A β-Helical Antifreeze Protein Isoform with Increased Activity

The insect spruce budworm (Choristoneura fumiferana)(Cf) produces a number of isoforms of its highly active antifreeze protein (CfAFP). Although most of the CfAFP isoforms are in the 9-kDa range, isoforms containing a 30- or 31-amino acid insertion have also been identified. Here we describe the functional and structural analysis of a selected long isoform, CfAFP-501. X-ray crystal structure determination reveals that the 31-amino acid insertion found in CfAFP-501 forms two additional loops within its highly regular β-helical structure. This effectively extends the area of the two-dimensional Thr array and ice-binding surface of the protein. The larger isoform has 3 times the thermal hysteresis activity of the 9-kDa CfAFP-337. As well, a deletion of the 31-amino acid insertion within CfAFP-501 to form CfAFP-501-Δ-2-loop, results in a protein with reduced activity similar to the shorter CfAFP isoforms. Thus, the enhanced antifreeze activity of CfAFP-501 is directly correlated to the length of its β-helical structure and hence the size of its ice-binding face. The insect spruce budworm (Choristoneura fumiferana)(Cf) produces a number of isoforms of its highly active antifreeze protein (CfAFP). Although most of the CfAFP isoforms are in the 9-kDa range, isoforms containing a 30- or 31-amino acid insertion have also been identified. Here we describe the functional and structural analysis of a selected long isoform, CfAFP-501. X-ray crystal structure determination reveals that the 31-amino acid insertion found in CfAFP-501 forms two additional loops within its highly regular β-helical structure. This effectively extends the area of the two-dimensional Thr array and ice-binding surface of the protein. The larger isoform has 3 times the thermal hysteresis activity of the 9-kDa CfAFP-337. As well, a deletion of the 31-amino acid insertion within CfAFP-501 to form CfAFP-501-Δ-2-loop, results in a protein with reduced activity similar to the shorter CfAFP isoforms. Thus, the enhanced antifreeze activity of CfAFP-501 is directly correlated to the length of its β-helical structure and hence the size of its ice-binding face. antifreeze protein(s) antifreeze glycoprotein(s) Choristoneura fumiferana Antifreeze proteins (AFPs)1 and antifreeze glycoproteins (AFGPs) are present in many organisms that must survive subzero temperatures. These proteins bind to seed ice crystals and inhibit their growth through an adsorption-inhibition mechanism (1Raymond J.A. DeVries A.L. Proc. Natl. Acad. Sci. U. S. A. 1977; 74: 2589-2593Crossref PubMed Scopus (681) Google Scholar). The structures of these proteins have proven to be remarkably varied (2Jia Z. Davies P.L. Trends Biochem. Sci. 2002; 27: 101-106Abstract Full Text Full Text PDF PubMed Scopus (330) Google Scholar), yet they perform the same freeze-protecting function. In all documented examples to date, an AFP-protected organism produces only one or two types of antifreeze, but they are typically comprised of multiple, independently active isoforms. The AFGP-producing fishes of the Arctic and Antarctic regions make a range of antifreeze glycoproteins (Mr ∼3,000–26,000) that differ mainly in the number of their glycotripeptide units (3DeVries A.L. Vandenheede J. Feeney R.E. J. Biol. Chem. 1971; 246: 305-308Abstract Full Text PDF PubMed Google Scholar, 4Feeney R.E. Yeh Y. Adv. Protein Chem. 1978; 32: 191-282Crossref PubMed Scopus (108) Google Scholar). The winter flounder (Pleuronectes americanus) produces two sets of tissue-specific isoforms. The skin and liver types of α-helical AFPs vary in both sequence and length (5DeVries A.L. Lin Y. Biochim. Biophys. Acta. 1977; 495: 388-392Crossref PubMed Scopus (139) Google Scholar, 6Fourney R.M. Fletcher G.L. Hew C.L. Gen. Comp. Endocrinol. 