A Ca 2+ dependent bacterial antifreeze protein domain has a novel beta-helical ice-binding fold

• Published in: Biochemical Journal
• Main Authors: Garnham, CP, Gilbert, JA, Hartman, CP, Campbell, RL, Laybourn-Parry, J, Davis, PL
• Format: Article in Journal/Newspaper
• Language: English
• Published: Oxford University Press 2008
• Subjects: Biological Sciences; Microbiology; Microbial Ecology; Antarctic; The Antarctic; Antarc*
• Online Access: http://www.biochemj.org/bj/default.htm; https://doi.org/10.1042/BJ20071372; http://www.ncbi.nlm.nih.gov/pubmed/18095937; http://ecite.utas.edu.au/53340

AFPs (antifreeze proteins) are produced by many organismsthat inhabit ice-laden environments. They facilitate survivalat sub-zero temperatures by binding to, and inhibiting, thegrowth of ice crystals in solution. The Antarctic bacterium Marinomonas primoryensis produces an exceptionally large(>1 MDa) hyperactive Ca 2+ -dependent AFP. We have cloned,expressed and characterized a 322-amino-acid region of theprotein where the antifreeze activity is localized that showssimilarity to the RTX (repeats-in-toxin) family of proteins. Therecombinant protein requires Ca 2+ for structure and activity, and itis capable of depressing the freezing point of a solution in excessof 2C at a concentration of 0.5 mg/ml, therefore classifying itas a hyperactive AFP. We have developed a homology-guidedmodel of the antifreeze region based partly on the Ca 2+ -bound β-roll from alkaline protease. The model has identified both a novelβ-helical fold and an ice-binding site. The interior of the !-helixcontains a single row of bound Ca 2+ ions down one side of thestructure and a hydrophobic core down the opposite side. The icebindingsurface consists of parallel repetitive arrays of threonineand aspartic acid/asparagine residues located down the Ca 2+ -bound side of the structure. The model was tested and validatedby site-directed mutagenesis. It explains the Ca 2+ -dependency ofthe region, as well its hyperactive antifreeze activity. This is thefirst bacterial AFP to be structurally characterized and is one ofonly five hyperactive AFPs identified to date.