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abstracts
Glypicans are heparan sulfate proteoglycans that are attached to the cell membrane surface by glycosylphosphatidylinositol anchorage. Glypican-1 (Gpc-1) is the predominant heparan sulphate proteoglycan in the developing and adult human brain and is involved in developmental morphogenesis, growth factor and cytokine signalling. We determined the crystal structure of N-glycosylated human glypican-1 core protein at 2.55 Å resolution, which revealed a cylindrical, all α-helical fold (dimensions 120 x 30 x 30 Å), decorated with three major loops and containing the 14 cysteine residues conserved in all members of the glypican family (1). The Gpc-1 crystals were delicate, highly fragile plates, which displayed poor isomorphism, with cell dimensions varying between different crystals. These crystals also diffracted anisotropically, reflected in a Wilson B factor that was twice as large in the c* direction as in the a* and b* directions, which limited the effective resolution to 2.9 Å in the c* direction. Recently we have shown Gpc-1 crystals to be a successful case for improvement in diffraction properties by controlled crystal dehydration using the humidity control device (HC1b), which delivers a humidified air stream of a precise relative humidity that can be used to alter the solvent content inside the crystals (2). The optimal dehydration protocol was developed by investigation of the parameters: final relative humidity RHf, dehydration rate and total incubation time Tinc. Of these, the most important was shown to be Tinc. After dehydration using the optimal protocol, the diffraction quality of the Gpc-1 crystals was clearly improved, with significant reduction in the anisotropy. This generated better, less noisy electron density maps, which allowed the building of previously disordered parts of the model and displayed well-defined side chains.
abstracts
Ribonucleotide reductases (RNRs) catalyze the reduction of ribonucleotides to deoxyribonucleotides, the building blocks for DNA synthesis, and are found in all but a few organisms. RNRs use radical chemistry to catalyze the reduction reaction. Despite RNR having evolved several different mechanisms for generation of different kinds of essential radicals across a large evolutionary time frame, for over 30 years the paradigm has been that this initial radical is always channeled to a strictly conserved cysteine residue directly adjacent to the substrate for initiation of substrate reduction. Such a cysteine residue has been present in the structure of each of the many RNRs determined to date. We present the crystal structure of an anaerobic RNR from the extreme thermophile Thermotoga maritima (tmNrdD), both alone and in complex with allosteric effector dATP and substrate CTP. Remarkably, tmNrdD lacks a cysteine for radical transfer to the substrate, and is the first structurally or biochemically characterized RNR to do so. However in many other respects tmNrdD appears to be a normal anaerobic RNR, including gene structure, expression levels, metal cofactor and binding of allosteric effectors and substrates in the expected conformations. Furthermore, it is possible to generate a glycyl radical as expected. We present evidence that the structure of tmNrdD is representative for the new RNR subclass IIIh, present in all Thermotoga species plus a wider group of bacteria from the distantly related phyla Firmicutes, Bacteroidetes and Proteobacteria, all lacking the canonical cysteine residue. The wide distribution provides further evidence that the subclass IIIh is a functional RNR. Taken together, the results imply that an alternative initiation route for the RNR reduction reaction must exist that do not require channeling through a cysteine side chain.