1984; 54: 392-401Crossref PubMed Scopus (9) Google Scholar, 7Davies P.L. Gene. 1992; 112: 163-170Crossref PubMed Scopus (13) Google Scholar, 8Gong Z. Ewart K.V., Hu, Z. Fletcher G.L. Hew C.L. J. Biol. Chem. 1996; 271: 4106-4112Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar), and sequence variation within each tissue-specific isoform is less than that between AFPs from different tissues. As well the ocean pout (Macrozoarces americanus) produces many isoforms of globular type III AFP (9Li X.M. Trinh K.Y. Hew C.L. Buettner B. Baenziger J. Davies P.L. J. Biol. Chem. 1985; 260: 12904-12909Abstract Full Text PDF PubMed Google Scholar, 10Hew C.L. Wang N.C. Joshi S. Fletcher G.L. Scott G.K. Hayes P.H. Buettner B. Davies P.L. J. Biol. Chem. 1988; 263: 12049-12055Abstract Full Text PDF PubMed Google Scholar), which are nearly the same size (7 kDa) but may be as much as 50% different in amino acid sequence. AFPs have been characterized in three insects, and each species contains multiple AFP isoforms. Thus far, eight Tenebrio molitor beetle AFP cDNAs have been identified coding for either 84, 96, or 120 amino acids (11Liou Y.C. Thibault P. Walker V.K. Davies P.L. Graham L.A. Biochemistry. 1999; 38: 11415-11424Crossref PubMed Scopus (98) Google Scholar), whereas 13 isoforms of the homologous pyrochroid beetle (Dendroides canadensis) AFP have been cloned (12Andorfer C.A. Duman J.G. J. Ins. Phys. 2000; 46: 365-372Crossref PubMed Scopus (68) Google Scholar), again varying in size and sequence. Similarly, the insect spruce budworm (Choristoneura fumiferana (Cf)) contains isoforms of its potent antifreeze protein (13Doucet D. Tyshenko M.G. Kuiper M.J. Graether S.P. Sykes B.D. Daugulis A.J. Davies P.L. Walker V.K. Eur. J. Biochem. 2000; 267: 6082-6088Crossref PubMed Scopus (60) Google Scholar). The isolation and sequencing of cDNA isoforms of C. fumiferana antifreeze proteins (CfAFPs) revealed the presence of several 9-kDa isoforms along with three isoforms containing an ∼30 amino acid insertion resulting in a 12-kDa protein (13Doucet D. Tyshenko M.G. Kuiper M.J. Graether S.P. Sykes B.D. Daugulis A.J. Davies P.L. Walker V.K. Eur. J. Biochem. 2000; 267: 6082-6088Crossref PubMed Scopus (60) Google Scholar). Among various isoforms of CfAFP, one 9-kDa isoform has been characterized in great detail (namely 337 or CfAFP-337) and has been the focus of recombinant expression and structure determination by NMR and x-ray crystallography (14Gauthier S.Y. Kay C.M. Sykes B.D. Walker V.K. Davies P.L. Eur. J. Biochem. 1998; 258: 445-453Crossref PubMed Scopus (53) Google Scholar, 15Leinala E.K. Davies P.L. Jia Z. Structure. 2002; 10: 619-627Abstract Full Text Full Text PDF PubMed Scopus (103) Google Scholar, 16Graether S.P. Kuiper M.J. Gagne S.M. Walker V.K. Jia Z. Sykes B.D. Davies P.L. Nature. 2000; 406: 325-328Crossref PubMed Scopus (379) Google Scholar). These investigations showed that CfAFP is a left-handed β-helix with a two-dimensional Thr array on one face of the protein that forms the ice-binding site. The antifreeze activity of another 9-kDa isoform (339) has also been assayed and found to be equivalent to that of isoform 337 (13Doucet D. Tyshenko M.G. Kuiper M.J. Graether S.P. Sykes B.D. Daugulis A.J. Davies P.L. Walker V.K. Eur. J. Biochem. 2000; 267: 6082-6088Crossref PubMed Scopus (60) Google Scholar). The long isoform 501 of CfAFP is only 66% identical to the characterized 337 isoform. Using CfAFP-501 as a representative of the longer isoforms, we were interested in determining whether the 31-amino acid insertion correlates with higher activity and, if so, what is the molecular and structural basis for the enhancement. To this end, we have expressed and measured the activity of the protein and determined its x-ray crystallographic structure. In addition, deletion of the 31amino acid insertion (representing two turns of the β-helix) in a so-called “loopectomy” procedure has been carried out to further probe the activity-structure relationship. The cDNA of CfAFP-501 was cloned into the pET20(b) vector (Novagen) for recombinant expression inEscherichia coli. AFP expressed in BL21(DE3) cells was present in inclusion bodies requiring refolding performed in a manner similar to that of CfAFP-337 (14Gauthier S.Y. Kay C.M. Sykes B.D. Walker V.K. Davies P.L. Eur. J. Biochem. 1998; 258: 445-453Crossref PubMed Scopus (53) Google Scholar). Refolded protein was purified using fast protein liquid chromatography and high pressure liquid chromatography column chromatography as reported for CfAFP-337 (14Gauthier S.Y. Kay C.M. Sykes B.D. Walker V.K. Davies P.L. Eur. J. Biochem. 