz NMR spectrometer
(Unity Inova-500, Varian) for the structural determination. Interproton
distance restraints were obtained from the two-dimensional
NOESY, 15N-edited NOESY (17), and simultaneous
15N/13C-edited 3D NOESY experiments (18)
performed at 4 °C. The mixing time dependence of the transient NOE
was determined from two-dimensional NOESY spectra to assess the effects
of spin diffusion; subsequently, the mixing time was set to 50 ms for
the NOESY experiments used to obtain experimental nuclear Overhauser
enhancement (NOE) restraints. The intensities of two-dimensional and 3D
NOESY data were calibrated on the basis of NOEs corresponding to a
known distance such as Tyr H
-H
(2.48 Å), and an error of 50%
was assumed for the NOE peak intensities. The following distance
constraints were used to calibrate the 3D spectra:
HNi-H
i = 2.70-3.05 Å (for residues with
negative
value); HNi-H
i-1 = 1.7-3.6 Å; H-C-C-H = 2.2-3.1 Å; H-C-CH3 = 2.5-2.7 Å; and H-C-H = 1.7-1.8 Å. In cases in which direct
calibration was not possible, the distance constraints were
overestimated. For NOEs found only in the NOESYs with a mixing time of
120 ms, the upper bound was set to 6 Å. For all proton-proton restraints, the lower bound was set to 1.7 Å. Dihedral angle
restraints for the
angle were estimated from the 3D HNHA
experiment, using a correction factor of 1.1. A 25% error on the peak
intensities was assumed, and the minimum
angle restraint
range was set to ±10°. The initial set of restraints containing no
dihedral angle restraints and a fraction of NOEs are listed in Table I.
From this initial set of NOE restraints and starting from an extended structure, 100 structures were generated with the simulated annealing protocol in X-PLOR 3.851 (19) using 12,000 high-temperature steps (60 ps at 1,000 K) and 6,000 cooling steps (30 ps, final temperature of 100 K). Of the 100 calculated structures, 75 were converged and folded
properly but had significantly high total energy, which is generally
due to assignment errors and a lack of input structural constraints.
Hence, the input constraints were refined by analysis of the
inconsistency between the constraints and the coordinates of the
calculated structures. The calculated structures were then used for a
new round of peak picking of NOESY spectra that enabled the assignment
of larger numbers of NOEs. With refined distance constraints and the
addition of dihedral
angle constraints, the next round
of structure calculation was performed starting with the 75 converged
structures using the simulated annealing protocol with 6,000 high-temperature steps (30 ps) and 4,000 cooling steps (20 ps).
Refinement of the input constraints with structure calculations was
repeated over 30 times. The set of structures presented in this paper
includes the 15 (see Fig. 2) and 40 (see Fig. 4b) lowest
energy structures selected from the 50 structures obtained in the last
round of refinement.
 |
RESULTS AND DISCUSSION |
Recombinant RD3 protein generated by bacterial expression gives
rise to thermal hysteresis and a bipyramidal shape for ice crystals as
typically observed for all AFP variants. The noncolligative increase in
antifreeze activity of RD3 is compared with that of type III AFP
monomer (RD3-Nl; Ref. 20) in the range of 0-2.0 mM protein
(Fig. 1a). RD3 was initially
reported to show 1.9-fold higher activity than type III AFP monomer
(11), which is verified in the present study when the concentrations of
RD3 and monomer are 0.5 mM (Fig. 1, a and
b). For AFP concentrations of 0-0.5 mM, a
significant 5.9-fold enhancement of antifreeze activity is observed for
RD3 compared with monomer (RD3-N1; Fig. 1b). Although RD3
consists of two type III AFP monomers, the increased antifreeze activity of RD3 does not correspond to simple additive activity of two
monomers under nonsaturating conditions. It has been suggested that
AFPs bind irreversibly to ice crystal nuclei according to the
adsorption-inhibition mechanism at the ice-water interface (8), on
which convex ice surfaces are created between the bound proteins. The
height and curvature of the convex ice surface increases because of the
Kelvin effect at subzero temperatures (21). The convex ice surface is
energetically unfavorable for water to join the ice lattice and results
in depression of the freezing point (
Tf; Ref.
