Site-directed mutation studies on Rcd-1 and hRcd-1 ΔNC. An approximate twofold axis runs nearly vertically in the plane of the paper intersecting both the Mn 2+ ion (gray sphere) and water molecule (red sphere). Residues from one of the dimers make up the left side of the image, while residues from the other dimer make up the right side of the image. The electron density is shown in light gray. The residue type and position from adjacent monomers are labeled. ( B) Stereo diagram showing the electron density (2 F o- F c) around the Mn 2+ ion found at the interface between the dimers within the unit cell a 3σ cutoff is used to emphasize the metal density and tight coordination of the liganding atoms. Individual arm three-helix repeats in each monomer are identified by a different color. Monomer A is colored from dark red to indigo (for helices), while monomer B is colored from dark blue to dark green. The twofold axis runs approximately horizontal to the page. Rcd-1's ability to bind to nucleic acids, in addition to the previously reported protein-protein interaction with NOT1, suggests a new feature in Rcd-1's role in regulation of overall cellular differentiation processes. Mutation of an arginine residue within the cleft strongly reduced or abolished oligonucleotide binding. Prompted by this finding, we established that Rcd-1 can bind to single- and double-stranded oligonucleotides in vitro with the affinity of G/C/T > A. The monomer is made up of six armadillo repeats forming a solvent-accessible, positively-charged cleft 21-22 A wide that, in contrast to other armadillo proteins, stays fully exposed in the dimer. Here, we present the 2.2 A X-ray structure of the highly conserved region of human Rcd-1 and investigate possible functional abilities of this and the full-length protein. Mammalian homologs are involved in various cellular differentiation processes including retinoic acid-induced differentiation and hematopoetic cell development. Rcd-1, a protein highly conserved across eukaryotes, was initially identified as a factor essential for nitrogen starvation-invoked differentiation in fission yeast, and its Saccharomyces cerevisiae homolog, CAF40, has been identified as part of the CCR4-NOT transcription complex, where it interacts with the NOT1 protein.
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