Restriction endonucleases that cleave RNA/DNA heteroduplexes bind dsDNA in A-like conformation.

Restriction endonucleases naturally target DNA duplexes. Systematic screening has identified a small minority of these enzymes that can also cleave RNA/DNA heteroduplexes and that may therefore be useful as tools for RNA biochemistry. We have chosen AvaII (G↓GWCC, where W stands for A or T) as a representative of this group of restriction endonucleases for detailed characterization. Here, we report crystal structures of AvaII alone, in specific complex with partially cleaved dsDNA, and in scanning complex with an RNA/DNA hybrid. The specific complex reveals a novel form of semi-specific dsDNA readout by a hexa-coordinated metal cation, most likely Ca2+ or Mg2+. Substitutions of residues anchoring this non-catalytic metal ion severely impair DNA binding and cleavage. The dsDNA in the AvaII complex is in the A-like form. This creates space for 2'-OH groups to be accommodated without intra-nucleic acid steric conflicts. PD-(D/E)XK restriction endonucleases of known structure that bind their dsDNA targets in the A-like form cluster into structurally similar groups. Most such enzymes, including some not previously studied in this respect, cleave RNA/DNA heteroduplexes. We conclude that A-form dsDNA binding is a good predictor for RNA/DNA cleavage activity.

Expression and Subcellular Distribution of GFP-Tagged Human Tetraspanin Proteins in Saccharomyces cerevisiae.

Tetraspanins are integral membrane proteins that function as organizers of multimolecular complexes and modulate function of associated proteins. Mammalian genomes encode approximately 30 different members of this family and remotely related eukaryotic species also contain conserved tetraspanin homologs. Tetraspanins are involved in a number of fundamental processes such as regulation of cell migration, fusion, immunity and signaling. Moreover, they are implied in numerous pathological states including mental disorders, infectious diseases or cancer. Despite the great interest in tetraspanins, the structural and biochemical basis of their activity is still largely unknown. A major bottleneck lies in the difficulty of obtaining stable and homogeneous protein samples in large quantities. Here we report expression screening of 15 members of the human tetraspanin superfamily and successful protocols for the production in S. cerevisiae of a subset of tetraspanins involved in human cancer development. We have demonstrated the subcellular localization of overexpressed tetraspanin-green fluorescent protein fusion proteins in S. cerevisiae and found that despite being mislocalized, the fusion proteins are not degraded. The recombinantly produced tetraspanins are dispersed within the endoplasmic reticulum membranes or localized in granule-like structures in yeast cells. The recombinantly produced tetraspanins can be extracted from the membrane fraction and purified with detergents or the poly (styrene-co-maleic acid) polymer technique for use in further biochemical or biophysical studies.

Crystallization of the photosystem II core complex and its chlorophyll binding subunit CP43 from transplastomic plants of Nicotiana tabacum.

Photosystem II from transplastomic plants of Nicotiana tabacum with a hexahistidine tag at the N-terminal end of the PsbE subunit (α-chain of the cytochrome b(559)) was purified according to the protocol of Fey et al. (BBA 12:1501-1509, 2008). The protein sample was then subjected to two additional gel filtration runs in order to increase its homogeneity and to standardize the amount of detergent. Large three dimensional crystals of the core complex were obtained. Crystals of one of its chlorophyll binding subunits (CP43) in isolation grew in very similar conditions that differed only in the concentration of the detergent. Diffraction of Photosystem II and CP43 crystals at various synchrotron beamlines was limited to a resolution of 7 and 14 Å, respectively. In both cases the diffraction quality was insufficient for an unambiguous assignment of the crystallographic lattice or space group.

Pseudomonas aeruginosa LD-carboxypeptidase, a serine peptidase with a Ser-His-Glu triad and a nucleophilic elbow.

LD-Carboxypeptidases (EC 3.4.17.13) are named for their ability to cleave amide bonds between l- and d-amino acids, which occur naturally in bacterial peptidoglycan. They are specific for the link between meso-diaminopimelic acid and d-alanine and therefore degrade GlcNAc-MurNAc tetrapeptides to the corresponding tripeptides. As only the tripeptides can be reused as peptidoglycan building blocks, ld-carboxypeptidases are thought to play a role in peptidoglycan recycling. Despite the pharmaceutical interest in peptidoglycan biosynthesis, the fold and catalytic type of ld-carboxypeptidases are unknown. Here, we show that a previously uncharacterized open reading frame in Pseudomonas aeruginosa has ld-carboxypeptidase activity and present the crystal structure of this enzyme. The structure shows that the enzyme consists of an N-terminal beta-sheet and a C-terminal beta-barrel domain. At the interface of the two domains, Ser(115) adopts a highly strained conformation in the context of a strand-turn-helix motif that is similar to the "nucleophilic elbow" in alphabeta-hydrolases. Ser(115) is hydrogen-bonded to a histidine residue, which is oriented by a glutamate residue. All three residues, which occur in the order Ser-Glu-His in the amino acid sequence, are strictly conserved in naturally occurring ld-carboxypeptidases and cannot be mutated to alanines without loss of activity. We conclude that ld-carboxypeptidases are serine peptidases with Ser-His-Glu catalytic triads.