Antifreeze protein from freeze-tolerant grass has a beta-roll fold with an irregularly structured ice-binding site

Adam J. Middleton; Christopher B. Marshall; Frédérick Faucher; Maya Bar-Dolev; Ido Braslavsky; Robert L. Campbell; Virginia K. Walker; Peter L. Davies

doi:10.1016/j.jmb.2012.01.032

Antifreeze protein from freeze-tolerant grass has a beta-roll fold with an irregularly structured ice-binding site

Adam J. Middleton, Christopher B. Marshall, Frédérick Faucher, Maya Bar-Dolev, Ido Braslavsky, Robert L. Campbell, Virginia K. Walker, Peter L. Davies^*

^*Corresponding author for this work

Institute of Biochemistry, Food Science and Nutrition

Research output: Contribution to journal › Article › peer-review

112 Scopus citations

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.

Original language	English
Pages (from-to)	713-724
Number of pages	12
Journal	Journal of Molecular Biology
Volume	416
Issue number	5
DOIs	https://doi.org/10.1016/j.jmb.2012.01.032
State	Published - 9 Mar 2012

Bibliographical note

Funding Information:
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 their home-source beams and Qilu Ye for aiding with initial crystal screening. P.L.D. holds a Canada Research Chair in Protein Engineering, A.J.M. was supported by a Natural Sciences and Engineering Research Council of Canada Postgraduate Scholarship ( PGS-D2 ), F.F. was supported by a postdoctoral fellowship from Fond de la Recherche en Santé du Québec , V.K.W. was supported by a Queen's Research Chair and M.B. was supported by the Lady Davis Fellowship Trust and by the Israel Science Foundation .

Keywords

X-ray crystallography
beta-helix
ice recrystallization inhibition
thermolabile
water

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10.1016/j.jmb.2012.01.032

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title = "Antifreeze protein from freeze-tolerant grass has a beta-roll fold with an irregularly structured ice-binding site",

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.",

keywords = "X-ray crystallography, beta-helix, ice recrystallization inhibition, thermolabile, water",

author = "Middleton, {Adam J.} and Marshall, {Christopher B.} and Fr{\'e}d{\'e}rick Faucher and Maya Bar-Dolev and Ido Braslavsky and Campbell, {Robert L.} and Walker, {Virginia K.} and Davies, {Peter L.}",

note = "Funding Information: 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 their home-source beams and Qilu Ye for aiding with initial crystal screening. P.L.D. holds a Canada Research Chair in Protein Engineering, A.J.M. was supported by a Natural Sciences and Engineering Research Council of Canada Postgraduate Scholarship ( PGS-D2 ), F.F. was supported by a postdoctoral fellowship from Fond de la Recherche en Sant{\'e} du Qu{\'e}bec , V.K.W. was supported by a Queen's Research Chair and M.B. was supported by the Lady Davis Fellowship Trust and by the Israel Science Foundation .",

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doi = "10.1016/j.jmb.2012.01.032",

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T1 - Antifreeze protein from freeze-tolerant grass has a beta-roll fold with an irregularly structured ice-binding site

AU - Middleton, Adam J.

AU - Marshall, Christopher B.

AU - Faucher, Frédérick

AU - Bar-Dolev, Maya

AU - Braslavsky, Ido

AU - Campbell, Robert L.

AU - Walker, Virginia K.

AU - Davies, Peter L.

N1 - Funding Information: 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 their home-source beams and Qilu Ye for aiding with initial crystal screening. P.L.D. holds a Canada Research Chair in Protein Engineering, A.J.M. was supported by a Natural Sciences and Engineering Research Council of Canada Postgraduate Scholarship ( PGS-D2 ), F.F. was supported by a postdoctoral fellowship from Fond de la Recherche en Santé du Québec , V.K.W. was supported by a Queen's Research Chair and M.B. was supported by the Lady Davis Fellowship Trust and by the Israel Science Foundation .

PY - 2012/3/9

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N2 - 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.

AB - 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.

KW - X-ray crystallography

KW - beta-helix

KW - ice recrystallization inhibition

KW - thermolabile

KW - water

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JF - Journal of Molecular Biology

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ER -

Antifreeze protein from freeze-tolerant grass has a beta-roll fold with an irregularly structured ice-binding site

Abstract

Bibliographical note

Keywords

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