Icefishes (suborder Notothenioidei; family Channichthyidae) are the only vertebrates that lack functional haemoglobin genes and red blood cells. Here, we report a high-quality genome assembly and linkage map for the Antarctic blackfin icefish Chaenocephalus aceratus, highlighting evolved genomic features for its unique physiology. Phylogenomic analysis revealed that Antarctic fish of the teleost suborder Notothenioidei, including icefishes, diverged from the stickleback lineage about 77 million years ago and subsequently evolved cold-adapted phenotypes as the Southern Ocean cooled to sub-zero temperatures. Our results show that genes involved in protection from ice damage, including genes encoding antifreeze glycoprotein and zona pellucida proteins, are highly expanded in the icefish genome. Furthermore, genes that encode enzymes that help to control cellular redox state, including members of the sod3 and nqo1 gene families, are expanded, probably as evolutionary adaptations to the relatively high concentration of oxygen dissolved in cold Antarctic waters. In contrast, some crucial regulators of circadian homeostasis (cry and per genes) are absent from the icefish genome, suggesting compromised control of biological rhythms in the polar light environment. The availability of the icefish genome sequence will accelerate our understanding of adaptation to extreme Antarctic environments.

Background: Ice-binding proteins improve the cold tolerance of cells by inhibiting ice growth and recrystallization. Results: Crystal structure and mutagenesis data of LeIBP suggests the B face as an ice-binding site. Conclusion: LeIBP structure adopts a ␤-helical fold and the aligned Thr/Ser/Ala residues are critical for ice binding. Significance: LeIBP structure can serve as a structural model for a large number of IBPs.

Antifreeze proteins (AFPs) are biological antifreezes with unique properties, including thermal hysteresis (TH), ice recrystallization inhibition (IRI), and interaction with membranes and/or membrane proteins. These properties have been utilized in the preservation of biological samples at low temperatures. Here, we review the structure and function of marine-derived AFPs, including moderately active fish AFPs and hyperactive polar AFPs. We also survey previous and current reports of cryopreservation using AFPs. Cryopreserved biological samples are relatively diverse ranging from diatoms and reproductive cells to embryos and organs. Cryopreserved biological samples mainly originate from mammals. Most cryopreservation trials using marine-derived AFPs have demonstrated that addition of AFPs can improve post-thaw viability regardless of freezing method (slow-freezing or vitrification), storage temperature, and types of biological sample type.

Ice-binding proteins (IBPs) inhibit ice growth through direct interaction with ice crystals to permit the survival of polar organisms in extremely cold environments. FfIBP is an ice-binding protein encoded by the Antarctic bacterium Flavobacterium frigoris PS1. The X-ray crystal structure of FfIBP was determined to 2.1 Å resolution to gain insight into its ice-binding mechanism. The refined structure of FfIBP shows an intramolecular disulfide bond, and analytical ultracentrifugation and analytical size-exclusion chromatography show that it behaves as a monomer in solution. Sequence alignments and structural comparisons of IBPs allowed two groups of IBPs to be defined, depending on sequence differences between the α2 and α4 loop regions and the presence of the disulfide bond. Although FfIBP closely resembles Leucosporidium (recently re-classified as Glaciozyma) IBP (LeIBP) in its amino-acid sequence, the thermal hysteresis (TH) activity of FfIBP appears to be tenfold higher than that of LeIBP. A comparison of the FfIBP and LeIBP structures reveals that FfIBP has different ice-binding residues as well as a greater surface area in the ice-binding site. Notably, the ice-binding site of FfIBP is composed of a T-A/G-X-T/N motif, which is similar to the ice-binding residues of hyperactive antifreeze proteins. Thus, it is proposed that the difference in TH activity between FfIBP and LeIBP may arise from the amino-acid composition of the ice-binding site, which correlates with differences in affinity and surface complementarity to the ice crystal. In conclusion, this study provides a molecular basis for understanding the antifreeze mechanism of FfIBP and provides new insights into the reasons for the higher TH activity of FfIBP compared with LeIBP.

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The Shank/proline-rich synapse-associated protein family of multidomain proteins is known to play an important role in the organization of synaptic multiprotein complexes. For instance, the Shank PDZ domain binds to the C termini of guanylate kinase-associated proteins, which in turn interact with the guanylate kinase domain of postsynaptic density-95 scaffolding proteins. Here we describe the crystal structures of Shank1 PDZ in its peptide free form and in complex with the C-terminal hexapeptide (EAQTRL) of guanylate kinaseassociated protein (GKAP1a) determined at 1.8-and 2.25-Å resolutions, respectively. The structure shows the typical class I PDZ interaction of PDZ-peptide complex with the consensus sequence -X-(Thr/Ser)-X-Leu. In addition, Asp-634 within the Shank1 PDZ domain recognizes the positively charged Arg at ؊1 position and hydrogen bonds, and salt bridges between Arg-607 and the side chains of the ligand at ؊3 and ؊5 positions contribute further to the recognition of the peptide ligand. Remarkably, whether free or complexed, Shank1 PDZ domains form dimers with a conserved ␤B/␤C loop and N-terminal ␤A strands, suggesting a novel model of PDZ-PDZ homodimerization. This implies that antiparallel dimerization through the N-terminal ␤A strands could be a common configuration among PDZ dimers. Within the dimeric structure, the two-peptide binding sites are arranged so that the N termini of the bound peptide ligands are in close proximity and oriented toward the 2-fold axis of the dimer. This configuration may provide a means of facilitating dimeric organization of PDZ-target assemblies.Multidomain Shank, proline-rich synapse-associated protein, and somatostatin receptor-interacting protein scaffold proteins bind to various membrane and cytoplasmic proteins within the PSDs 1 in excitatory synapses (1, 2). It has been suggested that Shank links N-methyl-D-aspartate receptor-PSD-95 complexes to the actin cytoskeleton, thereby playing a critical role in the organization of cytoskeletal signaling complexes at excitatory synapses (1, 2). The three known members of the Shank family (Shank1-3) all contain multiple sites for alternative splicing and show distinct tissue distributions (2). Although shank proteins vary in molecular mass, they share a common domain organization consisting of seven N-terminal ankyrin repeats followed by an SH3 domain, a PDZ domain, a long proline-rich region, and a SAM domain. All of these motifs are potentially involved in protein-protein interactions. For instance, the proline-rich region commonly acts as a binding site for SH3, EVH1, and WW domains and SAM domains can bind to each other in homomeric and heteromeric fashion, enabling oligomerization of Shank and its interacting proteins (3).PDZs are globular domains containing ϳ80 -100 amino acids (4). The Shank PDZ domain is a class I PDZ recognizing the C-terminal sequence X-(Thr/Ser)-X-Leu (where X represents any amino acid), which enables it to bind a variety of integral membrane proteins; however, it most specifically binds to ...

