No abstract

By binding to the surface of ice crystals, type III antifreeze protein (AFP) can depress the freezing point of fish blood to below that of freezing seawater. This 7-kDa globular protein is encoded by a multigene family that produces two major isoforms, SP and QAE, which are 55% identical. Disruptive mutations on the ice-binding site of type III AFP lower antifreeze activity but can also change ice crystal morphology. By attaching green fluorescent protein to different mutants and isoforms and by examining the binding of these fusion proteins to single-crystal ice hemispheres, we show that type III AFP has a compound ice-binding site. There are two adjacent, flat, ice-binding surfaces at 150° to each other. One binds the primary prism plane of ice; the other, a pyramidal plane. Steric mutations on the latter surface cause elongation of the ice crystal as primary prism plane binding becomes dominant. SP isoforms naturally have a greatly reduced ability to bind the prism planes of ice. Mutations that make the SP isoforms more QAE-like slow down the rate of ice growth. On the basis of these observations we postulate that other types of AFP also have compound ice-binding sites that enable them to bind to multiple planes of ice.

a b s t r a c tThe antifreeze protein of Lolium perenne, a perennial ryegrass, was previously modeled as a beta-roll with two extensive flat beta-sheets on opposite sides of the molecule. Here we have validated the model with a series of nine site-directed steric mutations in which outward-pointing short sidechain residues were replaced by tyrosine. None of these disrupted the fold. Mutations on one of the beta-sheets and on the sides of the protein retained 70% or greater activity. Three mutations that clustered on the other flat surface lost up to 90% of their antifreeze activity and identify this betasheet as the ice-binding face.

No abstract

The post-translational modification of proteins by ubiquitin is central to the regulation of eukaryotic cells. Substrate-bound ubiquitin chains linked by lysine 11 and 48 target proteins to the proteasome for degradation and determine protein abundance in cells, while other ubiquitin chain linkages regulate protein interactions. The specificity of chain-linkage type is usually determined by ubiquitin-conjugating enzymes (E2s). The degradative E2, Ube2K, preferentially catalyses formation of Lys48-linked chains, but like most E2s, the molecular basis for chain formation is not well understood. Here we report the crystal structure of a Ube2K~ubiquitin conjugate and demonstrate that even though it is monomeric, Ube2K can synthesize Lys48-linked ubiquitin chains. Using site-directed mutagenesis and modelling, our studies reveal a molecular understanding of the catalytic complex and identify key features required for synthesis of degradative Lys48-linked chains. The position of the acceptor ubiquitin described here is likely conserved in other E2s that catalyse Lys48-linked ubiquitin chain synthesis.