letters to nature
25. Porse, B. T., Leviev, I., Mankin, A. S. & Garrett, R. A. The antibiotic thiostrepton inhibits a
functional transition within protein L11 at the ribosomal GTPase center. J. Mol. Biol. 276, 391±404
(1998).
26. Bretscher, M. S. Translocation in protein synthesis: A hybrid structure model. Nature 218, 675±677
(1968).
27. Spirin, A. S. A model of the functioning ribosome: locking and unlocking of the ribosome
subparticles. Cold Spring Harbor Symp. Quant. Biol. 34, 197±207 (1969).
28. Woese, C. Molecular mechanics of translation: a reciprocating ratchet mechanism. Nature 226, 817±
820 (1970).
29. Moazed, D. & Noller, H. F. Intermediate states in the movement of transfer RNA in the ribosome.
Nature 342, 142±148 (1989).
30. Agrawal, R. K. & Burma, D. P. Sites of ribosomal RNAs involved in the subunit association of tight and
loose couple ribosomes. J. Biol. Chem. 271, 21285±21291 (1996).
31. Frank, J. et al. SPIDER and WEB: processing and visualization of images in 3D electron microscopy
and related ®elds. J. Struct. Biol. 116, 190±199 (1996).
Supplementary information is available on Nature's World-Wide Web site
(http://nature.com).
Acknowledgements
This work was supported by grants from the National Institutes of Health. We thank A.
Heagle for preparing the illustrations and animated sequence, and I. Gabashvili and
P. Penczek for help with image processing.
Correspondence and requests for materials should be addressed to J.F.
.................................................................
Mimicry of ice structure by
surface hydroxyls and water
of a b-helix antifreeze protein
Yih-Cherng Liou, Ante Tocilj, Peter L. Davies & Zongchao Jia
Department of Biochemistry, Queen's University, Kingston, Ontario K7L 3N6,
Canada
..............................................................................................................................................
Insect antifreeze proteins (AFP) are much more effective than ®sh
AFPs at depressing solution freezing points by ice-growth
inhibition1,2. AFP from the beetle Tenebrio molitor is a small
protein (8.4 kDa) composed of tandem 12-residue repeats3
(TCTxSxxCxxAx). Here we report its 1.4-AÊ resolution crystal
structure, showing that this repetitive sequence translates into
an exceptionally regular b-helix. Not only are the 12-amino-acid
loops almost identical in the backbone, but also the conserved side
chains are positioned in essentially identical orientations, making
this AFP perhaps the most regular protein structure yet observed.
The protein has almost no hydrophobic core but is stabilized by
numerous disulphide and hydrogen bonds. On the conserved side
of the protein, threonine-cysteine-threonine motifs are arrayed to
form a ¯at b-sheet, the putative ice-binding surface. The threonine side chains have exactly the same rotameric conformation
and the spacing between OH groups is a near-perfect match to the
ice lattice. Together with tightly bound co-planar external water,
three ranks of oxygen atoms form a two-dimensional array,
mimicking an ice section.
AFP from the beetle Tenebrio molitor (TmAFP) is shaped like a
slightly ¯attened cylinder, 32 AÊ in length, and 6.5 ´ 14 AÊ in the
pseudo-rectangular cross section (Fig. 1). The underlying architecture of this 8.4-kDa, threonine- and cysteine-rich protein is that of
an extremely regular parallel b-helix formed from seven 12-aminoacid turns or loops. This structure is designed to present a ¯at, rigid
b-sheet along one side of the molecule, which we propose is the icebinding site. With the exception of the amino-terminal turn, each
helix turn contributes a short b-strand to the sheet, typically TCT,
with the threonine residues projecting outwards in two precisely
aligned parallel arrays (Fig. 1b). The right-handed b-helix is
322
Figure 1 Ribbon illustrations of TmAFP. a, Side view of the TmAFP b-helix with the bsheets (TCT sequences) indicated by green arrows and the disulphide bonds in yellow.
Threonine side chains on the b-sheet surface are shown with oxygen atoms in red. b, Endon view of the b-helix with the N terminus proximal, showing the alignment of conserved
threonine, cysteine, serine and alanine side chains and internal water. We note that the
shorter dimension of the pseudo-rectangular cross-section in TmAFP is longer than the
disulphide linkage, but the ¯atness of the b-sheet is maintained by the opposite side
being pulled inwards. The ¯atness of the b-sheet is probably due to the shortness of the
b-strands, the disulphide bonds and the presence of favourable van der Waals
interactions between stacked threonine side chains. The N and C termini are shown. All
diagrams were generated using MOLSCRIPT25 unless otherwise stated.
stabilized by an extensive network of hydrogen bonds parallel to
the helix axis, linking peptide CO and NH groups of adjacent turns.
