Cwc2 is the only NTC-associated protein that has known RNA binding domains. RRM and ZnF are vital in co-ordination of the conformational changes that are involved during that activation of the B complex (see Introduction).
The two domains form a single folding unit, stabilised through extensive hydrophobic interactions and hydrogen bonding. Both RRM and ZnF are the most studied motifs in eukaryotic cells. A pull-down assay revealed a Phe194 residue of the α-helix 10 primarily mediates the interactions between the RRM and ZnF domain.
The two domains form a single folding unit, stabilised through extensive hydrophobic interactions and hydrogen bonding. Both RRM and ZnF are the most studied motifs in eukaryotic cells. A pull-down assay revealed a Phe194 residue of the α-helix 10 primarily mediates the interactions between the RRM and ZnF domain.
Figure 6: Crystal tertiary structure of the single folding unit, taken from Lu et al ( 2012)
Figure 7: The secondary structure of Cwc2, taken from PDB
Structural features of the RRM domain:
The RRM binding domain is one of two RNA binding domains. It has a canonical βαββαβ topology, consisting of residues 134-226, which takes up a total of 40% of the total Cwc2 structure:
- Four characteristic beta sheets packed against two alpha helices. The β-strands contain positively charged residues, essential for their binding function.
- There are two ‘insert’ regions of variable size and sequence, both highlighted in the crystal structure. The red region represents an insert region that encompasses α -helix 7, whereas the orange region represents that second insert domain encompassing a 310 α -helix 5.
- RRM contains two well conserved domains ( RNP1, RNP2 ) which are also found in the Rbm22 homologous protein. The axis running between these domains is found to be the central region for RNA binding. The aromatic residues highlighted, F183 and Y138 aid in this binding through stacking interactions. In addition, a positively charged residue, Lys 179, is located in close proximity to RNP1, which is required for hydrogen bonding. These residues involved in the RNA-binding interactions are exposed to the external solvent, further confirming their role with external interactions.
Figure 8: Illustrates the crystal structure of Cwc2ΔC. The main structural aspects are highlighted.
Figure 9: A 3-dimensional view of the RRM domain. The coloured domains correspond to the same domains highlighted in Figure 10. The aromatic residues have been labelled.
Structural features of the ZnF domain:
Zinc finger domains are relatively small protein motifs which contain multiple finger-like protrusions essential for creating vital interactions. This ZnF domain adopts the common CCCH circular conformation of the CX7CX5CX3H type:
- Seven short α-helices
- There are three cysteine residues and one histidine residue co-ordinating a single Zn atom, in a tetrahedral conformation. A short α-helix 4 is located between first and second co-ordinating cysteine residue and a 310 α-helix 3 is connecting the second and third cysteine residues.
Figure 10: The crystal structure of the ZnF domain. Taken from Lu et al. (2010)
- The flanking regions of the ZnF domain adopt the conformation of a protruding loop containing a short α-helix 6. This protrusion is only found in one of the two molecules of an asymmetric unit, which is possibly to enhance the molecules flexibility.
- This loop conformation is probably stabilised through salt bridge interactions between neighbouring residues E122, D123, and K224 found in the β -sheet 4 of the symmetrically related molecule of this asymmetric unit. Hydrophobic contacts also have a part to play in the stabilisation of this particular conformation.
- A shorter crystal structure exhibits same fold and topography but does not contain the loop, suggesting that this structure is more important to function, rather than creation and stabilisation of the conformational fold of the tertiary structure.
Figure 11: Illustrating the salt bridge interactions between specific, conserved residues. Taken from Schmitzova et al.
Figure 12: A 3-dimensional view of the ZnF domain. The loop region is visible as the extended purple region. The yellow areas are the E122, D123 residues which are labelled. The cysteine and histidine residues are also labelled. The short α-helix 4 and 310 α-helix 3 are represented as orange and light blue respectively.
Homologous domains of ZnF:
ZnF domain adopts a similar structure to the typical ZnF domains found in TIS11d protein, which contains a tandem zinc finger (TZF) domain. This protein specifically binds to the 3' untranslated region of target mRNAs and promotes their de-adenylation and degradation.
MBNL1 similarly contains tandem CCCH zinc-finger (ZnF) domains, and these Muscleblind-like (MBNL) proteins specifically target pre-mRNAs containing YGCU(U/G)Y sequence elements to regulate programmed alternative splicing:
Figure 11: A Superposition of the ZnF domain of MBNL1 (purple) on to Cwc2 (blue) .
