The Structure of the C4C4 RING Finger of Human NOT4 Reveals Features Distinct from those of C3HC4 RING Fingers

Abstract

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 finger 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. NMR experiments using a 113Cd-substituted hNOT4 RING finger showed that two metal ions are bound through cysteine residues in a cross-brace manner. The three-dimensional structure of the hNOT4 RING finger was refined with root mean square deviation values of 0.58 ± 0.13 Å for the backbone atoms and 1.08 ± 0.12 A for heavy atoms. The hNOT4 RING finger consists of an α-helix and three long loops that are stabilized by zinc coordination. The overall folding of the hNOT4 RING finger is similar to that of the C 3HC4 RING fingers. The relative orientation of the two zinc-chelating loops and the α-helix is well conserved. However, for the other regions, the secondary structural elements are distinct.