PDZ domains bind to short segments within target proteins in a sequence-specific fashion. Glutamate receptor-interacting protein (GRIP)/ABP family proteins contain six to seven PDZ domains and interact via the sixth PDZ domain (class II) with the C termini of various proteins including liprin-␣. In addition the PDZ456 domain mediates the formation of homo-and heteromultimers of GRIP proteins. To better understand the structural basis of peptide recognition by a class II PDZ domain and PDZ-mediated multimerization, we determined the crystal structures of the GRIP1 PDZ6 domain alone and in complex with a synthetic C-terminal octapeptide of human liprin-␣ at resolutions of 1.5 and 1.8 Å, respectively. Remarkably, unlike other class II PDZ domains, Ile-736 at ␣B5 rather than conserved Leu-732 at ␣B1 makes a direct hydrophobic contact with the side chain of the Tyr at the ؊2 position of the ligand. Moreover, the peptide-bound structure of PDZ6 shows a slight reorientation of helix ␣B, indicating that the second hydrophobic pocket undergoes a conformational adaptation to accommodate the bulkiness of the Tyr side chain, and forms an antiparallel dimer through an interface located at a site distal to the peptide-binding groove. This configuration may enable formation of GRIP multimers and efficient clustering of GRIP-binding proteins.Synaptic localization and clustering of ion channels and receptors is often mediated by scaffolding molecules containing the protein-protein interaction motifs called PDZ (Postsynaptic density-95/Discs large/Zona occludens-1) domains (1). One of the most abundant molecular recognition elements, these globular domains each contain two ␣-helices and six ␤-strands. They usually bind selectively to the C terminus or a short internal segment of interacting proteins (1) and are categorized into four classes according to their specificity for the C-terminal target sequences (2). Class I PDZ domains bind to a C-terminal motif with the sequence X-Ser/Thr-X-Val/Leu-COOH, where X represents any residue, while class II PDZ domains prefer X-⌽-X-⌽-COOH, where ⌽ is usually a large hydrophobic residue. Both class I and II domains have a preference for a hydrophobic residue at the 0 position of the ligand. Class III PDZ domains prefer the sequence X-Asp-X-Val-COOH in which a negatively charged amino acid is at the Ϫ2 position (3), while class IV domains prefer the sequence X-⌿-Asp/Glu-COOH in which an acidic residue is at the C-terminal position and where ⌿ represents an aromatic residue (4). In addition, there are other classes of PDZ domains that do not fall into any of the aforementioned classes (5, 6), and there are minor discrepancies in the proposed classifications of PDZ domains (7,8).Members of the GRIP 1 family proteins (GRIP1 and GRIP2/ ABP) contain six to seven PDZ domains (9, 10, 11). GRIP PDZ45, which is classified as a class II PDZ domain (1), binds to the C terminus of the GluR2/3 subunit of AMPA glutamate receptors (9, 10, 12), while GRIP PDZ6, also a class II PDZ domain, interacts with the ...

Motor proteins not actively involved in transporting cargoes should remain inactive at sites of cargo loading to save energy and remain available for loading. KIF1A/ Unc104 is a monomeric kinesin known to dimerize into a processive motor at high protein concentrations. However, the molecular mechanisms underlying monomer stabilization and monomer-to-dimer transition are not well understood. Here, we report an intramolecular interaction in KIF1A between the forkhead-associated (FHA) domain and a coiled-coil domain (CC2) immediately following the FHA domain. Disrupting this interaction by point mutations in the FHA or CC2 domains leads to a dramatic accumulation of KIF1A in the periphery of living cultured neurons and an enhancement of the microtubule (MT) binding and self-multimerization of KIF1A. In addition, point mutations causing rigidity in the predicted flexible hinge disrupt the intramolecular FHA-CC2 interaction and increase MT binding and peripheral accumulation of KIF1A. These results suggest that the intramolecular FHA-CC2 interaction negatively regulates KIF1A activity by inhibiting MT binding and dimerization of KIF1A, and point to a novel role of the FHA domain in the regulation of kinesin motors.