Role of Ca²⁺ in folding the tandem β-sandwich extender domains of a bacterial ice-binding adhesin

FEBS J. 2013 Nov;280(22):5919-32. doi: 10.1111/febs.12518. Epub 2013 Oct 11.


A Ca(2+) -dependent 1.5-MDa antifreeze protein present in an Antarctic Gram-negative bacterium, Marinomonas primoryensis (MpAFP), has recently been reassessed as an ice-binding adhesin. The non-ice-binding region II (RII), one of five distinct domains in MpAFP, constitutes ~ 90% of the protein. RII consists of ~ 120 tandem copies of an identical 104-residue sequence. We used the Protein Homology/analogy Recognition Engine server to define the boundaries of a single 104-residue RII construct (RII monomer). CD demonstrated that Ca(2+) is required for RII monomer folding, and that the monomer is fully structured at a Ca(2+) /protein molar ratio of 10 : 1. The crystal structure of the RII monomer was solved to a resolution of 1.35 Å by single-wavelength anomalous dispersion and molecular replacement methods with Ca(2+) as the heavy atom to obtain phase information. The RII monomer folds as a Ca(2+) -bound immunoglobulin-like β-sandwich. Ca(2+) ions are coordinated at the interfaces between each RII monomer and its symmetry-related molecules, suggesting that these ions may be involved in the stabilization of the tandemly repeated RII. We hypothesize that > 600 Ca(2+) ions help to rigidify the chain of 104-residue repeats in order to project the ice-binding domain of MpAFP away from the bacterial cell surface. The proposed role of RII is to help the strictly aerobic bacterium bind surface ice in an Antarctic lake for better access to oxygen and nutrients. This work may give insights into other bacterial proteins that resemble MpAFP, especially those of the large repeats-in-toxin family that have been characterized as adhesins exported via the type I secretion pathway.

Keywords: Ca2+-dependent; bacterial immunoglobulin (Ig)-like fold; crystal structure; extender domain; ice-binding adhesin.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Adhesins, Bacterial / chemistry*
  • Adhesins, Bacterial / genetics
  • Adhesins, Bacterial / metabolism
  • Amino Acid Sequence
  • Antarctic Regions
  • Antifreeze Proteins / chemistry*
  • Antifreeze Proteins / genetics
  • Antifreeze Proteins / metabolism
  • Bacterial Proteins / chemistry*
  • Bacterial Proteins / genetics
  • Bacterial Proteins / metabolism
  • Binding Sites
  • Calcium / metabolism
  • Crystallography, X-Ray
  • Ice
  • Marinomonas / genetics
  • Marinomonas / metabolism
  • Models, Molecular
  • Molecular Sequence Data
  • Protein Folding
  • Protein Stability
  • Protein Structure, Tertiary
  • Structural Homology, Protein


  • Adhesins, Bacterial
  • Antifreeze Proteins
  • Bacterial Proteins
  • Ice
  • Calcium

Associated data

  • PDB/4KDV
  • PDB/4KDW