Magainins are antimicrobial peptides that selectively disrupt bacterial cell membranes. In an effort to determine the propensity for oligomerization of specific highly active magainin analogues in membrane mimetic systems, we studied the structures and lipid interactions of two synthetic variants of magainins (MSI-78 and MSI-594) originally designed by Genaera Corp. Using NMR experiments on these peptides solubilized in dodecylphosphocholine (DPC) micelles, we found that the first analogue, MSI-78, forms an antiparallel dimer with a "phenylalanine zipper" holding together two highly helical protomers, whereas the second analogue, MSI-594, whose phenylalanines 12 and 16 were changed into glycine and valine, respectively, does not dimerize under our experimental conditions. In addition, magic angle spinning solid-state NMR experiments carried out on multilamellar vesicles were used to corroborate the helical conformation of the peptides found in detergent micelles and support the existence of a more compact structure for MSI-78 and a pronounced conformational heterogeneity for MSI-594. Since magainin activity is modulated by oligomerization within the membrane bilayers, this study represents a step forward in understanding the role of self-association in determining magainin function.

Methylation of CpG dinucleotides in DNA is a common epigenetic modification in eukaryotes that plays a central role in maintenance of genome stability, gene silencing, genomic imprinting, development, and disease. Kaiso, a bifunctional Cys 2 His 2 zinc finger protein implicated in tumor-cell proliferation, binds to both methylated CpG (mCpG) sites and a specific nonmethylated DNA motif (TCCTGCNA) and represses transcription by recruiting chromatin remodeling corepression machinery to target genes. Here we report structures of the Kaiso zinc finger DNA-binding domain in complex with its nonmethylated, sequence-specific DNA target (KBS) and with a symmetrically methylated DNA sequence derived from the promoter region of E-cadherin. Recognition of specific bases in the major groove of the core KBS and mCpG sites is accomplished through both classical and methyl CH···O hydrogen-bonding interactions with residues in the first two zinc fingers, whereas residues in the C-terminal extension following the third zinc finger bind in the opposing minor groove and are required for high-affinity binding. The C-terminal region is disordered in the free protein and adopts an ordered structure upon binding to DNA. The structures of these Kaiso complexes provide insights into the mechanism by which a zinc finger protein can recognize mCpG sites as well as a specific, nonmethylated regulatory DNA sequence.protein-DNA interaction | NMR spectroscopy | X-ray crystallography | folding upon binding | intrinsic disorder I n eukaryotes, DNA methylation is a common epigenetic modification that is central to the maintenance of genome stability, gene silencing, genomic imprinting, development, and disease (1, 2). Methyl-CpG-binding proteins (MBPs) mediate these processes by binding to methylated DNA signals and recruiting chromatin remodeling corepressor complexes, resulting in compaction of chromatin into its transcriptionally inactive state (3). To date, three classes of MBPs that recognize 5-methyl cytosine (5mC) have been identified. The SRA domain family has specificity for hemimethylated sites and is required for maintenance methylation during DNA replication (4). In contrast, the methyl CpG-binding domain (MBD) and Kaiso families of MBPs function as essential mediators of epigenetically controlled gene silencing by recognizing symmetrically methylated CpG sites (5). Kaiso is a MBP belonging to the BTB/POZ (broad complex, tramtrak, bric à brac/pox virus and zinc finger) subfamily of transcription factors that recognize cognate DNA sequences through a C-terminal zinc finger domain; the N-terminal BTB/POZ domain mediates protein-protein interactions (6). Kaiso is a POZ protein that participates in both methyl-dependent and sequence-specific transcriptional repression, using its three Cys 2 His 2 zinc fingers to recognize either two consecutive symmetrically methylated CpG dinucleotides (mCpG) (7, 8) or a TCCTGCNA consensus (termed "KBS") (9). Although originally it was reported that Kaiso only requires zinc fingers 2 and 3 for DNA recog...

DNA methylation is an epigenetic mark that is essential for the development of mammals; it is frequently altered in diseases ranging from cancer to psychiatric disorders. The presence of DNA methylation attracts specialized methyl-DNA binding factors that can then recruit chromatin modifiers. These methyl-CpG binding proteins (MBPs) have key biological roles and can be classified into three structural families: methyl-CpG binding domain (MBD), zinc finger, and SET and RING finger-associated (SRA) domain. The structures of MBD and SRA proteins bound to methylated DNA have been previously determined and shown to exhibit two very different modes of methylated DNA recognition. The last piece of the puzzle has been recently revealed by the structural resolution of two different zinc finger proteins, Kaiso and ZFP57, in complex with methylated DNA. These structures show that the two methyl-CpG binding zinc finger proteins adopt differential methyl-CpG binding modes. Nonetheless, there are similarities with the MBD proteins suggesting some commonalities in methyl-CpG recognition across the various MBP domains. These fresh insights have consequences for the analysis of the many other zinc finger proteins present in the genome, and for the biology of methyl-CpG binding zinc finger proteins.

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