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


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Abstract

A Ca2+-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 Ca2+ is required for RII monomer folding, and that the monomer is fully structured at a Ca2+/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 Ca2+ as the heavy atom to obtain phase information. The RII monomer folds as a Ca2+-bound immunoglobulin-like β-sandwich. Ca2+ 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 Ca2+ 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.DatabaseStructural data are available in the Protein Data Bank under the accession numbers 4KDW (P1 structure) and 4KDV (P21 structure).The104-residue domain that occurs ˜120 times as a direct tandem repeat in a 1.5 MDa ice-binding adhesin (MpAFP) has a Ca2+-dependent Ig-like fold. Based on the crystal structure we suggest Ca2+ also bridges these extender domains together to help project the ice-binding domain away from the surface of the host Antarctic bacterium towards ice.

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