The high frequency of internal structural symmetry in common protein folds is presumed to reflect their evolutionary origins from the repetition and fusion of ancient peptide modules, but little is known about the primary sequence and physical determinants of this process. Unexpectedly, a sequence and structural analysis of symmetric subdomain modules within an abundant and ancient globular fold, the β-trefoil, reveals that modular evolution is not simply a relic of the ancient past, but is an ongoing and recurring mechanism for regenerating symmetry, having occurred independently in numerous existing β-trefoil proteins. We performed a computational reconstruction of a β-trefoil subdomain module and repeated it to form a newly three-fold symmetric globular protein, ThreeFoil. In addition to its near perfect structural identity between symmetric modules, ThreeFoil is highly soluble, performs multivalent carbohydrate binding, and has remarkably high thermal stability. These findings have far-reaching implications for understanding the evolution and design of proteins via subdomain modules.

Galectin-1, a β-galactoside binding lectin involved in immunoregulation and cancer, binds natural and many synthetic multivalent glycoconjugates with an apparent glycoside cluster effect, that is, affinity above and beyond what would be expected from the concentration of the determinant sugar. Here we have analyzed the mechanism of such cluster effects in solution at physiological concentration using a fluorescence anisotropy assay with a novel fluorescent high-affinity galectin-1 binding probe. The interaction of native dimeric and monomeric mutants of rat and human galectin-1 with mono- and divalent small molecules, fetuin, asialofetuin, and human serum glycoproteins was analyzed. Surprisingly, high-affinity binding did not depend much on the dimeric state of galectin-1 and thus is due mainly to monomeric interactions of a single carbohydrate recognition domain. The mechanism for this is unknown, but one possibility includes additional interactions that high-affinity ligands make with an extended binding site on the carbohydrate recognition domain. It follows that such weak additional interactions must be important for the biological function of galectin-1 and also for the design of galectin-1 inhibitors.

Background: Alg44 regulates the production of alginate in Pseudomonas aeruginosa via c-di-GMP binding. Results:The structure of the PilZ domain of Alg44 in complex with c-di-GMP reveals residues that control c-di-GMP/Alg44 stoichiometry. Conclusion: Binding of dimeric c-di-GMP is required for alginate biosynthesis.Significance: This is the first example of a receptor requiring a specific form of c-di-GMP for activation.

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Class I ␣1,2-mannosidases (glycosylhydrolase family 47) are key enzymes in the maturation of N-glycans. This protein family includes two distinct enzymatically active subgroups. Subgroup 1 includes the yeast and human endoplasmic reticulum (ER) ␣1,2-mannosidases that primarily trim Man 9 GlcNAc 2 to Man 8 GlcNAc 2 isomer B whereas subgroup 2 includes mammalian Golgi ␣1,2-mannosidases IA, IB, and IC that trim Man 9 GlcNAc 2 to Man 5 GlcNAc 2 via Man 8 GlcNAc 2 isomers A and C. The structure of the catalytic domain of the subgroup 2 ␣1,2-mannosidase from Penicillium citrinum has been determined by molecular replacement at 2.2-Å resolution. The fungal ␣1,2-mannosidase is an (␣␣) 7