Structural basis for the superior activity of the large isoform of snow flea antifreeze protein.
The snow flea (Hypogastrum harveyi) is protected from freezing at sub-zero temperatures by a glycine-rich antifreeze protein (AFP) that binds to seed ice crystals and prevents them from growing larger. This AFP is hyperactive and comprises two isoforms [Graham, L. A., and Davies, P. L. (2005) Science 310, 461]. The larger isoform (15.7 kDa) exhibits several-fold higher activity than the smaller isoform (6.5 kDa), although it is considerably less abundant. To establish the molecular basis for this difference in activity, we determined the sequence of the large isoform. The primary sequences of these two isoforms are surprisingly divergent. However, both contain tripeptide repeats and turn motifs that enabled us to build a three-dimensional model of the large isoform based upon the six-polyproline helix structure of the small isoform. Our model contains 13 polyproline type II helices connected by proline-containing loops stacked into two flat sheets oriented antiparallel to one another. The structure is strictly amphipathic, with a hydrophilic surface on one side and a hydrophobic, putative ice-binding surface on the other. The putative ice-binding site is approximately twice as large in area as that of the small isoform, providing an explanation for the difference in activity that is consistent with other examples noted. By tagging the recombinant AFP with green fluorescent protein, we observed its binding to multiple planes of ice, especially the basal plane. This finding supports the correlation between AFP hyperactivity and basal plane binding first observed with spruce budworm AFP.
Mok
Yee-Foong
YF
Department of Biochemistry and Protein Function Discovery Group, Queen's University, Kingston, Ontario K7L 3N6, Canada.
Lin
Feng-Hsu
FH
Graham
Laurie A
LA
Celik
Yeliz
Y
Braslavsky
Ido
I
Davies
Peter L
PL
D000595:Amino Acid Sequence; D000818:Animals; D021301:Antifreeze Proteins / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D001181:Arthropods / Q000737:chemistry; D005998:Glycine; D057927:Hydrophobic and Hydrophilic Interactions; D008958:Models, Molecular; D008969:Molecular Sequence Data; D008970:Molecular Weight; D011392:Proline; D020033:Protein Isoforms / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D017433:Protein Structure, Secondary
D021301:Antifreeze Proteins; D020033:Protein Isoforms; D011392:Proline; D005998:Glycine