22). The freezing point depression was assumed to follow the
relationship
Tf = constant × h/D2, where h is the
height of the convex ice surface, and D is the average
distance between the ice-bound AFPs on the ice surface (21). Assuming
that the average separation distance (D) for RD3 is less
than the distance between RD3-Nl monomers because of the unique
interdomain topology of RD3, this equation predicts an increased
Tf for RD3 compared with type III AFP
monomer. It should be noted that this equation was proposed under the
assumption that type I AFP is cylindrically shaped. Thus, modifications
or additional hypotheses, or both, may be necessary to describe the ice
binding properties of RD3. In any case, more coverage of the ice
surface area or more effective ice binding of RD3 compared with monomer
(RD3-Nl), or both, is thought to alter efficiently the convex ice
surface between ice-bound protein molecules especially at low protein
concentrations (0.1-0.5 mM AFP). At higher AFP concentrations (0.5-2 mM), saturating amounts of AFP are
bound to the ice crystal surface; thus no new convex ice surfaces can be formed, leading to an upper limit for
Tf
(Fig. 1a). Assuming that the average distance between RD3 is
shorter than that between RD3-Nl molecules, a smaller amount of RD3
compared with RD3-Nl is enough to saturate the ice surface. Hence, the
Tf curve becomes a plateau even at the low
concentration of RD3. The validity of this idea requires more
experimental evidence and consideration, and detailed information about
the structural difference between RD3 and the type III AFP monomer
would have a significant contribution in this regard.

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Fig. 1.
a, noncolligative freezing point
depression ( Tf) of RD3 (circles)
and type III AFP monomer (RD3-Nl; Ref. 20; rectangles).
Freezing point depression attributable to hen egg white lysozyme is
also plotted (triangles) as an example of colligative
freezing point depression. b, ratio of
Tf for RD3 compared with
Tf for RD3-Nl as a function of protein
concentration. RD3 possesses a maximum of 5.9-fold higher activity than
RD3-Nl in the protein concentration range of 0-0.5
mM.
|
|
The NMR solution structure of intact RD3 (Fig.
2) has been determined on the basis of
complete assignments of the 1H, 13C, and
15N resonances of RD3 at pH 6.8 and 4 °C, which is
approximately the freezing point of the solvent (BioMagResBank
accession number 4449; Ref. 16). The determined structures have high
stereochemical quality as well as sufficiently small values for NOE
violations and root mean squared atomic coordinate differences (Table
I; restraints and coordinates are
available from the Research Collaboratory for Structural
Bioinformatics, http://www.rcsb.org/pdb). The N and C domains of
RD3 (residues 1-64 and 74-134) have highly similar compact folds
(Fig. 3). These folds are characterized
by an internal two-fold symmetry motif or so-called "pretzel fold"
(14) consisting of two antiparallel loop-shaped elements (residues
8-23 and 40-55 for the N domain and residues 78-93 and 110-125 for
the C domain). A large part of the secondary structure of each domain
is due to the
-strand (48%). The elements of secondary structure
for the N domain consist of eight short
-strands (
1-
8;
residues 3-7, 9-13, 15-18, 22-26, 31-33, 43-45, 47-49, and
53-55; Fig. 3a) followed by a type III turn. The eight
-strands are connected through a type II turn (residues 40-43), a
type III turn (residues 18-21), and an
-helix (
1; residues
36-39). The eight
-strands are also identified in the corresponding
regions of the C domain (
9-
16; residues 73-77, 79-83, 85-88,
92-96, 101-103, 113-115, 117-119, and 122-125). Similar to the N
domain, the other C domain secondary structural elements are an
-helix (
2; residues 106-109), a type II turn (residues
110-113), and two type III turns (residues 88-91 and 126-129). For
the type III AFP monomer (QAE isoform), there are coordinates available
for the NMR solution structure (Protein Data Bank code 1KDF; Refs. 11,
12) and the x-ray structure (Protein Data Bank code 1MSI; Ref. 13).