1998; 258: 445-453Crossref PubMed Scopus (53) Google Scholar) with the exception that the majority of the CfAFP-501 protein eluted in a single, well folded peak from both the fast protein liquid chromatography and high pressure liquid chromatography columns without the splitting into incompletely folded and well folded peaks as seen with CfAFP-337. To excise the 31-amino acid insertion found in the long isoform CfAFP-501 and to produce CfAFP-501-Δ-2 loop, the pET20(b) vector containing the cDNA for CfAFP-501 was cleaved with the restriction enzyme ClaI whose natural cleavage sites were found on either side of the cDNA encoding the 31-amino acid segment. Religation of the remaining vector backbone and cDNA produced an expression vector for the CfAFP-501-Δ-2 loop. Protein was recombinantly produced using the cDNA containing pET20(b) vector in BL21(DE3) E. coli cells. Again, the protein was present in inclusion bodies, and the refolding, expression, and purification were performed as in the case of CfAFP-337 (14Gauthier S.Y. Kay C.M. Sykes B.D. Walker V.K. Davies P.L. Eur. J. Biochem. 1998; 258: 445-453Crossref PubMed Scopus (53) Google Scholar) and CfAFP-501 described above. Crystals of CfAFP-501 were grown at room temperature using the hanging drop vapor diffusion method. The protein was crystallized using a solution of 18% polyethylene glycol-4000 and 20% isopropanol in sodium citrate buffer (100 mm, pH 4.8–5.6) producing thin plate-like crystals. These crystallization conditions are different in both temperature and solution components used for CfAFP-337. Due to structural microheterogeneity found in recombinantly produced CfAFP-337, higher temperatures were required to reduce this heterogeneity and produce crystals of CfAFP-337. 2Leinala, E. K., Davies, P. L., and Jia, Z. (2002) Acta Crystallogr. Biol. Crystallogr. Sect. D58,1081–1083 This conformational microheterogeneity is not seen in the preparation of CfAFP-501. Due to poor diffraction and high mosaicity of the thin crystals, diffraction quality of most crystals was unsatisfactory. After screening many CfAFP-501 crystals (between 50–75) under cryogenic conditions, a data set was eventually collected to a 2.45-Å resolution using a copper Rigaku rotating anode x-ray generator and a Mar Research imaging plate. The diffraction images were processed using the HKL program suite (18Otwinowski Z. Minor W. Methods Enzymol. 1997; 276: 307-326Crossref PubMed Scopus (38617) Google Scholar), and data statistics are summarized in Table I.Table ICfAFP-501 Structure Determination StatisticsData collection statistics Wavelength λ (Å)1.54 Resolution (Å)2.45 Rsym (%)7.8 (35.6) 〈I/ςΣ1-aSignal to noise ration of intensities.11.5 (2.8) Completeness (%)85.7 (71.3) Total reflections567 512 Unique reflections12 991Refinement statistics Resolution (Å)50–2.45 Reflections10 750 Space groupP2(1) Unit cell (Å)a = 43.9°; b = 57.4°; c = 70.13°; β = 92.1° Protein atoms3424 Solvent molecules134 Solvent content (%)33 R-value (%)22.1 Free R-value1-bTen percent of reflections were randomly chosen for calculation of free R-value. (%)29.1Average B-factor (Å2) Overall27.4 Solvent23.5Deviation from ideal geometry1-cRoot mean squared deviation from ideal geometry (29). Bond lengths (Å)0.006 Bond angles (°)1.3Ramachandran1-dPercentage of residues in regions of the Ramachandran plot (17). Most favoured regions (%)85 Additional allowed regions (%)15 Generously allowed regions (%)0.0 Disallowed regions (%)0.0Values in parentheses are for highest resolution shells. The coordinates of CfAFP-501 structure have been deposited in Protein Data Bank (PDB code 1M8N).1-a Signal to noise ration of intensities.1-b Ten percent of reflections were randomly chosen for calculation of free R-value.1-c Root mean squared deviation from ideal geometry (29Engh R.A. Huber R. Acta Crystallogr. Sect. A. 1991; 47: 392-400Crossref Scopus (2548) Google Scholar).1-d Percentage of residues in regions of the Ramachandran plot (17Laskowski R.A. Macarthur M.W. Moss D.S. Thornton J.M. J. Appl. Crystallogr. 1993; 26: 283-291Crossref Google Scholar). Open table in a new tab Values in parentheses are for highest resolution shells. The coordinates of CfAFP-501 structure have been deposited in Protein Data Bank (PDB code 1M8N). The positions of four molecules in the asymmetric unit of the CfAFP-501 crystals were determined by molecular replacement using the program EPMR (version 3.1) (19Kissinger C.R. Gehlhaar D.K. Smith B.A. Bouzida D. Acta Crystallogr. Sect. D Biol. Crystallogr. 