Nicotinamide adenine dinucleotides have emerged as key signals of the cellular redox state. Yet the structural basis for allosteric gene regulation by the ratio of reduced NADH to oxidized NAD(+) is poorly understood. A key sensor among Gram-positive bacteria, Rex represses alternative respiratory gene expression until a limited oxygen supply elevates the intracellular NADH:NAD(+) ratio. Here we investigate the molecular mechanism for NADH/NAD(+) sensing among Rex family members by determining structures of Thermus aquaticus Rex bound to (1) NAD(+), (2) DNA operator, and (3) without ligand. Comparison with the Rex/NADH complex reveals that NADH releases Rex from the DNA site following a 40 degrees closure between the dimeric subunits. Complementary site-directed mutagenesis experiments implicate highly conserved residues in NAD-responsive DNA-binding activity. These rare views of a redox sensor in action establish a means for slight differences in the nicotinamide charge, pucker, and orientation to signal the redox state of the cell.
Activation-induced deaminase (AID) uses base deamination for class-switch recombination and somatic hypermutation and is related to the mammalian RNA-editing enzyme apolipoprotein B editing catalytic subunit 1 (APOBEC-1). CDD1 is a yeast ortholog of APOBEC-1 that exhibits cytidine deaminase and RNA-editing activity. Here, we present the crystal structure of CDD1 at 2.0-Å resolution and its use in comparative modeling of APOBEC-1 and AID. The models explain dimerization and the need for trans-acting loops that contribute to active site formation. Substrate selectivity appears to be regulated by a central active site ''flap'' whose size and flexibility accommodate large substrates in contrast to deaminases of pyrimidine metabolism that bind only small nucleosides or free bases. Most importantly, the results suggested both AID and APOBEC-1 are equally likely to bind single-stranded DNA or RNA, which has implications for the identification of natural AID targets.A ntibody diversity is generated during B cell development through class-switch recombination (CSR) and somatic hypermutation (SHM) (1), and in many vertebrates by gene conversion (2). Expression of activation-induced deaminase (AID) is critical for all three processes (3-5) and ectopic AID expression was sufficient to activate SHM in B cell lines, hybridomas, and fibroblasts (6-9), gene conversion in B cells (4, 5), as well as CSR in fibroblast cell lines (10). Mutations in the AID protein have been detected in humans with hyper-IgM type 2 syndrome (11). Expression of AID in Escherichia coli caused deoxycytidine-todeoxyuridine deamination in actively transcribed host genes that was enhanced in strains deficient in the deoxyuridine repair enzyme DNA uracil N-glycosylase (12). DNA uracil N-glycosylase deficiency in mammals resulted in altered patterns of CSR and SHM (13,14). In vitro studies revealed that AID catalyzed deamination on single-stranded DNA at WRCY hot spots, but not on doublestranded DNA, RNA-DNA hybrids, or single-stranded RNA (15,16). Cytidine deamination on single-stranded DNA within transcription bubbles (17, 18) and the observation that transcription rates influenced SHM activity (17)(18)(19) suggested that the biological substrate for AID is DNA.In contrast, the homology of AID to the mRNA-editing enzyme APOBEC-1 (3), suggested AID might edit RNA (3). This idea was intriguing because edited mRNAs either encode novel proteins or lose the ability to express proteins (20,21). Consistent with expression of a novel protein from edited mRNA, de novo protein synthesis was required for CSR subsequent to AID activation (22).APOBEC-1 and AID have proven difficult to purify at levels sufficient for structural studies. CDD1 from Saccharomyces cerevisiae (ScCDD1) can be purified readily, which is relevant due to its orthology with APOBEC-1 at the level of both cytidine deaminase (CDA) sequence similarity (27%) and mRNA-editing activity on apolipoprotein B (apoB) substrates (23). We determined the crystal structure of ScCDD1 to 2.0 Å resolution, which ...
Bactofilins are novel cytoskeleton proteins that are widespread in Gram-negative bacteria. Myxococcus xanthus, an important predatory soil bacterium, possesses four bactofilins of which one, BacM (Mxan_7475) plays an important role in cell shape maintenance. Electron and fluorescence light microscopy, as well as studies using over-expressed, purified BacM, indicate that this protein polymerizes in vivo and in vitro into ~3 nm wide filaments that further associate into higher ordered fibers of about 10 nm. Here we use a multipronged approach combining secondary structure determination, molecular modeling, biochemistry, and genetics to identify and characterize critical molecular elements that enable BacM to polymerize. Our results indicate that the bactofilin-determining domain DUF583 folds into an extended β-sheet structure, and we hypothesize a left-handed β-helix with polymerization into 3 nm filaments primarily via patches of hydrophobic amino acid residues. These patches form the interface allowing head-to-tail polymerization during filament formation. Biochemical analyses of these processes show that folding and polymerization occur across a wide variety of conditions and even in the presence of chaotropic agents such as one molar urea. Together, these data suggest that bactofilins are comprised of a structure unique to cytoskeleton proteins, which enables robust polymerization.
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