There are slight inconsistencies in the secondary structure assignment
between the NMR and x-ray structural coordinates. For example, one
-strand (residues 2-13) identified in the x-ray structure (1MSI)
was assigned as two strands (residues 3-7 and 9-13) in the NMR
structure (1KDF). Our determination of the lengths and locations of the
-helices and
-strands for each globular domain of RD3 are similar to those identified in the NMR structure (1KDF; Ref. 12). The
structures of the type II turns at residues 40-43 and 110-113 were
not identified in the QAE isoform and are due, in part, to the presence
of residues Gly42 and Gly112 of RD3, which
favor turns. The root mean square deviation value between the globular
domains of RD3, 1KDF, and 1MSI is 1.17 ± 0.23 Å for the
backbone atoms, indicating that the overall structural motif
of the N- and C-terminal globular domains of RD3 is typical of type III
AFP monomer.

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Fig. 2.
Solution structure of RD3 determined at
4 °C. a, stereo view showing 15 of the lowest energy
structures of RD3 superimposed on residues 4-64 (N domain).
b, stereo view showing 15 of the lowest energy structures
superimposed on residues 74-131 (C domain). When the calculated
structures of one domain are superimposed (black), the
structures for the counter-domain are dispersed (gray). This
implies that the flexible linker region (residues 65-73) causes
divergence in the relative orientation of the N and C domains with
respect to each other, although each domain is well converged with
sufficiently small root mean square deviation values (see Table I). The
structural quality of the two globular domains is comparable with the
high-precision NMR structure of type III AFP monomer (QAE isoform; Ref.
12).
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Fig. 3.
a, amino acid sequence of RD3 with the
location of the 16 -strands and two -helices shown. b,
stereo view of the minimized average structure of RD3 shown in the
ribbon representation. The secondary structural assignments were
performed using the program VADAR (28). All figures were produced with
the programs Molscript (29) and Raster3D (30).
|
|
The side chain oxygen atoms of Gln9, Asn14,
Thr15, Thr18, and Gln44 and main
chain carbonyl oxygen atoms of Asn14 and Ala16
are clustered together and form a flat surface plane on the outer face
of the two-fold symmetry motif of the N domain (Fig.
4a). These atoms were
identified as putative ice binding residues for type III AFP monomer.
Mutation of these residues reduces protein thermal hysteresis activity
and causes changes in morphology of the ice crystal (13). The
corresponding ice binding residues of the C domain of RD3 are
Gln79, Asn84 (O'), Thr85,
Ala86, Thr88, and Gln114 (Fig.
4a). It appears that the nonpolar side chain groups of Pro12, Thr15, Ala16,
Thr18, Ile20, Met21, and
Gly42 fill the gaps between the ice binding residues of the
N domain. The corresponding residues of the C domain are
Pro82, Thr85, Ala86,
Thr88, Ile90, Met91, and
Gly112. The location of these nonpolar residues in each
domain is thought to stabilize the spaced polar groups and the
planarity of the ice binding planes to achieve appropriate ice binding
function (12, 13). The side chain groups of the key ice binding
residues Asn14 in the N domain, and the corresponding
residue Asn84 in the C domain are located slightly behind
the ice binding plane (Fig. 4a). For the type III AFP
monomer, Asn15 plays a pivotal role in initiation of
binding to transition points between prism and basal ice planes (13).
The 9-residue linker of RD3 (residues 65-73) forms a bent structure,
as found in RD3-Nl (20), that places the N and C domains in close
proximity but does not allow for direct association. This short linker
locates the two ice binding planes of each domain laterally with an
angle of 32 ± 12° between the planes (Fig. 4, a and
b). To our knowledge, this interdomain topology is
structurally unique among smaller proteins (<30 kDa) that comprise two
similarly sized domains. One well-known, small intramolecular dimer is
calmodulin (148 residues), a dumbbell-shaped calcium regulatory
protein. Calmodulin is composed of two homologous N- and C-terminal
globular domains connected through a 28-residue linker that forms a
long
-helix (23). Disparate from RD3, the two homologous domains of
calmodulin have no preferred orientation with respect to each other
(24). The domains have the propensity to come together and "grip"
the target protein, much like two hands capturing a rope (25). The N
and C termini of RD3 (both type III AFP monomers) point to almost opposite directions in the ice-free state and presumably in the ice-bound state as well. On the other hand, the N and C termini of
calmodulin point to nearly the same direction when bound to the target
protein; i.e. the functional difference between RD3 and
calmodulin may be ascribed to the difference in the interdomain topologies.

