Antifreeze Protein from Freeze-Tolerant Grass Has a Beta-Roll Fold with an Irregularly Structured Ice-Binding Site

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Abstract

The grass Lolium perenne produces an ice-binding protein (LpIBP) that helps this perennial tolerate freezing by inhibiting the recrystallization of ice. Ice-binding proteins (IBPs) are also produced by freeze-avoiding organisms to halt the growth of ice and are better known as antifreeze proteins (AFPs). To examine the structural basis for the different roles of these two IBP types, we have solved the first crystal structure of a plant IBP. The 118-residue LpIBP folds as a novel left-handed beta-roll with eight 14- or 15-residue coils and is stabilized by a small hydrophobic core and two internal Asn ladders. The ice-binding site (IBS) is formed by a flat beta-sheet on one surface of the beta-roll. We show that LpIBP binds to both the basal and primary-prism planes of ice, which is the hallmark of hyperactive AFPs. However, the antifreeze activity of LpIBP is less than 10% of that measured for those hyperactive AFPs with convergently evolved beta-solenoid structures. Whereas these hyperactive AFPs have two rows of aligned Thr residues on their IBS, the equivalent arrays in LpIBP are populated by a mixture of Thr, Ser and Val with several side-chain conformations. Substitution of Ser or Val for Thr on the IBS of a hyperactive AFP reduced its antifreeze activity. LpIBP may have evolved an IBS that has low antifreeze activity to avoid damage from rapid ice growth that occurs when temperatures exceed the capacity of AFPs to block ice growth while retaining the ability to inhibit ice recrystallization.

Graphical Abstract

Highlights

► Some ice-binding (antifreeze) proteins (IBPs) help organisms tolerate freezing. ► The first structure of a plant IBP has been solved by X-ray crystallography. ► IBP from grass has a novel beta-roll fold with eight similar coils. ► The IBP's IBS is irregular compared to most AFPs. ► An irregular IBS may favor inhibition of ice recrystallization.

Introduction

Antifreeze proteins (AFPs) are found in diverse organisms ranging from vertebrates to bacteria that are exposed to sub-freezing environments at some point in their life histories.1, 2, 3, 4 Two frequently measured activities of AFPs are depression of the freezing point relative to the melting point, referred to as thermal hysteresis (TH),5 and ice recrystallization inhibition (IRI).6 TH activity halts ice crystal growth, allowing freeze-intolerant organisms (such as fish and some insects) to supercool and avoid freezing. IRI activity is more relevant in freeze-tolerant organisms, which are unable to avoid freezing and therefore must minimize the damage caused by recrystallization of ice in frozen tissue.7 For example, some overwintering plants such as carrots and grasses produce proteins that show weak TH activity but have IRI activity matching or exceeding that of fish and insect AFPs.8, 9, 10 The structural basis for the differences in these activities is one of the issues addressed in this work. Because some proteins with TH activity function as IRI proteins rather than antifreezes, we introduce the more inclusive term “ice-binding protein” (IBP) to refer to proteins that serve in either capacity.11

IBPs irreversibly adsorb to the surface of ice crystals and inhibit their growth by the Gibbs–Thomson effect,12 in which the addition of water molecules to the ice lattice is limited to the space between bound IBPs, resulting in surface curvature that makes the further addition of water thermodynamically unfavorable.13 Recent work suggests that IBPs adsorb to ice through an ordered ice-like array of waters assembled by the protein's ice-binding site (IBS), which merges with and freezes to the ice surface.14, 15, 16 The spacing of these so-called “clathrate” waters, anchored to the IBP by hydrogen bonds, matches one or more planes of ice. In this anchored-clathrate mechanism, the IBP essentially preforms part of its ligand before irreversibly adhering to it.

AFPs were recognized in certain insects over 30 years ago.17, 18, 19 More recently, characterization of purified insect AFPs revealed that their specific TH activities exceed those of fish AFPs by an order of magnitude;20, 21 thus, they were termed “hyperactive.” The heightened TH of hyperactive versus moderately active AFPs has been attributed to their unique affinity for the basal plane of ice.22 Moderately active AFPs bind only to prism and/or pyramidal planes, leaving the basal planes unprotected and vulnerable to growth.23, 24, 25, 26, 27 Although the addition of water to the basal planes of ice is less favorable than to a prism surface, this readily occurs under supercooled conditions when the TH gap of moderately active AFPs is exceeded, resulting in rapid crystal growth along the c-axis. Adsorption of AFPs to the basal plane provides substantially better coverage of the ice crystal and therefore provides greater TH activity.