The extreme similarity of each helix turn enables inter-loop hydrogen bonds to form at many points around the helix, not just in the
b-strand region. Indeed, the root mean square deviation (r.m.s.d.)
between six of the seven helical turns (excluding the N-terminal
one) is only 0.48 6 0.02 AÊ, demonstrating the nearly identical
backbone structures. TmAFP is additionally stabilized or constrained by disulphide bonds, which run like rungs of a ladder
between opposite sides of the helix (Fig. 1b), making it one of the
most extensively disulphide-bonded proteins. As previously
predicted4, all 16 cysteines of recombinant TmAFP are paired to
form disulphide bridges, C3-C12, C9-C19, C16-C22, C28-C34,
C40-C46, C52-C58, C64-C70, and C76-C82, equivalent to those
seen in the native AFP from the bark beetle, Dendroides
canadensis5,6. Six of the eight disulphide bonds are spaced six
residues apart and are in near-perfect alignment with each other
(Fig. 1). In the N-terminal region the other two disulphides (C3C12, C9-C19) do not ®t this pattern, but remarkably this region
folds like the rest of the molecule to continue the repetitive b-helix
structure with 12 residues per loop (Fig. 1b).
The tight b-helical turns that form the building blocks of TmAFP
correspond to the repeated-in-tandem consensus sequence3,
TCTxSxxCxxAx. Its identi®cation as a structural unit with an
internal disulphide bond is consistent with the observation that
different isoforms of TmAFP contain insertions or deletions of
this 12-amino-acid repeat3,4. The TCT (or ACT) residues of the
repeat represent the b-strand region of the turn, in which the
inward-pointing cysteine is disulphide bonded to the other conserved cysteine in order to form a highly constrained seven-residue
closed circular loop within the turn (Fig. 2). The extraordinary
tightness of the 12-amino-acid turns is also facilitated by intraloop hydrogen bond connections between backbone CO and NH
groups. At all four corners of the pseudo-rectangular cross-section,
regular secondary turn structures are formed (g-, g-, g- and
b-turns).
Although a number of parallel b-helix structures have been
© 2000 Macmillan Magazines Ltd
NATURE | VOL 406 | 20 JULY 2000 | www.nature.com
letters to nature
Figure 2 Substructure of a 12-amino-repeat from TmAFP (white) forming one complete
loop of the b-helix. Conserved amino acids are identi®ed by their one-letter code and
variable residues by X. Numbers indicate their position in the 12-amino-acid repeat.
Backbone intra-loop hydrogen bonds (red dashed lines) denote g- and b-turns. A portion
of the preceding repeat is shown (orange) to illustrate the inter-loop hydrogen-bond
connections formed by the internal water and serine side chains. Nitrogen atoms are
shown in blue and oxygen atoms in red.
reported in bacteria and fungi, including the right-handed b-helices
of pectate lyase7, pectin lyase8, rhamnogalacturonase A9 and tailspike endorhamnosidase of phage P2210, and the left-handed
b-helix domain of N-acetylglucosamine acyltransferase11, none of
these structures is as compressed or regular as TmAFP. These
enzymes contain a larger number of residues per turn of b-helix
(typically from 22 to 27)12, and always have a hydrophobic core. In
contrast, with only 12 amino acids per turn, TmAFP is the smallest
b-helix and has a very narrow bore, which is constricted and further
bisected by disulphide bonds to form two channels, leaving no room
for a hydrophobic core. Thus, the few hydrophobic residues in
TmAFPÐV26, V35, F59 and Y71 (the site of iodination)Ðall have
their side chains projecting outwards to the solvent.
In TmAFP, there is room only for the relatively small side
chains of the conserved serine and alanine to project into the
core, on either side of the bisecting disulphide bridge. The serine
side chains are perpendicular to the disulphide bridge (Figs 1b and
2), with their hydroxyls arrayed in exactly the same orientation (x1 =
-64.0 6 1.08) at an average distance of 4.73 6 0.05 AÊ apart. These
serine hydroxyl groups tilt towards the N terminus and form interturn hydrogen bonds to two backbone NH groups on the Nterminal neighbouring turn. In the channel opposite serine, all
alanine side chains are perpendicular to the disulphide bond, with
their methyl groups aligned and separated by an average distance of
5.39 6 0.05 AÊ. Within the alanine-containing channel there are also
®ve bound waters regularly spaced between the six carboxy-terminal
loops. These bound waters play a similar stabilizing role to the OH
group of the internal serine residues, each forming hydrogen bonds
to three residues. With an average B-factor of 14.3 AÊ2 (as compared
to the average B of 16.6 AÊ2 for protein atoms and the average B
of 35.4 AÊ2 for all other waters), these internal water molecules are
very tightly bound and can be regarded as an intrinsic part of the
protein.
