Abstract
A novel role for antifreeze proteins (AFPs) may reside in an exceptionally large 1.5-MDa adhesin isolated from an Antarctic Gram-negative bacterium, Marinomonas primoryensis. MpAFP was purified from bacterial lysates by ice adsorption and gel electrophoresis. We have previously reported that two highly repetitive sequences, region II (RII) and region IV (RIV), divide MpAFP into five distinct regions, all of which require mM Ca2+ levels for correct folding. Also, the antifreeze activity is confined to the 322-residue RIV, which forms a Ca2+-bound beta-helix containing thirteen Repeats-In-Toxin (RTX)-like repeats. RII accounts for approximately 90% of the mass of MpAFP and is made up of ∼120 tandem 104-residue repeats. Because these repeats are identical in DNA sequence, their number was estimated here by pulsed-field gel electrophoresis. Structural homology analysis by the Protein Homology/analogY Recognition Engine (Phyre2) server indicates that the 104-residue RII repeat adopts an immunoglobulin beta-sandwich fold that is typical of many secreted adhesion proteins. Additional RTX-like repeats in RV may serve as a non-cleavable signal sequence for the type I secretion pathway. Immunodetection shows both repeated regions are uniformly distributed over the cell surface. We suggest that the development of an AFP-like domain within this adhesin attached to the bacterial outer surface serves to transiently bind the host bacteria to ice. This association would keep the bacteria within the upper reaches of the water column where oxygen and nutrients are potentially more abundant. This novel envirotactic role would give AFPs a third function, after freeze avoidance and freeze tolerance: that of transiently binding an organism to ice.
Highlights
Antifreeze proteins (AFPs) were initially characterized in marine fishes [1], [2] where they protect their hosts from freezing by binding to, and preventing the growth of, seed ice crystals [3]
A series of peaks showing only low antifreeze activity eluted from DEAEcellulose at .0.5 M NaCl and these were contaminated with nucleic acids
E. coli fixed to glass slides stained green with SYTO 9 but showed negligible blue fluorescence from the anti-region II (RII) and anti-region IV (RIV) antibodies used in conjunction with the labeled anti-rabbit second antibody (Fig. S3). These results demonstrate that MpAFP is uniformly distributed over the Marinomonas primoryensis cell surface and that both the large repetitive RII and the antifreeze domain are exposed to the extracellular environment, consistent with what would be expected for an adhesion protein
Summary
Antifreeze proteins (AFPs) were initially characterized in marine fishes [1], [2] where they protect their hosts from freezing by binding to, and preventing the growth of, seed ice crystals [3]. We recently solved the X-ray crystal structure of MpAFP Region IV to 1.7 A [9] This segment of the protein folds as an extended, Ca2+-bound right-handed beta-helix whose ice-binding site (IBS) consists of a flat, repetitive array of outwardprojecting Thr and Asx residues. Experimental observation of these ordered surface waters provided strong physical evidence for a mechanism of ice binding that was originally predicted by molecular modeling [10], [11], [12], [13] This may well be a general mechanism of action for all ice-binding proteins where the IBS orders surface waters into an ice-like ‘‘anchored clathrate’’ pattern that helps ‘‘freeze’’ the AFPs to the ice surface [9]
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