Structural basis of antifreeze activity of a bacterial multi-domain antifreeze protein

November Structural basis of antifreeze activity of a bacterial multi-domain antifreeze protein Chen Wang 0 1 Svetlana Pakhomova 0 1 Marcia E. Newcomer 0 1 Brent C. Christner 0 1 Bing- Hao Luo 0 1 ☯ These authors contributed equally to this work. 1 1 0 Department of Biological Sciences, Louisiana State University , Baton Rouge , Louisiana, United States of America, 2 Department of Microbiology and Cell Science, Biodiversity Institute, University of Florida , Gainesville, Florida , United States of America 1 Editor: Eugene A. Permyakov, Russian Academy of Medical Sciences , RUSSIAN FEDERATION Antifreeze proteins (AFPs) enhance the survival of organisms inhabiting cold environments by affecting the formation and/or structure of ice. We report the crystal structure of the first multi-domain AFP that has been characterized. The two ice binding domains are structurally similar. Each consists of an irregular β-helix with a triangular cross-section and a long αhelix that runs parallel on one side of the β-helix. Both domains are stabilized by hydrophobic interactions. A flat plane on the same face of each domain's β-helix was identified as the ice binding site. Mutating any of the smaller residues on the ice binding site to bulkier ones decreased the antifreeze activity. The bulky side chain of Leu174 in domain A sterically hinders the binding of water molecules to the protein backbone, partially explaining why antifreeze activity by domain A is inferior to that of domain B. Our data provide a molecular basis for understanding differences in antifreeze activity between the two domains of this protein and general insight on how structural differences in the ice-binding sites affect the activity of AFPs. - Funding: This work was supported by the U.S. Department of Energy (DE-AC02-98CH10886). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing interests: The authors have declared that no competing interests exist. Introduction Ice binding proteins (IBPs) are characterized by their ability to specifically bind to one or multiple planes of ice crystals [ 1 ]. Antifreeze proteins (AFPs) are a class of IBPs that have been documented in a number of cold-tolerant fish [ 2, 3 ], insect [4], bacterial [ 5, 6 ], fungal [7], and plant [ 8 ] species, and this phenotype permits them to prevent and/or control ice crystal formation. When bound to the ice surface, AFPs depress the freezing point without significantly altering the melting point [ 9 ]. The difference between the freezing and melting point, referred to as the thermal hysteresis (TH) gap, is often used as an indicator of AFP activity [ 10 ]. It is thought that TH is caused by the Kelvin effect because AFP binding to the ice surface generates a micro-convex structure that is thermodynamically less favorable for water molecules to bind compared with a flat ice surface [ 11, 12 ]. At subzero temperatures, small ice crystals recrystallize into larger ones to minimize the surface energy (i.e., Ostwald ripening). Importantly, ice recrystallization damages cell membranes, and therefore is one of the most lethal stresses a cell encounters under frozen conditions [13]. AFPs significantly inhibit this process after binding to ice (RI, recrystallization inhibition) [14, 15], either by preventing water molecules from leaving the ice crystals or acting as a surfactant to reduce the surface tension. Since this activity conserves the boundaries among ice grains, AFPs are hypothesized to enhance microbial survival in ice matrices, such as those found in deep Antarctic glacial ice [ 5, 16, 17 ]. While all AFPs share the similar function of ice binding, their sequences and structures differ widely, making it difficult to infer their molecular detail responsible for this property. The AFPs of Antarctic fish were the first to be discovered [ 2 ] and have been studied extensively. Based on their structural features, four types of fish AFPs are recognized [18]. The type I fish AFPs have the simplest structures and may consist of a single Ala rich α-helix [19]. Recently, Sun et al. reported the crystal structure of an isoform of type I fish AFP isoform, Maxi, which consists of a four helical bundle that retain 400 water molecules inside its core [20]. Type II and type III fish AFPs are both relatively small globular proteins. Type II fish AFPs are stabilized by disulfide bonds [21], while type III fish AFPs are held together mainly through a hydrophobic core [22]. There is currently no structure of type IV fish AFPs reported. Most of the structurally characterized AFPs adopt a β-solenoid / helical structure with various cross sections [23], contain repeating motifs, and have well aligned side chains on their ice binding sites [24±29]. However, there are several β-helical AFPs that deviate from this structural regularity and conservation [30±34]. In general, AFPs form three-dimension (...truncated)