The b-helix structure of TmAFP is very different from any of the
deduced or predicted ®sh AFP structures13. But interestingly, it is
similar to the structure of another insect AFP determined by NMR2.
This equally hyperactive 9-kDa spruce budworm AFP (sbwAFP)
also forms a b-helix, probably as a result of convergent evolution.
The main structural feature that these two insect AFPs have in
common is the regular array of TXT sequences on one side of the
molecule. In sbwAFP, this has been identi®ed as the ice-binding site
by site-directed mutagenesis2. In TmAFP, the six short b-strands
(Fig. 1b) form a remarkably ¯at b-sheet that lacks the typical twist
NATURE | VOL 406 | 20 JULY 2000 | www.nature.com
Figure 3 Dimer of TmAFP and organization of external water. a, A TmAFP pair dimerized
through hydrogen bonding to two ranks of ordered waters. The ®rst TmAFP is shown in
blue; the second in pink. The oxygen atoms of the waters are shown as blue and magenta
spheres. b, A surface presentation (GRASP26) of TmAFP with the closest rank of water
molecules (red spheres) bound, illustrating their regularity and the ¯atness of the resulting
surface when one rank of the water molecules is in place.
seen in other b-sheet proteins. Our high-resolution TmAFP structure shows that the threonine residues are precisely positioned and
fully exposed to the solvent, with their side chains in near-perfect
alignment (x1 = -58.2 6 0.88). The average distance between
hydroxyls within the TCT motif is 7.44 6 0.05 AÊ. At right angles
to this dimension, the average distance between equivalent hydroxyls of TCT motifs in adjacent loops is 4.64 6 0.04 AÊ. The twodimensional array of threonine side chains makes a remarkably
good match to the repeated spacing between oxygen atoms in the ice
lattice on the primary prism plane (7.35 AÊ and 4.52 AÊ), and a
reasonable match to the basal plane (7.83 AÊ and 4.52 AÊ). This lattice
matching is highly suggestive of its ice-binding function.
Although TmAFP is monomeric in solution14, the protein crystallized as a dimer with twofold symmetry. The two monomers are
essentially identical with an r.m.s.d. of only 0.33 AÊ (including all
regular internal and external waters). The dimerization of TmAFP
occurs along the surface of the parallel b-sheets, with both monomers in the same (that is, parallel) orientation (Fig. 3a). The two
monomers are at least 8 AÊ apart and do not directly interact with
each other. Rather, contact between the two monomers is mediated
by two highly ordered ranks of water molecules that form an
extensive hydrogen-bonding network with the threonine residues
on one side of the TCT motifs (not shown). The remarkable
regularity of the water molecules (Fig. 3b) is re¯ected by their
near-identical positions in both monomers (0.46 AÊ r.m.s.d.). Each
water molecule forms two hydrogen bonds to the closer monomer
and one hydrogen bond to the more distant monomer. These
external water molecules are tightly bound and highly ordered,
with an average B factor of only 16.5 AÊ2.
Because of the positional regularity, the distance between two
adjacent waters is 4.64 6 0.20 AÊ, the same distance as between
threonine residues (4.64 6 0.23 AÊ). A good two-dimensional
match to the ice lattice, including all three ranks of oxygen atoms
(two from threonine and one from water), can be achieved with
,0.5 AÊ offset or deviation, implying that the ordered water molecules could act as part of an ice surface and directly participate in the
AFP±ice interaction (Fig. 4). Importantly, this match requires no
adjustment of any threonine side chain and methyl groups of
threonine, and side chains of adjacent residues do not present
steric interference (Fig. 4a). Hence the regular array formed by
three ranks of oxygen atoms can be seen as a small piece of onelayer-thick ice to be incorporated into a large ice lattice, an idea ®rst
proposed by Knight et al.15. Regardless of whether the bound water
© 2000 Macmillan Magazines Ltd
323
letters to nature
determine handedness21. To improve the map, non-crystallographic averaging and density
modi®cation were performed. Model building was carried out using the program O22, and
®nal re®nement was carried out using the program CNS23 with the 1.4-AÊ data collected at
the X8C beamline of Brookhaven National Laboratory. An individual isotropic Bfactor model with non-crystallographic symmetry restraint was used in the initial
re®nement. The R factor of the initial model with 154 water molecules was improved
from 36.0% to 23.54% (Rfree = 25.24%). A further re®nement was then carried out
using SHELX-9719. After the re®nement of anisotropic B-factors and occupancies of
iodine atoms were introduced, the R-factor dropped to 16.99% (Rfree = 20.73%). The
R-factor of the ®nal model, containing 164 amino acid residues of the dimer (Nterminal methionine and C-terminal glycine-histidine were invisible) and 229 water
molecules, is 16.05% (Rfree = 19.96%). The structure geometry is excellent with no
Ramachandran violations24. Calculations of r.m.s.d. included all main-chain atoms,
where appropriate.
