title: A Ca2+-dependent bacterial antifreeze protein domain has a novel beta-helical ice-binding fold.
abstract: AFPs (antifreeze proteins) are produced by many organisms that inhabit ice-laden environments. They facilitate survival at sub-zero temperatures by binding to, and inhibiting, the growth of ice crystals in solution. The Antarctic bacterium Marinomonas primoryensis produces an exceptionally large(>1 MDa) hyperactive Ca2+-dependent AFP. We have cloned,expressed and characterized a 322-amino-acid region of the protein where the antifreeze activity is localized that shows similarity to the RTX (repeats-in-toxin) family of proteins. The recombinant protein requires Ca2+ for structure and activity, and it is capable of depressing the freezing point of a solution in excess of 2 degrees C at a concentration of 0.5 mg/ml, therefore classifying it as a hyperactive AFP. We have developed a homology-guided model of the antifreeze region based partly on the Ca2+-bound beta-roll from alkaline protease. The model has identified both a novel beta-helical fold and an ice-binding site. The interior of the beta-helix contains a single row of bound Ca2+ ions down one side of the structure and a hydrophobic core down the opposite side. The ice binding surface consists of parallel repetitive arrays of threonine and aspartic acid/asparagine residues located down the Ca2+-bound side of the structure. The model was tested and validated by site-directed mutagenesis. It explains the Ca2+-dependency of the region, as well its hyperactive antifreeze activity. This is the first bacterial AFP to be structurally characterized and is one of only five hyperactive AFPs identified to date.AFPS
authors: Protein Function Discovery Group and the Department of Biochemistry, Queen's University, Kingston, ON, Canada K7L 3N6.
authors: Christopher P
authors: CP
authors: Garnham
authors: Jack A
authors: JA
authors: Gilbert
authors: Christopher P
authors: CP
authors: Hartman
authors: Robert L
authors: RL
authors: Campbell
authors: Johanna
authors: J
authors: Laybourn-Parry
authors: Peter L
authors: PL
authors: Davies
mesh_terms: D021301:Antifreeze Proteins / Q000737:chemistry* / Q000235:genetics; D001426:Bacterial Proteins / Q000737:chemistry*; D001665:Binding Sites; D002118:Calcium / Q000737:chemistry*; D003001:Cloning, Molecular; D007053:Ice; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary
chemical_list: D021301:Antifreeze Proteins; D001426:Bacterial Proteins; D007053:Ice; D002118:Calcium