Bernard A. Brown

Proteolytic dissection of Zab, the Z-DNA-binding domain of human ADAR1

Zα is a peptide motif that binds to Z-DNA with high affinity. This motif binds to alternating dC-dG sequences stabilized in the Z-conformation by means of bromination or supercoiling, but not to B-DNA. Zα is part of the N-terminal region of double-stranded RNA adenosine deaminase (ADAR1) , a candidate enzyme for nuclear pre-mRNA editing in mammals. Zα is conserved in ADAR1 from many species; in each case, there is a second similar motif,Zβ, separated from Zα by a more divergent linker. To investigate the structure-function relationship ofZα, its domain structure was studied by limited proteolysis. Proteolytic profiles indicated that Zα is part of a domain, Zab, of 229 amino acids (residues 133–361 in human ADAR1). This domain contains both Zα and Zβas well as a tandem repeat of a 49-amino acid linker module. Prolonged proteolysis revealed a minimal core domain of 77 amino acids (positions 133–209), containing only Zα, which is sufficient to bind left-handed Z-DNA; however, the substrate binding is strikingly different from that of Zab. The second motif, Zβ, retains its structural integrity only in the context of Zab and does not bind Z-DNA as a separate entity. These results suggest that Zαand Zβ act as a single bipartite domain. In the presence of substrate DNA, Zab becomes more resistant to proteases, suggesting that it adopts a more rigid structure when bound to its substrate, possibly with conformational changes in parts of the protein.

Structurally Conserved Nop56/58 N-terminal Domain Facilitates Archaeal Box C/D Ribonucleoprotein-guided Methyltransferase Activity

Background: Box C/D RNPs direct site-specific 2′-O-methylation of rRNA. Results: The Nop56/58 and fibrillarin core proteins establish a very stable dimer with Nop56/58 contributing to methyltransferase activity. Conclusion: The Nop56/58 core protein plays a role not only in RNP assembly, but also methyltransferase activity. Significance: Our observations reveal a novel role for the Nop56/58 core protein in box C/D RNP function. Box C/D RNA-protein complexes (RNPs) guide the 2′-O-methylation of nucleotides in both archaeal and eukaryotic ribosomal RNAs. The archaeal box C/D and C′/D′ RNP subcomplexes are each assembled with three sRNP core proteins. The archaeal Nop56/58 core protein mediates crucial protein-protein interactions required for both sRNP assembly and the methyltransferase reaction by bridging the L7Ae and fibrillarin core proteins. The interaction of Methanocaldococcus jannaschii (Mj) Nop56/58 with the methyltransferase fibrillarin has been investigated using site-directed mutagenesis of specific amino acids in the N-terminal domain of Nop56/58 that interacts with fibrillarin. Extensive mutagenesis revealed an unusually strong Nop56/58-fibrillarin interaction. Only deletion of the NTD itself prevented dimerization with fibrillarin. The extreme stability of the Nop56/58-fibrillarin heterodimer was confirmed in both chemical and thermal denaturation analyses. However, mutations that did not affect Nop56/58 binding to fibrillarin or sRNP assembly nevertheless disrupted sRNP-guided nucleotide modification, revealing a role for Nop56/58 in methyltransferase activity. This conclusion was supported with the cross-linking of Nop56/58 to the target RNA substrate. The Mj Nop56/58 NTD was further characterized by solving its three-dimensional crystal structure to a resolution of 1.7 Å. Despite low primary sequence conservation among the archaeal Nop56/58 homologs, the overall structure of the archaeal NTD domain is very well conserved. In conclusion, the archaeal Nop56/58 NTD exhibits a conserved domain structure whose exceptionally stable interaction with fibrillarin plays a role in both RNP assembly and methyltransferase activity.

Structure of the Aeropyrum pernix L7Ae multifunctional protein and insight into its extreme thermostability

Archaeal ribosomal protein L7Ae is a multifunctional RNA-binding protein that directs post-transcriptional modification of archaeal RNAs. The L7Ae protein from Aeropyrum pernix (Ap L7Ae), a member of the Crenarchaea, was found to have an extremely high melting temperature (>383 K). The crystal structure of Ap L7Ae has been determined to a resolution of 1.56 Å. The structure of Ap L7Ae was compared with the structures of two homologs: hyperthermophilic Methanocaldococcus jannaschii L7Ae and the mesophilic counterpart mammalian 15.5 kD protein. The primary stabilizing feature in the Ap L7Ae protein appears to be the large number of ion pairs and extensive ion-pair network that connects secondary-structural elements. To our knowledge, Ap L7Ae is among the most thermostable single-domain monomeric proteins presently observed.

Crystallization of the Zα domain of the human editing enzyme ADAR1 complexed with a DNA–­RNA chimeric oligonucleotide in the left-­handed Z-conformation

The Zalpha domain of human double-stranded RNA adenosine deaminase (ADAR1) has been crystallized with a hexanucleotide containing alternating deoxyribose and ribose furanose sugars. Solution circular dichroism experiments show that this double-stranded chimera (dCrG)(3) initially adopts the right-handed A-conformation. However, addition of stoichiometric amounts of Zalpha causes a rapid transition to the Z-conformation. Raman spectroscopy of crystals of the Zalpha-(dCrG)(3) complex confirm that the chimeric oligonucleotide is stabilized in the Z-conformation. A complete data set has been obtained at 2.5 A resolution. The Zalpha-(dCrG)(3) crystals belong to the tetragonal I422 space group, with unit-cell parameters a = b = 104.2, c = 117.6 A. Work is under way to solve the structure by molecular replacement.