Received 9 March; accepted 15 May 2000.
Figure 4 Lattice matching/occupation model for TmAFP binding to ice. a, End-on view of
the b-helix with the C terminus proximal, showing the occupation of ice oxygen atoms
(blue) on the prism plane of ice by three ranks of oxygen atoms from threonine and bound
water (red perimeter). We note the absence of steric clashes with the threonine methyl
groups. b, Side view of the b-helix with the C terminus to the right, showing the
occupation of ice oxygen atoms on the prism plane of ice by threonine from six loops. Ice
c-axis and a-axis directions are indicated by the arrows.
molecules are considered to be part of the protein or part of the ice,
their arrangement (along with the threonine residues) to match the
ice lattice represents the ®rst direct observation, to our knowledge,
of the structure of an AFP±ice interface.
Dimerization of TmAFP may have fortuitously stabilized the
bound water to give us this ®rst glimpse and approximation of
the elusive AFP±ice structure. Certainly, bound water matching the
ice lattice has not been seen before in the X-ray structures of the two
®sh AFPs, types I and III (refs 16, 17). In comparison to the single
a-helix structure of ®sh type I AFP, which contains one array of
regularly spaced threonine residues, TmAFP possesses multiple
ranks of regularly spaced threonine residues and bound water
molecules. If it is remarkable that lattice-matching regularity can
occur in one dimension, as in type I AFP, then it is even more
surprising for a protein to achieve the extreme regularity in two
dimensions that is seen in TmAFP. A highly constrained b-helix is
perhaps an ideal platform for the design of two-dimensional
regularity.
M
Methods
We have previously reported the preparation and crystallization of iodinated recombinant
TmAFP18. The structure determined is that of the Y71-iodinated TmAFP. X-ray data
including anomalous re¯ections were collected to 1.95-AÊ resolution using a rotating
anode X-ray generator with an imaging plate. TmAFP crystals belonged to a P65 space
group, with unit cell dimensions a = b = 73.1 and c = 53.1 AÊ. There are two molecules per
asymmetric unit and there is a twofold non-crystallographic symmetry. Using direct
methods implemented19 in SHELX-97, the positions of four iodine atoms were determined and the correct space group P65 was assigned. The iodine sites were re®ned, the
phases were subsequently obtained using the method of single anomalous scattering and a
solvent-¯attened map (51% solvent) was generated using the program SHARP20. In
addition, ®gures of merit generated from two opposite enantiomers were examined to
324
1. Tyshenko, M. G., Doucet, D., Davies, P. L. & Walker, V. K. The antifreeze potential of the spruce
budworm thermal hysteresis protein. Nature Biotechnol. 15, 887±890 (1997).
2. Graether, S. P. et al. b-Helix structure and ice-binding properties of a hyperactive antifreeze protein
from an insect. Nature 406, 325±328 (2000).
3. Graham, L. A., Liou, Y. -C., Walker, V. K. & Davies, P. L. Hyperactive antifreeze protein from beetles.
Nature 388, 727±728 (1997).
4. Liou, Y. -C., Thibault, P., Davies, P. L., Walker, V. K. & Graham, L. A. A complex family of highly
heterogeneous and internally repetitive hyperactive antifreeze proteins from the beetle Tenebrio
molitor. Biochemistry 38, 11415±11424 (1999).
5. Li, N., Chibber, B. A. K., Castellino, F. J. & Duman, J. G. Mapping of disul®de bridges in antifreeze
proteins from overwintering larvae of the beetle Dendroides canadensis. Biochemistry 37, 6343±6350
(1998).
6. Duman, J. G. et al. Molecular characterization and sequencing of antifreeze proteins from larvae of the
beetle Dendroides canadensis. J. Comp. Physiol. B 168, 225±232 (1998).
7. Yoder, M. D., Keen, N. T. & Jurnak, F. New domain motif: structure of pectate lyase C, a secreted plant
virulence factor. Science 260, 1503±1507 (1993).