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Fig. 4.
a, stereo view of the minimized average
structure of RD3. Shown on the ice binding planes are all polar side
chain atoms assumed to be involved in complementary hydrogen bonds with
water oxygen atoms of the ice lattice {10 0} prism plane. The
ice binding O and N atoms are represented in
Corey-Pauling-Koltun (red and blue,
respectively). b, stereo view of the 40 calculated
structures of RD3 superimposed on the C domain (residues 74-131). The
various solid lines for the N domain indicate the range of
the movement of this domain allowed by the flexible linker. The region
from Val45 to Glu64 is not drawn to avoid
congestion. The oxygen atoms of the C domain essential for ice binding
(a) are shown in the Corey-Pauling-Koltun representation.
The backbone of the minimized average structure is shown as a
thick black rod. The ice binding plane of the structure with
a backbone represented by the thick blue rod is nearly
aligned with that of the C domain, whereas the structure represented by
the red rod is located farthest behind the ice binding plane
of the C domain. The average values of the backbone N-NH bond vector
order parameter (S2) are 0.84 ± 0.08 and
0.87 ± 0.08 for the N and C domains (residues 4-64 and 74-131),
respectively. The overall correlation times
( m) were estimated separately for each domain
(11.38 ± 1.09 ns for the N domain and 11.29 ± 1.03 ns for
the C domain; Refs. 31, 32). The S2 profile of
the linker (residues 65-73) shows a V shape as found in RD3-Nl (20).
The average S2 value for the linker (0.60 ± 0.15) indicates that the backbone atoms of the linker are more
flexible compared with the globular domains.
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The lateral orientation of the N- and C-terminal domains of RD3 directs
the faces of two ice binding planes in nearly the same direction with a
slight difference in their alignments. The ice binding plane of the N
domain is located ~3.5 Å behind that of the C domain in the
minimized average structure (Fig. 4a). This orientation of
the two domains suggests that the C domain ice binding face of RD3
attaches to the ice crystal first, which follows the second attachment
of the ice binding face of the N-domain in a cooperative manner.
Presumably, the ice-bound form of RD3 would have the ice binding faces
of each domain oriented nearly identically with respect to the ice
surface. Indeed, an "aligned" form of RD3 was identified in one of
the present solution structures (Fig. 4b, thick blue rod).
If a structural change occurs between ice-free and ice-bound states of
RD3, it is likely that the conformation of the 9-residue linker changes
between the two states. Consistent with a view of conformational
flexibility between ice-free and ice-bound forms of RD3, greater
flexibility for the backbone in the linker region was identified by
15N NMR relaxation measurements, and fewer experimental NMR
constraints were found compared with the structured regions (Fig.
4b).
Manual docking of RD3 to ice indicated that interatomic distances
between key atoms of ice binding residues of the two domains of RD3
form a close match to the spacing of water oxygen atoms in the ice
{10
0} prism plane of hexagonal ice crystal
(Ih: c-axis = 7.361Å;
a-axis = 4.507Å; Ref. 26). For example, the distance between the oxygen atoms of Thr88 and Ala86
(CO) is 4.5 Å, and that of Ala86 and Gln114 is
7.4 Å. The corresponding residues in the N domain are
Thr18, Ala16, and Gln44. These
atoms are located in line in each domain and match the positions of
three water oxygen atoms lined up in the
0001
direction. The
placement of the C domain in this direction leads to the positioning of
Asn84 (Fig. 4a) at the intersection between the
prism and basal planes of the ice crystal. These results are in good
agreement with the ice-docking model proposed for type III AFP monomer
(12, 13). Assuming that the C domain binds ice first in the
0001
direction, one possible target ice surface for the N domain might be
the same prism plane in the
0101
direction. Respective ice
binding in the
0001
and
0101
directions would be in good
agreement with the 32° difference in alignment of the two ice binding
planes. In such an ice-bound model for RD3, close proximity is still
identified between the oxygen atoms of the N domain plane and the water
oxygen atoms in the prism plane. The positioning of the nonplanar
residue Asn14 to the intersection between prism and basal
planes is unexpected in this model because of steric hindrance by two
globular domains. Note that the two ice binding planes can be manually
aligned in the
0001
direction to achieve better interoxygen space
matching of the two domains by adjustment of backbone
/
angles of the linker sequence (residues
67-69).