The perennial ryegrass Lolium perenne is a freeze-tolerant forage grass planted worldwide28 that thrives in cold environments.29, 30 An IBP isolated from L. perenne (LpIBP) was reported to have high IRI but low TH activity.8 LpIBP TH activity is an order of magnitude lower than that of hyperactive AFPs and slightly less than that of moderately active fish type I, II and III AFPs.31 However, it has superior IRI activity.8, 10 Subsequently, several isoforms and homologues of this IBP have been found in various grasses in the sub-family Pooideae.32 The protein originally isolated constitutes the highly repetitive C-terminal “IRI” domain of a longer protein. On the basis of the DNA sequence, the complete protein includes an N-terminal secretion signal sequence and a leucine-rich repeat domain, but the ice-binding activity is limited to the IRI domain, which may represent the fully processed form of the protein. LpIBP is secreted from plant cells and functions in the apoplast, where it acts to minimize frost damage to the plant tissue, largely through its IRI activity.28

The sequence of the highly repetitive C-terminal domain of LpIBP suggested that its structure might resemble other beta-solenoid proteins, in particular the hyperactive AFPs discovered in the insects Tenebrio molitor (TmAFP) and Choristoneura fumiferana (CfAFP).33, 34 This was supported by a computer model of this protein as a right-handed beta-roll with two flat beta-sheets on opposite sides of the solenoid termed “a” and “b” faces.35 These flat surfaces resembled the IBSs of the insect AFPs, and it was originally proposed that both sheets might simultaneously bind ice on opposite sides of LpIBP. However, it was subsequently determined experimentally that only one of the two flat surfaces is responsible for TH activity.31 The introduction of single steric point mutations on the a face of LpIBP reduced the TH and IRI activities of the protein by up to 90%, whereas mutation of the b face had only a small effect.

Here, we report the X-ray crystal structure of LpIBP, which is the first such description of a plant IBP. Its structure resembles that of some of the hyperactive AFPs but with a less regular IBS, which we argue is the basis for its low TH activity. Like the hyperactive AFPs, LpIBP binds the basal plane of ice. Together, these analyses suggest that LpIBP has developed the ability to control ice growth at high sub-zero temperatures, which is compatible with a role in promoting freeze tolerance.

Section snippets

Crystallization and structure determination of LpIBP

LpIBP crystallized in the monoclinic C2 space group with two molecules per asymmetric unit. Crystals were grown at low pH (4.2) and high concentrations of ethanol (40–50%) at 4 °C over approximately 4 weeks. Data were collected at beamline X6A at National Synchrotron Light Source in Brookhaven National Laboratories. Two data sets obtained from two crystals were combined to obtain phases using single isomorphous replacement with anomalous scattering. After solvent flattening, the electron

Discussion

Here, we have presented the first structure of a plant “antifreeze protein,” which is in effect the first structure of an IBP that functions primarily in freeze tolerance by inhibiting ice recrystallization. The fish and insect AFP structures solved to date25, 33, 34, 42, 43, 44, 45, 46 all come from organisms that must avoid freezing. The repetitive, beta-helical LpIBP structure provides a flat regular ice-binding face that resembles several AFPs, most notably the hyperactive AFPs from

Purification and crystallization of LpIBP

Recombinant LpIBP with a C-terminal 6 × His-tag was purified in three steps as previously described.31 After removal of most contaminating Escherichia coli proteins by boiling, LpIBP was further purified by ice affinity purification,65 which also selected for properly folded LpIBP. Finally, Ni-Agarose affinity chromatography was performed, mainly to concentrate the His-tagged product. The pure protein was eluted and dialyzed against 50 mM Tris–HCl (pH 7.8), 100 mM NaCl and 1 mM

Acknowledgements

This research was funded by a grant to P.L.D. from the Canadian Institutes of Health Research. The authors thank Debborah Fass for the gift of GFP-TmAFP, Christopher Garnham for help growing ice crystal hemispheres, Jean Jakoncic and Vivian Stojanoff from beamline X6A at Brookhaven National Laboratory for beamline support, Margaret Daley and Brian Sykes for validating the folding of the TmAFP point mutations, Sherry Gauthier for technical support, Zongchao Jia and John Allingham for access to

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