2001; 57: 1474-1479Crossref PubMed Scopus (72) Google Scholar). A theoretical search model was used for molecular replacement and was composed of the crystal structure of CfAFP-337 (15Leinala E.K. Davies P.L. Jia Z. Structure. 2002; 10: 619-627Abstract Full Text Full Text PDF PubMed Scopus (103) Google Scholar) (PDB code 1L0S) with two extra β-helical loops manually modeled into the structure using Sybyl (Tripos, St. Louis, MO) and Turbo-Frodo (20Jones T.A. J. Appl. Crystallogr. 1978; 11: 268-272Crossref Google Scholar). These two loops were residues 29–59 of CfAFP-337 inserted between residues 28 and 29. Amino acids in the model were mutated in silico to those of CfAFP-501 without additional energy minimization. Structure refinement was performed using the CNS program package (21Brunger A.T. Adams P.D. Clore G.M. DeLano W.L. Gros P. Grosse-Kunstleve R.W. Jiang J.S. Kuszewski J. Nilges M. Pannu N.S. Read R.J. Rice L.M. Simonson T. Warren G.L. Acta Crystallogr. Sect. D Biol. Crystallogr. 1998; 54: 905-921Crossref PubMed Scopus (16979) Google Scholar) with gradual reduction of non-crystallographic symmetry restraints. Ribbon diagrams in figures were generated using MolScript (22Kraulis P.J. J. Appl. Crystallogr. 1991; 24: 946-950Crossref Google Scholar). Measurement of AFP activity was performed according to a previously established routine process (23Chakrabartty A. Hew C.L. Eur. J. Biochem. 1991; 202: 1057-1063Crossref PubMed Scopus (143) Google Scholar) in the presence of 0.1 m ammonium bicarbonate employing a nanoliter osmometer (Clifton Technical Physics). CfAFP-501 retains the extremely regular left-handed β-helical structure seen with CfAFP-337 (15Leinala E.K. Davies P.L. Jia Z. Structure. 2002; 10: 619-627Abstract Full Text Full Text PDF PubMed Scopus (103) Google Scholar) (Table I). The β-helix is formed by a series of 15-amino acid turns, which result in an elongated protein with a triangular cross-section (Fig.1). The 31-amino acid insertion found in the CfAFP-501 sequence forms two extra loops (16 + 15 amino acids) within the central region of the β-helix (Fig. 1), extending the length of the β-helical structure from 31 Å (in CfAFP-337) to 39.6 Å. Although each turn of the CfAFP β-helix is usually 15 amino acids in length, one of the additional loops in CfAFP-501 contains 16 residues. An extra Ser within this repeat unit is accommodated within a corner of the triangular β-helix, not within the putative ice-binding site, and is directed away from the core structure of the protein. Interior and exterior amino acid positions form stacks of similar residues that contribute to the rigidity of the β-helical structure. One side of the exterior of the protein features two stacks of Thr residues (Fig. 2A), while three interior stacks are composed of hydrophobic residues (Fig.2B). Interior corner residues are mainly Ser, Thr, and Cys residues producing a hydrogen-bonding network to form the sharp 60 ° interior turns or corners required for the formation of the triangular cross-section of the protein (Fig. 2C). By analogy with CfAFP-337, the side of CfAFP-501 containing the repetitive Thr-Xaa-Thr array forms the ice-binding face of the protein. On this face of CfAFP-501, Val residues replace Thr-5 and Thr-52, whereas Thr-37 is substituted with an Ile. These Val and Ile residues have side-chain χ1 torsion angles of 177 ° and −60 °, respectively, whereas the Thr side-chains are at a χ1 torsion angle of −60 °. In terms of the definition of χ1 torsion angles for Val, Ile, and Thr residues these values represent geometrically equivalent rotamers. The positioning of the side-chains places all the residues on this face of the protein in the same orientation arrangement enabling the continued formation of the regularly spaced ice-binding platform (Fig.3). As in CfAFP-337 (15Leinala E.K. Davies P.L. Jia Z. Structure. 2002; 10: 619-627Abstract Full Text Full Text PDF PubMed Scopus (103) Google Scholar), this face forms an ice-binding site with extensive surface complementarity to ice, and the addition of two extra loops within CfAFP-501 increases the size of the interfacial surface between the AFP and ice by ∼34%. A theoretical model of CfAFP-501 was used in the molecular replacement stage of CfAFP-501 x-ray structure determination. Because CfAFPs have such a repetitive protein backbone this procedure is a very rational approach to structure determination. Although there has been some success in using theoretical models for molecular replacement, in most cases the probing models have been built from the combination of a number of core structures from homologous proteins, and these models were usually not larger than the starting templates. However, in this case a larger model had to be constructed instead of employing the usual approach of trimming the original structure. The theoretical model was based on the structure of CfAFP-337 (15Leinala E.K. Davies P.L. Jia Z. Structure. 