Hyperactive antifreeze protein from an Antarctic sea ice bacterium Colwellia sp. has a compound ice‐binding site without repetitive sequences

Abstract

Antifreeze proteins (AFPs) are structurally diverse macromolecules that bind to ice crystals and inhibit their growth to protect the organism from injuries caused by freezing. An AFP identified from the Antarctic bacterium Colwellia sp. strain SLW05 (ColAFP) is homologous to AFPs from a wide variety of psychrophilic microorganisms. To understand the antifreeze function of ColAFP, we have characterized its antifreeze activity and determined the crystal structure of this protein. The recombinant ColAFP exhibited thermal hysteresis activity of approximately 4°C at a concentration of 0.14 mM, and induced rapid growth of ice crystals in the hexagonal direction. Fluorescence-based ice plane affinity analysis showed that ColAFP binds to multiple planes of ice, including the basal plane. These observations show that ColAFP is a hyperactive AFP. The crystal structure of ColAFP determined at 1.6 A resolution revealed an irregular b-helical structure, similar to known homologs. Mutational and molecular docking studies showed that ColAFP binds to ice through a compound ice-binding site (IBS) located at a flat surface of the b-helix and the adjoining loop region. The IBS of ColAFP lacks the repetitive sequences that are characteristic of hyperactive AFPs. These results suggest that ColAFP exerts antifreeze activity through a compound IBS that differs from the characteristic IBSs shared by other hyperactive AFPs. This study demonstrates a novel method for protection from freezing by AFPs in psychrophilic microorganisms. DatabaseStructural data for ColAFP have been submitted to the Protein Data Bank (PDB) under accession number 3WP9.

Interaction of ice binding proteins with ice, water and ions

“…An a-helix lies alongside the b-helix axis. [59][60][61] Yet other IBP structures are found in insect RiAFP and sfAFP. The structure of RiAFP of the beetle Rhagium inquisitor is formed by two closely packed b-sheets [ Fig.…”

“…69 While for most IBPs the IBS is localized on one face of the protein that can bind to either one or several ice crystal plane(s), in some cases, a "compound" IBS is identified. 59,67 Type III AFP from notched fin eel pout and ocean pout have an IBS composed of two regions positioned at an angle of roughly 150 with respect to each other. One binds the primary prism plane of ice; the other a pyramidal plane [ Fig.…”

“…Both ice binding sites could be docked onto several ice crystal planes, including the basal plane. 59 The IBS of several AFPs contains residues arranged with remarkably high order (Fig. 3).…”

“…Despite this lack regularity in the IBS these proteins are able to bind both basal and prism planes. [59][60][61]65 Crystal structures of various AFPs reveal ordered waters associated with the protein IBS, 48,56,58,65 which are thought to play an important role in ice-binding (see Sec. VI E for more details).…”

Coarse grained simulation reveals antifreeze properties of hyperactive antifreeze protein from Antarctic bacterium Colwellia sp.

“…1 AFPs were found to be expressed in fishes, [2][3][4] insects, [5][6][7] plants 8,9 and bacteria [10][11][12] living in extreme cold environments. AFPs exhibit ice inhibition mechanism by interacting through the gaps of ice cascade and preventing the ice crystals from linking to each other.…”