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, 391404 (1998). 26. Bretscher, M. S. Translocation in protein synthesis: A hybrid structure model. Nature 218, 675677 (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, 197207 (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, 142148 (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, 2128521291 (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, 190199 (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

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.

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 resolution crystal (TCTxSxxCxxAx). Here we report its 1.4-A 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 in length, and 6.5 14 A in the slightly attened cylinder, 32 A 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

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 , demonstrating the nearly identical one) is only 0.48 6 0.02 A 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 identication 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
NATURE | VOL 406 | 20 JULY 2000 | www.nature.com

2000 Macmillan Magazines Ltd

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 identied 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.

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.

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 TmAFPV26, V35, F59 and Y71 (the site of iodination)all 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 = apart. These -64.0 6 1.08) at an average distance of 4.73 6 0.05 A 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 . Within the alanine-containing channel there are also 5.39 6 0.05 A 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 2 (as compared to three residues. With an average B-factor of 14.3 A 2 to the average B of 16.6 A for protein atoms and the average B 2 for all other waters), these internal water molecules are of 35.4 A 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 identied 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

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 . At right angles hydroxyls within the TCT motif is 7.44 6 0.05 A to this dimension, the average distance between equivalent hydro . The twoxyls of TCT motifs in adjacent loops is 4.64 6 0.04 A dimensional array of threonine side chains makes a remarkably good match to the repeated spacing between oxygen atoms in the ice and 4.52 A ), and a lattice on the primary prism plane (7.35 A and 4.52 A ). This lattice reasonable match to the basal plane (7.83 A 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 (including all essentially identical with an r.m.s.d. of only 0.33 A 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 apart and do not directly interact with monomers are at least 8 A 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 reected by their r.m.s.d.). Each near-identical positions in both monomers (0.46 A 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, 2. with an average B factor of only 16.5 A Because of the positional regularity, the distance between two , the same distance as between adjacent waters is 4.64 6 0.20 A ). A good two-dimensional threonine residues (4.64 6 0.23 A match to the ice lattice, including all three ranks of oxygen atoms (two from threonine and one from water), can be achieved with offset or deviation, implying that the ordered water mole,0.5 A cules could act as part of an ice surface and directly participate in the AFPice 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
323

2000 Macmillan Magazines Ltd

letters to nature
determine handedness21. To improve the map, non-crystallographic averaging and density modication were performed. Model building was carried out using the program O22, and data collected at nal renement was carried out using the program CNS23 with the 1.4-A the X8C beamline of Brookhaven National Laboratory. An individual isotropic Bfactor model with non-crystallographic symmetry restraint was used in the initial renement. The R factor of the initial model with 154 water molecules was improved from 36.0% to 23.54% (Rfree = 25.24%). A further renement was then carried out using SHELX-9719. After the renement 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. 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, 887890 (1997). 2. Graether, S. P. et al. b-Helix structure and ice-binding properties of a hyperactive antifreeze protein from an insect. Nature 406, 325328 (2000). 3. Graham, L. A., Liou, Y. -C., Walker, V. K. & Davies, P. L. Hyperactive antifreeze protein from beetles. Nature 388, 727728 (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, 1141511424 (1999). 5. Li, N., Chibber, B. A. K., Castellino, F. J. & Duman, J. G. Mapping of disulde bridges in antifreeze proteins from overwintering larvae of the beetle Dendroides canadensis. Biochemistry 37, 63436350 (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, 225232 (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, 15031507 (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, 677689 (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, 533544 (1997). 10. Steinbacher, S. et al. Crystal structure of P22 tailspike protein: interdigitated subunits in a thermostable trimer. Science 265, 383386 (1994). 11. Raetz, C. R. & Roderick, S. L. A left-handed parallel beta helix in the structure of UDP-Nacetylglucosamine acyltransferase. Science 270, 9971000 (1995). 12. Jenkins, J., Mayans, O. & Pickersgill, R. Structure and evolution of parallel beta-helix proteins. J. Struct. Biol. 122, 236246 (1998). 13. Davies, P. L. & Sykes, B. D. Antifreeze proteins. Curr. Opin. Struct. Biol. 7, 828834 (1997). 14. Liou, Y.-C. et al. Folding and structural characterization of highly disulde-bonded beetle antifreeze protein produced in bacteria. Protein Expr. Purif. 19, 148157 (2000). 15. Knight, C. A., Driggers, E. & DeVries, A. L. Adsorption to ice of sh antifreeze glycopeptides 7 and 8. Biophys. J. 64, 252259 (1993). 16. Sicheri, F. & Yang, D. S. C. Ice-binding structure and mechanism of an antifreeze protein from winter ounder. Nature 375, 427431 (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, 285288 (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, 354356 (2000). 19. Sheldrick, G. M. & Schneider, T. R. in Methods in Enzymology Vol. 276 (eds Carter, C. W. & Sweet, R. M.) 319343 (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.) 472494 (Academic Press, New York, 1997). determined 21. Chen, L. Q. et al. Crystal structure of a bovine neurophysin II dipeptide complex at 2.8 A from the single-wavelength anomalous scattering signal of an incorporated iodine atom. Proc. Natl Acad. Sci. USA 88, 42404244 (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, 110119 (1991). nger, A. T. et al. Crystallography & NMR system (CNS): A new software suite for macromolecular 23. Bru structure determination. Acta Cryst. D 54, 905921 (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, 283291 (1993). 25. Kraulis, P. J. MOLSCRIPT: a program to produce both detailed and schematic plots of protein structures. J. Appl. Crystallogr. 24, 946950 (1991). 26. Nicholls, A., Sharp, K. & Honig, B. Protein folding and association: insights from the interfacial and thermodynamic properties of hydrocarbons. Proteins 11, 281296 (1991).

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 AFPice interface. Dimerization of TmAFP may have fortuitously stabilized the bound water to give us this rst glimpse and approximation of the elusive AFPice 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
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 resolution using a rotating including anomalous reections were collected to 1.95-A anode X-ray generator with an imaging plate. TmAFP crystals belonged to a P65 space . There are two molecules per group, with unit cell dimensions a = b = 73.1 and c = 53.1 A 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 rened, 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

Methods

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: daviesp@post.queensu.ca). The coordinates have been deposited in the Protein Data Bank under accession code 1EZG.

324

2000 Macmillan Magazines Ltd

NATURE | VOL 406 | 20 JULY 2000 | www.nature.com