It is likely that the ice binding of the C domain induces the specific
ice binding of the N domain to the prism surface. In addition, recent
proposals implicate flatness of the ice binding plane and
hydrophobicity as important determinants of ice binding (12-14). These
determinants are also expected to contribute to the specificity of ice
binding of the N domain. DeLuca et al. (27) reported that
genetically expressed type III AFP connected through the N terminus to
thioredoxin (12 kDa) or maltose-binding protein (42 kDa) possess
~2-3-fold higher antifreeze activity than type III AFP monomer on a
molar basis. Thioredoxin and maltose-binding protein do not possess
antifreeze activity, and the higher activities of the fusion proteins
were ascribed to ~3-8-fold wider coverage (without ice binding) of
the ice surface. For RD3 we can assume just a 2-fold increase in
coverage of the surface area compared with the monomer. Hence, the
observed 5.9-fold increased activity of RD3 compared with the monomer
cannot be explained on the basis of increased ice surface coverage of
RD3 but can be explained on the basis of increased ice binding strength
of the molecule. It is likely that the on rate for ice binding of the
type III AFP monomer is a diffusion-controlled process. The same
process is likely to dominate ice binding of the C domain of RD3,
whereas the on rate of ice binding of the N domain is influenced by its close proximity to the ice surface because of initial ice binding of
the C domain. Hence, the affinity of intact RD3 for ice would not be a
simple sum of the affinity of two type III AFP monomers but, rather,
would be greater than the sum of the monomer affinities, leading to the
6-fold activity enhancement of RD3. We conclude that specific ice
binding of the two domains, guided by the unique structural topology of
RD3, is an important factor in determination of the high antifreeze
activity of RD3. The 9-residue linker presumably plays a key role in
connecting the N- and C-terminal AFP domains laterally and potentially
affords simultaneous ice binding of the two domains. An artificial AFP
consisting of multiple type III AFP monomers connected through
9-residue linker sequences may possess highly enhanced antifreeze
function compared with unconnected monomers of type III AFP.
 |
ACKNOWLEDGEMENTS |
We acknowledge Dr. Brian Sykes for critical
reading of the manuscript. We also thank Drs. Peter Davies, Steffen
Graether, M. Odaira, K. Nitta, and N. Matsushima for encouragement and
valuable discussions and Dr. Lewis Kay for providing NMR pulse
sequences. The atomic coordinates for the hexagonal ice crystal
(Ih) were kindly provided by Dr. H. Itoh (Tokyo
Institute of Technology).
 |
FOOTNOTES |
*
The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
The atomic coordinates and the structure factors (code 1C8A and 1C89) have been deposited in the Protein Data Bank, Research Collaboratory for Structural Bioinformatics, Rutgers University, New Brunswick, NJ (http://www.rcsb.org/).
To whom correspondence should be addressed; Bioscience and
Chemistry Division, Hokkaido National Industrial Research Institute, 2-17-2-1 Tsukisamu-Higashi, Toyohira, Sapporo 062-8517, Japan. Tel:
81-11-857-8912; Fax: 81-11-857-8983; E-mail: tsuda@hniri.go.jp.
Published, JBC Papers in Press, September 28, 2000, DOI 10.1074/jbc.M007902200
 |
ABBREVIATIONS |
The abbreviations used are:
AFP, antifreeze protein;
Tf, freezing temperature;
NOE, nuclear Overhauser enhancement;
NOESY, NOE spectroscopy;
3D, three-dimensional.
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