8. Mayans, O. et al. Two crystal structures of pectin lyase A from Aspergillus reveal a pH driven
conformational change and striking divergence in the substrate-binding clefts of pectin and pectate
lyases. Structure 5, 677±689 (1997).
9. Petersen, T. N., Kauppinen, S. & Larsen, S. The crystal structure of rhamnogalacturonase A from
Aspergillus aculeatus: a right-handed parallel beta helix. Structure 5, 533±544 (1997).
10. Steinbacher, S. et al. Crystal structure of P22 tailspike protein: interdigitated subunits in a
thermostable trimer. Science 265, 383±386 (1994).
11. Raetz, C. R. & Roderick, S. L. A left-handed parallel beta helix in the structure of UDP-Nacetylglucosamine acyltransferase. Science 270, 997±1000 (1995).
12. Jenkins, J., Mayans, O. & Pickersgill, R. Structure and evolution of parallel beta-helix proteins. J.
Struct. Biol. 122, 236±246 (1998).
13. Davies, P. L. & Sykes, B. D. Antifreeze proteins. Curr. Opin. Struct. Biol. 7, 828±834 (1997).
14. Liou, Y.-C. et al. Folding and structural characterization of highly disul®de-bonded beetle antifreeze
protein produced in bacteria. Protein Expr. Purif. 19, 148±157 (2000).
15. Knight, C. A., Driggers, E. & DeVries, A. L. Adsorption to ice of ®sh antifreeze glycopeptides 7 and 8.
Biophys. J. 64, 252±259 (1993).
16. Sicheri, F. & Yang, D. S. C. Ice-binding structure and mechanism of an antifreeze protein from winter
¯ounder. Nature 375, 427±431 (1995).
17. Jia, Z., DeLuca, C. I., Chao, H. & Davies, P. L. Structural basis for the binding of a globular antifreeze
protein to ice. Nature 384, 285±288 (1996).
18. Liou, Y. -C, Davies, P. L. & Jia, Z. Crystallization and preliminary X-ray analysis of insect antifreeze
protein from the beetle, Tenebrio molitor. Acta Cryst. D 56, 354±356 (2000).
19. Sheldrick, G. M. & Schneider, T. R. in Methods in Enzymology Vol. 276 (eds Carter, C. W. & Sweet,
R. M.) 319±343 (Academic Press, New York, 1997).
20. de La Fortelle, E. & Bricogne, G. in Methods in Enzymology Vol. 276 (eds Carter, C. W. & Sweet, R. M.)
472±494 (Academic Press, New York, 1997).
21. Chen, L. Q. et al. Crystal structure of a bovine neurophysin II dipeptide complex at 2.8 AÊ determined
from the single-wavelength anomalous scattering signal of an incorporated iodine atom. Proc. Natl
Acad. Sci. USA 88, 4240±4244 (1991).
22. Jones, T. A., Zou, J. -Y., Cowan, S. W. & Kjeldgaard, M. Improved methods for building protein
models in electron density maps and the location of errors in these models. Acta Cryst. A 47, 110±119
(1991).
23. BruÈnger, A. T. et al. Crystallography & NMR system (CNS): A new software suite for macromolecular
structure determination. Acta Cryst. D 54, 905±921 (1998).
24. Laskowski, R. A., MacArthur, M. W., Moss, D. S. & Thornton, J. M. PROCHECK: a program to check
the stereochemical quality of protein structures. J. Appl. Crystallogr. 26, 283±291 (1993).
25. Kraulis, P. J. MOLSCRIPT: a program to produce both detailed and schematic plots of protein
structures. J. Appl. Crystallogr. 24, 946±950 (1991).
26. Nicholls, A., Sharp, K. & Honig, B. Protein folding and association: insights from the interfacial and
thermodynamic properties of hydrocarbons. Proteins 11, 281±296 (1991).
Acknowledgements
We would like to thank Q. Ye, M. Kuiper and S. Gauthier for excellent technical assistance,
and staff at the X8C beamline of Brookhaven National Laboratory for help with
synchrotron data collection. This work was supported by grants from MRC of Canada to
Z. J and P. L. D; Z. J. is an MRC Scholar. P.L.D. is a Killam Research Fellow.
Correspondence and requests for materials should be addressed to P.L.D.
(e-mail: [email protected]). The coordinates have been deposited in the Protein
Data Bank under accession code 1EZG.
© 2000 Macmillan Magazines Ltd
NATURE | VOL 406 | 20 JULY 2000 | www.nature.com