A Ca2+-dependent bacterial antifreeze protein domain has a novel beta-helical ice-binding fold.

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

[1]  T. Blundell,et al.  Comparative protein modelling by satisfaction of spatial restraints. , 1993, Journal of molecular biology.

[2]  Michael J. Kuiper,et al.  β-Helix structure and ice-binding properties of a hyperactive antifreeze protein from an insect , 2000, Nature.

[3]  B. Glick,et al.  Cloning and Expression of afpA , a Gene Encoding an Antifreeze Protein from the Arctic Plant Growth-Promoting Rhizobacterium Pseudomonas putida GR 12-2 † , 2004 .

[4]  B. Sykes,et al.  Cold survival in freeze‐intolerant insects , 2004 .

[5]  Gerrit Groenhof,et al.  GROMACS: Fast, flexible, and free , 2005, J. Comput. Chem..

[6]  Bernard R. Glick,et al.  Cloning and Expression of afpA, a Gene Encoding an Antifreeze Protein from the Arctic Plant Growth-Promoting Rhizobacterium Pseudomonas putida GR12-2 , 2004, Journal of bacteriology.

[7]  Zongchao Jia,et al.  Antifreeze proteins: an unusual receptor-ligand interaction. , 2002, Trends in biochemical sciences.

[8]  R. Meier,et al.  A Calcium-gated Lid and a Large β-Roll Sandwich Are Revealed by the Crystal Structure of Extracellular Lipase from Serratia marcescens* , 2007, Journal of Biological Chemistry.

[9]  R. Hodges,et al.  Antifreeze protein from shorthorn sculpin: Identification of the ice‐binding surface , 2001, Protein science : a publication of the Protein Society.

[10]  M. Uemura,et al.  A Comparison of Freezing Injury in Oat and Rye: Two Cereals at the Extremes of Freezing Tolerance , 1994, Plant physiology.

[11]  J. Coote.  Structural and functional relationships among the RTX toxin determinants of gram-negative bacteria. , 1992, FEMS microbiology reviews.

[12]  S. Petersen,et al.  NMR Structure of the R-module , 2006, Journal of Biological Chemistry.

[13]  W. Delano.  The PyMOL Molecular Graphics System , 2002 .

[14]  Zongchao Jia,et al.  Mimicry of ice structure by surface hydroxyls and water of a β-helix antifreeze protein , 2000, Nature.

[15]  Bing Liu,et al.  Significance of conservative asparagine residues in the thermal hysteresis activity of carrot antifreeze protein. , 2004, The Biochemical journal.

[16]  B. Sykes,et al.  Identification of the ice‐binding face of antifreeze protein from Tenebrio molitor , 2002, FEBS letters.

[17]  U. Baumann,et al.  Three‐dimensional structure of the alkaline protease of Pseudomonas aeruginosa: a two‐domain protein with a calcium binding parallel beta roll motif. , 1993, The EMBO journal.

[18]  J. Bujnicki,et al.  Structure and Evolutionary Origin of Ca2+-Dependent Herring Type II Antifreeze Protein , 2007, PloS one.

[19]  J. Raymond,et al.  An ice-binding protein from an Antarctic sea ice bacterium. , 2007, FEMS microbiology ecology.

[20]  P. Davies,et al.  A theoretical model of a plant antifreeze protein from Lolium perenne. , 2001, Biophysical journal.

[21]  Jack A Gilbert,et al.  A hyperactive, Ca2+-dependent antifreeze protein in an Antarctic bacterium. , 2005, FEMS microbiology letters.

[22]  Jack A Gilbert,et al.  Demonstration of antifreeze protein activity in Antarctic lake bacteria. , 2004, Microbiology.

[23]  J. Madura,et al.  Antifreeze proteins at the ice/water interface: three calculated discriminating properties for orientation of type I proteins. , 2007, Biophysical journal.

[24]  Alasdair C. Steven,et al.  β‐Rolls, β‐Helices, and Other β‐Solenoid Proteins , 2006 .

[25]  K. Sharp,et al.  Hydrophobic tendency of polar group hydration as a major force in type I antifreeze protein recognition , 2005, Proteins.

[26]  Donald E. Wohlschlag,et al.  Freezing Resistance in Some Antarctic Fishes , 1969, Science.

[27]  Erlend Kristiansen,et al.  The mechanism by which fish antifreeze proteins cause thermal hysteresis. , 2005, Cryobiology.

[28]  A. Chakrabartty,et al.  The effect of enhanced α‐helicity on the activity of a winter flounder antifreeze polypeptide , 1991 .

[29]  P. Davies,et al.  The antifreeze potential of the spruce budworm thermal hysteresis protein , 1997, Nature Biotechnology.

[30]  J. Gilbert,et al.  The basis for hyperactivity of antifreeze proteins. , 2006, Cryobiology.

[31]  R. Hodges,et al.  New ice‐binding face for type I antifreeze protein , 1999, FEBS letters.

[32]  C. Hew,et al.  The role of Ca2+-coordinating residues of herring antifreeze protein in antifreeze activity. , 2004, Biochemistry.

[33]  Daniel S. C. Yang,et al.  Theoretical study of interaction of winter flounder antifreeze protein with ice , 2001, Protein science : a publication of the Protein Society.

[34]  C. Knight,et al.  Plant thermal hysteresis proteins. , 1992, Biochimica et biophysica acta.

[35]  B. McConkey,et al.  Ordered surface carbons distinguish antifreeze proteins and their ice-binding regions , 2006, Nature Biotechnology.