2002; 10: 619-627Abstract Full Text Full Text PDF PubMed Scopus (103) Google Scholar) with residues 29–59 repeated and incorporated between residues 28 and 29. The amino acid residues were changed to that of CfAFP-501 with no additional energy minimization. The actual resulting root mean square deviation value between the theoretical search model and the final structure of CfAFP-501 is 1.53 Å for all atoms and 0.63 Å for the backbone atoms, respectively. The latter value is also representative of the overall backbone structural similarity between CfAFP-501 and 337, even though the amino acid identity between is only To the on antifreeze activity of the length of the β-helical structure and the overall area of the putative ice-binding thermal hysteresis were This the of the AFP to the of a solution by the growth of a seed ice was assayed a range of from to CfAFP-501 was active than CfAFP-337 all (Fig. with thermal hysteresis values to at a of mm, CfAFP-337 has a thermal hysteresis value of whereas CfAFP-501 has an activity of To whether the antifreeze activity of CfAFP-501 was to sequence between the two isoforms or to the length of CfAFP-501, a shorter CfAFP-501 was recombinantly This the deletion of a 31-amino acid from CfAFP-501 at the cDNA to form the protein CfAFP-501-Δ-2 in which two loops of the β-helix were from the protein. Antifreeze activity were performed on the shorter CfAFP-501-Δ-2 resulting in thermal hysteresis values that are similar to those of the 9-kDa 337 isoform (Fig. This result that than its sequence the size of CfAFP-501 is for its The results of this that the size of the ice-binding face of CfAFP is an of its antifreeze These those a longer isoform of the α-helical type containing four ice-binding as with the usual was found to be a potent antifreeze protein Kay C.M. S.Y. Davies P.L. Protein Sci. 1996; PubMed Scopus Google Scholar). The protein had a of at whereas the protein had a thermal hysteresis activity of at the same Although in the case of type AFP there has been no structure determined for the longer isoform, the regular α-helical structure in shorter isoforms D.S. Nature. PubMed Scopus Google Scholar) not be if the longer isoform the same repetitive α-helical By the same of two ice-binding from the isoform a 15-amino acid that had all its thermal hysteresis activity but the to the ice crystal Sykes B.D. Kay C.M. Davies P.L. J. Biol. Chem. 1998; Full Text Full Text PDF PubMed Scopus Google Scholar). An additional between AFP length and activity has also been described for the of the thermal hysteresis activity of various molecular isoforms of determined that molecular not amino acid was for the reduced activity of S.M. DeVries A.L. Biochim. Biophys. Acta. PubMed Scopus Google Scholar). the of these C.A. Feeney R.E. S. Yeh Y. J. Protein PubMed Scopus Google Scholar) the molecular isoforms are length with that the higher activity of CfAFP-501, as with the shorter CfAFP-337, is to the length of the β-helical platform and the ice-binding also that as long as the β-helical is a of sequence variation in the Thr-Xaa-Thr be A that from this is what are the to the length of the AFP β-helical Although be that there is a that be as described is to that such a with some structural The overall of a or β-helix has to be by the which has no additional with of the overall structure as usually seen in many As the length of the the the of ice much on the of AFP have on the length be a or between the ice-binding area and structural To directly out the between antifreeze activity and AFP length surface a series of AFPs of varying must be This be with the addition or deletion of loops within CfAFP or be directly using the beetle which contains three different lengths of its natural isoforms. for in the of the CfAFP-501.

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