The structure of the C4C4 RING finger of human NOT4 reveals features distinct from those of C3HC4 RING fingers

The NOT4 protein is a component of the CCR4.NOT complex, a global regulator of RNA polymerase II transcription. Human NOT4 (hNOT4) contains a RING fin ger motif of the C4C4 type. We expressed and purified the N-terminal region of hNOT4 (residues 1-78) encompassing the RING finger motif and determined the solution structure by heteronuclear NMR. MMR experiments using a Cd-11 3-substituted hNOT4 RING finger showed that two metal ions are bound throug h cysteine residues in a cross-brace manner. The three-dimensional structur e of the hNOT4 RING finger was refined with root mean square deviation valu es of 0.58 +/- 0.13 Angstrom for the backbone atoms and 1.08 +/- 0.12 Angst rom for heavy atoms. The hNOT4 RING finger consists of an cw-helix and thre e long loops that are stabilized by zinc coordination. The overall folding of the hNOT4 RING finger is similar to that of the C3HC4 RING fingers. The relative orientation of the two zinc-chelating loops and the alpha -helix i s well conserved. However, for the other regions, the secondary structural elements are distinct.